吡嗪酰胺对人肝细胞L02的毒性及作用机制研究

doi:10.19803/j.1672-8629.2021.11.10

摘要/Abstract

摘要： 目的评价吡嗪酰胺（pyrazinamide, PZA）的肝脏毒性,并探讨其导致肝损伤的作用机制。方法将L02细胞分为对照组,PZA给药组（25、125、625、3 125 µg/mL）,N-乙酰-L-半胱氨酸（N-acetlg-L-cysteine, NAC）（10 mmol/L）+ PZA（25、125、625、3 125 µg/mL）给药组,分别给予对应培养基及不同浓度PZA、NAC;采用流式细胞仪定量检测L02细胞凋亡数量、细胞内线粒体膜电位（mitochondrial membrane potential, MMP）;采用Western-blot法检测L02细胞内Fas、FADD、Cyt-c、Cleaved caspase-3的蛋白表达。结果与对照组比较,各浓度PZA给药组凋亡细胞比例均显著升高,且中晚期凋亡细胞比例较早期凋亡细胞比例明显增多;25、125 µg/mL PZA给药组MMP分别降低32.23%、24.79%,3 125 µg/mL PZA给药组MMP位升高31.40%;3 125 µg/mL PZA给药组Fas蛋白表达升高;125、625 µg/mL PZA给药组Cleaved caspase-3蛋白表达µg升高。PZA联用NAC后,各浓度组凋亡细胞比例低于单用PZA组,25、125、3 125 µg/mL组下降明显;25、625、3 125 µg/mL组与单用PZA组比较,MMP分别增加了21.95%、33.48%、22.33%;3 125 µg/mL组与单用PZA组比较Fas蛋白表达量降低47.50%;125、625、3 125 µg/mL组Cleaved caspase-3蛋白表达量分别降低66.13%、73.91%、41.67%。结论 PZA可能是通过死亡受体途径、线粒体途径诱导L02细胞凋亡从而导致肝损伤,联用NAC后可明显缓解PZA导致的肝损伤。

关键词: 吡嗪酰胺, 细胞凋亡, 细胞内线粒体膜电位, N-乙酰-L-半胱氨酸, 肝损伤

Abstract: Objectiv eTo evaluate the liver toxicity of pyrazinamide and to explore its mechanism of liver injury. Methods L02 cells were divided into the control group, PZA group (25, 125, 625, 3 125 µg/mL), and N-acetlg-L-cysteine (NAC) (10 mmol/L) + PZA (25, 125, 625, 3 125 µg/mL) group. The corresponding culture medium and different concentrations of PZA and NAC were given respectively. Flow cytometry was used to quantitatively detect the degree of apoptosis and intracellular mitochondrial membrane potential (MMP) of L02 cells. The Western-blot method was used to detect the protein expressions of Fas, Fas-related protein (FADD), cytochrome C (Cyt-c), and activated caspase-3 (Cleaved caspase-3). Results Compared with the control group, the proportion of apoptotic cells in the PZA administration group at each concentration was significantly increased, and the proportion of cells at the mid-late stage of apoptosis was significantly higher than that of cells at the early stage of apoptosis. MMP in the 25 and 125 µg/mL PZA administration groups was decreased by 32.23% and 24.79%, respectively, compared with 31.40% in the 3 125 µg/mL PZA administration group. The expression of Fas protein in the 3 125 µg/mL PZA administration group increased, so did the expression of Cleaved caspase-3 protein in the 125, 625 µg/mL PZA administration group. After PZA was combined with NAC, the proportion of apoptotic cells in each concentration group was not only lower than that of the PZA alone group, but decreased significantly in the 25, 125 and 3 125 μg/mL groups. Compared with the PZA alone group, MMP was increased by 21.95%, 33.48%, and 22.33% respectively in the 25, 625, and 3 125 µg/mL groups. Compared with the PZA alone group, Fas protein expression decreased by 47.50% in the 3 125 µg/mL group. The expression of Cleaved caspase-3 protein in the 125, 625, 3 125 µg/mL groups decreased by 66.13%, 73.91%, and 41.67%, respectively. Conclusion PZA may induce apoptosis of L02 cells through the death receptor pathway and mitochondrial pathway, resulting in liver injury. The combination with NAC can significantly alleviate liver injury induced by PZA.

Key words: pyrazinamide, apoptosis, MMP, NAC, liver injury

中图分类号:

R965

刘梦醒, 刘媛, 孙辉, 刘幸, 杨敏, 彭江丽, 陈洁. 吡嗪酰胺对人肝细胞L02的毒性及作用机制研究[J]. 中国药物警戒, 2021, 18(11): 1043-1047.

LIU Mengxing, LIU Yuan, SUN Hui, LIU Xing, YANG Min, PENG Jiangli, CHEN Jie. Toxicity and Mechanism of Pyrazinamide in Human Hepatocyte L02[J]. Chinese Journal of Pharmacovigilance, 2021, 18(11): 1043-1047.

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