寒痉汤对冷刺激诱导主动脉平滑肌细胞氧化应激的影响及机制

doi:10.6040/j.issn.1671-7554.0.2023.0017

摘要/Abstract

摘要： 目的观察寒痉汤对冷刺激诱导血管平滑肌细胞氧化应激的保护作用及其作用机制。方法将实验细胞分为空白组、模型组、寒痉汤组及富马酸二甲酯组,以富马酸二甲酯为阳性对照,各组血管平滑肌细胞(A7r5)培养24 h后,4 ℃冷刺激4 h。采用CCK-8法检测各组细胞活力;采用化学法检测细胞超氧化物歧化酶(SOD)、丙二醛(MDA)表达水平;采用DCFH-DA荧光法检测细胞活性氧(ROS)的表达;采用Elisa法检测细胞白细胞介素1β(IL-1β)、核苷酸结合寡聚结构域样受体蛋白3(NLRP3)表达水平;采用免疫印迹法检测细胞核因子E2相关因子2(Nrf2)、NAD(P)H醌氧化还原酶1(NQO1)、核因子κB(NF-κB)、人核因子κB抑制蛋白α(IκBα)蛋白表达水平;采用实时荧光定量PCR技术检测NQO1 mRNA、NF-κB mRNA表达水平。结果与空白组相比,模型组细胞活力、SOD表达降低(P<0.01),ROS、MDA、IL-1β、NLRP3表达升高(P<0.01);NF-κB、IκBα蛋白表达升高(P<0.01),Nrf2、NQO1蛋白表达降低(P<0.01);NF-κB mRNA表达升高(P<0.01),NQO1 mRNA表达降低(P<0.01)。与模型组相比,寒痉汤组细胞活力升高(P<0.01),SOD表达升高(P<0.01);ROS表达降低(P<0.01);MDA表达降低(P<0.05),IL-1β、NLRP3表达降低(P<0.01),Nrf2、NQO1蛋白表达升高(P<0.01),NF-κB、IκBα蛋白表达降低(P<0.05,P<0.01);NF-κB mRNA表达降低(P<0.01),NQO1 mRNA表达升高(P<0.05)。结论寒痉汤提高冷刺激主动脉平滑肌细胞活力,调节细胞ROS表达,抑制炎症因子表达,改善氧化应激反应,其机制可能与Nrf2/NF-κB通路有关。

关键词: 冷刺激, 氧化应激, 寒痉汤, 核因子E2相关因子2, 核因子κB

Abstract: Objective To observe the effects and mechanism of Hanjingtang on the oxidative stress induced by cold stimulation in aortic vascular smooth muscle cells. Methods The experimental cells were divided into blank control group, model group, Hanjingtang(HJT)group and dimethyl fumarate(DMF)group. The DMF group was defined as the positive control. After vascular smooth muscle cells(A7r5)were cultured for 24 hours, they received cold stimulation at 4 ℃ for 4 hours. Cell viability was detected with CCK-8; expressions of superoxide dismutase(SOD)and malonaldehyde(MDA)were determined with immunochemistry; expression of active oxygen(ROS)was detected with DCFH-DA fluorescence; expressions of interleukin-1β(IL-1β)and nucleotide-binding oligomeric domain-like receptor protein 3(NLRP3)were assessed with Elisa; protein expressions of cellular nuclear factor E2-associated factor 2(Nrf 2), NAD(P)H quinone oxidoreductase 1(NQO1), nuclear factor κB(NF-κB), and human nuclear factor κB inhibitor protein α(IκBα)were determined with Western blotting; mRNA expressions of NQO1 and NF-κB were detected with quantitative real-time PCR. Results Compared with the blank control group, the model group had decreased cell viability and SOD(P<0.01); increased ROS, MDA, IL-1β and NLRP3 levels; increased protein expressions of NF-κB and IκBα, but decreased protein expressions of Nrf2 and NQO1(P<0.01); increased mRNA expression of NF-κB, but decreased mRNA expression of NQO1(P<0.01). Compared with the model group, the HJT group had increased cell viability and SOD(P<0.01); decreased ROS, IL-1β, NLRP3(P<0.01), and MDA(P<0.05)levels; increased protein expressions of Nrf2 and NQO1(P<0.01), but decreased protein expressions of NF-κB and IκBα(P<0.05, P<0.01); decreased mRNA expression of NF-κB(P<0.01), but increased mRNA expression of NQO1(P<0.05). Conclusion The mechanism of enhancing cold-stimulated aortic smooth muscle cell vitality, regulating cellular ROS expression, inhibiting inflammatory factor expression, and improving oxidative stress response may be related to Nrf2/NF-κB pathway.

Key words: Cold stimulation, Oxidative stress, Hanjingtang, Nuclear factor E2-associated factor 2, Nuclear factor κB

中图分类号:

R259

刘洋,陈贵海. 寒痉汤对冷刺激诱导主动脉平滑肌细胞氧化应激的影响及机制[J]. 山东大学学报 (医学版), 2023, 61(8): 24-30.

LIU Yang, CHEN Guihai. Effects and mechanism of Hanjingtang on the oxidative stress induced by cold stimulation in aortic vascular smooth muscle cells[J]. Journal of Shandong University (Health Sciences), 2023, 61(8): 24-30.

参考文献

[1] Wang B, Liu J, Lei R, et al. Cold exposure, gut microbiota, and hypertension: a mechanistic study [J]. Sci Total Environ, 2022, 833: 155199. doi: 10.1016/j.scitotenv.2022.155199.
[2] Omi T, Kumada M, Kamesaki T, et al. An intronic variable number of tandem repeat polymorphisms of the cold-induced autoinflammatory syndrome 1(CIAS1)gene modifies gene expression and is associated with essential hypertension [J]. Eur J Hum Genet, 2006, 14(12): 1295-1305.
[3] Greaney JL, Kenney WL, Alexander LM. Neurovascular mechanisms underlying augmented cold-induced reflex cutaneous vasoconstriction in human hypertension [J]. J Physiol, 2017, 595(5): 1687-1698.
[4] Humphrey JD. Mechanisms of vascular remodeling in hypertension [J]. Am J Hypertens, 2021, 34(5): 432-441.
[5] Qiu S, Li P, Zhao H, et al. Maresin 1 alleviates dextran sulfate sodium-induced ulcerative colitis by regulating NRF2 and TLR4/NF-kB signaling pathway [J]. Int Immunopharmacol, 2020, 78: 106018. doi: 10.1016/j.intimp.2019.106018.
[6] Xu L, Yu Y, Sang R, et al. Protective effects of taraxasterol against ethanol-induced liver injury by regulating CYP2E1/Nrf2/HO-1 and NF-κB signaling pathways in mice [J]. Oxid Med Cell Longev, 2018, 2018: 8284107. doi: 10.1155/2018/8284107.
[7] 张洁晗. 国医大师李士懋教授寒痉汤治疗寒凝证型高血压病机制探讨[D]. 石家庄: 河北医科大学, 2018.
[8] 刘洋. 寒痉汤对寒凝型高血压大鼠CGRP、RLX及血管平滑肌细胞UCP2、RXFP2的影响[D]. 石家庄: 河北中医学院, 2019.
[9] Xu R, Shi C, Wei J, et al. Cause-specific cardiovascular disease mortality attributable to ambient temperature: a time-stratified case-crossover study in Jiangsu Province, China [J]. Ecotoxicol Environ Saf, 2022, 236: 113498. doi: 10.1016/j.ecoenv.2022.113498.
[10] Stewart S, Keates AK, Redfern A, et al. Seasonal variations in cardiovascular disease [J]. Nat Rev Cardiol, 2017, 14(11): 654-664.
[11] Liu C, Yavar Z, Sun Q. Cardiovascular response to thermoregulatory challenges [J]. Am J Physiol Heart Circ Physiol, 2015, 309(11): H1793-H1812.
[12] 张静, 赵外荣, 施雯婷, 等. 中医药调控平滑肌细胞增殖抑制动脉粥样硬化的研究进展[J]. 中华中医药杂志, 2021, 36(9): 5429-5432. ZHANG Jing, ZHAO Wairong, SHI Wenting, et al. Research process of inhibition of smooth muscle cell proliferation against atherosclerosis by traditional Chinese medicine [J]. China Journal of Traditional Chinese Medicine and Pharmacy, 2021, 36(9): 5429-5432.
[13] 邬雨洁, 张明泉, 纪永利, 等. 寒痉汤及其拆方对寒凝证高血压大鼠血清炎症因子、血管内皮功能及纤维化的影响[J]. 山东大学学报(医学版), 2022, 60(6): 10-18. WU Yujie, ZHANG Mingquan, JI Yongli, et al. Effects of Hanjingtang and its disassembled Prescriptions on serum inflammatory factors, vascular endothelial function and fibrosis in hypertensive rats with cold coagulation syndrome [J]. Journal of Shandong University(Health Science), 2022, 60(6): 10-18.
[14] Hajleh MNA, Khleifat KM, Alqaraleh M, et al. Antioxidant and antihyperglycemic effects of Ephedra foeminea aqueous extract in streptozotocin-induced diabetic rats [J]. Nutrients, 2022, 14(11): 2338.
[15] Seif M, Deabes M, El-Askary A, et al. Ephedra sinica mitigates hepatic oxidative stress and inflammation via suppressing the TLR4/MyD88/NF-κB pathway in fipronil-treated rats [J]. Environ Sci Pollut Res Int, 2021, 28(44): 62943-62958.
[16] Tarkhan MM, Balamsh KS, El-Bassossy HM. Cinnamaldehyde protects from methylglyoxal-induced vascular damage: effect on nitric oxide and advanced glycation end products [J]. J Food Biochem, 2019, 43(7): e12907.
[17] Xue C, Lu H, Liu Y, et al. Trans-ferulic acid-4-β-glucoside alleviates cold-induced oxidative stress and promotes cold tolerance [J]. Int J Mol Sci, 2018, 19(8): E2321.
[18] Tian M, Wang L, Dong Z, et al. Preparation, structural characterization, antioxidant activity and protection against cisplatin-induced acute kidney injury by polysaccharides from the lateral root of Aconitum carmichaelii [J]. Front Pharmacol, 2022, 13: 1002774. doi: 10.3389/fphar.2022.1002774.
[19] Zhang QQ, Chen FH, Wang F, et al. A novel modulator of the renin-angiotensin system, benzoylaconitine, attenuates hypertension by targeting ACE/ACE2 in enhancing vasodilation and alleviating vascular inflammation [J]. Front Pharmacol, 2022, 13: 841435. doi: 10.3389/fphar.2022.841435.
[20] Machado RJ, Junior LG, Monteiro NK, et al. Homology modeling, vasorelaxant and bradykinin-potentiating activities of a novel hypotensin found in the scorpion venom from Tityus stigmurus [J]. Toxicon, 2015, 101: 11-18. doi: 10.1016/j.toxicon.2015.04.003. JP
[21] Wang JS, Li X, Chen ZL, et al. Effect of leech-centipede medicine on improving erectile function in DIED rats via PKC signalling pathway-related molecules [J]. J Ethnopharmacol, 2021, 267: 113463. doi: 10.1016/j.jep.2020.113463.
[22] Li Z, Chen C, Zhu X, et al. Glycyrrhizin suppresses RANKL-induced osteoclastogenesis and oxidative stress through inhibiting NF-κB and MAPK and activating AMPK/Nrf2 [J]. Calcif Tissue Int, 2018, 103(3): 324-337.
[23] Gao Y, Lv XH, Yang HH, et al. Isoliquiritigenin exerts antioxidative and anti-inflammatory effects via activating the KEAP-1/Nrf2 pathway and inhibiting the NF-κB and NLRP3 pathways in carrageenan-induced pleurisy [J]. Food Funct, 2020, 11(3): 2522-2534.
[24] Li C, Li J, Jiang F, et al. Vasculoprotective effects of ginger(Zingiber officinale Roscoe)and underlying molecular mechanisms [J]. Food Funct, 2021, 12(5): 1897-1913.
[25] Das S, Zhang E, Senapati P, et al. A novel angiotensin II-induced long noncoding RNA giver regulates oxidative stress, inflammation, and proliferation in vascular smooth muscle cells [J]. Circ Res, 2018, 123(12): 1298-1312.
[26] Bruder-Nascimento T, Callera GE, Montezano AC, et al. Atorvastatin inhibits pro-inflammatory actions of aldosterone in vascular smooth muscle cells by reducing oxidative stress [J]. Life Sci, 2019, 221: 29-34. doi: 10.1016/j.lfs.2019.01.043.
[27] Camargo LL, Montezano AC, Hussain M, et al. Central role of c-Src in NOX5- mediated redox signalling in vascular smooth muscle cells in human hypertension [J]. Cardiovasc Res, 2022, 118(5): 1359-1373.
[28] Burger F, Baptista D, Roth A, et al. NLRP3 inflammasome activation controls vascular smooth muscle cells phenotypic switch in atherosclerosis [J]. Int J Mol Sci, 2021, 23(1): 340.
[29] Kim EJ, Park SY, Baek SE, et al. HMGB1 increases IL-1β production in vascular smooth muscle cells via NLRP3 inflammasome [J]. Front Physiol, 2018, 9: 313. doi: 10.3389/fphys.2018.00313.
[30] Wang Y, Huang Z, Zhong H, et al. MiR-1929-3p overexpression alleviates murine Cytomegalovirus-induced hypertensive myocardial remodeling by suppressing ednra/NLRP3 inflammasome activation [J]. Biomed Res Int, 2020, 2020: 6653819. doi: 10.1155/2020/6653819.
[31] Zhang J, Cai W, Fan Z, et al. MicroRNA-24 inhibits the oxidative stress induced by vascular injury by activating the Nrf2/HO-1 signaling pathway [J]. Atherosclerosis, 2019, 290: 9-18. doi: 10.1016/j.atherosclerosis.2019.08.023.
[32] Qiu L, Hu L, Liu X, et al. Physalin B inhibits PDGF-BB-induced VSMC proliferation, migration and phenotypic transformation by activating the Nrf2 pathway [J]. Food Funct, 2021, 12(21): 10950-10966.
[33] Pascale CL, Martinez AN, Carr C, et al. Treatment with dimethyl fumarate reduces the formation and rupture of intracranial aneurysms: role of Nrf2 activation [J]. J Cereb Blood Flow Metab, 2020, 40(5): 1077-1089.
[34] Oh CJ, Park S, Kim JY, et al. Dimethylfumarate attenuates restenosis after acute vascular injury by cell-specific and Nrf2-dependent mechanisms [J]. Redox Biol, 2014, 2: 855-864. doi: 10.1016/j.redox.2014.06.003.
[35] Cao YJ, Zhang YM, Qi JP, et al. Ferulic acid inhibits H2O2-induced oxidative stress and inflammation in rat vascular smooth muscle cells via inhibition of the NADPH oxidase and NF-κB pathway [J]. Int Immunopharmacol, 2015, 28(2): 1018-1025.

多维度评价

Viewed

Full text

Abstract

Cited

Shared

Discussed