Catalpol Inhibits Homocysteine-induced Oxidation and Inflammation via Inhibiting Nox4/NF-κB and GRP78/PERK Pathways in Human Aorta Endothelial Cells
Hyperhomocysteinemia (HHCY) has been recognized as an independent risk factor for atherosclerosis and plays a vital role in the development of atherosclerosis. Catalpol, an iridoid glucoside extracted from the root of Rehmannia glutinosa, can produce anti-inflammatory, anti-oxidant, anti-tumor, and dopaminergic neurons protecting effects. This study aimed to determine the protecting effects of catalpol against homocysteine (HCY)-induced injuries in human aortic endothelial cells (HAECs) and uncover the underlying mechanisms: 1. HAECs were cultured with different concentrations of HCY (3 mM) and catalpol (7.5 μΜ, 15 μΜ, 30 μΜ) for 24 h. (1) The level of MDA and GSH as well as LDH release was measured with colorimetric assay. (2) Reactive oxygen species (ROS) were detected by flow cytometry analysis. (3) Western blotting analysis was performed to detect the expression of Nox4, p22phox, ICAM-1, MCP-1, VCAM-1, IκB, nucleus p65, p65 phosphorylation, caspase-3, −9, bax, bcl-2, and ER stress-related proteins. (4) The expressions of CHOP, ATF4 were measured by qRT-PCR. (5) Mitochondrial membrane potential in HCY-treated HAECs was measured by rhodamine 123 staining, and the samples were observed by confocal laser scanning microscopy. 2. DPI, PDTC, and TUDCA were used to determine the interaction among Nox4/ROS, NF-κB, and endoplasmic reticulum stress. 3. TUDCA or Nox4 siRNA were used to investigate whether the effect of catalpol inhibiting the over-production of ROS were associated with inhibiting ER stress and Nox4 expression. Catalpol significantly suppressed LDH release, MDA level, and the reduction of GSH. Catalpol reduced HCY-stimulated ROS over-generation, inhibited the NF-κB transcriptional activation as well as the protein over-expressions of Nox4, ICAM-1, VCAM-1, and MCP-1. Catalpol elevated bcl-2 protein expression and reduced bax, caspase-3, −9 protein expressions in the HCY-treated HAECs. Simultaneously, catalpol could also inhibit the activation of ER stress-associated sensors GRP78, IRE1α, ATF6, P-PERK, P-eIF2α, CHOP, and ATF4 induced by HCY. In addition, the extent of catalpol inhibiting ROS over-generation and NF-κB signaling pathway was reduced after inhibiting Nox4 or ER stress with DPI or TUDCA. The inhibitor of NF-κB PDTC also reduced the effects of catalpol inhibiting the expressions of Nox4 and GRP78. Furthermore, the effect of catalpol inhibiting the over-generation of ROS was reduced by Nox4 siRNA. Catalpol could ameliorate HCY-induced oxidation, cells apoptosis and inflammation in HAECs possibly by inhibiting Nox4/NF-κB and ER stress.
KEY WORDScatalpol homocysteine HAECs ER stress Nox4 NF-κB
Endothelial dysfunction is considered as a preliminary event of pathophysiologic importance in the development of atherosclerosis (AS) [1, 2], and provides a significant link between diseases, including hypertension, diabetes, and other high-risk cardiovascular diseases. Homocysteine (HCY) is an intermediate , derived from sulfur-containing amino acid metabolism. Elevated level of circulating HCY, called hyperhomocysteinemia (HHCY), is a common and an independent risk factor for atherogenesis and diabetic cardiovascular diseases (CVDs) [4, 5, 6, 7]. Previous studies showed that HCY-induced endothelial dysfunction is associated with oxidative stress and ER stress, probably due to stimulating inflammatory response as well as causing disturbance in the anti-thrombotic activities of the endothelium .
Pieces of evidence indicate that HHCY is linked with increased risk of oxidative stress injury. The pathophysiological level of HCY is implicated to reduce bioavailability of NO, accelerate superoxide and peroxynitrite generation, and inhibit anti-oxidant defense in cardiovascular system [9, 10, 11]. Reactive oxygen species (ROS) have been identified as mediators in the thiol of HCY auto-oxidation . Therefore, administration of anti-oxidant is widely involved in attenuating the impairment of cardiovascular function induced by HHCY.
The endoplasmic reticulum (ER) and mitochondria are viewed as important cellular organelles to preserve cellular homeostasis, and ER and mitochondria interact with each other physically and functionally . Recent studies suggested that HCY could trigger ER stress by perturbing disulfide bond formation and inducing unfolded protein response (UPR) . One crucial response of UPR signaling is to elevate protein synthesis to perturb homeostasis, such as PERK, eIF2α, and IRE1α. The other is to activate a coordinately regulated transcriptional network, including ATF4, ATF6, and XBP-1 . Simultaneously, HCY could activate ER-mitochondria coupling and mitochondrial metabolic reprogramming, containing mitochondrial ROS generation, calcium signal and membrane potential .
Catalpol is an iridoid glycoside extracted from the fresh roots of radix rehmannia. It has been demonstrated to exert anti-inflammatory, anti-oxidant, anti-apoptotic, and other neuroprotective properties, and has a role in neuroprotection against ischemic injuries and neurodegenerative diseases. Catalpol can reduce oxidative damage, regulate endocrine function, and enhance anti-inflammatory properties . Catalpol can also protect against cardiovascular injuries by attenuating free radicals, lipid peroxidation, and cell apoptosis . However, the underlying mechanism of catalpol on endothelial dysfunction induced by HCY in HAECs has not been fully elucidated. In the current study, we investigated the causative role of catalpol on HCY-induced injuries in HAECs. Furthermore, we also analyzed the involvement of Nox4/NF-κB and GRP78/PERK pathways in mediating the protecting effects of catalpol in HAECs.
MATERIALS AND METHOD
Reagent and Antibodies
Human aorta endothelial cells (HAECs) were purchased from Shanghai Bioleaf Biotech Co, Ltd. (Shanghai, China). Cell culture media (DMEM) and fetal bovine serum (FBS) were obtained from the Gibco-BRP Company (Gaithersburg, MD, USA). Catalpol (at a purity of ≥ 99.5%) was bought from Nanjing Jingzhu Biotechnology Co., Ltd. (Jiangsu, China). DL-homocysteine (HCY) was purchased from Sigma-Aldrich (St Louis, MO, USA). DCFH-DA fluorescent probe and ECL Plus were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Rabbit anti-human polyclonal antibodies recognizing Nox4, p65, bcl-2, cleaved caspase-3, caspase-9, cytochrome c, ATF6, CHOP, and IκB polyclonal antibodies were obtained from Proteintech Group, Inc. Rabbit anti-GRP78 polyclonal antibody was purchased from Wuhan Boster Bio-Engineering Limited Company. Rabbit anti-PERK antibody and rabbit anti-phospho-PERK were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. Polyclonal antibodies specific for eIF2α, phospho-eIF2α were purchased from Beyotime Biotechnology (Jiangsu, China). Antibodies specific for IRE1, phosph-IRE1 (phosphor S724) were purchased from Abcam (Hong Kong) Ltd. Mouse anti-beta actin monoclonal antibody and gold anti-rabbit IgG-HRP were purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China).
Cell Culture Studies
HAECs were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 1 U/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated at 37 °C in humidified atmosphere with 5% CO2. The cultured cells were made quiescent by incubation with serum-free basal medium for 24 h before use.
SOD, MDA, GSH, and LDH Assays
In order to estimate the damage level of endothelial cells, we applied a colorimetric assay kit (Beyotime, Nanjing, China) to measure malonaldehyde (MDA), glutathione (GSH), and lactate dehydrogenase (LDH). In summary, HAECs were incubated in 6-well plates at a density of 1 × 105 each well. The cells were then cultivated for 24 h with different concentrations of catalpol (7.5, 15, 30 μM) and HCY (3 mM) for 24 h.
Measurement of Intracellular ROS
ROS level was measured by the fluorescent probe, 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe as previously described . HAECs were cultured in 6-well plates at a density of 2 × 105 per well, while the cells were incubated with different concentrations of catalpol for 23 h at 37 °C, HCY was followed for 1 h. At the end of treatment, the culture medium was changed to serum-free medium with DCFH-DA (10 μM) at 37 °C. After 30 min, the fluorescence intensity was quantified by a BD FACSCalibur Flow Cytometer (Becton, Dickinson and Company, USA).
HAECs were seeded in 6-well plates at a density of 1 × 105/well. The cells were treated with catalpol (7.5, 15, and 30 μM) and HCY (3 mM) at 37 °C for 24 h. At the end of treatment, the cells were fixed with 4% paraformaldehyde for 15 min, rinsed with PBS, and mounted in mounting medium with DAPI. The samples were observed by an inverted fluorescence microscope.
Terminal Deoxynucleotide Transferase dot Nick-End Labeling (TUNEL) Assay
Apoptosis was measured by TUNEL assay using TUNEL apoptosis detection kit (Bio tool, Houston, TX, USA). Briefly, after treatment with different concentrations of catalpol (7.5, 15, 30 μM) for 24 h, the cells were fixed with 4% paraformaldehyde for 15 min and rinsed with PBS. After permeabilization with 0.5% tween 20, HAECs were incubated at room temperature for 1 h in a moist chamber with TUNEL mixture as recommended by the manufacturer. Then, the samples were observed by an inverted fluorescence microscope.
Measurement of Mitochondrial Transmembrane Potential
Changes in mitochondrial membrane potential were detected in the presence of the fluorescent rhodamine 123 (rh123). After treatment with different concentrations of catalpol (7.5, 15, 30 μM) for 24 h, the HAECs were cultivated with rh123 (1 mg ml−1 in dimethyl sulfoxide) at 37 °C for 1 h and rinsed with PBS. The cells were collected and analyzed by confocal laser scanning microscope (Leica microsystems).
RNA Isolation and Real-Time RT-PCR Analysis
The transcript level of cells damage was determined in the cDNA sample by using quantitative real-time PCR. Total RNA was extracted with RNAiso plus reagent following the manufacturer’s instructions (Takara Biotechnology, Dalian, China) and cDNA was synthesized with the Primescript RT reagent Kit with gDNA Eraser (Takara Biotechnology, Dalian, China). Then, the cDNA was supplemented with primers and PCR-mix according to the manufacturers’ protocol. Real-time PCR amplification and detection were performed using the ABI PRISM 7500 real-time PCR system (Applied Biosystems, USA) with the SYBR Premix EX Taq™. Relative mRNA amount was calculated by the 2(−delta delta CT) method using β-actin as an internal control for each sample.
The primer sequences for CHOP:
The primer sequences for ATF4:
The primer sequences for β-actin:
Western Blotting Analysis
The samples were homogenized in a whole cell extract buffer. Equal amounts of protein was resolved on a SDS-PAGE (8%, 10%, 15%) and transferred onto polyvinyl difluoride (PVDF) membranes. The reacted band was visualized by chemiluminescence (ECL plus, Beyotime Institute of Biotechnology, Shanghai, China). The blots were analyzed with Gel-Pro analyzer software according to manufacturer’s instruction. To account for possible difference in the protein load, the density of each band was divided by the density of the respective of β-actin.
siRNA against Nox4 was purchased from Life Technologies (CA, USA) for transfection. The sense and anti-sense strands of the Nox4 siRNA were 5′-CCAUGUGCCGAACACUCUUTT-3′ and 5′-AAGAGUGUUCGGCACAUGGT-3′, respectively. The siRNA transfection was performed with Lipofectamine™ 2000 (Life Technologies, CA, USA), according to the manufacture’s guide.
The results were collected and analyzed using SPSS software package (version 19.0, SPSS), and all of the values were expressed as the mean ± SD. Comparison of quantitative variables was performed by ANOVA followed by the Student-Newman-Keuls (SNK) test. P-values < 0.05 (two-tailed) were considered statistically significant.
Catalpol Decreased the Level of MDA and LDH Release as well as Increased GSH Generation in HCY-Treated HAECs
Catalpol Attenuated HCY-Induced ROS over-Generation and NADPH Oxidase over-Expression
Catalpol Ameliorated HCY-Induced ROS over-Generation via Inhibiting Nox4 Activation in HAECs
Catalpol Down-Regulated HCY-Induced Inflammatory Response in HAECs
Catalpol Inhibited HCY-Induced Apoptosis of HAECs
Catalpol Improved the Expression of bcl-2 and Inhibited the Protein Levels of bax, Cleaved Caspase-3, Caspase-9, and Cytochrome c
Catalpol Protected Against ER Stress-Mediated Apoptotic Processes in HCY-Treated HAECs
Catalpol Relieved the Over-Expression and Activation of ER Stress-Associated Marker Proteins in HAECs Induced by HCY
Catalpol Inhibited the Degradation of IκB and Transcriptional Activation of NF-κB Induced by HCY
Catalpol Attenuated HCY-Induced Activation of NF-κB Pathway and ER Stress via Inhibiting Nox4 in HAECs
Catalpol Alleviated HCY-Induced over-Expression of Nox4 and Activation of ER Stress via Blocking NF-κB/p65 in HAECs
Catalpol Relieved HCY-Induced ROS over-Generation and the Protein over-Expressions of Nox4 and NF-κB/p65 through Inhibiting ER Stress in HAECs
Homocysteine is an anechogenic sulfurated amino acid, which is rooted in ingested methionine including eggs, cheese, fish, meat, and poultry . Hyperhomocysteinemia is a pathological process characterized by an elevation in plasma concentration of total homocysteine . Recent studies observed that HHCY is an indicator of adverse cardiovascular events among the patients of ischemic heart disease and stroke [33, 34]. Homocysteine could cause oxidative stress and ER stress, leading to inflammatory response, growth arrest, and programmed cell death in cultured human vascular endothelial cells.
It is well known that HCY yields superoxide and hydrogen peroxidase by auto-oxidation to trigger oxidative stress to blood vessels . The present study observed that HCY inhibited the generation of glutathione peroxidase and enhanced intracellular production of superoxide, including MDA level and LDH release. Given that catalpol could activate the expression of anti-oxidant enzymes, catalpol could preliminarily prevent atherogenesis by inhibiting oxidative stress. NAD (P) H oxidases are the major sources of intracellular ROS production in Hcy-induced endothelial cells and NAD (P) H oxidases consist of multiple subunits, containing membrane-associated Nox1–4 and p22phox subunits, with Nox4 playing the key role in vasculopathies [36, 37]. In this study, we found that Hcy-induced Nox4 and p22phox over-expressions were both down-regulated by catalpol. Catalpol also enhanced the superoxide anion scavenging ROS capacity and reduced endothelial cell injury. This provided that catalpol could protect against the oxidative damage of cytotoxic oxygen radical and improve the function of cardiovascular system.
Evidence has shown that an excess of HCY could induce accumulation of misfolded proteins and trigger the ER stress . GRP78 is the typical ER chaperone protein that binds to ATF6, PERK, and IRE1α in normal condition. When the cells are under stress, these transmembrane transducers will be disaggregated from GRP78 and activate down-stream signaling genes to initiate UPR . Our results showed that the expressions of ER stress-associated proteins including GRP78, ATF6, IRE1α, and PERK phosphorylation were increased in the presence of HCY. Catalpol pre-treatment significantly alleviated the over-expressions of ER stress-related proteins and recovered the homeostasis. In addition, previous studies have reported that ER stress and oxidative stress are relevant . This finding is consistent with our results showing that both Nox4 and ER stress-specific inhibitors (DPI and TUDCA) suppressed the effects of catalpol on inhibiting oxidative stress and the expression level of NF-κB/p65.
A progressive inflammatory response is the major contributor to atherosclerosis, and NF-κB, as a pro-inflammatory factor, involved in atherosclerotic lesion from cardiovascular disease [40, 41]. The activation of NF-κB pathway induced by HCY generally increased the expression of cytokines, chemokines, and cellular adhesion molecules . In the present study, we investigated that HCY stimulated the activation of NF-κB pathway, the expressions of MCP-1, VCAM-1, and ICAM-1, leading to inflammatory reaction in cultured endothelial cells. Catalpol can exert its anti-inflammatory effects via multiple routes, including suppression of the overproduction of focal inflammatory mediators, and the protection of tissues by mediating cytokines. In our present study, catalpol exerted its anti-inflammatory property through reducing the expressions of pro-inflammatory factors. Many studies have verified that the translocation of NF-κB is associated with programmed cell death via activating caspase family . Indeed, our results suggested that catalpol relieved the cleaved caspase-3, caspase-9, and bax expression, as well as elevated the expression of bcl-2. Taking together, the effects of catalpol might be achieved by maintaining endothelial function via reducing oxidative stress and ER stress and inflammation.
In conclusion, these results indicated that HCY could cause endothelial impairment and mitochondrial dysfunction in HAECs, including decreased membrane mitochondrial potential and increased oxidative stress, ER stress as well as inflammation. Catalpol pre-treatment significantly alleviated oxidative stress, inflammatory response and maintained the ER and mitochondrial integrity in HCY-induced HAECs. Consequently, the actions of catalpol protecting against HCY-induced injuries in HAECs by inhibiting Nox4/ROS-NF-κB pathway and ER stress might be used in the treatment and/or prevention of atherosclerosis. Catalpol would be a potentially novel drug for the treatment and prevention of AS.
Catalpol can ameliorate HCY-induced ER stress and oxidation then reduced cells apoptosis and inflammation in HAECs. These results suggested catalpol played a key role in protecting endothelial dysfunction in HHCY-induced cardiovascular diseases.
This study was supported in part by Grants from the National Natural Science Foundation of China (No. 81273508) and Medical Research Project of Dalian municipal commission of Health and Family Planning (No. 17Z2002).
Compliance with Ethical Standards
Conflict of Interest
The authors report no declarations of interest.
- 3.Korai, M., K.T. Kitazato, Y. Tada, T. Miyamoto, K. Shimada, N. Matsushita, Y. Kanematsu, J. Satomi, T. Hashimoto, and S. Nagahiro. 2016. Hyperhomocysteinemia induced by excessive methionine intake promotes rupture of cerebral aneurysms in ovariectomized rats. Journal of Neuroinflammation 13: 165.CrossRefGoogle Scholar
- 6.Fang, P., D. Zhang, Z. Cheng, C. Yan, X. Jiang, W.D. Kruger, S. Meng, E. Arning, T. Bottiglieri, E.T. Choi, Y. Han, X.F. Yang, and H. Wang. 2014. Hyperhomocysteinemia potentiates hyperglycemia-induced inflammatory monocyte differentiation and atherosclerosis. Diabetes 63: 4275–4290.CrossRefGoogle Scholar
- 7.Wang, H., X. Jiang, F. Yang, J.W. Gaubatz, L. Ma, M.J. Magera, X. Yang, P.B. Berger, W. Durante, H.J. Pownall, and A.I. Schafer. 2003. Hyperhomocysteinemia accelerates atherosclerosis in cystathionine beta-synthase and apolipoprotein E double knock-out mice with and without dietary perturbation. Blood 101: 3901–3907.CrossRefGoogle Scholar
- 9.Timkova, V., Z. Tatarkova, J. Lehotsky, P. Racay, D. Dobrota, and P. Kaplan. 2016. Effects of mild hyperhomocysteinemia on electron transport chain complexes, oxidative stress, and protein expression in rat cardiac mitochondria. Molecular and Cellular Biochemistry 411: 261–270.CrossRefGoogle Scholar
- 22.Zhang, A., S. Hao, J. Bi, Y. Bao, X. Zhang, L. An, and B. Jiang. 2009. Effects of catalpol on mitochondrial function and working memory in mice after lipopolysaccharide-induced acute systemic inflammation. Experimental and toxicologic pathology. Official journal of the Gesellschaft fur Toxikologische Pathologie 61: 461–469.CrossRefGoogle Scholar
- 24.Ren, H., J. Mu, J. Ma, J. Gong, J. Li, J. Wang, T. Gao, P. Zhu, S. Zheng, J. Xie, and B. Yuan. 2016. Selenium inhibits homocysteine-induced endothelial dysfunction and apoptosis via activation of AKT. Cellular Physiology and Biochemistry: International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology 38: 871–882.CrossRefGoogle Scholar
- 36.Xu, S., S.M. Nam, J.H. Kim, R. Das, S.K. Choi, T.T. Nguyen, X. Quan, S.J. Choi, C.H. Chung, E.Y. Lee, I.K. Lee, A. Wiederkehr, C.B. Wollheim, S.K. Cha, and K.S. Park. 2015. Palmitate induces ER calcium depletion and apoptosis in mouse podocytes subsequent to mitochondrial oxidative stress. Cell Death & Disease 6: e1976.CrossRefGoogle Scholar
- 40.Brand, K., S. Page, G. Rogler, A. Bartsch, R. Brandl, R. Knuechel, M. Page, C. Kaltschmidt, P.A. Baeuerle, and D. Neumeier. 1996. Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. The Journal of Clinical Investigation 97: 1715–1722.CrossRefGoogle Scholar
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