沈阳药科大学学报

2023, v.40;No.324(01) 63-74

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二氢杨梅素通过线粒体途径及影响ROS-NF-κB信号通路诱导心肌细胞AC16凋亡
Dihydromyricetin induces apoptosis of myocardial AC16 cells through mitochondrial pathway and ROS-NF-κB signaling pathway

林志康,赵宇侠,方明,宁忠平,葸灵娟,黄煜茹,李新明
LIN Zhikang,ZHAO Yuxia,FANG Ming,NING Zhongping,XI Lingjuan,HUANG Yuru,LI Xinming

摘要(Abstract):

目的 探讨二氢杨梅素(dihydromyricetin, DMY)对人源正常心室肌细胞AC16毒性作用及其机制。方法 采用CCK-8法检测不同质量浓度的DMY对心肌细胞AC16存活率的影响,后选取低、中、高3个质量浓度(30、60、90 mg·L~(-1))DMY处理AC16细胞,采用流式细胞术(flow cytometry, FCM)检测细胞凋亡率、细胞活性氧(reactive oxygen species, ROS)生成水平。酶标仪检测细胞外液中乳酸脱氢酶(lactate dehydrogenase, LDH)、谷草转氨酶(aspartate transaminase, AST)、细胞内超氧化物歧化酶(superoxide dismutase, SOD),以及过氧化氢酶(catalase, CAT)变化。通过激光共聚焦及FCM共同检测细胞线粒体膜电位变化,蛋白印迹法检测相关蛋白包括核转录因子-κB(nuclear factor kappa B,NF-κB)p65、p-NF-κB p65、核转录因子-κB抑制蛋白α(inhibitor protein α of kappa B,IκBα)、p-IκBα及凋亡相关蛋白包括B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl2相关蛋白x(Bcl2-associated x, Bax)及剪切型半胱氨酸蛋白水解酶-3(cleaved-cysteinyl aspartate specific proteinase-3,cleaved-caspase-3)的表达水平。结果 DMY致心肌损伤的最佳处理时间为24 h, AC16细胞的存活率降低,凋亡率增加,细胞外液中LDH及AST含量的上调、细胞内SOD及CAT酶活性的降低均呈现与浓度相关的剂量依赖效应,线粒体膜电位下降伴随ROS释放的增加提示线粒体功能受损严重。此外,细胞凋亡率的增加伴随p-IκBα、p-NF-κB p65、cleaved-caspase-3表达水平的显著上调及Bcl-2/Bax比值下调趋势。结论 二氢杨梅素通过ROS-NF-κB信号通路轴及线粒体途径诱导心肌细胞凋亡,造成细胞毒性。
Objective To investigate the toxic effects of dihydromyricetin(DMY)on human normal ventricular myocytes(AC16)and its mechanism.Methods CCK-8 assay was used to detect the effects of different concentrations of DMY(0,20,40,60,80 and 100 mg·L~(-1))on the survival rates of AC16 cardiomyocytes, and the changes of cell morphology were observed.AC16 cells were treated with low, medium and high concentrations(30,60 and 90 mg·L~(-1))according to the cell half inhibition rate(IC_(50))obtained by proliferation assay.Flow cytometry(FCM)was used to detect the apoptosis rate and the generation level of reactive oxygen species(ROS).The changes of lactate dehydrogenase(LDH)and aspartate aminotransferase(AST)in extracellular nutrient solution and superoxide dismutase(SOD)and catalase(CAT)in intracellular fluid were detected by microplate plate analyzer.The changes of mitochondrial membrane potential were detected by laser confocal and FCM.Western blotting was used to detect the expression levels of related proteins including nuclear factor-κB(NF-κB)p65,p-NF-κB p65,inhibitor protein α of kappa B(IκBα),p-IκBα and apoptosis-related proteins B-cell lymphoma-2(Bcl-2),Bcl2-associated x(Bax)and cleaved-cysteinyl aspartate specific proteinase-3(cleaved-caspase-3).Results The optimal treatment time of myocardial injury induced by DMY was 24 h.The survival rate of AC16 cells was decreased, the apoptosis rate was increased, the contents of LDH and AST in extracellular fluid were up-regulated, and the activities of SOD and CAT in the cells were decreased in dose-dependent manner.The decrease of mitochondrial membrane potential accompanied by the increase of ROS release suggested that mitochondrial function was severely impaired.In addition, the increase of apoptosis rate was accompanied by significant up-regulation of p-IκBα,p-NF-κB p65,cleaved-caspase-3 expression levels and down-regulation of Bcl-2/Bax ratio.Conclusion Dihydromyricetin induces apoptosis of cardiomyocytes through ROS-NF-κB signaling pathway and mitochondrial pathway, resulting in cytotoxicity.

关键词(KeyWords): 二氢杨梅素;心肌毒性;凋亡;氧化应激;NF-κB
dihydromyricetin;myocardial toxicity;apoptosis;oxidative stress;NF-κB

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金资助项目(21307031);; 上海市浦东新区高峰学科建设项目(PWYgf2021-04);; 上海市浦东新区重点学科群建设项目(PWZxq2022-11);; 上海市浦东新区心房颤动流行病学调查及分级诊疗模式下房颤全程管理前瞻性队列研究(PKJ2021-Y33)

作者(Author): 林志康,赵宇侠,方明,宁忠平,葸灵娟,黄煜茹,李新明
LIN Zhikang,ZHAO Yuxia,FANG Ming,NING Zhongping,XI Lingjuan,HUANG Yuru,LI Xinming

DOI: 10.14066/j.cnki.cn21-1349/r.2021.0366

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