截断逆挽方调控AMPK改善HepG2细胞线粒体损伤的机制  

Jieduan Niwan Formula regulates AMPK to alleviate mitochondrial damage in HepG2 cells

作　　者：马瑞旻 王瀚婧 张文馨 马重阳 张秋云[1] 杜宇琼[1] MA Ruimin;WANG Hanjing;ZHANG Wenxin;MA Chongyang;ZHANG Qiuyun;DU Yuqiong(Capital Medical University,Beijing 100069,China;Central Medical Branch of Chinese PLA Hospital,Beijing 100120,China)

机构地区：[1]首都医科大学,北京100069 [2]中国人民解放军总医院京中医疗区

出　　处：《北京中医药大学学报》2025年第2期193-204,共12页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(No.82074237,No.81573767);北京市中医药科技发展基金项目(No.JJ-2020-51)。

摘　　要：目的 探讨截断逆挽方含药血清对D-氨基半乳糖(D-GalN)诱导的人肝癌细胞株G2型(HepG2细胞)线粒体功能的保护作用及对单磷酸腺苷激活的蛋白激酶(AMPK)介导的线粒体质量控制(MQC)的调控作用。方法 将20只Wistar雄性大鼠随意分为空白对照组和截断逆挽方含药血清组,每组10只。截断逆挽方含药血清组给予截断逆挽方(21.7 g/kg)灌胃,空白对照组给予生理盐水灌胃,灌胃3 d。腹主动脉取血制备截断逆挽方含药血清及空白对照血清。综合评估细胞活力、线粒体损伤指标及MQC通路蛋白表达水平以筛选截断逆挽方的最优体积分数。将HepG2细胞分为对照组、D-GalN组、二甲基亚砜(DMSO)组、AMPK抑制剂组、截断逆挽方含药血清组、截断逆挽方含药血清加AMPK抑制剂组,分别给予对应的药物干预。使用CCK-8法检测细胞活力,荧光探针JC-1检测线粒体膜电位,荧光探针DCFH-DA检测细胞内活性氧(ROS)水平,WST-8法检测超氧化物歧化酶(SOD)活性,TBA法检测丙二醛(MDA)含量等线粒体损伤指标,蛋白免疫印迹法检测磷酸化AMPK(p-AMPK)、AMPK、过氧化物酶体增殖受体γ辅激活因子α(PGC-1α)、核呼吸因子1(NRF1)、线粒体转录因子A(TFAM)、线粒体融合蛋白2(MFN2)、动力蛋白相关蛋白1(DRP1)等MQC通路蛋白表达水平。结果 5%截断逆挽方含药血清对D-GalN诱导的HepG2细胞的细胞活力、线粒体损伤指标及MQC通路蛋白表达的恢复最明显,后续实验选择5%截断逆挽方含药血清干预。与对照组比较,D-GalN组、DMSO组、AMPK抑制剂组的细胞活力和SOD活性下降(P<0.01),线粒体膜电位异质性、ROS、MDA含量上升(P<0.01),p-AMPK、PGC-1α、NRF1、TFAM、MFN2蛋白表达降低(P<0.01),DRP1蛋白表达降低(P<0.01);与DMSO组比较,截断逆挽方含药血清干预后,细胞活力和SOD活性上升(P<0.01),线粒体膜电位异质性、ROS、MDA含量下降(P<0.01),p-AMPK、PGC-1α、NRF1、TFAM、MFN2蛋白表达升高Objective To investigate the regulatory effect of Jieduan Niwan Formula(JDNWF) drug-containing serum on AMPK-mediated mitochondrial quality control in D-GalN-induced HepG2 cells.Methods Twenty male Wistar rats were randomly divided into blank control and JDNWF-containing serum groups,10 rats per group.The JDNWF-containing serum group was gavaged with JDNWF(21.7 g/kg),whereas the blank control group was gavaged with saline.Blood was collected to prepare JDNWF-containing and blank control serum.Cell viability,mitochondrial damage indicators,and MQC pathway protein expression levels were evaluated to determine the optimal volume fraction of JDNWF.HepG2 cells were divided into control,D-GalN,DMSO,AMPK inhibitor,JDNWF drug-containing serum,and JDNWF drug-containing serum plus AMPK inhibitor groups,and corresponding drug interventions were administered to each group.Cells were collected after the interventions,and the CCK-8 assay was used to measure cell viability,the 2′-7′-dichlorodihydrofluorescein diacetate fluorescent probe was used to detect reactive oxygen species(ROS) levels,JC-1 was used to detect mitochondrial membrane potential,thiobarbituric acid was used to measure malondialdehyde(MDA) levels,WST-8 was used to measure superoxide dismutase(SOD) activity,and western blotting was used to detect the expression levels of mitochondrial quality control-related proteins,including p-AMPK,AMPK,PGC-1α,NRF1,TFAM,MFN2,and DRP1.Results 5% JDNWF drug-containing serum most significantly restored cell viability,mitochondrial damage markers,and MQC pathway protein expression in the model group.Therefore,it was chosen for intervention in subsequent experiments.Compared to the control group,the cell viability of the D-GalN,DMSO,and AMPK inhibitor groups was significantly reduced(P<0.01).In contrast,the heterogeneity of mitochondrial membrane potential,ROS,and MDA levels was significantly increased(P<0.01),and SOD activity was significantly decreased(P<0.01).The p-AMPK,PGC-1α,NRF1,TFAM,MFN2,and DRP1 protein expression lev

关 键 词：截断逆挽方 肝细胞损伤 线粒体质量控制 单磷酸腺苷激活的蛋白激酶 慢加急性肝衰竭 

分 类 号：R285.5[医药卫生—中药学]