N‑乙酰半胱氨酸通过调节线粒体自噬减轻H9c2心肌细胞缺氧/复氧损伤  

N‑acetylcysteine alleviates hypoxia/reoxygenation‑induced injury in H9c2 cells by regulating mitophagy

作　　者：金振帅 高雷 纪延炜 文昕郁 唐赫鹏 雷少青[1] Jin Zhenshuai;Gao Lei;Ji Yanwei;Wen Xinyu;Tang Hepeng;Lei Shaoqing(Department of Anesthesiology,Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院麻醉科,武汉430060

出　　处：《国际麻醉学与复苏杂志》2024年第3期233-238,共6页International Journal of Anesthesiology and Resuscitation

基　　金：国家自然科学基金(81772049)。

摘　　要：目的探讨抗氧化剂N‑乙酰半胱氨酸(NAC)减轻H9c2心肌细胞缺氧/复氧(H/R)损伤的可能机制及线粒体自噬在其中的作用。方法体外培养H9c2心肌细胞,按随机数字表法分为3组(每组3个复孔):对照组(NC组)、缺氧/复氧组(H/R组)、缺氧/复氧+N‑乙酰半胱氨酸组(H/R+NAC组)。NC组细胞正常培养,H/R组细胞进行缺氧4 h复氧4 h处理,H/R+NAC组细胞进行缺氧4 h复氧4 h的同时给予终浓度为1.5 mmol/L的NAC。细胞计数试剂盒(CCK‑8)检测细胞活性,酶联免疫吸附试验(ELISA)法检测细胞培养液中乳酸脱氢酶(LDH)水平,2,7‑二氯荧光素二乙酸酯(DCFH‑DA)荧光探针法检测细胞内活性氧(ROS)水平,5,5′,6,6′‑四氯‑1,1′,3,3′‑四乙基苯并咪唑羰花青碘化物(JC‑1)法检测线粒体膜电位水平,免疫印迹法(Western blot)检测线粒体自噬分子蛋白P62、微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)、磷酸酯酶与张力蛋白同源物诱导假定激酶1(PINK1)、帕金森病蛋白(Parkin)的蛋白水平,流式细胞仪检测细胞凋亡水平。结果与NC组比较:H/R组细胞存活率降低(P<0.05),LDH和ROS水平升高(均P<0.05),线粒体膜电位水平和P62、LC3Ⅱ、PINK1、Parkin蛋白水平降低(均P<0.05),细胞凋亡率升高(P<0.05)。与H/R组比较:H/R+NAC组细胞存活率升高(P<0.05),LDH和ROS水平降低(均P<0.05),线粒体膜电位水平和P62、LC3Ⅱ、PINK1、Parkin蛋白水平升高(均P<0.05),细胞凋亡率降低(P<0.05)。与NC组比较,H/R+NAC组细胞存活率,LDH、ROS、线粒体膜电位水平,P62、LC3Ⅱ、PINK1、Parkin蛋白水平和细胞凋亡率差异均无统计学意义(均P>0.05)。结论NAC可以通过促进线粒体自噬水平从而减轻H/R对H9c2心肌细胞的损伤。Objective To explore the possible mechanism by which the antioxidant N‑acetylcysteine(NAC)reduces hypoxia/reoxygenation(H/R)injury in H9c2 cardiomyocytes and the role of mitophagy in the process.Methods H9c2 cardiomyocytes were cultured in vitro.According to the random number table method,they were divided into three groups(each group had three replicate wells):a control(NC)group,a hypoxia/reoxygenation(H/R)group,and a hypoxia/reoxygenation+N‑acetylcysteine(H/R+NAC)group.Cells in the NC group were normally cultured.Those in the H/R group were subjected to hypoxia for 4 h,followed by reoxygenation for 4 h.The H/R+NAC group were subjected to hypoxia for 4 h,followed by reoxygenation for 4 h,with the presence of NAC at a final concentration of 1.5 mmol/L.The cell counting kit‑8(CCK‑8)was used to detect cell viability,while the enzyme‑linked immunosorbent assay(ELISA)was used to detect lactate dehydrogenase(LDH)levels in culture media.In addition,the 2,7‑dichlorodihydrofluorescein‑diacetate(DCFH‑DA)fluorescent probe method was used to measure the levels of intracellular reactive oxygen species(ROS).The 5,5',6,6'‑tetrachloro‑1,1',3,3'‑tetraethylbenzimidazolecarbocyanine iodide(JC‑1)method was used to monitor the levels of mitochondrial membrane potential.Western blot was used to detect the protein levels of mitophagy molecule proteins,P62,microtubule‑associated protein 1 light chain3Ⅱ(LC3Ⅱ),PTEN‑induced putative kinase 1(PINK1),and Parkinson's disease protein(Parkin).Flow cytometry was conducted to estimate apoptotic levels.Results Compared with the NC group,the H/R group showed reduced cell survival rate(P<0.05),increased LDH and ROS levels(all P<0.05),reduced levels of mitochondrial membrane potential and P62,LC3Ⅱ,PINK1,and Parkin proteins (all P<0.05), with an increased cell apoptosis rate (P<0.05). Compared with the H/R group, the H/R+NAC group exhibitedincreased cell survival rate (P<0.05), decreased LDH and ROS levels (all P<0.05), increased levels of mitochondrial membrane pote

关 键 词：N‑乙酰半胱氨酸 H9C2心肌细胞 缺氧/复氧 线粒体自噬 

分 类 号：R614[医药卫生—麻醉学]