湖南中医药大学学报2025，Vol.45Issue(5)：845-855,11.DOI:10.3969/j.issn.1674-070X.2025.05.009
基于PINK1/Parkin通路探讨温阳解毒化瘀方含药血浆对肝细胞损伤的影响
Effects of plasma containing Wenyang Jiedu Huayu Formula on hepatocyte injury based on the PINK1/Parkin signaling pathway
摘要
Abstract
Objective To investigate the effects of plasma containing Wenyang Jiedu Huayu Formula(WYJDHYF)on hepatocyte injury based on the PTEN-induced kinase 1(PINK1)/E3 ubiquitin ligase(Parkin)pathway.Methods A hepatocellular injury model was established using D-galactosamine(D-GalN)combined with lipopolysaccharide(LPS).Experimental groups included control group,model group,blank plasma group,WYJDHYF plasma group,3-MA group,and WYJDHYF+3-MA group.The optimal concentration and intervention duration of plasma was determined via CCK-8 assay.Cell apoptosis was assessed using Annexin V-APC/PI double staining.Mitochondrial membrane potential was measured by JC-1 staining,and reactive oxygen species(ROS)levels were checked using MitoSOX Red staining.Biochemical methods were employed to evaluate superoxide dismutase(SOD)and malondialdehyde(MDA)levels.Hepatocyte ultrastructure was observed by transmission electron microscopy(TEM).RT-qPCR was used to quantify mRNA levels of PINK1,Parkin,microtubule-associated protein 1 light chain 3(LC3),and sequestosome 1(SQSTM1/P62).The protein expression levels of PINK1,Parkin,LC3,p62/SQSTM1,B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),cleaved-Caspase-3,and Caspase-3 were analyzed by Western blot.Results The CCK-8 assay indicated that intervention with 15％drug-containing plasma for 12 hours was the optimal concentration and duration for the experiment Compared with the control group,the model group showed increased apoptosis rate of cells at all stages(P＜0.01);decreased mitochondrial membrane potential(P＜0.01);elevated levels of intracellular ROS and MDA(P＜0.01)and reduced SOD level(P＜0.01).TEM revealed severely disordered mitochondrial structures and fewer autolysosomes.The mRNA expression levels of PINK1,Parkin,and LC3 decreased(P＜0.01),while P62 mRNA expression increased(P＜0.01).The protein expressions of PINK1,Parkin,Bcl-2,and the ratio of LC3 Ⅱ/Ⅰ decreased(P＜0.01),whereas the P62 and Bax protein expressions and the cleaved-Caspase-3/Caspase-3 ratio were elevated(P＜0.01).Compared to the model group,the WYJDHYF plasma group exhibited reduced apoptosis rates at all stages(P＜0.01),increased mitochondrial membrane potential(P＜0.01),decreased intracellular ROS and MDA levels(P＜0.01),and increased SOD levels(P＜0.01).Alleviated mitochondrial structural disruption and increased autolysosomes were revealed.Upregulated PINK1,Parkin,and LC3 mRNA(P＜0.01)and downregulated P62 mRNA(P＜0.01)levels were shown.Protein expressions of PINK1,Parkin,and Bcl-2,as well as the LC3 Ⅱ/Ⅰ ratio,were demonstrated elevated(P＜0.01 or P＜0.05),whereas the expressions of P62 and Bax proteins and the cleaved-Caspase-3/Caspase-3 ratio decreased(P＜0.01 or P＜0.05).Compared to the WYJDHYF plasma group,the WYJDHYF+3-MA group exhibited markedly increased apoptosis rate(P＜0.01),decreased mitochondrial membrane potential(P＜0.01),elevated ROS and MDA levels(P＜0.01),reduced SOD levels(P＜0.01).Electron microscopy showed worsened mitochondrial disorganization and reduced autolysosome numbers.mRNA expressions of PINK1,Parkin,and LC3 decreased(P＜0.01),while P62 mRNA expression increased(P＜0.01).Protein expressions of PINK1,Parkin,Bcl-2 protein,and LC3-Ⅱ/Ⅰ ratio reduced(P＜0.01),whereas expressions of P62 and Bax proteins and Cleaved-Caspase-3/Caspase-3 ratio increased(P＜0.01 or P＜0.05).Conclusion The plasma containing WYJDHYF can alleviate ROS accumulation and oxidative stress,inhibit mitochondrial intrinsic apoptosis,and exert a protective effect against D-GalN/LPS-induced hepatocyte injury.Its protective mechanism may involve enhancing PINK1/Parkin pathway-mediated mitophagy and improving mitochondrial function.关键词
慢加急性肝衰竭/肝损伤/温阳解毒化瘀方/PINK1/Parkin通路/线粒体自噬Key words
acute-on-chronic liver failure/liver injury/Wenyang Jiedu Huayu Formula/PINK1/Parkin pathway/mitophagy分类
中医学引用本文复制引用
黄玉,张涛,丁琳,孙克伟..基于PINK1/Parkin通路探讨温阳解毒化瘀方含药血浆对肝细胞损伤的影响[J].湖南中医药大学学报,2025,45(5):845-855,11.基金项目
国家自然科学基金项目(81973833) （81973833）
国家中医药管理局高水平中医药重点学科项目 （）
湖南中医药大学双一流学科建设项目(2018[03]). （2018[03]）
