健脾益气方调控Pink1/Parkin通路干预人腹膜间皮细胞损伤  

Jianpi Yiqi Formula Interfering with Human Peritoneal Mesothelial Cell Injury by Regulating Pink1/Parkin Pathway

作　　者：孟立锋[1] 邓湘雨 史伟[1] MENG Lifeng;DENG Xiangyu;SHI Wei(The First Affiliated Hospital to Guangxi University of Chinese Medicine,Nanning Guangxi China 530022;Guangxi University of Chinese Medicine,Nanning Guangxi China 530200)

机构地区：[1]广西中医药大学第一附属医院,广西南宁530022 [2]广西中医药大学,广西南宁530200

出　　处：《中医学报》2025年第2期248-254,共7页Acta Chinese Medicine

基　　金：广西自然科学基金面上项目(2020GXNSFAA297200);国家中医药管理局第五批全国中医临床优秀人才研修项目{国中医药人教函[2022]1号}。

摘　　要：目的:基于Pink1/Parkin通路探讨健脾益气方干预人腹膜间皮细胞HMrSV5损伤的作用机制。方法:采用转化生长因子-β1(transforming growth factor-β1,TGF-β1)干预HMrSV5细胞48 h构建损伤模型。将HMrSV5细胞分为空白组(10%胎牛血清)、模型组(10μg·L^(-1) TGF-β1)、健脾益气方组(10μg·L^(-1) TGF-β1+160 mg·L^(-1)健脾益气方含药血清)、抑制剂组(10μg·L^(-1) TGF-β1+160 mg·L^(-1)健脾益气方含药血清+50μmmol·L^(-1) Mdivi-1)。采用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡率,JC-1荧光探针检测线粒体膜电位(mitochondrial membrane potential, MMP)。采用免疫荧光法和Western blot法检测细胞Parkin、Pink1、微管相关蛋白1轻链3-Ⅱ(microtubule-associated protein light chain 3-Ⅱ,LC3-Ⅱ)、P62、cleaved Caspase-3表达水平。结果:CCK-8检测显示,与空白组比较,模型组和抑制剂组细胞存活率降低(P<0.05)。流式细胞术显示,与空白组比较,模型组细胞凋亡率和MMP升高(P<0.000 1);与模型组比较,健脾益气方组和抑制剂组细胞凋亡率降低(P<0.000 1)。与模型组比较,健脾益气方组细胞MMP降低(P<0.000 1);与健脾益气方组比较,抑制剂组细胞MMP升高(P<0.001)。免疫荧光显示,与空白组比较,模型组和抑制剂组细胞Parkin、Pink1、LC3-Ⅱ表达减少,P62、cleaved Caspase-3表达增加。与模型组和抑制剂组比较,健脾益气方组细胞Parkin、Pink1、LC3-Ⅱ表达增加,P62、cleaved Caspase-3表达减少。Western blot显示,与空白组比较,模型组细胞Parkin、Pink1表达水平降低(P<0.01),LC3-Ⅱ/LC3-Ⅰ、P62、cleaved Caspase-3表达水平升高(P<0.05)。与模型组比较,健脾益气方组细胞Parkin、Pink1、LC3-Ⅱ/LC3-Ⅰ表达水平升高(P<0.05),P62、cleaved Caspase-3表达水平降低(P<0.000 1)。与健脾益气方组比较,抑制剂组细胞Parkin、Pink1、LC3-Ⅱ/LC3-Ⅰ表达水平降低(P<0.05),P62、cleaved Caspase-3表达水平升高(P<0.05)。结论:健脾益气方可能通过�Objective:To explore the mechanism of action of Jianpi Yiqi Formula in intervening human peritoneal mesothelial cell HMrSV5 injury based on Pink1/Parkin pathway.Methods:HMrSV5 cells were treated with transforming growth factorβ1(TGF-β1)for 48 h to construct an injury model.HMrSV5 cells were divided into a blank group(10%fetal bovine serum),a model group(10μg·L^(-1) TGF-β1),a Jianpi Yiqi Formula group(10μg·L^(-1) TGF-β1+160 mg·L^(-1) spleen-healthy formula),and an inhibitor group(10μg·L^(-1) TGF-β1+160 mg·L^(-1) Jianpi Yiqi Formula+50μmmol·L^(-1) Mdivi-1).Cell viability was detected by CCK-8 method,apoptosis rate was detected by flow cytometry,and mitochondrial membrane potential(MMP)was detected by JC-1 fluorescent probe.The expression levels of Parkin,Pink1,microtubule-associated protein light chain 3-II(LC3-II),P62,and cleaved caspase-3 were detected by immunofluorescence and Western blot.Results:CCK-8 assay showed that cell survival was reduced in the model and inhibitor groups compared with the blank group(P<0.05).Flow cytometry showed that the apoptosis rate and MMP were elevated in the model group compared with the blank group(P<0.0001);the apoptosis rate was reduced in Jianpi Yiqi Formula group and the inhibitor group compared with the model group(P<0.0001).Compared with the model group,the cellular MMP was decreased in Jianpi Yiqi Formula group(P<0.0001);compared with the spleen-enhancing and qi-beneficial formula group,the cellular MMP was elevated in the inhibitor group(P<0.001).Immunofluorescence showed that the expression of Parkin,Pink1 and LC3II decreased,and the expression of P62 and cleaved caspase-3 increased in the cells of the model and inhibitor groups compared with the blank group.Compared with the model and inhibitor groups,the expression of Parkin,Pink1 and LC3II in cells of Jianpi Yiqi Formula group was increased,and the expression of P62 and cleaved caspase-3 was decreased.Western blot showed that the expression levels of Parkin and Pink1 in cells of the model group were de

关 键 词：健脾益气方 腹膜间皮细胞 Pink1/Parkin通路 线粒体自噬 HMrSV5 

分 类 号：R285.5[医药卫生—中药学]