吴茱萸碱介导线粒体凋亡途径保护H_(2)O_(2)所致L-O2肝细胞损伤  

Evodiamine-mediated mitochondrial apoptosis pathway protects L-O2 liver cell injury induced by H_(2)O_(2)

作　　者：陈祺松 田港[4] 吴燕 阮凌燕 CHEN Qi-song;TIAN Gang;WU Yan;RUAN Ling-yan(Medical Department,Ningbo Hospital of Urology and Nephropathy,Ningbo 315100,Zhejiang Province,China;Department of Clinical Pharmacy,Ningbo Hospital of Urology and Nephropathy,Ningbo 315100,Zhejiang Province,China;Department of Endocrinology,Ningbo Hospital of Urology and Nephropathy,Ningbo 315100,Zhejiang Province,China;Department of Personnel,Zhejiang University of Chinese Medicine,Hangzhou 311400,Zhejiang Province,China)

机构地区：[1]宁波市泌尿肾病医院医务科,浙江宁波315100 [2]宁波市泌尿肾病医院临床药学部,浙江宁波315100 [3]宁波市泌尿肾病医院内分泌科,浙江宁波315100 [4]浙江中医药大学人事处,浙江杭州311400

出　　处：《中国临床药理学杂志》2023年第18期2635-2639,共5页The Chinese Journal of Clinical Pharmacology

基　　金：宁波市鄞州区农业与社会发展科技基金资助项目(20191YZQ010017)。

摘　　要：目的探究吴茱萸碱对过氧化氢(H_(2)O_(2))所致L-O2肝细胞损伤的保护作用及其作用机制。方法将L-O2肝细胞分为对照组、H_(2)O_(2)组、低剂量实验组、中剂量实验组、高剂量实验组;对照组细胞为正常培养的L-O2细胞,H_(2)O_(2)组细胞用120μmol·L^(-1)的H_(2)O_(2)处理细胞6 h,低、中、高剂量实验组细胞分别用4、8和16μmol·L^(-1)的吴茱萸碱处理细胞24 h后,再用120μmol·L^(-1)的H_(2)O_(2)继续处理细胞6 h。用细胞计数试剂盒(CCK-8)法检测各组细胞存活率,用流式细胞术法检测各组细胞凋亡情况,用JC-1染色法检测各组细胞线粒体膜电位,用化学发光仪检测三磷酸腺苷(ATP)水平,用蛋白质印迹法检测各组细胞相关蛋白表达水平。结果对照组、H_(2)O_(2)组、高剂量实验组细胞存活率分别为(100.00±3.92)%、(64.09±2.69)%和(94.89±5.78)%,细胞凋亡率分别为(3.05±0.27)%、(19.75±1.47)%和(5.58±0.34)%,JC-1红/绿荧光比值分别为2.55±0.37、0.55±0.03和2.11±0.11,裂解的半胱氨酸天冬氨酸蛋白酶-3(Cl Caspase-3)蛋白表达水平分别为0.34±0.02、1.07±0.05和0.40±0.04,细胞色素(Cyt C)蛋白表达水平分别为0.30±0.02、0.91±0.09和0.35±0.03,磷酸化C-Jun氨基端激酶(p-JNK)蛋白表达水平分别为0.32±0.03、1.04±0.04和0.52±0.05,线粒体裂变因子(Mff)蛋白表达水平分别为0.31±0.04、0.94±0.15和0.45±0.05。以上指标,H_(2)O_(2)组与对照组比较,高剂量实验组与H_(2)O_(2)组比较,差异均有统计学意义(均P<0.05)。结论吴茱萸碱预处理可通过改变线粒体膜电位介导线粒体凋亡途径,抑制细胞凋亡,从而减轻H_(2)O_(2)所致的L-O2肝细胞损伤,这可能与JNK/Mff通路有关。Objective To explore the protective effect and mechanism of evodiamine on L-O2 hepatocyte injury induced by hydrogen peroxide(H_(2)O_(2)).Methods L-O2 hepatocytes were divided into control group,H_(2)O_(2) group,experimental-L,-M,-H groups.Normal L-02 cells were cultured in the control group.Cells in the H,O2 group were treated with 120μmol·L^(-1)H_(2)O_(2)for 6 h.Cells in the experimental-L,-M,-H groups were treated with 4,8 and 16μmol·L^(-1) evodiamine for 24 h,and then treated with 120μmol.L^(-1)H_(2)O_(2)for 6 h.Cell survival rate of each group was detected by cell counting kit-8(CCK-8)method,cell apoptosis was detected by flow cytometry,mitochondrial membrane potential of each group was detected by JC-1 staining,adenosine triphosphate(ATP)level was detected by chemiluminescence analyzer,and expression level of related proteins was detected by Western blot.ResultsTThe cell survival rates of control group,H,02 group,experimental-H group were(100.00±3.92)%,(64.09±2.69)%and(94.89±5.78)%,respectively;the apoptosis rates were(3.05±0.27)%,(19.75±1.47)%and(5.58±0.34)%,respectively;the red/green fluorescence ratios of JC-1 were 2.55±0.37,0.55±0.03 and 2.11±0.11,respectively;cleaved cysteine aspartate proteinase-3(Cl-Caspase-3)protein levels were 0.34±0.02,1.07±0.05 and 0.40±0.04,respectively;cytochrome C(Cyt C)protein levels were 0.30±0.02,0.91±0.09 and 0.35±0.03,respectively;phosphorylated c-Jun N-terminal protein kinase(p-JNK)protein levels were 0.32±0.03,1.04±0.04 and 0.52±0.05,respectively;mitochondrial fission factor(Mff)protein levels were 0.31±0.04,0.94±0.15 and 0.45±0.05,respectively.The above indexes were compared between H,O2 group and control group,experimental-H group compared with H_(2)O_(2) group,and the differences were statistically significant(all P<0.05).Conclusion Evodiamine can reduce L-02 hepatocyte injury induced by H_(2)O_(2)by changing mitochondrial membrane potential,mediating mitochondrial apoptosis pathway and inhibiting cell apoptosis,this may be related to the JN

关 键 词：吴茱萸碱 过氧化氢 肝损伤 线粒体凋亡 C-Jun氨基端激酶/线粒体裂变因子通路 

分 类 号：R285[医药卫生—中药学]