Propofol Suppresses LPS-induced BBB Damage by Regulating miR-130a-5p/ZO-1 Axis

The blood-brain barrier (BBB) is a highly selective semi-permeable barrier that separates circulating blood from the extracellular fluid of the brain and central nervous system, which is crucial for maintaining brain homeostasis. This study aimed to explore the role of propofol in BBB damage and further evaluate the underlying molecular mechanism. Lipopolysaccharide (LPS) was administered to mice to create an in vivo BBB damage mice model. Additionally, hCMEC/D3 cells as brain microvascular endothelial cells (BMECs) were treated with LPS to establish the in vitro BBB damage cell model. Subsequently, propofol was used for the BBB damage model. Evans blue staining and fluorescein sodium were utilized in the in vivo experiments to demonstrate BBB leakage and BBB permeability. Cell counting kit-8 (CCK-8) assay was used to assess cell viability and the trans-endothelial electrical resistance (TEER) value was measured using an epithelial voltmeter. Furthermore, enzyme-linked immunosorbent assay was performed to measure the levels of the inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α). The levels of miR-130a-5p and zonula occludens-1 (ZO-1) in brain tissues and cells were detected using reverse transcription-quantitative polymerase chain reaction, western blot, or immunofluorescence staining. Furthermore, a dual-luciferase reporter assay was used to demonstrate the association between miR-130a-5p and ZO-1. Propofol treatment suppressed BBB leakage, the amount of fluorescein sodium, and the levels of IL-1β and TNF-α in the LPS-induced BBB damage mice model. Meanwhile, propofol treatment increased the TEER value in the LPS-induced hCMEC/D3 cells. Additionally, propofol treatment significantly down-regulated miR-130a-5p and up-regulated ZO-1. More importantly, miR-130a-5p directly targeted ZO-1 and negatively regulated ZO-1 expression in hCMEC/D3 cells. Furthermore, miR-130a-5p mimic partially reversed the effect of propofol on the TEER value and the levels of inflammatory cytokines such as IL-1β and TNF-α in the LPS-induced hCMEC/D3 cells. Propofol suppressed LPS-induced BBB damage by regulating miR-130a-5p/ZO-1 axis. These findings suggested a potentially effective treatment approach for BBB damage.


Introduction
The blood-brain barrier (BBB) is a highly selective, semipermeable barrier that regulates the exchange of substances between the blood and the central nervous system [1].BBB integrity is significant in maintaining the stability of the brain environment and central nervous system function [2, the transmembrane transport of water ions and macromolecules via paracellular pathways [4].Several studies have demonstrated that changes in tight junction protein (TJP) expression, location distribution, and structural and functional abnormalities may impair the integrity of tight junctions and alter BBB permeability [7,8].Zonula occludens-1 (ZO-1), also known as TJP1, is a major component of tight junctions, which indicates its significance as a regulator of paracellular permeability in epithelia and endothelia [8][9][10].
Propofol is the most commonly [11] utilized intravenous anesthetic in the clinic [12].Propofol has been reported to have neuroprotective and anti-neuroinflammatory properties that protect neurons in ischemic brain tissue [13,14].For instance, propofol protects astrocytes from oxidative stress and promotes astrocyte-mediated neuronal protection [15,16].The use of propofol has attracted immense research interests worldwide and emerging evidence suggests that propofol enhances tissue and organ function [11,17,18].To the best of our knowledge, the mechanisms of propofol on BBB damage have not been thoroughly described.
MiRNA is a highly conserved non-coding short-chain RNA with 19-24 nucleotides [19].By binding to the 3'-UTR regions of the target gene mRNA, miRNA inhibits the translation process and regulates the expression of the target genes [19].MiRNA is used to regulate and control cell metabolism, proliferation, differentiation, and death.Evidence has suggested that miRNA is crucial for maintaining BBB integrity under inflammatory conditions.miR-130a-5p has been reported to be associated with BBB.A clinical study demonstrated that patients with brain edema following BBB injury had elevated serum levels of miR-130a-5p [20].Another study also demonstrated that miR-130a-5p impaired BBB function and increased BBB permeability by inhibiting Homeobox A5 expression [21].However, it is unclear whether propofol attenuated BBB damage by regulating miR-130a-5p.
This study aimed to investigate the exact functions and mechanisms of propofol on BBB damage.The results revealed that propofol suppressed lipopolysaccharide (LPS)induced BBB damage by regulating the miR-130a-5p/ZO-1 axis, which provided a novel approach for the treatment of BBB damage.

Animal Studies
The male C57BL/C mice (age, 4-6 weeks) were purchased from Jiangsu Animal Laboratory (Jiangsu, China).The mice were housed under 12 h light/dark cycles at 24-26 ℃ with ad libitum access to sterile food and water.Thirty C57BL/6J mice were divided into three groups: the Control group (n = 10), the LPS group (n = 10), and the LPS + Propofol group (n = 10).The mice in the LPS + Propofol group were intravenously administered 20 mg/kg/h propofol for 30 min via the tail vein.Meanwhile, phosphate-buffered solution (PBS, Sigma, USA) was administered to the mice in the control and LPS groups as a negative control.After 7 days of pre-treatment, the mice in the LPS and the LPS + Propofol groups were administered intraperitoneally 5 mg/kg LPS (Sigma, USA) for 12 h before the mice were processed.The mice were then intraperitoneally injected with 200 mg/ kg sodium pentobarbital (Sigma, USA), and the serum and brain tissues were collected.This experiment was approved by the Animal Ethics Committee of The Sixth People's Hospital Affiliated with the School of Medicine of Shanghai Jiaotong University (NO.: 2017-0089).

Evans Blue Staining
The mice were intravenously injected with 4 mL/kg 2% Evans blue dye at 2 h before euthanasia as previously described [22].The mice were then infused with heparinized saline through the cardiac ventricle until colorless infusion fluid was obtained from the atrium.The brains were removed and frozen.Frozen slices with a thickness of 10 μm were harvested and observed under an epifluorescence microscope (Leica, Germany).

Blood-Brain Barrier Permeability Measurement
To assess BBB permeability, the mice were intraperitoneally injected with 200 mg/kg sodium fluorescein 1 h before euthanasia as previously described [23].The brains were removed and extracted using 30% trichloroacetic acid.The fluorescence intensity was measured using a microplate reader (excitation 440 nm, emission 525 nm, Molecular Devices, USA).

Cell Treatment and Transfection
To mimic the BBB dysfunction model in vitro, hCMEC/ D3 cells were treated with 30 µg/mL LPS (Sigma, USA) for 24 h.hCMEC/D3 cells were treated with PBS (Sigma, USA) as the negative control.

Trans-Endothelial Electrical Resistance (TEER)
The TEER value was measured using an epithelial voltmeter (World Precision Instruments, USA).Briefly, the medium was removed from the upper chamber, and the cell culture insert was transferred to a new well and washed with PBS.The measurement procedure includes measuring the blank resistance (without cells) and measuring the resistance across the cells.Besides, the values were expressed as Ω*cm 2 .When the values reached 300 Ω*cm 2 , the in vitro BBB model was considered established.

Western Blot
The total protein from tissues or cells was extracted with ice-cold radio-immunoprecipitation assay lysis buffer (Beyotime, China) plus phenylmethylsulphonyl fluoride (Beyotime, China), and the protein concentrations were detected by bicinchoninic acid kit (Beyotime, China).Subsequently, LPS-induced BBB damage in vivo and controlled the levels of miR-130a-5p and ZO-1.
the protein samples were separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into polyvinylidene fluoride membranes.The membranes were incubated with primary antibodies ZO-1 (ab190085, 1:1000, Abcam, USA) or GAPDH (ab9485, 1:2500, Abcam, USA) at 4 ℃ overnight.Afterward, the membranes were incubated with secondary antibodies (Abcam, USA) for 1 h at 25℃.Finally, an enhanced chemiluminescence kit (ThermoFisher, USA) was used to visualize the membranes and the protein bands were analyzed by ImageJ software.

Statistical Analysis
All experiments were repeated thrice independently.Data were analyzed using the Prism Graphpad 8.0 software and presented as means ± standard deviation.A paired student's t-test was employed to compare two groups, and a oneway analysis of variance followed by Tukey's poc host was performed to analyze differences among multiple groups.P < 0.05 was considered statistically significant.

Propofol Alleviates LPS-induced BBB Damage, Inhibits miR-130a-5p, and Promotes ZO-1 Expression in Vivo
Propofol was injected into the LPS-induced BBB damage mice model to investigate its influence on BBB damage.Evans blue staining revealed that BBB permeability was higher in the LPS group than in the control group; however, it was lower in the LPS group following propofol administration (Fig. 1A).Additionally, LPS increased the amount of fluorescein sodium in the brain tissue, depicting that the BBB was damaged and that the permeability had increased (P < 0.001), whereas propofol treatment reversed this effect (Fig. 1B, P < 0.01).According to ELISA results, LPS significantly increased the levels of IL-1β and TNF-α in brain tissues and serum (P < 0.001), which were further reduced by propofol treatment (Fig. 1C-F, P < 0.05).We then determined the expression levels of miR-130a-5p and ZO-1 in the LPS-induced BBB damage mice model.RT-qPCR assay revealed that miR-130a-5p level was significantly increased in the LPS group compared with that in the control group (P < 0.01); however, propofol administration dramatically down-regulated miR-130a-5p (Fig. 1G, P < 0.01).Furthermore, RT-qPCR and western blot assays demonstrated that LPS dramatically suppressed ZO-1 expression (P < 0.001), while propofol treatment reversed this reduction (Fig. 1H-J, P < 0.01).These data demonstrated that propofol reduced miR-130a-5p mimic.The data validated that propofol treatment increased the TEER value (P < 0.01) when compared to the LPS group, whereas miR-130a-5p mimic partially reversed the impact of propofol (Fig. 4A, P < 0.01).RT-qPCR assay demonstrated that miR-130a-5p was downregulated in propofol treated-hCMEC/D3 cells compared

miR-130a-5p Reverses the Protective Role of Propofol in LPS-Induced BBB Damage
To determine whether propofol contributed to LPS-induced BBB damage by regulating miR-130a-5p, hCMEC/D3 cells were co-treated with LPS and propofol and transfected with  # P < 0.05, ## P < 0.01, ### P < 0.001 vs. LPS group LPS, the primary component of Gram-negative bacteria, triggers an inflammatory response that damages neurons [33].Several studies have reported that LPS causes systemic inflammation and the release of inflammatory cytokines such as IL-1β and TNF-α, which ultimately disrupts the integrity of BBB [33,34].Tight junctions are the dynamic structures present between BMECs [4].The primary structure of the barrier is mainly formed by claudin-1, and occludin and ZO-1 increase its stability [35,36].Wei CC et al. demonstrated that alterations in claudin-1, occludin, and ZO-1 expression and distribution altered the stability of tight junctions, resulting in brain injury [37].Previous studies have revealed that LPS dramatically reduces the levels of TJPs such as occludin and ZO-1 and changes their distribution [38].ZO-1 is an extremely significant structural protein in the tight junction complex and is the first tight junction adhesive protein that has been verified [39].To contrast, ZO-1 links to transmembrane proteins.It links with the cytoskeleton protein on the inner side of the cell, forming an essential junction.ZO-1 can be employed as an evaluation indicator of BBB damage and function since its abnormal expression is correlated with the severity of BBB injury [34].In the study, LPS was used to create an in vivo and in vitro BBB damage model.We discovered that LPS triggered injury in hCMEC/D3 cells and caused BBB damage in C57BL/C mice.

Discussion
The BBB plays a crucial role in maintaining the physiological state of the central nervous system [1].Once BBB is destroyed, a substantial amount of material enters the brain parenchyma and BBB permeability changes [28].BMEC injury, tight junction opening, increased vesicle transport, and astrocyte swelling are the major factors contributing to alterations in BBB permeability [29].It has been reported that BBB damage is closely associated with several degenerative diseases, such as ischemic stroke, Alzheimer's disease, and vascular cognitive impairments [30][31][32].Therefore, improving our understanding of the potential mechanisms of BBB degradation is crucial for creating better treatment strategies.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, 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 changes were made.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/4.0/.
of inflammatory cytokines IL-1β, IL-6, and TNF-α, and played an anti-I/R injury role [40].Additionally, Chen W et al. suggested that controlling ZO-1 protected against BBB integrity caused by hypoxia [41].In this study, propofol intervention dramatically reduced BBB leakage, suppressed inflammatory response, and preserved the structural integrity of BBB.Moreover, propofol significantly reduced BBB damage to brain tissue in mice and BMECs.The underlying mechanisms may be associated with the down-regulation of miR-130a-5p and up-regulation of ZO-1.
Numerous studies have demonstrated that miRNA contributes to the preservation of BBB integrity [42,43].To illustrate, it has been noted that the inhibition of the miR-132/212 cluster increases infarct volume and damages BBB [42].Wu J et al. hypothesized that miR-9-5p ameliorated the BBB damage and neuroinflammatory response caused by oxygen-glucose deprivation (OGD) by targeting Ptch-1 [43].Currently, the bioinformatics prediction approach of miRNA target genes relies on the principle of base matching between the miRNA sequence and the corresponding target mRNA sequence.In this study, TargetScan and dual-luciferase reporter assay validated the direct targeting of ZO-1 by miR-130a-5p.Recently, some studies have demonstrated that miR-130a-5p has multiple biological functions, such as regulating cell behaviors including cell growth, apoptosis, and angiogenesis [44,45].Sui S et al. revealed that miR-130a-5p promoted cell viability and angiogenesis in OGD induced-BMECs [44].In this study, miR-130a-5p reversed the protective role of propofol on LPS-induced BBB injury.

Conclusion
Propofol exhibits a significant protective impact on LPSinduced BBB damage.The mechanism of propofol includes decreased miR-130a-5p and increased ZO-1, thus, reducing the release of inflammatory cytokines and maintaining the integrity of BBB.