Nrf2在黄芩素抑制氧化应激诱导心肌细胞凋亡中的作用  

Effects of baicalein on inhibition of oxidative stress-induced apoptosis of cardiomyocytes via Nrf2

作　　者：徐由财 丁文俊 陈思[1] 陈俊邦 陈婷芳 曾科峰 刘彬[1,2] 张竞之[1,2] XU You-cai;DING Wen-jun;CHEN Si;CHEN Jun-bang;CHEN Ting-fang;ZENG Ke-feng;LIU Bin;ZHANG Jing-zhi(Department of Traditional Chinese Medicine,The Second Hospital Affiliated to Guangzhou Medical University,Guangzhou 510260,China;Institute of Integrative Medicine,Guangzhou Medical University,Guangzhou 510180,China)

机构地区：[1]广州医科大学附属第二医院中医科,广东广州510260 [2]广州医科大学中西医结合研究所,广东广州510180

出　　处：《中成药》2022年第5期1434-1440,共7页Chinese Traditional Patent Medicine

基　　金：国家自然科学基金项目(81774100,81874418,81973830);广东省中医药局科研项目(20213012);广东省基础与应用基础研究基金项目(2019A1515110367,2019B1515120026)。

摘　　要：目的探索核因子E2相关因子(Nrf2)在黄芩素抑制氧化应激诱导心肌细胞凋亡中的作用。方法采用MTS、TUNEL细胞凋亡染色观察黄芩素对叔丁基过氧化氢(TBHP)诱导H9c2心肌细胞凋亡的影响,分子对接、分子动力学及表面等离子体共振技术(SPR)探索黄芩素与Nrf2的相互作用,Western blot法检测Nrf2、HO-1、NQO1蛋白表达,试剂盒检测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性,Nrf2抑制剂ML385验证Nrf2在黄芩素抑制氧化应激诱导心肌细胞凋亡中的作用。结果黄芩素能剂量依赖性地抑制TBHP诱导的心肌细胞活力降低和凋亡率升高(P<0.01),抑制TBHP诱导的SOD、GSH-Px活性降低(P<0.01)。分子对接发现,黄芩素与Keap1的Kelch位点结合,结合力为-9.0 kcal/mol,与Keap1的氨基酸残基形成氢键。分子动力学与SPR研究发现,黄芩素与Keap1结合的平衡解离常数(K_(D))为6.54×10^(-7)mol/L,具有很强的结合能力,黄芩素剂量依赖性地上调Nrf2、HO-1、NQO1的表达(P<0.05),ML385抑制了黄芩素对TBHP诱导心肌细胞凋亡的保护作用。结论黄芩素能抑制氧化应激导致的心肌细胞凋亡,其机制与激活Nrf-2信号通路有关。AIM To explore the effects of baicalein on inhibition of oxidative stress-induced apoptosis of cardiomyocytes via Nrf2.METHODS MTS and TUNEL cell apoptosis staining were used to observe the effect of baicalein on apoptosis of H9c2 induced by tert-butyl hydroperoxide(TBHP).Molecular docking,molecular dynamics and surface plasmon resonance(SPR)were used to explore the interaction between baicalein and Nrf2.Western blot was used to detect the protein expressions of Nrf2,HO-1 and NQO1.The kit was used to detect the activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px).The Nrf2 inhibitor ML385 was used to verify the role of Nrf2 in baicalein’s inhibition on oxidative stress-induced cardiomyocyte apoptosis.RESULTS Baicalein dose-dependently inhibited TBHP-induced decrease of myocardial cell activity and increase of apoptosis rate(P<0.01);and reduction of SOD and GSH-Px activities(P<0.01)as well.Molecular docking found that baicalein bound to the Kelch site of Keap1 at a binding force of-9.0 kcal/mol,and formed hydrogen bonds with the amino acid residues of Keap1.Molecular dynamics simulations demonstrated the stable combination of baicalein and Keap1.The SPR study showed the value of 6.54×10^(-7)mol/L equilibrium dissociation constant(K_(D))of baicalein and Keap1.Baicalein dose-dependently up-regulated the expressions of Nrf2,HO-1 and NQO1(P<0.05).ML385 inhibited the protective effect of baicalein on TBHP-induced cardiomyocyte apoptosis.CONCLUSION Being an inhibitor to oxidative stress-induced cardiomyocyte apoptosis,baicalein works via the activation of Nrf-2 signaling pathway.

关 键 词：黄芩素 氧化应激 心肌细胞 NRF2 

分 类 号：R285.5[医药卫生—中药学]