目的 研究葛根素（puerarin, Pur）对H9c2心肌细胞低氧损伤的保护作用。方法 采用低氧工作站模拟低氧环境（0.5% O2）构建H9c2 心肌细胞低氧损伤模型,设置常氧对照组(normoxia)、低氧组（hypoxia）及低氧给药组（h+1 Pur、h+2.5 Pur、h+5 Pur）,每组设置3个复孔;常氧对照组在37℃、5%CO2 浓度的条件下进行培养,低氧给药组给药干预12 h后与低氧组一同转入低氧工作站,培养48 h;检测H9c2细胞活力、活性氧（reactive oxygen species,ROS）、乳酸脱氢酶（lactate dehydrogenase,LDH）、丙二醛（malondialdehyde,MDA）、超氧化物歧化酶（superoxide dismutase, SOD）与细胞上清液肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)、白细胞介素（interleukin, IL）-1β和IL-6 水平,蛋白免疫印迹（Western blot）检测焦亡相关蛋白表达水平。结果 与常氧对照组比较,H9c2细胞经低氧处理后,细胞活力与SOD活性显著下降,细胞ROS、LDH、MDA水平显著升高, Caspase-1、NLRP3、GSDMD-N蛋白表达显著上调,差异均有统计学意义（P<0.05）;与低氧组比较,给予Pur处理后H9c2细胞活力与SOD活性显著提升,LDH、ROS、MDA水平显著降低,NLRP3、GSDMD-N、IL-18蛋白表达下调,差异均有统计学意义（P<0.05）。结论 Pur可通过调控H9c2细胞NLRP3/Caspase-1/GSDMD通路,缓解细胞凋亡改善低氧诱导的氧化应激损伤。
Abstract
Objective To investigate the protective effect of puerarin (Pur) against hypoxia-induced pyroptosis and oxidative stress in H9c2 cardiomyocytes. Methods An in vitro model of hypoxia injury was established using H9c2 cardiomyocytes cultured in a hypoxic workstation (0.5% O₂). Cells were divided into five groups: normoxic control (normoxia), hypoxia (hypoxia), and hypoxia with Pur treatment at concentrations of 1 µmol/L (h+1Pur), 2.5 µmol/L (h+2.5Pur), and 5 µmol/L (h+5Pur), each with three replicates. After a 12-hour pre-treatment with Pur, the hypoxia groups were subjected to hypoxia for 48 hours. Cell viability, intracellular reactive oxygen species (ROS), and supernatant levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), were measured. Additionally, the expression of pyroptosis-related proteins (NLRP3, Caspase-1, GSDMD-N, and IL-18) was assessed by Western blot. Results Compared with the normoxic control, hypoxia significantly reduced cell viability and SOD activity (P<0.05), and markedly increased intracellular ROS, LDH, and MDA levels (P<0.05). Moreover, the protein levels of NLRP3, Caspase-1, and GSDMD-N were significantly up-regulated (P<0.05). Pur treatment significantly improved cell viability and SOD activity, and reduced ROS, LDH, and MDA levels (P<0.05). Importantly, Pur markedly down-regulated the expression of NLRP3, GSDMD-N, and IL-18 (P<0.05) compared with the hypoxia group. Conclusion Puerarin effectively attenuates hypoxia-induced pyroptosis and oxidative stress in H9c2 cells, partly through inhibition of the NLRP3/Caspase-1/GSDMD signaling pathway.
关键词
葛根素 /
心肌细胞 /
低氧 /
细胞焦亡 /
氧化应激
Key words
puerarin /
cardiomyocytes /
hypoxia /
pyroptosis /
oxidative stress
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] Geraets IME, Glatz JFC, Luiken JJFP, et al. Pivotal role of membrane substrate transporters on the metabolic alterations in the pressure-overloaded heart[J]. Cardiovasc Res,2019,115:1000–1012.
[2] Tejero J, Shiva S, Gladwin MT.Sources of vascular nitric oxide and reactive oxygen species and their regulation[J]. Physiol Rev,2019,99:311–379.
[3] Qiu X, Yao Y, Chen Y, et al. TRPC5 promotes intermittent hypoxia-induced cardiomyocyte injury through oxidative stress[J]. Nat Sci Sleep,2024,16:2125–2141.
[4] Huang H, Ruan Y, Li C, et al. Hypoxia microenvironment preconditioning attenuated myocardial ischemia-reperfusion injury via Stc1-mediating cardiomyocyte self-protection and neutrophil polarization[J]. Adv Sci (Weinh),2025,12:e2411880.
[5] Mauro AG, Bonaventura A, Mezzaroma E, et al. NLRP3 inflammasome in acute myocardial infarction[J]. J Cardiovasc Pharmacol. 2019,74:175–187.
[6] Yanpiset P, Maneechote C, Sriwichaiin S, et al. Gasdermin D-mediated pyroptosis in myocardial ischemia and reperfusion injury: cumulative evidence for future cardioprotective strategies[J]. Acta Pharm Sin B,2023;13:29–53.
[7] 韩磊,贾娟. 药食同源葛根的生物活性及其在食品中应用的研究[J]. 粮食与食品工业,2025,32:51–54.
[8] Ritterhoff J, Tian R.Metabolic mechanisms in physiological and pathological cardiac hypertrophy: new paradigms and challenges[J]. Nat Rev Cardiol, 2023, 20:812–829.
[9] Tao Z, Ge Y, Zhou N, et al. Puerarin inhibits cardiac fibrosis via monocyte chemoattractant protein (MCP)-1 and the transforming growth factor-β1 (TGF-β1) pathway in myocardial infarction mice[J]. Am J Transl Res,2016,8:4425–4433.
[10] 程安然,王钰滢,华健,等. 黄芪-葛根药对治疗糖尿病心肌病的理论探讨[J]. 中草药,2023,54:7853–7861.
[11] Li D, Wang X, Huang Q, et al. Cardioprotection of CAPE-oNO2 against myocardial ischemia/reperfusion induced ROS generation via regulating the SIRT1/eNOS/NF-κB pathway in vivo and in vitro[J]. Redox Biol, 2018;15:62–73.
[12] Ward JPT.Physiological redox signalling and regulation of ion channels: implications for pulmonary hypertension[J]. Exp Physiol, 2017;102(9):1078–1082.
[13] Irarrázaval S, Allard C, Campodónico J, et al. Oxidative stress in acute hypobaric hypoxia[J]. High Alt Med Biol,2017,1128–134.
[14] Ma ZG, Yuan YP, Xu SC, et al. CTRP3 attenuates cardiac dysfunction, inflammation, oxidative stress and cell death in diabetic cardiomyopathy in rats[J]. Diabetologia,2017,60:1126–1137.
[15] Wang F, Liang Q, Ma Y, et al. Silica nanoparticles induce pyroptosis and cardiac hypertrophy via ROS/ NLRP3/Caspase-1 pathway[J]. Free Radic Biol Med,2022,182:171–181.
[16] Li C, Liu Q, Chen Q, et al. Effects of Chinese medicine on pyroptosis in coronary heart disease[J]. Am J Chin Med,2023,5:1653–1673.
[17] Liu X, Zhang Z, Ruan J, et al. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores[J]. Nature,2016,535:153–158.
