Abstract
Under high-altitude hypoxia, neuroinflammation contributes to cognitive impairment, though the underlying mechanisms remain unclear. In this study, we established rat and astrocyte models of hypoxic exposure. We found that hypoxia induced significant alterations in blood biochemistry, widespread neuronal and glial damage, and impaired spatial learning and memory in rats, which were associated with the abnormal accumulation of p-Tau and Aβ. Hypoxia also triggered neuroinflammation, increasing the levels of inflammatory mediators and activating microglia and astrocytes. Targeted metabolomics and molecular analyses revealed disrupted oxidized lipid metabolism, including reduced synthesis of key metabolites such as arachidonic acid derivatives, accompanied by downregulation of cytochrome P450 (CYP450) expression. In vitro, hypoxia enhanced astrocyte inflammation, promoted Aβ/p-Tau accumulation, increased apoptosis, and suppressed CYP450. Inhibition of CYP450 (particularly epoxygenase) exacerbates hypoxia-induced inflammatory responses and promotes abnormal accumulation of cognition-related proteins by negatively regulating the NF-κB inflammatory signaling pathway. Furthermore, CYP450 downregulation was associated with DNA methylation changes. These findings highlight the role of DNA methylation-mediated CYP450 and oxidative lipid metabolic dysregulation in hypoxia-induced neuroinflammation and cognitive deficits, offering new insights for the development of neuroprotective strategies targeting the CYP450-oxidized lipid axis.
Similar content being viewed by others
Introduction
High-altitude environments are characterized by low oxygen levels. The reduction in oxygen partial pressure leads to decreased tissue oxygen utilization, which ultimately disrupts the body’s internal balance. Consequently, for travelers at high altitudes, mountaineers, and special military personnel in high-altitude regions, hypoxia is the primary source of physiological harm. High-altitude hypoxia can inflict considerable harm on the respiratory, cardiovascular, and circulatory systems, along with the central nervous system (CNS), with especially pronounced consequences. Although comprising merely 2% of total body weight, brain tissue utilizes up to 20% of the body’s total oxygen, rendering it highly susceptible to hypoxic conditions [1]. Exposure to mild hypoxia can induce a series of adverse neurological reactions. These initially manifest primarily as emotional disorders, such as anxiety and depression, accompanied by the activation of oxidative stress and neuroinflammatory responses. These changes can lead to alterations in synaptic plasticity and damage the microstructure of the brain, which can ultimately result in cognitive decline. As the severity of hypoxia increases or the exposure time is prolonged, these pathological changes may progress to high-altitude-specific brain injury. This can include high-altitude headache (HAH), acute mountain sickness (AMS), and, in severe cases, potentially fatal high-altitude cerebral edema (HACE) [2, 3].
The brain possesses a high concentration of polyunsaturated fatty acids (PUFAs), rendering brain tissue particularly vulnerable to lipid metabolism abnormalities in hypoxic settings, thereby facilitating the onset of hypoxic neurological impairment [4, 5]. Bioactive lipid mediators originating from the metabolism of arachidonic acid (AA) and associated PUFAs are termed oxidized lipids. These lipids are predominantly synthesized via three metabolic pathways: oxidation by cyclooxygenases (COX-1 and COX-2), yielding prostaglandins (PGs) and thromboxane compounds (TXs); oxidation by lipoxygenase (LOX), resulting in leukotrienes, lipoxins (LXs), and hydroxy-eicosatetraenoic acids (HETEs); and metabolism by cytochrome P450 (CYP450), producing eicosapentaenoic acids (EETs) and HETEs. Oxidized lipids exist throughout the body as free radicals. They influence cellular functions through autocrine or paracrine processes by binding to G protein-coupled receptors (GPCRs) or nuclear receptors located on cell membrane surfaces [6, 7]. These oxidized lipids can elicit a diverse array of biological consequences. As principal regulators of disease pathology and significant mediators of inflammatory responses, they regulate diverse activities, including sleep, memory, learning functions, neuroinflammatory responses, and neurodegenerative and neuropsychiatric diseases [8, 9]. Consequently, oxidized lipids are regarded as biomarkers that can clarify the phases of tissue damage and disease progression. CYP450 is a crucial catalytic enzyme in lipid oxidation synthesis whose expression is directly influenced by hypoxia. Its metabolites, including EETs and HETEs, have been demonstrated to play a role in regulating brain function under hypoxic conditions by modulating vascular tension and inflammatory responses [10, 11]. However, there is no direct evidence that the CYP450-oxidized lipid axis facilitates the cognitive impairment and neuroinflammation associated with high-altitude hypoxia.
Epigenetic regulation, particularly DNA methylation, may be a key link between hypoxic stress and the dysregulation of CYP450 expression. DNA methylation refers primarily to the process by which methyl groups are added to the C5 position of the cytosine ring within CpG dinucleotides via DNA methyltransferases. The promoter regions of several CYP450 genes, such as CYP1A2, CYP2E1, CYP2C9, CYP2C19, and CYP3A4, contain CpG island structures, making their expression highly susceptible to dynamic regulation by DNA methylation [12,13,14]. Recently, numerous studies have shown that hypoxic stress can significantly induce genome-wide reprogramming of DNA methylation, thereby affecting gene expression patterns. This epigenetic regulatory mechanism is important for the body’s adaptation to hypoxia [15, 16]. Notably, previous studies have confirmed the abnormal methylation of specific CYP450 promoter regions, such as that of CYP2S1, under hypoxic conditions [17]. Nonetheless, whether DNA methylation also affects the expression of additional CYP450 members under hypoxic conditions remains to be further examined.
This study employed ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technology combined with animal behavioral assessment and epigenetic detection to systematically investigate the cascading regulatory mechanism of the DNA methylation-CYP450-oxidized lipid axis in hypoxia-induced brain injury on the basis of the above scientific questions. These findings not only bridge a knowledge gap regarding the molecular processes of brain damage caused by high-altitude hypoxic environments but also provide a crucial theoretical foundation for the development of neuroprotective treatments aimed at the CYP450-oxidized lipid pathway.
Materials and methods
Chemicals and reagents
All oxidized lipid standards were purchased from Cayman Chemical (Ann Arbor, MI, USA). Enzyme-linked immunosorbent assay (ELISA) kits for HIF-1α, IL-6, NF-κB, iNOS, TNF-α, IL-1β, and 5-mC were obtained from Shanghai Kexing Trading (Shanghai, China). The following primary antibodies were used: anti-amyloid-β42 polyclonal antibody (Aβ) (Immunoway, Cat: YT0226, Newark, DE, USA); polyclonal total Tau antibody (Immunoway, Cat: YT4546, Newark, DE, USA); polyclonal phospho-Tau (p-Tau) (Bioss, Cat: bs-3489R, Beijing, China); monoclonal ionophore-binding protein 1 (IBA1) (zenbio, Cat: R382207, Chengdu, China); glial fibrillary acidic protein (GFAP) (Servicebio, Cat: GB11096, Wuhan, China); polyclonal β-actin (Immunoway, Cat: YT0099, Newark, DE, USA); polyclonal CYP2C23 (Proteintech, Cat: 16546-1-AP, Wuhan, China); polyclonal CYP2J3 (Proteintech, Cat: 13562-1-AP, Wuhan, China); polyclonal CYP2C11 (Biorbyt, Cat: Orb5951, Cambridge, UK); polyclonal CYP4F1 (Biorbyt, Cat: Orb214800, Cambridge, UK); monoclonal CYP2C22 (Abcam, Cat: Ab137015, Cambridge, UK); and monoclonal CYP4A2 (Abcam, Cat: Ab140635, Cambridge, UK). The reactive oxygen species (ROS) assay kit (Cat: 040-1000T) was purchased from Fluorescence (Beijing, China). The RNA extraction kit (Cat: R30922), ReverTra Ace qPCR RT Master Mix (Cat: Q20620), and TransScript One-Step gDNA Removal and cDNA Synthesis kit (Cat: R10905) were purchased from TransGen Biotech (Beijing, China). An Annexin V-FITC/PI cell apoptosis detection kit (Cat: G1511) was purchased from Servicebio (Wuhan, China). A rapid DNA extraction kit (Cat: B518221) was purchased from Sangon Biotech (Shanghai, China). The EZ DNA Methylation-Gold™ kit (Cat: D5005) was purchased from ZYMO RESEARCH (CA, USA). The 17-ODYA (Cat: HY-101016), MS-PPOH (Cat: HY-114759), and 5-azacitidine-2’-deoxycytidine (5-Aza-dC) (Cat: HY-A0004) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).
Treatment of animals
Male Sprague-Dawley rats, with a body weight range of 180–220 g and an age range of 6–8 weeks, were procured from the Laboratory Animal Center of Xi’an Jiaotong University Medical College (License No. SCXK (Shaanxi) 2023-002). All the rats were housed in separate rooms per cage with a constant temperature (22 ± 2 °C), constant humidity (55 ± 10%), and a 12 h light/12 h dark cycle. All experimental procedures were performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the protocol was approved by the Animal Ethics Committee of Qinghai University (Approval No. PJ-202302-12).
Rats were randomly divided into four groups: low altitude control group (LAC), high altitude hypoxia for 7 days group (HAH-7), high altitude hypoxia for 30 days group (HAH-30), and high-altitude hypoxia for 90 days group (HAH-90). The rats in the LAC group were housed in Xi’an City, Shaanxi Province, China (altitude 390 m, PaO2 20.2 kPa). The rats in the HAH-7, HAH-30, and HAH-90 groups were transported by air from Xi’an City to Xining City in Qinghai Province, China. They were then transported by car to Maduo County in the Guoluo Tibetan Autonomous Prefecture of Qinghai Province (altitude 4300 m, PaO2 12.4 kPa), with a total transit time of 10 h. The rats in the HAH-7, HAH-30, and HAH-90 groups were subjected to a hypoxic exposure period of 7, 30, or 90 days after arriving in Maduo County. The samples were promptly frozen and preserved in liquid nitrogen after post-collection before being dispatched to the Plateau Medicine Research Center at Qinghai University for analysis.
Morris water maze (MWM) experiment
The MWM apparatus is a circular pool with a height of 50 cm, a diameter of 180 cm, and a depth of 30 cm. The water temperature in the MWM test was maintained at 22 ± 1 °C. The pool was partitioned into four equal quadrants, featuring a circular platform with a diameter of 10 cm positioned at the center of quadrant 2 and submerged 2 cm beneath the water surface. The pool was surrounded by sufficient visual cues to serve as references. The actions of the rats in the water maze were recorded via the BAS-100 animal behavioral experiment analysis system (TECHMAN, Chengdu, China). Acquisitive training was conducted for 5 days, 4 times per day, in which the rats were sequentially placed into the water from the first, second, third, and fourth quadrants facing the wall of the pool, and the time it took for the rats to find a safe platform was recorded; if the rats did not find the platform within 2 min, they were guided to the platform for 20 s. The day after the final acquisition training session, the platform was removed and the rats were allowed to explore freely. The rats were placed in the water from the opposite side to the original location of the platform and were allowed to swim freely for 120 s (Fig. 3A). The time spent in the quadrant where the platform was located, the average swimming speed, the number of times the rats entered the platform area, and the path of movement were recorded to test the ability of the rat to memorize space.
Determination of physiological and biochemical indices
The rats in each group were anesthetized via an intraperitoneal injection of 20% urethane (1 g/kg) before blood collection. 1 mL of whole blood was drawn from the main abdominal vein into an anticoagulant tube containing EDTA-K2, and the following routine blood parameters were measured via an XN-10 automatic hematology analyzer (Sysmex Corporation, Tokyo, Japan): red blood cell count (RBC), hemoglobin (HGB), white blood cell count (WBC), platelet count (PLT), hematocrit (HCT), mean corpuscular volume (MCV), and mean platelet volume (MPV). 2 mL of whole blood was centrifuged in a tube without anticoagulant and centrifuged, and the serum was extracted. The following blood biochemical parameters were measured via an AU5800 automatic biochemistry analyzer (Olympus Corporation, Tokyo, Japan): alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), lactate dehydrogenase (LDH), total bilirubin (TBIL), globulin (GLOB), uric acid (UA), creatinine (CREA), glucose (GLU), cholesterol (CHOL), and triglycerides (TG).
Hematoxylin-Eosin (HE) staining
The rats were anesthetized via the intraperitoneal injection of 20% urethane (1 g/kg). The abdominal cavity of each animal was exposed with surgical scissors and forceps to reveal the heart. A needle was inserted into the right atrium, and the sinusoidal vein was clamped with arterial forceps. The animals were then perfused with saline (for approximately 30 min) until their livers turned white and then with 4% paraformaldehyde until their livers hardened and their tails stiffened. After complete fixation, the head of the rat was cut off, and the brain was removed. The rat brains were soaked in 4% paraformaldehyde for 1 day, then dehydrated and paraffin-embedded. Coronal sections (5 μm) of the hippocampal region were stained with hematoxylin and eosin and placed under a Pannoramic 250 digital section scanner (3DHISTECH, Hungary) for image acquisition.
Nissl staining
Rat brain tissues fixed with 4% paraformaldehyde were taken, paraffin-embedded, and sectioned in the hippocampal region. The sections were stained with 1% toluidine blue at 56 °C for 20 min, soaked in 70% alcohol for 1 min, and differentiated in 95% alcohol until the positive expression was shown clearly. The tissues were subsequently dehydrated in 75%, 85%, 95%, and 100% ethanol, each for 1 min, and then blocked with a neutral resin after transparency was achieved using xylene. The sections were positioned under a Pannoramic 250 digital section scanner (3DHISTECH, Hungary) for image acquisition.
Transmission Electron Microscopy (TEM)
Samples prefixed with 3% glutaraldehyde were refixed with 1% osmium tetroxide, dehydrated in series with acetone, infiltrated with Epox 812 for a longer time, and embedded. The semithin sections were stained with methylene blue, and the ultrathin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate. The sections were examined with a JEM-1400FLASH transmission electron microscope (JEOL, Tokyo, Japan).
Immunohistochemical analysis
Rat brain tissue fixed with 4% paraformaldehyde was taken, paraffin-embedded, and sectioned in the cortical area. The sections were immersed in citrate buffer (pH 6.0) for antigen retrieval, subsequently followed by endogenous peroxidase blocking with 3% hydrogen peroxide at room temperature in the dark. Following three washes with PBS, the sections were blocked with bovine serum at room temperature for 20 min. The primary antibody was subsequently applied and incubated overnight at 4 °C. After being washed with PBS, the sections were incubated with the secondary antibody at 37 °C for 30 min and rewashed with PBS. DAB was used for chromogenic detection, and hematoxylin was used for restaining. The sections were dehydrated via a graded ethanol series, cleared in xylene, and subsequently mounted with neutral resin. Sections were subjected to 400× microscopic image acquisition using a BA400Digital microcamera system (Motic China Group Co., Ltd., Xiamen, China). Three animals were selected for each group, 1 slice per animal, and 3 non-overlapping fields of view within a specific area of the cortex were randomly selected by the system for photographing and analyzing. The percentage of positive area for p-Tau and Aβ, and the number of positive cells counted for IBA1 and GFAP, were analyzed using the Halo 101-WL-HALO-1 data analysis system (Indica Labs, Albuquerque, USA).
Targeted oxidized lipid metabolomics analysis
Each group of rats was euthanized by decapitation, and their brains were swiftly collected and stored at -80 °C. 50 mg of rat brain tissue was weighed accurately. After the addition of 600 µL of extract solution (80% methanol/H2O (v/v), precooled at -40 °C, containing an isotopically labeled internal standard mixture), homogenize and sonicate in an ice bath. Then, an aliquot of the supernatant was transferred to an EP tube, and water was added. After vortexing for 30 s, the sample was further purified with SPE. The SPE cartridges were equilibrated with 1 mL of MeOH and 1 mL of water. After loading a sample, the samples were eluted with MeOH, and then the eluent was evaporated to dryness under a gentle stream of nitrogen and reconstituted in 30% ACN/H2O (v/v). After vortexing the recombinant solution, homogenize it using ultrasound. After centrifugation, transfer the recombinant solution to an EP tube with a filter membrane and centrifuge again. Take the supernatant for UHPLC-MS/MS analysis.
The UHPLC separation was carried out using an ACQUITY Premier (Waters, Milford, Massachusetts, USA), equipped with a Waters ACQUITY UPLC BEH C18 column (150 × 2.1 mm, 1.7 μm, Waters). The mobile phase A was 0.01% formic acid in water, and the mobile phase B was 0.01% formic acid in acetonitrile. The column temperature was set at 50 °C. The autosampler temperature was set at 4 °C, and the injection volume was 10 µL.
A SCIEX Triple Quad™ 6500 + mass spectrometer (Sciex), equipped with an IonDrive Turbo V electrospray ionization (ESI) interface, was applied for assay development. Typical ion source parameters were as follows: curtain gas = 40 psi, ion spray voltage = -4500 V, temperature = 500 °C, ion source gas 1 = 30 psi, and ion source gas 2 = 30 psi. SCIEX Analyst Work Station Software (Version 1.6.3) and Multiquant 3.03 software were employed for MRM data acquisition and processing.
Isolation and culture of primary astrocytes
1-2-day-old SD rats were removed and sterilized with an alcohol spray. The heads were clipped, and the cerebral cortex was stripped into a small dish containing precooled dissection fluid. The vascular membrane was stripped, and the brains were preserved. The tissue was minced and digested with 0.25% EDTA-free trypsin. Then, a serum-containing medium was added to terminate digestion. The tissue was blown repeatedly with a pipette until dispersed. It was then filtered through a 70 μm filter membrane, centrifuged, and resuspended in DMEM/F12 medium containing 10% fetal bovine serum. Finally, it was inoculated at a density of 1 × 10⁶/mL into poly-lysine (1 µg/mL)-coated T25 culture flasks. The cells were cultured at 37 °C and 5% CO2, with the first full fluid change taking place after 24 h. Thereafter, the fluid was changed every 1–2 days, and the cells were cultured for approximately 7–10 days. After the astrocyte layer had become almost confluent, the cells were purified by placing the culture flasks in a constant-temperature shaker at 37 °C and oscillating them at 200 rpm for 48 h to remove microglia and other cells from the upper layer. The supernatant was discarded, and the culture medium was replaced with fresh medium after PBS washing to obtain high-purity astrocytes. These were identified by GFAP immunofluorescence and were > 95% pure. Purified astrocytes in good condition were used for subsequent experiments. The control group was placed in a normoxic incubator (21% O2), and the hypoxic group was placed in a triple-gas incubator (2% O2). They were treated for 3, 6, 12, 24, and 48 h, respectively.
Immunofluorescence analysis
The cell smears were fixed with 4% paraformaldehyde and then washed with a PBS buffer solution. The slides were incubated in a wet box with blocking serum at 37 °C for 60 min to prevent nonspecific binding. Following the blocking, the primary antibody was applied at the appropriate concentration, and the slides were subsequently incubated overnight at 4 °C. Following PBS washing, the fluorescently labeled secondary antibody was applied, and the slides were incubated at 37 °C in the dark for 1 h. Subsequently, another wash with PBS was conducted. DAPI solution was added dropwise, and the samples were incubated in the dark for 10 min to stain the nuclei. The samples were subsequently washed with PBS, mounted with glycerol, and observed and imaged immediately under a DM3000 fluorescence microscope (Leica, Germany).
ROS detection
Before loading the probe, the DCFH-DA storage solution was diluted with serum-free medium at a ratio of 1:1000 to prepare a DCFH-DA working solution with a final concentration of 10 µM. After aspirating the cells from the medium and washing them with PBS, the appropriate volume of the diluted DCFH-DA working solution was added, and the cells were incubated in a cell culture incubator at 37 °C and protected from light for 30 min. After the incubation period, the probe solution was aspirated and discarded. The cells were then washed with serum-free culture medium 1 ~ 2 times to adequately remove any DCFH-DA that had not entered the cells. The cells were then observed immediately under a DM3000 fluorescence microscope (Leica, Germany), and images were captured. The average fluorescence intensity of single cells was then measured using ImageJ software. After deducting the background, statistical analysis was performed between groups.
Flow cytometry
Following digestion with pancreatic enzymes, the mixture was centrifuged to isolate the cells. Following washing with PBS, the cells were resuspended in prechilled 1× binding buffer. The cell concentration was modified, and Annexin V-FITC and PI were added. The mixture was mixed gently and incubated at room temperature in the dark for 10 min. Subsequently, prechilled 1× binding buffer was added, and immediate analysis was conducted with a BeamCyte-1026 flow cytometer (BeamCyte Biotechnology, Changzhou, China).
ELISA
Rat brain cortical region tissue samples were minced and added to cold physiological saline, homogenized in an ice bath, and centrifuged at 3000 rpm for 10 min, after which the supernatant was collected. Astrocytes were centrifuged at 3000 rpm for 10 min to remove particles and polymers, and the supernatant was collected. The tissue homogenate and cell supernatant were then subjected to ELISA testing according to the manufacturer’s experimental instructions.
Western blot
Total protein was extracted from the brains of rats and astrocytes using RIPA lysis, and the bicinchoninic acid method was used for protein quantification. SDS-PAGE was used to separate the protein samples, which were subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was immersed in TBST containing 5% skim milk powder and blocked on a room temperature shaker for 2 h. The corresponding primary antibodies were diluted with the blocking solution, as followes: CYP2C23 (1:2000), CYP2C11 (1:1000), CYP2C22 (1:1000), CYP2J3 (1:2000), CYP4A2 (1:4000), CYP4F1 (1:500), and β-actin (1:1000), and the PVDF membrane was immersed in the primary antibody incubation solution and incubated at 4 °C overnight. The PVDF membrane was washed with TBST 5 times and incubated with appropriate secondary antibodies for 2 h at room temperature. The PVDF membrane was washed with TBST 5 times. The membranes were imaged using an Amersham Imager 600 ELC system (General Electric, Boston, USA).
Reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR)
Total RNA from rat brain tissue and astrocytes was extracted according to the kit instructions, and the purity of the RNA solution was checked using a NanoDrop 2000c spectrophotometer (Thermo, USA). The cDNA was synthesized by reverse transcription using the TransScript One-Step gDNA Removal and cDNA Synthesis Supermix Kit. The cDNA product from reverse transcription was amplified in three steps using a Roche Light Cycler 96 Real-Time Fluorescent Quantitative PCR Instrument (Roche, Switzerland), with the following reaction procedure: 94 °C for 30 s, followed by 94 °C for 5 s, 50‒60 °C for 15 s, and 72 °C for 10 s, and the lysis curve was added after 45 cycles. The relative expression of the target gene was expressed as the 2−ΔΔCt value of the target protein and β-actin. The amplification primers for the target and internal reference genes are listed in Table 1.
Bisulfite-Sequencing PCR (BSP)
DNA was extracted from rat brain tissue using a rapid DNA extraction kit. Using the MethPrimer online tool (http://www.urogene.org/methprimer/), CpG islands were predicted based on the rat CYP2C11 gene sequence, and BSP primers were designed. The upstream primer sequence was 5’-AATGTAGGTAATAAAAGTAAAATTTTAAG-3’ and the downstream primer sequence was 5’-ACAAAAACTCTAACTCCTCTTTCAAA-3’. The PCR amplification process was as follows: 95 °C predenaturation for 5 min, followed by 35 cycles of 94 °C denaturation for 30 s, 55 °C annealing for 30 s, and 72 °C extension for 40 s, with a final extension at 72 °C for 8 min. After amplification, the PCR products were purified by gel electrophoresis and ligated into the pUC18-T vector system. The ligation reaction was incubated overnight at 16 °C, followed by transformation into competent cells. The bacteria were cultured overnight at 37 °C on plates containing ampicillin that had been pre-coated with 100 mM IPTG and 20 mg/mL X-gal. PCR was performed using a single colony as a template. The resulting bands were then purified and recovered for first-generation sequencing. The obtained sequences were analyzed via the quantitative methylation analysis tool (QUMA; https://www.quma.cdb.riken.jp/).
Data analysis
Data were processed using SPSS 27.0 statistical software, and results were expressed as mean ± standard deviation (SD). Independent-samples t-tests were used to compare two groups. Differences among multiple groups were analyzed using one-way or two-way analysis of variance (ANOVA). For one-way ANOVA, intergroup comparisons were subsequently performed using Dunnett’s post hoc test. For the two-way ANOVA, after assessing main effects and their interaction, multiple comparisons were conducted using a Bonferroni correction. For the hidden platform training task of the MWM test, the escape latency was analyzed by repeated-measures ANOVA, based on the calculated total escape latency period. Intergroup comparisons were performed using one-way ANOVA and LSD tests. P < 0.05 indicates a statistically significant difference.
Results
High-altitude hypoxia induces neuroinflammation and cognitive impairment
Physiological and biochemical parameters and histomorphological changes in rats under high-altitude hypoxia
High-altitude hypoxia significantly affected the hematological and biochemical parameters in rats. A routine blood analysis indicated time-dependent variations in the HAH group relative to the LAC group. HGB, RBC, and HCT levels exhibited a gradual increase in the HAH-7, HAH-30, and HAH-90 groups, whereas WBC levels showed a consistent decline (P < 0.05). Furthermore, the MCV significantly decreased in the HAH-90 group (P < 0.05), whereas the MPV significantly increased in both the HAH-7 and HAH-90 groups (P < 0.05) (Fig. 1A). These alterations may be linked to oxidative stress induced by hypoxia, immune regulation, and erythropoiesis. The biochemical parameters indicated that, in comparison to the LAC group, the levels of ALP, GLU, and TG gradually decreased in the HAH-7, HAH-30, and HAH-90 groups (P < 0.05). In contrast, the level of CRP gradually increased. ALT, AST, and ALB significantly increased in the HAH-90 group (P < 0.05), whereas TP, GLOB, and LDH significantly increased in both the HAH-30 and HAH-90 groups (P < 0.05). CREA exhibited dynamic changes: a decrease was observed in the HAH-7 group (P < 0.05), whereas increases were noted in the HAH-30 and HAH-90 groups (P < 0.05) (Fig. 1B). Those findings suggest that high-altitude hypoxia impacts metabolic and organ functions in rats.
Changes in physiological and biochemical parameters in rats under high-altitude hypoxia. (A) Changes in the blood parameters of the rats. (B) Changes in the biochemical parameters of the rats. (LAC: low-altitude control group, HAH-7: high-altitude hypoxia 7-day group, HAH-30: high-altitude hypoxia 30-day group, HAH-90: high-altitude hypoxia 90-day group. n = 10. Data are shown as the mean ± SD. Statistical analyses were performed with one-way ANOVA with Dunnett’s multiple comparisons test. *P < 0.05 vs. LAC group)
The HE staining results indicated that the soft meningeal structure of the brain tissue from the LAC group remained intact and rich in blood vessels, exhibiting no significant inflammatory exudation. The cortical and hippocampal regions had dense and neatly arranged pyramidal cells. In the HAH group, as the duration of hypoxia increased, the cell bodies of dark-colored neurons in the cortical and hippocampal regions gradually decreased in size, their color gradually darkened, and their internal structures became blurred. Distinct axon-like structures were observed at the posterior region of the cell bodies (Fig. 2A). Further Nissl staining of neurons revealed that those in the hippocampus region of the brains of rats in the LAC group exhibited a similar morphology, with plump neurons arranged in a regular pattern and distributed uniformly. Their cytoplasm contained abundant tiger-striped bodies and granular Nissl bodies. In contrast, rats exposed to a high-altitude hypoxic environment exhibited a sparse distribution of neurons in brain tissue, altered morphology, and indistinct nuclear morphology. The number of Nissl bodies in surviving neurons was reduced. Furthermore, the extent of neuronal damage worsened as the duration of hypoxia increased. Nissl-positive neuronal counts indicated that, compared with the LAC group, the HAH-90 group exhibited a significant reduction in positive neurons (P < 0.05) (Fig. 2B). TEM revealed that the morphological structure of microglia and astrocytes in the hippocampus and cortical regions of the LAC group was normal and that the structure of neurons and synapses was intact. However, under high-altitude hypoxic conditions, significant pathological changes were observed in these cells: the perinuclear spaces of microglia were widened, their mitochondria were swollen with matrix dissolution and reduced electron density, and their rough endoplasmic reticulum was expanded with ribosomal detachment. Astrocytes exhibit similar mitochondrial damage, accompanied by increased autophagosomes and glial filaments. The neurons exhibited cell body shrinkage, widened perinuclear spaces, and abnormal chromatin. The synaptic structures were markedly abnormal, characterized by a reduced contact area between the presynaptic and postsynaptic membranes, thickening of the postsynaptic dense structures, blurred gaps, and fewer synaptic vesicles (Fig. 2C). These findings indicate that high-altitude hypoxia results in extensive damage to neurons and glial cells in brain tissue.
Histological changes in the rat brain under high-altitude hypoxia. (A) Rat brain tissue HE staining (400×). (B) Neuronal Nissl staining and counting results for rat hippocampal tissue (400×). (C) TEM analysis of the ultrastructures of the hippocampal and cortical regions (25000×). (LAC: low-altitude control group, HAH-7: high-altitude hypoxia 7-day group, HAH-30: high-altitude hypoxia 30-day group, HAH-90: high-altitude hypoxia 90-day group. n = 3. Data are shown as the mean ± SD. Statistical analyses were performed with one-way ANOVA with Dunnett’s multiple comparisons test)
High-altitude hypoxia induces learning and memory impairment in rats
We used the MWM to assess the effects of prolonged high-altitude hypoxia on cognitive function. Repeated-measures ANOVA revealed significant main effects of group and time on escape latency, but no significant interaction. Subsequent LSD tests, based on the significant group main effect, demonstrated that all hypoxic exposure groups (HAH-7, HAH-30, and HAH-90) exhibited significantly prolonged overall escape latency compared to the LAC group (P < 0.05) (Fig. 3B and F). In the probe test, compared with the LAC group, the HAH-30 and HAH-90 groups presented a significant reduction in the number of times they crossed the platform (P < 0.05), and the HAH-90 groups presented a significant reduction in the time spent in the target quadrant (P < 0.05). The average swimming speed of the hypoxic groups did not differ significantly from that of the LAC group (Fig. 3C, D and E, and G). These findings demonstrate that high-altitude hypoxia impairs learning and memory in rats, with longer exposure durations correlating with more pronounced effects on these cognitive functions.
Effects of high-altitude hypoxia on memory and learning ability in rats. (A) The procedure for the Morris water maze experiment. (B) The escape latency of rats in the training trials of the hidden platform test. (C) Number of times the rat crossed the platform in the probe test. (D) Relative time spent by the rats in the target quadrant in the probe test. (E) The average swimming speed of the rats in the probe test. (F) The representative search traces of rats in the hidden platform test. (G) The representative search traces of rats in the probe test. (H) Western blot analysis of p-Tau, total tau, and Aβ protein in the brain cortex of high-altitude hypoxic rats. (I) Immunohistochemical results of p-Tau and Aβ in the brain cortex of high-altitude hypoxic rats. (LAC: low-altitude control group, HAH-7: high-altitude hypoxia 7-day group, HAH-30: high-altitude hypoxia 30-day group, HAH-90: high-altitude hypoxia 90-day group. n = 3. Data (B-E, H, I) are shown as the mean ± SD. Statistical analyses in (C-E, H, I) were performed with one-way ANOVA with Dunnett’s multiple comparisons test; statistical analyses in (B) were performed with repeated-measures ANOVA with LSD test)
Aβ deposition and the hyperphosphorylation of tau proteins are known to be key molecular pathological events that lead to cognitive deficits. To explore the underlying pathological basis of these behavioral deficits, we examined the expression of the relevant proteins. Immunohistochemical results showed that the areas stained positively for p-Tau and Aβ were significantly increased in the brains of the HAH-7, HAH-30, and HAH-90 groups compared with the LAC group (P < 0.05) (Fig. 3H). Quantitative analysis by Western blot further confirmed that the levels of p-Tau and Aβ proteins were significantly elevated under high-altitude hypoxia (P < 0.05) and did not cause significant changes in total Tau protein levels (Fig. 3I). This suggests that the increase in p-Tau originated from a specific elevation in the level of its phosphorylation modification. These results imply that long-term exposure to high-altitude hypoxia not only induces time-dependent spatial memory impairment but also exacerbates elevated levels of Aβ and p-Tau proteins in the brain. This suggests that hypoxia may contribute to cognitive dysfunction by promoting the accumulation of Aβ and p-Tau proteins in the brain.
High-altitude hypoxia induces neuroinflammation in rats
This study examined the influence of hypoxia on neuroinflammation by quantifying the expression levels of hypoxia-inducible factor (HIF-1α), proinflammatory cytokines (IL-6, IL-1β, and TNF-α), and inflammatory regulatory factors (NF-κB and iNOS) in rat brain cortex. The findings indicated that, in contrast to those in the LAC group, the levels of HIF-1α in rat brain tissue were significantly elevated after exposure to a high-altitude hypoxic environment (P < 0.05), confirming the occurrence of hypoxic stress. Moreover, the levels of IL-1β, IL-6, TNF-α, NF-κB, and iNOS were dramatically increased (P < 0.05), with more pronounced alterations observed as the duration of hypoxia increased (Fig. 4A), indicating the activation of the inflammatory response under hypoxic conditions. To further investigate the regulatory role of hypoxia in neuroinflammation, immunohistochemistry was used to determine the expression of the microglial marker IBA1 and the astrocyte marker GFAP in brain tissue. The findings indicated that, in comparison with the LAC group, the HAH-30 and HAH-90 groups presented substantial increases in IBA1-positive cell counts (P < 0.05). The HAH-90 groups presented an increase in GFAP-positive cell counts (P < 0.05) (Fig. 4B). These results indicate that high-altitude hypoxia promotes the expression of inflammatory factors and the inflammatory activation of glial cells.
Effect of high-altitude hypoxia on the level of neuroinflammation in rats. (A) Expression levels of inflammatory factors in the brain cortex of high-altitude hypoxic rats. (B) Immunohistochemical results of IBA1 and GFAP in the brain cortex of high-altitude hypoxic rats. (LAC: low-altitude control group, HAH-7: high-altitude hypoxia 7-day group, HAH-30: high-altitude hypoxia 30-day group, HAH-90: high-altitude hypoxia 90-day group. n = 3. Data are shown as the mean ± SD. Statistical analyses were performed with one-way ANOVA with Dunnett’s multiple comparisons test. *P < 0.05 vs. LAC group)
High-altitude hypoxia induces lipid metabolism disorders and downregulates CYP450 expression in rat brain tissue
High-altitude hypoxia induces lipid metabolism disorders in rat brain tissue
To investigate the role of oxidized lipids in hypoxia-induced neuroinflammation and cognitive impairment, this study employed targeted metabolomics to systematically analyze the differential profiles of oxidized lipids in rat brain tissues. Principal component analysis (PCA) was used to preliminarily examine the metabolite levels in each sample. The results revealed that the levels of oxidized lipid metabolites were relatively similar within each group. Additionally, as the duration of hypoxia increased, the three groups of rats exposed to high-altitude hypoxia trended to shift to the right in the PCA plot (Fig. 5A). Further analysis using orthogonal partial least squares discriminant analysis (OPLS-DA) revealed no overlap between the LAC group and the HAH-30 and HAH-90 groups, with significant differences and clear distinctions (Fig. 5B). These findings suggest that the levels of oxidized lipid metabolites in the samples undergo regular changes with prolonged hypoxia, and these changes are sufficient to serve as biomarkers distinguishing normal from hypoxic states.
Effects of high-altitude hypoxia on oxidative lipid metabolites. (A) Score scatter plot for the PCA model. (B) Score scatter plot for the OPLS-DA model. (C) Heatmap of hierarchical clustering analysis results. (D) KEGG enrichment analysis of differential metabolites. (E) Effects of high-altitude hypoxia on the levels of oxidative lipid metabolites in the arachidonic acid pathway. (LAC: low-altitude control group, HAH-7: high-altitude hypoxia 7-day group, HAH-30: high-altitude hypoxia 30-day group, HAH-90: high-altitude hypoxia 90-day group, n = 5, *P < 0.05 vs. LAC group)
A total of 72 differentially expressed metabolites were detected through targeted oxidized lipidomes (Table 2), including ± 8-HDoHE, 9-OxoODE, ± 18-HETE, 8 S,15 S-DiHETE, and 16 S-HETE, which were the five metabolites co-upregulated in the HAH-7, HAH-30, and HAH-90 groups. There were 51 co-downregulated metabolites, including 12 S-HEPE, 15 S-HEPE, ± 5,6-DiHETrE, 13 S-HOTrE, 15-keto PGF1α, 8-iso PGF2α, 19 S-HETE, EPA, and 13,14-dihydro-15-keto PGD2, among others. Based on PUFA substrates, these metabolites can be grouped into 44 AA metabolites, 12 DHA metabolites, 7 LA metabolites, 4 DGLA metabolites, 3 EPA metabolites, and 2 ALA metabolites. Based on metabolic pathways, these metabolites can be classified into 20 CYP450 metabolites, 29 LOX metabolites, 19 COX metabolites, and 4 metabolites from other pathways.
Following bidirectional cluster analysis of the samples and metabolic products, the heatmap clearly revealed distinct color blocks clustered in different regions. This finding indicates that oxidative lipid metabolism disorders are present in rat models of hypoxia-induced cognitive impairment and neuroinflammation, as manifested by the widespread downregulation of most PUFA metabolites (Fig. 5C). Further KEGG pathway annotation of the measured metabolites revealed that the differentially metabolized oxidized lipids in rat brain tissue under hypoxia were enriched primarily in the AA metabolic pathway (Fig. 5D). Univariate statistical analysis of the metabolites identified 25 AA pathway metabolites with P < 0.05 in the U-test. These metabolites were categorized by metabolic pathway into 12 metabolites from the COX pathway, 5 from the LOX pathway, and 8 from the CYP450 pathway (Fig. 5E). The above results indicate that oxidative lipid metabolism in rat brain tissue is significantly disrupted in a high-altitude hypoxic environment, with products of the CYP450 metabolic pathway accounting for a large proportion of this disruption. As CYP450 plays a crucial role in lipid oxidation metabolism, changes in its expression or activity could directly impact lipid metabolic homeostasis. Therefore, we further investigated the effects of hypoxia on CYP450 expression.
High-altitude hypoxia reduces CYP450 expression in brain tissue
To further investigate the regulatory role of CYP450 in lipid metabolism disorders and brain injury under hypoxic conditions, this study measured the mRNA and protein expression levels of 4 key CYP450 epoxygenases (CYP2C23, CYP2C11, CYP2C19, and CYP2J3) and 2 key ω-hydroxylases (CYP4A2 and CYP4F1). RT-qPCR results revealed that the mRNA expression levels of CYP2C11, CYP2C22, CYP2J3, and CYP4A2 were significantly reduced in brain tissue of the HAH-7, HAH-30, and HAH-90 groups compared with the LAC group (P < 0.05)(Fig. 6A). The results of the Western blot analysis further validated the decrease in CYP450 protein expression levels in hypoxic environments. Compared with the LAC group, CYP2C23 protein expression levels were significantly reduced in the HAH-7, HAH-30, and HAH-90 groups (P < 0.05). In the HAH-90 group, CYP2C11 protein expression was also significantly reduced (P < 0.05). Meanwhile, CYP2C22 protein expression was significantly downregulated in both the HAH-7 and HAH-90 groups (P < 0.05) (Fig. 6B). These observations indicate that exposure to a high-altitude hypoxic environment markedly reduces CYP450 expression, potentially disrupting the lipid oxidation balance.
Effects of hypoxia on the expression of cytochrome P450 in rats. A mRNA expression of cytochrome P450. B Protein expression of cytochrome P450 (LAC: low-altitude control group, HAH-7: high-altitude hypoxia 7-day group, HAH-30: high-altitude hypoxia 30-day group, HAH-90: high-altitude hypoxia 90-day group. n = 3. Data are shown as the mean ± SD. Statistical analyses were performed with one-way ANOVA with Dunnett’s multiple comparisons test)
Hypoxic stress modulates the inflammatory responses of astrocytes through the downregulation of CYP450
Hypoxic stress promotes astrocyte dysfunction and downregulates CYP450
To systematically evaluate the neurotoxic effects of hypoxia in an in vitro model, we cultured astrocytes under normoxic (21% O2) and hypoxic (2% O2) conditions for 3, 6, 12, 24, and 48 h, respectively. Cell viability assays revealed that astrocyte viability decreased in a time-dependent manner following hypoxia treatment. Notably, after 24 h at 2% O2, the cell viability decreased to below 50% (Fig. 7A). ELISA studies revealed that the expression levels of IL-1β, IL-6, TNF-α, NF-κB, iNOS, and HIF-1α continued to increase with prolonged exposure to hypoxia (P < 0.05) (Fig. 7B). Immunofluorescence assays revealed that the expression levels of the cognitive-related proteins Aβ and p-Tau were significantly higher in the 2% O2 hypoxia group than in the normoxic group (P < 0.05) (Fig. 7C). Additionally, the flow cytometry results revealed that the apoptosis rate in the hypoxic group was significantly higher than that in the normoxic group (P < 0.05) (Fig. 7D).
Effects of hypoxia on astrocytes. A Effect of hypoxia on astrocyte survival. B Effect of hypoxia on inflammatory factor expression. C Effects of hypoxia on Aβ and p-Tau expression. D Effect of hypoxia on apoptosis. E Effect of hypoxia on CYP450 mRNA expression. F Effect of hypoxia on CYP450 protein expression. (n = 3. Data are shown as the mean ± SD. Statistical analyses were performed with one-way ANOVA with Dunnett’s multiple comparisons test. *P < 0.05 vs. normoxia group)
To investigate the impact of hypoxic stress on the expression of CYP450 in astrocytes, we measured the mRNA and protein expression levels of key CYP450 subtypes at various time points during hypoxia. RT-qPCR analysis revealed that compared with the normoxic group, CYP2C23 mRNA expression levels were significantly upregulated after 3 h of hypoxia (P < 0.05), followed by a sustained decline between 6 and 48 h (P < 0.05). CYP2C11 expression also exhibited a significant decreasing trend during the 6 to 48 h hypoxia period (P < 0.05). CYP2C22 was transiently upregulated at 6 h, followed by downregulation from 12 to 48 h (P < 0.05). CYP2J3, CYP4A2 (except for upregulation at 3 h), and CYP4F1 exhibited sustained downregulation throughout all durations of hypoxia (P < 0.05) (Fig. 7E). The Western blot results revealed consistent overall protein expression patterns but with temporal differences: the CYP2C23 protein level decreased from 24 to 48 h (P < 0.05); both the CYP2C11 and CYP2J3 levels progressively decreased (from 6 to 48 h and from 3 to 48 h, respectively) (P < 0.05), while the CYP4A2 and CYP4F1 levels demonstrated late-phase suppression (from 24 to 48 h and from 12 to 48 h, respectively) (P < 0.05) (Fig. 7F). These results indicate that the expression of apoptosis, inflammatory factors, and cognition-related proteins significantly increases in a time-dependent manner with prolonged hypoxia, whereas CYP450 expression gradually declines. These findings correspond with those observed in an in vivo study. Notably, the survival rate of astrocytes was observed to be less than 50% under 2% O2 hypoxia for 24 h, with all indicators being substantial. Therefore, the subsequent hypoxia group was exposed to 2% O2 hypoxia for 24 h to simulate moderate to severe hypoxia damage.
Inhibition of CYP450 increases astrocyte inflammation and abnormal accumulation of Aβ/p-Tau under hypoxic conditions
To further investigate the role of CYP450 in neuroinflammation and cognitive impairment, we employed a specific inhibitor intervention strategy to systematically assess the impact of CYP450 on cellular inflammatory responses and cognition-related protein expression by inhibiting the activity of CYP450 epoxygenase (MS-PPOH) and ω-hydroxylase (17-ODYA). According to the literature data and toxicity experiments on cell proliferation (Fig. 8A), the concentrations of MS-PPOH and 17-ODYA employed in the experiments were established at 20 µM and 25 µM, respectively. Compared with the normoxia control group, the mRNA and protein expression levels of 6 CYP450 enzymes were significantly lower in the hypoxic group. Further analysis of the effects of two inhibitors under hypoxia conditions revealed that MS-PPOH selectively reduced the mRNA and protein expression levels of CYP2C23, CYP2C11, CYP2C22, and CYP2J3 (P < 0.05), with no significant changes observed in the expression levels of CYP4A2 and CYP4F1. 17-ODYA specifically reduced the mRNA and protein expression levels of CYP4A2 and CYP4F1 (P < 0.05), while the mRNA and protein expression levels of CYP450 epoxidase showed no significant change. These findings indicate that MS-PPOH exclusively inhibits CYP450 epoxygenase under hypoxic conditions, whereas 17-ODYA primarily inhibits ω-hydroxylase (Fig. 8B and C).
The effect of CYP450 inhibition on astrocytes under hypoxic conditions. (A) Reproductive toxicity testing of MS-PPOH and 17-ODYA on astrocytes. (B) Changes in CYP450 mRNA expression after CYP450 inhibition. (C) Changes in CYP450 protein expression after CYP450 inhibition. (D) Changes in inflammatory factor expression after CYP450 inhibition. (E) Immunofluorescence staining of Aβ, p-Tau, GFAP, and ROS expression following CYP450 inhibition (400×; green: Aβ/p-Tau/GFAP/ROS; blue: DAPI). (F) Western blot analysis of Aβ, p-Tau, and total Tau protein expression after CYP450 inhibition. (G) Changes in apoptosis after CYP450 inhibition. (n = 3. Data are shown as the mean ± SD. Statistical analyses were performed with two-way ANOVA with the Bonferroni multiple comparisons test. Significance: *P < 0.05)
Results from inflammatory factor assays revealed that hypoxia treatment significantly increased the expression levels of IL-1β, IL-6, TNF-α, NF-κB, iNOS, and HIF-1α in astrocytes (P < 0.05). Notably, intervention with either 17-ODYA or MS-PPOH further exacerbated the trend of increased expression of all these inflammatory factors under hypoxic conditions (P < 0.05) (Fig. 8D). Immunofluorescence analysis revealed that Aβ and p-Tau levels were significantly higher in the hypoxic group (P < 0.05), and treatment with 17-ODYA and MS-PPOH exacerbated the immunofluorescence intensity of both (P < 0.05) (Fig. 8E). The quantitative analysis of Western blots for Aβ and p-Tau proteins was consistent with the results of the immunofluorescence assay. Furthermore, although total Tau protein levels showed no significant change, the p-Tau/total Tau ratio increased significantly under hypoxic conditions (P < 0.05) (Fig. 8F). To comprehensively assess the effects of hypoxia on oxidative stress and neuroinflammation, we also examined the levels of ROS and GFAP. The results revealed that the immunofluorescence intensity of both ROS and GFAP was increased in the hypoxia group and that intervention with 17-ODYA and MS-PPOH exacerbated this increase further (P < 0.05) (Fig. 8E). Apoptosis analysis confirmed that both inhibitor treatments resulted in higher apoptosis rates compared to the hypoxia-only group (Fig. 8G). Notably, MS-PPOH had a greater inhibitory effect on inflammatory factors (IL-1β, TNF-α, NF-κB, and HIF-1α), cognitive-related proteins (Aβ and p-Tau), and apoptosis than did 17-ODYA. These findings suggest that reduced CYP450 epoxygenase activity is a key mechanism underlying hypoxia-induced astrocyte inflammation, abnormal Aβ and p-Tau accumulation, and increased apoptosis.
CYP450 inhibition exacerbates hypoxic-induced astrocyte injury by modulating the NF-κB inflammatory signaling pathway
To investigate whether CYP450 epoxygenase exerts its effects by modulating specific neuroprotective anti-inflammatory pathways, we employed the NF-κB inhibitor BAY 11-7082 to block this key inflammatory signaling pathway and combined it with the CYP450 inhibitor MS-PPOH. The results showed that BAY 11-7082 treatment effectively reversed hypoxic injury, significantly reducing the levels of NF-κB and its downstream factors (IL-1β, IL-6, TNF-α, iNOS) as well as HIF-1α, and decreasing Aβ/p-Tau accumulation, ROS, GFAP, and cell apoptosis (P < 0.05). This confirms that the NF-κB pathway is a central mechanism in hypoxic damage. Inhibition of CYP450 epoxygenase further exacerbated all injury indicators compared to the hypoxic group (P < 0.05). However, when BAY 11-7082 and MS-PPOH were administered simultaneously under hypoxic conditions, the levels of inflammatory factors, cognition-related proteins, and apoptosis, although still higher than in the BAY 11-7082-alone group, were significantly lower than in the MS-PPOH-alone group (P < 0.05) (Fig. 9). These findings indicate that pharmacological inhibition of the NF-κB pathway can partially counteract the exacerbation of injury induced by CYP450 inhibition. This suggests that under hypoxic conditions, one of the primary mechanisms through which CYP450 epoxygenase exerts its neuroprotective effects is by negatively regulating the NF-κB inflammatory signaling pathway.
NF-κB signaling pathway mediates the neuroprotective effects of CYP450 epoxygenase under hypoxia conditions. (A) Changes in inflammatory factor expression after CYP450 and NF-κB inhibition. (B) Changes in Aβ and p-Tau protein expression after CYP450 and NF-κB inhibition. (C) Changes in apoptosis after CYP450 and NF-κB inhibition. (D) Changes in GFAP and ROS expression after CYP450 and NF-κB inhibition. (400×; green: GFAP/ROS; blue: DAPI). (n = 3. Data are shown as the mean ± SD. Statistical analyses were performed with two-way ANOVA with the Bonferroni multiple comparisons test. Significance: *P < 0.05)
DNA methylation-mediated transcriptional repression of CYP450 under hypoxic conditions
Hypoxia-induced upregulation of DNMTs/MeCP2, along with hypermethylation of CYP2C11
Hypoxia can induce DNA methylation, and DNA methylation can regulate CYP450 expression. We investigated the effect of DNA methylation status on CYP450 expression under hypoxic conditions. ELISA analysis revealed that, compared with the LAC group, 5-mC expression levels were significantly higher in the HAH-7, HAH-30, and HAH-90 groups (P < 0.05) (Fig. 10A), suggesting that overall DNA methylation levels in the brain are elevated in a high-altitude hypoxic environment. Further measurements were conducted to determine the mRNA levels of the DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b), as well as the methylcytosine binding protein (MeCP2), in brain tissue under hypoxic conditions. Compared to the LAC group, the results revealed that hypoxia significantly increased the mRNA levels of DNMT1, DNMT3a, and MeCP2 in rat brain tissue (P < 0.05) (Fig. 10B), confirming hypoxia-induced epigenetic reprogramming.
The effects of high-altitude hypoxia on DNA methylation. (A) Effects of high-altitude hypoxia on the expression of 5-mC in rats. (B) Effects of high-altitude hypoxia on the expression of epigenetic markers in rats. (C) Predicted CpG islands in the CYP2C11 gene promoter region. (D) BSP analysis results for the methylation sites of CYP2C11. (E) Effect of hypoxia on the methylation rates of partial methylation sites in the CYP2C11 gene promoter region. (LAC: low-altitude control group, HAH-7: high-altitude hypoxia 7-day group, HAH-30: high-altitude hypoxia 30-day group, HAH-90: high-altitude hypoxia 90-day group. n = 3. Data are shown as the mean ± SD. Statistical analysis in (A and B) was performed with a one-way ANOVA with Dunnett’s multiple comparisons test. Statistical analysis in (E) utilized an independent samples t-test. *P < 0.05 vs. LAC group)
To confirm the role of DNA methylation in regulating CYP450, we measured its methylation levels in the brain tissue of rats in the HAH-90 group. We obtained the gene sequences of rat CYP2C11, CYP2C22, CYP2C23, CYP2J3, CYP4F1, and CYP4A2 from the NCBI database. Using the MethPrimer online tool, we identified a CpG island in the CYP2C11 promoter region (Fig. 10C). BSP detection results revealed that the number of methylated sites in the CYP2C11 gene promoter DNA in the brains of HAH-90 rats increased significantly compared with the LAC group, with the methylation rates of three sites (CpG#26, CpG#90, and CpG#142) being significantly higher (P < 0.05) (Fig. 10D and E). It is suggested that hypermethylation of specific CpG sites in the CYP2C11 promoter region may be an epigenetic mechanism for downregulating CYP2C11 gene expression under hypoxic conditions.
DNMT inhibition by 5-Aza-dC rescues hypoxia-induced CYP2C11 suppression and neuroinflammation
To validate the regulatory role of DNA methylation in CYP2C11 expression, we treated astrocytes cultured under hypoxic conditions with the DNA methyltransferase inhibitor 5-Aza-dC. The experimental results indicate that, at concentrations of less than 1 µM, 5-Aza-dC does not affect the survival rate of cells (P < 0.05) (Fig. 11A). The results revealed that, although CYP2C11 expression was significantly suppressed under hypoxic conditions (P < 0.05), treatment with the DNA methylation inhibitor 5-Aza-dC effectively reversed this inhibitory effect, restoring both mRNA and protein expression levels (P < 0.05) (Fig. 11B). Restoration of CYP2C11 expression was concurrent with the alleviation of hypoxia-induced astrocytic inflammatory responses and abnormal accumulation of Aβ and p-Tau proteins (P < 0.05) (Fig. 11C and D, and 11E). These results confirm that elevated CYP2C11 methylation levels are a key factor in inflammatory responses and the accumulation of cognition-related proteins under hypoxic conditions.
The effect of 5-Aza-dC inhibition on astrocytes under hypoxic conditions. (A) Reproductive toxicity testing of 5-Aza-dC on astrocytes. (B) Effects of 5-Aza-dC treatment on CYP2C11 protein and mRNA expression. (C) Effects of 5-Aza-dC treatment on inflammatory factor expression. (D) Effect of 5-Aza-dC treatment on the expression of Aβ and p-Tau by immunofluorescence assay (400×; green: Aβ and p-Tau; blue: DAPI). (E) Effect of 5-Aza-dC treatment on the expression of Aβ and p-Tau by Western blot. (n = 3. Data are shown as the mean ± SD. Statistical analyses were performed with two-way ANOVA with the Bonferroni multiple comparisons test. Significance: *P < 0.05)
Discussion
This study used a high-altitude hypoxia-exposed rat model and astrocyte hypoxia stress experiments to systematically elucidate the pathological mechanisms underlying hypoxia-induced neuroinflammation and cognitive impairment. Our results demonstrate that exposure to high-altitude hypoxia leads to neuronal damage and activation of glial cells in the brains of SD rats, accompanied by a time-dependent decline in spatial learning and memory ability. At a molecular level, the high-altitude hypoxic environment exacerbates the increase in Aβ and p-Tau levels in the brain, activating the NF-κB-mediated neuroinflammatory signaling pathway. Simultaneously, hypoxia induced disorders in lipid metabolism and significantly down-regulated the mRNA and protein expression of several CYP450 isoforms in the brain. In vitro studies confirmed that inhibiting CYP450 (especially epoxygenase activity) exacerbates the astrocyte inflammatory response, Aβ/p-Tau accumulation, and apoptosis induced by hypoxia, and that this effect is mainly mediated by negatively regulating the NF-κB inflammatory signaling pathway. Finally, in terms of mechanism, our study found for the first time that hypoxia induces overall DNA hypermethylation in brain tissue and leads to hypermethylation of specific CpG sites in the promoter region of CYP2C11, a key isoform of CYP450, and that inhibition of DNA methylation reverses hypoxia-induced inhibition of CYP2C11 expression and simultaneously attenuates inflammation and cognition-related protein accumulation. These findings innovatively establish a DNA methylation-dependent regulatory axis involving CYP450, the NF-κB inflammatory signaling pathway, and neuroinflammation/cognitive impairment (Fig. 12), providing a potential intervention strategy targeting CYP450 for the prevention and treatment of high-altitude-related neural damage.
The mechanism by which high-altitude hypoxia exacerbates neuroinflammation and cognitive impairment through the CYP450-oxidised lipid axis mediated by DNA methylation
The body undergoes various physiological and pathological changes in a high-altitude hypoxic environment. Among these changes, blood and serum biochemical indicators are important markers for assessing an individual’s health status. This study demonstrated that exposure to a hypoxic environment increased the levels of HGB, RBC, and HCT, which is a typical adaptive response to high-altitude hypoxia. This occurs because hypoxia induces the release of erythropoietin (EPO), thereby facilitating erythropoiesis in the bone marrow. This increases the RBC count and hemoglobin concentration, thereby increasing the oxygen-carrying capacity of the blood and enhancing oxygen delivery to tissues. However, excessive proliferation of RBCs can increase blood viscosity, which may cause microcirculatory disorders and increase the prevalence of cerebrovascular diseases in high-altitude settings [18]. WBCs can reflect the body’s immune status, as they are one of the body’s defense systems against foreign pathogens. In line with previous research [19], this study revealed that hypoxia at high altitudes decreased the WBC of rats. These findings suggest that hypoxic conditions may induce inflammatory responses and that a sustained decrease in WBC may make high-altitude residents more susceptible to infection. However, another study found that acute hypoxia can lead to an increase in the WBC count [20]. This difference may be attributed to factors such as hypoxia modeling methods and hypoxia duration. In addition, this study revealed that high-altitude hypoxia significantly altered the serum biochemical indicators in rats. Hypoxia significantly impacts rat liver function, which may affect the production and activity of drug-metabolizing enzymes and consequently impact drug metabolism in the body [20]. Furthermore, renal function experience is notably altered under hypoxic conditions. CREA initially decreases but then increases under hypoxic conditions, which is likely attributed to a compensatory increase in the glomerular filtration rate or a reduction in muscle metabolism during the early stages of hypoxia. However, renal function gradually deteriorates as hypoxia persists. Impairment of liver and kidney function may result in the accumulation of toxins, causing inflammation and oxidative stress in the brain. In addition, the results of this study demonstrated that energy and glucose-lipid metabolism are altered under severe hypoxic conditions. The sustained decrease in GLU and TG indicates increased glycolysis and lipolysis as the duration of hypoxia increases. Reduced glucose utilization under hypoxic conditions may result in an insufficient energy supply to brain tissue, affecting neuronal electrical activity and cognitive function. In agreement with earlier research [21], CRP is a reliable and sensitive systemic inflammatory marker. Our findings indicate that CRP levels remain elevated following hypoxic exposure, suggesting that chronic inflammatory responses induced by hypoxia exacerbate neurodegeneration. These changes may suggest potential pathological damage and reflect the body’s compensatory adaptation; however, the specific mechanisms necessitate further investigation.
The findings of this study first confirm that hypoxia is a key factor in neuroinflammation and cognitive impairment. Previous research indicates that acute and chronic hypoxia disrupts the body’s pro-inflammatory/anti-inflammatory balance. In severe cases, it may trigger varying degrees of cognitive impairment, and with severity correlated to altitude and duration of exposure [22,23,24,25]. Our data further support this view, showing that learning and memory deficits progressively worsen with prolonged exposure to hypoxia. This discovery corresponds to the findings of Rimoldi et al.‘s research on adolescents [26]. However, some studies have reported an initial decline in cognitive function followed by partial recovery with prolonged exposure to hypoxia. For example, Zhang et al. found that mice exposed to an altitude of 7000 m for 1, 3, or 7 days presented initial cognitive impairment during the first three days, which improved by day 7 [27]. Xu et al. observed a similar phenomenon: cognitive function declined within two days of exposure at an altitude of 3800 m but progressively improved from day 3 onward, largely recovering to baseline performance by days 5 to 7 [28]. Research suggests that the ‘decline-partial recovery’ pattern of cognitive function following exposure to hypoxia may be associated with the activation of the body’s hypoxic adaptation mechanisms. However, these studies focused only on short-term exposure to hypoxia within seven days and have failed to systematically assess the sustained effects of long-term exposure to hypoxia on cognitive function. In contrast, this study examined cognitive changes during the acute phase and further measured the dynamic evolution of cognitive function over the long term (30 and 90 days). These findings provide a more comprehensive understanding of patterns of cognitive function changes under different durations of hypoxia. This study design addresses the knowledge gap in the literature on the cognitive effects of long-term exposure to hypoxia, offering more comprehensive experimental evidence to improve our understanding of the mechanisms that compensate for and damage cognitive function in high-altitude hypoxic environments.
This study also identified the molecular mechanism by which cognitive impairment induced by hypoxia is accompanied by increased levels of Aβ and phosphorylated tau protein. This study focuses primarily on changes in the expression of Aβ42 under hypoxic conditions and their pathological significance. In Alzheimer’s disease (AD), Aβ42 exhibits greater hydrophobicity compared to other subtypes, making it more prone to aggregation. Currently, most AD pathology studies utilize Aβ42 deposition as a core assessment metric [29, 30]. Furthermore, Aβ can mediate the excessive phosphorylation of the tau protein, inducing neurofibrillary tangles and causing cognitive impairment through mechanisms such as neuroinflammation and oxidative stress. Consequently, tau phosphorylation and Aβ expression upregulation are commonly regarded as early biological markers of cognitive dysfunction. Our findings demonstrate that hypoxic exposure significantly increases Aβ expression, which is highly consistent with previous research [31, 32], suggesting that these pathological alterations may directly underpin cognitive dysfunction under hypoxic conditions. The accumulation of Aβ and the phosphorylation of tau protein induced by hypoxia are closely associated with the activation of HIF-1α. The overexpression of HIF-1α under hypoxic conditions significantly increases the levels of beta-secretase 1 (BACE1) mRNA and protein, ultimately leading to increased Aβ production [33, 34]. Similarly, HIF-1α regulates tau phosphorylation under hypoxic conditions. Lei et al. found that chronic hypoxia activates HIF-1α, which results in a deficiency of leucine carboxyl methyltransferase 1 (LCMT1) and protein phosphatase 2 A (PP2A). These effects mediate the abnormal hyperphosphorylation of tau, ultimately impairing cognitive function [35]. On the other hand, this study demonstrates that persistently elevated inflammatory mediators (such as IL-1β and TNF-α) exhibit synchronous changes with p-Tau/Aβ levels, supporting the notion that the inflammatory microenvironment can directly influence Aβ/Tau metabolic processes [36,37,38,39]. Notably, although this study found elevated levels of p-Tau/Aβ expression under hypoxic conditions, typical amyloid plaque deposition of Aβ was not observed in wild-type SD rats or in vitro models of primary astrocytes. This is consistent with the established fact that significant Aβ plaque deposition usually occurs in transgenic rats that overexpress a mutant form of the human amyloid precursor protein (APP), whereas wild-type or conventional experimental injury rat models rarely spontaneously form Aβ amyloid plaques that resemble those found in human AD [40, 41]. Furthermore, at the cellular level, although astrocytes can independently express their precursor proteins and processing-related enzyme systems to autonomously produce Aβ and phosphorylated Tau, these cells are not the primary sources of Aβ and Tau [42, 43]. Therefore, the absence of plaque deposition in the purified astrocyte system used in this study was also anticipated. However, this finding precisely demonstrates that chronic hypoxic stress alone, without exogenous genetic intervention, is sufficient to simultaneously activate both core pathological pathways of AD—abnormal Aβ metabolism and excessive tau phosphorylation—and that this process can occur independently of typical plaque formation. This discovery further supports the idea that hypoxia is an independent driver of AD-related molecular pathology, providing crucial experimental evidence to help us understand the mechanisms underlying hypoxia-associated cognitive impairment.
Our study used oxidized lipid metabolomics technology to systematically reveal a specific pattern of oxidized lipid dysregulation for the first time in a hypoxia-induced cognitive impairment rat model. This finding is consistent with previous reports of abnormalities in lipid metabolism in cognitive disorders such as AD [4, 32, 44]. Although the exact way in which these oxidized lipids cause cognitive impairment is unclear, previous research suggests that they may be involved in the pathological process by affecting the anti-inflammatory and vasoregulatory properties of eicosanoids. This study found that, during acute hypoxia, certain proinflammatory mediators (such as LTB₄ and 12 S-HETE) temporarily increase, whereas the levels of anti-inflammatory mediators (such as EETs and PGD₁) decrease. This exacerbates inflammatory damage. In contrast, during chronic hypoxia, the levels of most proinflammatory mediators (e.g., PGE₂ and 20-HETE) decrease persistently, whereas the levels of proresolution mediators (e.g., MaR2 and 15 S-HETE) remain deficient. This leads to delayed inflammatory resolution. These changes result in abnormal activation and delayed resolution of neuroinflammation. On the other hand, it may be related to its vascular regulatory function. EETs and HETEs are particularly important in this context, as they play a central role in vascular responses, regulating vascular tone under hypoxic conditions and promoting angiogenesis [45, 46]. In endothelial cells, EETs activate K+ Ca channels, induce smooth muscle cell hyperpolarization, and inhibit L-type Ca2+ channels. This process induces relaxation. Consequently, EETs act as endothelium-derived vasodilators, dilating blood vessels throughout the vascular system. This study found that exposure to high-altitude hypoxia reduced the biosynthesis of 8(9)-EET, 11(12)-EET, and 14(15)-EET in brain tissue during acute hypoxia. This may lead to cerebral vasoconstriction and reduced cerebral blood flow, thereby exacerbating the damage caused to the brain by hypoxia. HETEs reduce the probability of opening K+ Ca channels and inhibit Na-K-ATPase. This leads to the depolarization of smooth muscle cell membranes and the activation of L-type Ca2+ channels, inducing contraction. This study found that the biosynthesis of 20-HETE, which has a clear vasoconstrictive effect, decreased significantly with prolonged hypoxia, whereas the biosynthesis of 16 S-HETE and 18-HETE increased significantly. While the precise vascular effects of these two metabolites remain unclear, given that other members of the HETE family generally exhibit vasoconstrictive properties, this abnormal increase may also intensify the vasoconstrictive response. Although reducing 20-HETE may alleviate hypoxia-induced vascular vasoconstriction, increasing 16-HETE/18-HETE suggests that there are more complex regulatory mechanisms in cerebral blood vessels under hypoxic conditions. Therefore, clarifying the functional contributions of each subtype using selective inhibitors or agonists will be an important area of future research.
A key advancement of this study is the direct linking of hypoxic injury to the dysfunction of the CYP450-oxidized lipid metabolism axis. We identified disrupted oxidized lipid metabolism in a rat model of hypoxic-induced cognitive impairment. As a key enzyme in oxidized lipid synthesis, CYP450 exhibited significantly reduced mRNA and protein expression levels under hypoxic conditions. Indeed, previous studies have confirmed that hypoxia significantly affects CYP450 expression levels in brain tissue. Jacob et al. exposed human cerebral microvascular endothelial cell lines to hypoxic conditions for 6 h and found that the expression of CYP1A1 and CYP1B1 was significantly reduced [47]. Similarly, another study reported that, under hypoxic conditions, the activity of CYP19a1b, as well as the mRNA and protein expression levels of CYP19a1, decreased significantly in the hypothalamus of Atlantic croaker [48]. These studies suggest that changes in the expression of CYP450 in the brain under hypoxic conditions may be related to changes in brain function under hypoxic conditions, particularly hypoxia-induced cognitive impairment, which has been reported in previous studies. Wan et al. exposed male C57BL/6 mice to an altitude of 4300 m for 6 months and analyzed the proteins in their hippocampal tissue quantitatively. The results showed that differentially expressed proteins were enriched in the ‘drug metabolism-other enzymes’ and ‘drug metabolism-CYP450’ pathways [49]. This highlights the importance of CYP450 in hypoxia-induced cognitive impairment, a finding that was confirmed by our results. We found that the mRNA and protein expression of CYP450 was altered in the brains of rats with hypoxia-induced cognitive impairment and that CYP450 inhibition exacerbated the abnormal accumulation of inflammatory factors and cognition-related proteins under hypoxic conditions. These results provide new experimental evidence to help us understand the role of CYP450 in hypoxia-induced cognitive impairment and could inform the development of neuroprotective strategies targeting the CYP450 metabolic pathway.
In vivo experiments revealed that high-altitude hypoxia impairs the learning and memory abilities of SD rats, with the cortex serving as a critical brain region for executive functions, including working memory, decision-making, and cognitive flexibility [50, 51]. Furthermore, significant alterations in inflammatory factors and cognition-related proteins were observed within the cortex, which provides a potential neurobiological explanation for these behavioral deficits. To investigate these cortical pathological mechanisms at the cellular level, we selected cortical-derived astrocytes as an in vitro model. Based on literature reports and our prior research [52,53,54,55], we simulated chronic moderate hypoxia in vitro by treating cells with 2% O₂ for 24 hours. This enabled us to investigate whether CYP450 exerts its protective effects on astrocytes by regulating specific inflammatory signaling pathways. NF-κB acts as the ‘master switch’ of inflammatory responses in the central nervous system. Under stressful conditions such as hypoxia, it is rapidly activated, driving the expression of downstream pro-inflammatory factors. These molecules directly drive sustained astrocyte and microglia activation, forming a vicious inflammatory cycle [56, 57]. Furthermore, extensive research confirms that the abnormal activation of the NF-κB pathway is a critical initial event in the production of Aβ and the hyperphosphorylation of tau protein in neurodegenerative diseases such as AD [58, 59]. Our research indicates that, under hypoxia, CYP450 inhibition not only exacerbates inflammatory responses but is also accompanied by Aβ/p-Tau accumulation. This phenotype is highly consistent with excessive activation of the NF-κB pathway. Using NF-κB inhibitors, we found that inhibiting NF-κB significantly reversed the exacerbated inflammatory response and Aβ/p-Tau accumulation induced by the CYP450 inhibitor MSPPOH under hypoxic conditions. These results strongly suggest that one of the core molecular mechanisms by which CYP450 exerts its neuroprotective effects in hypoxic environments involves negative regulation of the NF-κB pathway, a key pro-inflammatory signaling pathway. However, it is noteworthy that blocking the NF-κB pathway did not eliminate all the damaging effects caused by CYP450 inhibition. This suggests that, in addition to the NF-κB pathway, CYP450 may exert its comprehensive neuroprotective functions through other parallel or intersecting signaling networks. Therefore, exploring the interactions between CYP450 and other signaling pathways, along with the roles of its specific metabolites (such as EET), is a crucial area for future research into the complete adaptive mechanism.
Our research revealed a novel mechanism by which hypoxia induces specific methylation of the CYP2C11 promoter via DNMTs and MeCP2, thereby inhibiting its expression. These findings expand the current understanding of the regulatory mechanisms of CYP450 under hypoxic conditions. Previous studies have shown that nuclear receptors such as the pregnane X receptor (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR), and hepatocyte nuclear factor (HNF) are involved in regulating CYP450 expression at the transcriptional level under hypoxic conditions [10]. Moreover, multiple cytokines (such as HIF-1α, Nrf2, IL-1β, and IL-6) and gut microbiota metabolites participate in this regulatory network [60, 61]. At the epigenetic level, although it has been confirmed that miRNAs regulate transporter function by modulating PXR under hypoxic conditions [55], their direct regulation of CYP450 remains unclear. Similarly, although it has been demonstrated that DNA methylation is an important epigenetic mechanism that regulates CYP450 expression, the specifics of this regulation under hypoxic conditions and its molecular basis are still unclear. This study is the first to link the suppression of CYP450 expression to methylation at specific CpG sites, revealing a novel mechanism for the regulation of CYP450 under hypoxic conditions. Notably, this study, by identifying key CpG sites and conducting 5-Aza-dC intervention experiments, confirmed the importance of epigenetic modifications in regulating CYP450 expression under hypoxic conditions and offered a novel perspective on the role of epigenetic regulation of CYP450 in neurological diseases. Comprehensive studies indicate that DNA methylation facilitates early central nervous system development, contributes to neuronal growth and differentiation, is crucial for adult neurogenesis, and regulates genes vital to learning and memory-related cognitive functions [62]. Research has elucidated that DNA methylation alterations of the CYP450 gene may significantly contribute to the pathogenesis of neurodegenerative diseases such as AD [63], and our investigation confirms this conclusion.
This study is the first to elucidate the regulatory axis involving DNA methylation-mediated CYP450, oxidized lipids, and neuroinflammation in the context of hypoxia. These findings provide a new theoretical basis for understanding the pathogenesis of cognitive impairment and neuroinflammation in high-altitude hypoxic environments. This research has significant scientific value. First, elucidating the association between metabolic characteristics and events of high-altitude hypoxic encephalopathy could provide new biomarkers for disease monitoring and diagnosis. Second, this study confirmed the critical function of CYP450 in hypoxic neuroprotection, offering a significant theoretical basis for the formulation of targeted pharmaceuticals. Finally, a novel mechanism of DNA methylation of CYP450 under hypoxic conditions in neural damage has been identified, offering a fresh perspective for the research and treatment of hypoxic brain injury disorders. However, this study has several limitations. First, although the use of SD rats and primary astrocytes provided consistency within the experimental framework, the conclusions necessitate additional validation through primate models or human samples to ascertain whether the correlation between CYP450 and neural damage under hypoxic conditions is species independent. Second, although our study identified CYP450 and oxidized lipid metabolites as crucial targets in the onset of inflammation and cognitive decline under hypoxic conditions, the molecular interactions between oxidized lipid metabolites and Aβ/p-Tau proteins remain ambiguous. Additional confirmation is necessary using in vitro co-incubation experiments and protein interaction analysis methods. Third, the main focus of this study was the methylation regulatory mechanism of CYP2C11. However, the reduced expression of other CYP450 subtypes under hypoxic conditions suggests the existence of additional regulatory pathways. Future research could combine gene-editing techniques, such as CRISPR-Cas9, with multiomics integrated analysis strategies to systematically elucidate the expression regulatory network of the CYP450 family under hypoxic conditions. This would provide valuable insight into the molecular mechanisms underlying high-altitude adaptive responses.
Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- AA:
-
Arachidonic acid
- AhR:
-
Aryl hydrocarbon receptor
- AMS:
-
Acute mountain sickness
- Aβ:
-
Amyloid-β
- BSP:
-
Bisulfite-sequencing PCR
- CAR:
-
Constitutive androstane receptor
- COX:
-
Cyclooxygenase
- CYP450:
-
Cytochrome P450
- EETs:
-
Eicosapentaenoic acids
- ELISA:
-
Enzyme-linked immunosorbent assay
- GFAP:
-
Glial fibrillary acidic protein
- HACE:
-
High-altitude cerebral edema
- HAH:
-
High-altitude headache
- HE:
-
Hematoxylin-eosin
- HETEs:
-
Eicosatetraenoic acids
- HIF-1α:
-
Hypoxia-inducible factor-1α
- HNF:
-
Hepatocyte nuclear factor
- IBA1:
-
Ionophore-binding protein 1
- LOX:
-
Lipoxygenase
- p-Tau:
-
Phospho-Tau
- PUFAs:
-
Polyunsaturated fatty acids
- PXR:
-
Pregnane X receptor
- ROS:
-
Reactive oxygen species
- RT-qPCR:
-
Reverse transcription quantitative real-time polymerase chain reaction
- TEM:
-
Transmission electron microscopy
- UHPLC-MS/MS:
-
Ultrahigh-performance liquid chromatography-tandem mass spectrometry
References
Erecińska M, Silver IA. Tissue oxygen tension and brain sensitivity to hypoxia. Respir Physiol. 2001;128(3):263–76.
Chen X, Zhang J, Lin Y, Li Y, Wang H, Wang Z, et al. Mechanism, prevention and treatment of cognitive impairment caused by high altitude exposure. Front Physiol. 2023;14:1191058.
Aboouf MA, Thiersch M, Soliz J, Gassmann M, Schneider Gasser EM. The brain at high altitude: from molecular signaling to cognitive performance. Int J Mol Sci. 2023;24(12):10179.
Dhillon VS, Thomas P, Lee SL, Deo P, Fenech M. Red blood cell fatty acid profiles are significantly altered in south australian mild cognitive impairment and alzheimer’s disease cases compared to matched controls. Int J Mol Sci. 2023;24(18):14164.
Chauhan G, Roy K, Kumar G, Kumari P, Alam S, Kishore K, et al. Distinct influence of COX-1 and COX-2 on neuroinflammatory response and associated cognitive deficits during high altitude hypoxia. Neuropharmacology. 2019;146:138–48.
Funk CD. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science. 2001;294(5548):1871–5.
Zhang G, Kodani S, Hammock BD. Stabilized epoxygenated fatty acids regulate inflammation, pain, angiogenesis and cancer. Prog Lipid Res. 2014;53:108–23.
Welty FK. Omega-3 fatty acids and cognitive function. Curr Opin Lipidol. 2023;34(1):12–21.
Bazinet RP, Layé S. Polyunsaturated fatty acids and their metabolites in brain function and disease. Nat Rev Neurosci. 2014;15(12):771–85.
Wang Q, Liu G, Duan Y, Duo D, Zhu J, Li X. Exploring cytochrome P450 under hypoxia: potential pharmacological significance in drug metabolism and protection against high-altitude diseases. Drug Metab Dispos. 2025;53(2):100026.
Halder SK, Milner R. The impact of chronic mild hypoxia on cerebrovascular remodelling; uncoupling of angiogenesis and vascular breakdown. Fluids Barriers CNS. 2021;18(1):50.
Penaloza CG, Cruz M, Germain G, Jabeen S, Javdan M, Lockshin RA, et al. Higher sensitivity of female cells to ethanol: methylation of DNA lowers Cyp2e1, generating more ROS. Cell Commun Signal. 2020;18(1):111.
Sukmawan R, Hoetama E, Suridanda Danny S, Giantini A, Listiyaningsih E, Gilang Rejeki V, et al. Increase in the risk of clopidogrel resistance and consequent TIMI flow impairment by DNA hypomethylation of CYP2C19 gene in STEMI patients undergoing primary percutaneous coronary intervention (PPCI). Pharmacol Res Perspect. 2021;9(2):e00738.
Jiang W, Wu H, Yu X, Wang Y, Gu W, Wei W, et al. Third-hand smoke exposure is associated with abnormal serum melatonin level via hypomethylation of CYP1A2 promoter: Evidence from human and animal studies. Environ Pollut. 2021;277:116669.
Lin Z, Lu Y, Yu G, Teng H, Wang B, Yang Y, et al. Genome-wide DNA methylation landscape of four Chinese populations and epigenetic variation linked to Tibetan high-altitude adaptation. Sci China Life Sci. 2023;66(10):2354–69.
Zhang S, Fu W, Jia X, Bade R, Liu X, Xie Y, et al. Hypoxic preconditioning modulates BDNF and its signaling through DNA methylation to promote learning and memory in mice. ACS Chem Neurosci. 2023;14(12):2320–32.
Jin T, Lu H, Zhang Z, Wang Y, Yang W, Wang Y, et al. CYP2S1 gene methylation among High-altitude pulmonary edema. Gene. 2022;834:146590.
Villafuerte FC, Simonson TS, Bermudez D, León-Velarde F. High-altitude erythrocytosis: mechanisms of adaptive and maladaptive responses. Physiol (Bethesda) 2022;37(4).
Yan C, Tian D, Zhang C, Zhang Q, Sun Y. Evaluation of blood cellular and biochemical parameters in rats under a chronic hypoxic environment at high altitude. Ann Med. 2023;55(1):898–907.
Zhu J, Duan Y, Duo D, Yang J, Bai X, Liu G, et al. High-altitude hypoxia influences the activities of the drug-metabolizing enzyme CYP3A1 and the pharmacokinetics of four cardiovascular system drugs. Pharmaceuticals (Basel). 2022;15(10):1303.
Hartmann G, Tschöp M, Fischer R, Bidlingmaier C, Riepl R, Tschöp K, et al. High altitude increases circulating interleukin-6, interleukin-1 receptor antagonist and C-reactive protein. Cytokine. 2000;12(3):246–52.
Jiang S, Fan F, Yang L, Chen K, Sun Z, Zhang Y, et al. Salidroside attenuates high altitude hypobaric hypoxia-induced brain injury in mice via inhibiting NF-κB/NLRP3 pathway. Eur J Pharmacol.2022;925:175015.
Xie N, Fan F, Jiang S, Hou Y, Zhang Y, Cairang N, et al. Rhodiola crenulate alleviates hypobaric hypoxia-induced brain injury via adjusting NF-κB/NLRP3-mediated inflammation. Phytomedicine. 2022;103:154240.
Tian L, Liu G, Zhao Q, Han J, Lin Y, Wang Q, et al. Pharmacokinetics of midazolam in plasma and brain tissue of rats after exposure to acute and chronic high altitude hypoxia. High Alt Med Biol. 2025;26(3):273–82.
Algaze I, Phillips L, Inglis P, Lathrop G, Gadbois J, Rizzolo K, et al. Incidence of mild cognitive impairment with ascending altitude. High Alt Med Biol. 2020;21(2):184–91.
Rimoldi SF, Rexhaj E, Duplain H, Urben S, Billieux J, Allemann Y, et al. Acute and chronic altitude-induced cognitive dysfunction in children and adolescents. J Pediatr. 2016;169:238–43.
Zhang H, Zhang X, Liu Z, Mei Y, Liu Y, Wei X, et al. Time-course effects and mechanisms of hypobaric hypoxia on nervous system in mice. Neurosci Lett. 2023;801:137163.
Xu S, Zheng ZY, Zhao GC, Lu ZH, Nie H, Wang XJ, et al. The study on effects of acute exposure to high altitude hypoxia on cognitive function in lowlander. Behav Brain Res. 2025;484:115515.
Upadhyay A, Chhangani D, Rao NR, Kofler J, Vassar R, Rincon-Limas DE, et al. Amyloid fibril proteomics of AD brains reveals modifiers of aggregation and toxicity. Mol Neurodegener. 2023;18(1):61.
Kamagata K, Andica C, Takabayashi K, Saito Y, Taoka T, Nozaki H, et al. Association of MRI indices of glymphatic system with amyloid deposition and cognition in mild cognitive impairment and Alzheimer disease. Neurology. 2022;99(24):e2648–60.
Krstic D, Madhusudan A, Doehner J, Vogel P, Notter T, Imhof C, et al. Systemic immune challenges trigger and drive Alzheimer-like neuropathology in mice. J Neuroinflammation. 2012;9:151.
Nasaruddin ML, Pan X, McGuinness B, Passmore P, Kehoe PG, Hölscher C, et al. Evidence that parietal lobe fatty acids may be more profoundly affected in moderate Alzheimer’s Disease (AD) pathology than in severe AD pathology. Metabolites. 2018;8(4):69.
Zhang X, Zhou K, Wang R, Cui J, Lipton SA, Liao FF, et al. Hypoxia-inducible factor 1alpha (HIF-1alpha)-mediated hypoxia increases BACE1 expression and beta-amyloid generation. J Biol Chem. 2007;282(15):10873–80.
Guglielmotto M, Aragno M, Autelli R, Giliberto L, Novo E, Colombatto S, et al. The up-regulation of BACE1 mediated by hypoxia and ischemic injury: role of oxidative stress and HIF1alpha. J Neurochem. 2009;108(4):1045–56.
Lei L, Feng J, Wu G, Wei Z, Wang JZ, Zhang B, et al. HIF-1α causes LCMT1/PP2A deficiency and mediates tau hyperphosphorylation and cognitive dysfunction during chronic hypoxia. Int J Mol Sci. 2022;23(24):16140.
Djelti F, Braudeau J, Hudry E, Dhenain M, Varin J, Bièche I, et al. CYP46A1 inhibition, brain cholesterol accumulation and neurodegeneration pave the way for Alzheimer’s disease. Brain. 2015;138(Pt 8):2383–98.
Maioli S, Båvner A, Ali Z, Heverin M, Ismail MA, Puerta E, et al. Is it possible to improve memory function by upregulation of the cholesterol 24S-hydroxylase (CYP46A1) in the brain? PLoS ONE. 2013;8(7):e68534.
Gjota-Ergin S, Gökçek-Saraç Ç, Adalı O, Jakubowska-Doğru E. Relationship between the hippocampal expression of selected cytochrome P450 isoforms and the animal performance in the hippocampus-dependent learning task. Neurosci Lett. 2018;673:104–10.
Zhang F, Li J, Na S, Wu J, Yang Z, Xie X, et al. The involvement of PPARs in the selective regulation of brain CYP2D by growth hormone. Neuroscience. 2018;379:115–25.
Liu L, Orozco IJ, Planel E, Wen Y, Bretteville A, Krishnamurthy P, et al. A transgenic rat that develops Alzheimer’s disease-like amyloid pathology, deficits in synaptic plasticity and cognitive impairment. Neurobiol Dis. 2008;31(1):46–57.
Isobe I, Yanagisawa K, Michikawa M. A possible model of senile plaques using synthetic amyloid beta-protein and rat glial culture. Exp Neurol. 2000;162(1):5–60.
Rostami J, Mothes T, Kolahdouzan M, Eriksson O, Moslem M, Bergström J, et al. Crosstalk between astrocytes and microglia results in increased degradation of α-synuclein and amyloid-β aggregates. J Neuroinflammation. 2021;18(1):124.
Lee JH, Yang DS, Goulbourne CN, Im E, Stavrides P, Pensalfini A, et al. Faulty autolysosome acidification in Alzheimer’s disease mouse models induces autophagic build-up of Aβ in neurons, yielding senile plaques. Nat Neurosci. 2022;25(6):688–701.
Shen Y, Wei C, Taishi Y, Zhang G, Su Z, Zhao P, et al. Association between the circulating very long-chain saturated fatty acid and cognitive function in older adults: findings from the NHANES. BMC Public Health. 2024;24(1):1061.
Michaelis UR, Xia N, Barbosa-Sicard E, Falck JR, Fleming I. Role of cytochrome P450 2 C epoxygenases in hypoxia-induced cell migration and angiogenesis in retinal endothelial cells. Invest Ophthalmol Vis Sci. 2008;49(3):1242–7.
Xia Y, Xia L, Jin Z, Jin R, Paudel O, Sham JSK. Cytochrome P450 epoxygenase-dependent activation of TRPV4 channel participates in enhanced serotonin-induced pulmonary vasoconstriction in chronic hypoxic pulmonary hypertension. Anal Cell Pathol (Amst). 2020;2020:8927381.
Jacob A, Potin S, Saubaméa B, Crete D, Scherrmann JM, Curis E, et al. Hypoxia interferes with aryl hydrocarbon receptor pathway in hCMEC/D3 human cerebral microvascular endothelial cells. J Neurochem. 2015;132(4):373–83.
Rahman MS, Thomas P. Molecular characterization and expression of cytochrome P450 aromatase in atlantic croaker brain: regulation by antioxidant status and nitric oxide synthase during hypoxia stress. Front Physiol. 2021;12:720200.
Wan Y, Ge RL, Cao Y, Luo L, Ji W. Chronic hypobaric hypoxia stimulates differential expression of cognitive proteins in hippocampal tissue. High Alt Med Biol. 2025;26(2):175–86.
Lima A, Sardinha VM, Oliveira AF, Reis M, Mota C, Silva MA, et al. Astrocyte pathology in the prefrontal cortex impairs the cognitive function of rats. Mol Psychiatry. 2014;19(7):834–41.
Delcourte S, Bouloufa A, Rovera R, Bétry C, Abrial E, Dkhissi-Benyahya O, et al. Chemogenetic activation of prefrontal astroglia enhances recognition memory performance in rat. Biomed Pharmacother. 2023;166:115384.
Horie N, So K, Moriya T, Kitagawa N, Tsutsumi K, Nagata I, et al. Effects of oxygen concentration on the proliferation and differentiation of mouse neural stem cells in vitro. Cell Mol Neurobiol. 2008;28(6):833–45.
Guo M, Ma X, Feng Y, Han S, Dong Q, Cui M, at al. In chronic hypoxia, glucose availability and hypoxic severity dictate the balance between HIF-1 and HIF-2 in astrocytes. FASEB J. 2019;33(10):11123–36.
Aldinucci C, Maiorca SM, De Rosa P, Palmi M, Sticozzi C, Ciccoli L, et al. The effects of hypoxia/reoxygenation on the physiological behaviour of U373-MG astrocytes. Neurochem Res. 2010;35(1):42–9.
Duan Y, Bai X, Yang J, Zhou Y, Gu W, Liu G, et al. Exposure to high-altitude environment is associated with drug transporters change: microRNA-873-5p-mediated alteration of function and expression levels of drug transporters under hypoxia. Drug Metab Dispos 2022;50(2):174–86.
Ageeva T, Rizvanov A, Mukhamedshina Y. NF-κB and JAK/STAT signaling pathways as crucial regulators of neuroinflammation and astrocyte modulation in spinal cord injury. Cells. 2024;13(7):581.
Jong Huat T, Camats-Perna J, Newcombe EA, Onraet T, Campbell D, Sucic JT, et al. The impact of astrocytic NF-κB on healthy and Alzheimer’s disease brains. Sci Rep. 2024;14(1):14305.
Sivamaruthi BS, Raghani N, Chorawala M, Bhattacharya S, Prajapati BG, Elossaily GM, et al. NF-κB Pathway and Its Inhibitors: A Promising Frontier in the Management of Alzheimer’s Disease. Biomedicines. 2023;11(9):2587.
Nayak V, Patra S, Rout S, Jena AB, Sharma R, Pattanaik KP, et al. Regulation of neuroinflammation in Alzheimer’s disease via nanoparticle-loaded phytocompounds with anti-inflammatory and autophagy-inducing properties. Phytomedicine. 2024;122:155150.
Liu M, Alkayed NJ. Hypoxic preconditioning and tolerance via hypoxia inducible factor (HIF) 1alpha-linked induction of P450 2C11 epoxygenase in astrocytes. J Cereb Blood Flow Metab. 2005;25(8):939–48.
Bai X, Yang J, Liu G, Zhu J, Wang Q, Gu W, et al. Regulation of CYP450 and drug transporter mediated by gut microbiota under high-altitude hypoxia. Front Pharmacol. 2022;13:977370.
Cui D, Xu X. DNA methyltransferases, DNA methylation, and age-associated cognitive function. Int J Mol Sci. 2018;19(5):1315.
Bahado-Singh RO, Vishweswaraiah S, Turkoglu O, Graham SF, Radhakrishna U. Alzheimer’s precision neurology: epigenetics of cytochrome P450 genes in circulating cell-free dna for disease prediction and mechanism. Int J Mol Sci. 2023;24(3):2876.
Acknowledgements
Not applicable.
Funding
This study was supported by the National Natural Science Foundation of China (No. 82260731, China), Qinghai University Research Ability Enhancement Project (2025KTST08, China), and Qinghai Provincial Department of Science and Technology (2024-ZJ-724, China).
Author information
Authors and Affiliations
Contributions
Qian Wang: Conceptualisation, Methodology, Investigation, Validation, Formal analysis, and Writing–original draft. Junjun Han: Methodology and Data curation. Guiqin Liu and Yabin Duan: Investigation and writing-review. Delong Duo and Junbo Zhu: Data curation, Software, and Formal analysis. Yue Lin and Yawen Xin: Resources, Visualization, and Investigation. Xiangyang Li: Writing-review & editing, Resources, Supervision, Project administration, and Funding acquisition. Ting Li: Writing-review, Supervision, and Project administration. All authors contributed to the article, agreed to be accountable for all aspects of the work, and approved the submitted version.
Corresponding authors
Ethics declarations
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Wang, Q., Han, J., Liu, G. et al. DNA methylation regulation of CYP450-lipid metabolism by high-altitude hypoxia: linking neuroinflammation to cognitive impairment. J Neuroinflammation 23, 145 (2026). https://doi.org/10.1186/s12974-026-03775-6
Received:
Accepted:
Published:
Version of record:
DOI: https://doi.org/10.1186/s12974-026-03775-6