雷公藤甲素诱导自噬促进人肾小管上皮HK-2细胞凋亡  

Triptolide upregulates autophagy to promote apoptosis in human proximal tubular epithelial HK-2 cells

作　　者：王春强 云阳 梁马丹 梁泰刚[2] WANG Chun-qiang;YUN Yang;LIANG Ma-dan;LIANG Tai-gang(Dept of Critical Care Medicine,the First Affiliated Hospital of Shanxi Medical University,Taiyuan 030001,China;School of Pharmacy,Shanxi Medical University,Jinzhong,Shanxi 030600,China;The First Affiliated Hospital of Shanxi Medical University,Jinzhong,Shanxi 030600,China)

机构地区：[1]山西医科大学第一医院重症医学科,山西太原030001 [2]山西医科大学药学院,山西晋中030600 [3]山西医科大学第一临床医学院,山西晋中030600

出　　处：《中国药理学通报》2023年第11期2133-2140,共8页Chinese Pharmacological Bulletin

基　　金：山西省基础研究计划(No 20210302123300);山西省“黄芪”资源产业化与产业国际化协同创新中心项目(No HQXTCXZXX2016-021);山西省“1331工程”创新团队建设(2018-4培育No 11);山西省自然科学基金(No 201601D011112);国家重点研发计划(No 2019YFC1710803)。

摘　　要：目的研究自噬在雷公藤甲素(triptolide,TP)诱导人肾近端小管上皮HK-2细胞凋亡中的作用。方法以HK-2细胞为研究对象,CCK-8法检测细胞存活率;倒置荧光显微镜观察细胞形态变化;流式细胞术检测细胞凋亡率和线粒体膜电位MMP变化;激光扫描共聚焦显微镜观察细胞内LC3荧光强度变化;Western blot检测细胞凋亡及自噬相关蛋白cleaved caspase 9、caspase 9、Bax、Bcl-2、LC3、p62和Beclin 1的表达;采用自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)和自噬激动剂雷帕霉素(rapamycin,Rap)研究自噬对TP诱导HK-2细胞凋亡的影响。结果TP降低HK-2细胞存活率,诱导细胞凋亡和自噬,降低MMP水平,增加胞内LC3Ⅱ荧光强度,上调Beclin 1表达,下调p62表达,升高cleaved caspase 9/caspase 9、Bax/Bcl-2和LC3Ⅱ/LC3Ⅰ比值。此外,与TP组相比,3-MA+TP组细胞内LC3Ⅱ/LC3Ⅰ和cleaved caspase 9/caspase 9比值降低,p62表达上升,细胞增殖的抑制作用和促凋亡作用减弱。相反,与TP组相比,Rap+TP组细胞内LC3Ⅱ/LC3Ⅰ和cleaved caspase 9/caspase 9比值上升、p62表达下降,细胞增殖的抑制作用和促凋亡作用增强。结论TP能够诱导HK-2细胞发生自噬和线粒体途径介导的细胞凋亡,抑制HK-2细胞自噬可降低TP诱导细胞凋亡的发生,因此抑制自噬可能是减轻TP肾毒性的有效干预策略。Aim To study the role of autophagy in triptolide(TP)-induced apoptosis in human proximal tubular epithelial HK-2 cells.Methods Cell viability of HK-2 cells was detected by the CCK-8 assay.The morphological changes of cells were observed by inverted fluorescence microscope.Cell apoptosis and mitochondrial membrane potential(MMP)were measured by flow cytometry(FCM).The fluorescence intensity of intracellular LC3 was observed by laser scanning confocal microscopy.Apoptosis-and autophagy-related proteins cleaved caspase 9,caspase 9,Bax,Bcl-2,LC3,p62(SQSTM1)and Beclin 1 were detected by Western blot.3-methyladenine(3-MA,autophagy inhibitor)and rapamycin(Rap,autophagy agonist)were used to study the effect of autophagy on TP-induced apoptosis in HK-2 cells.Results Compared with the control group,the viability of HK-2 cells decreased gradually with the increasing concentration and incubation time of TP.Besides,TP could decrease MMP level,increase fluorescence intensity of intracellular LC3 II,upregulate the ratios of cleaved caspase-9/caspase 9,Bax/Bcl-2 and LC3 II/LC3 I,enhance the expression of Beclin 1,and decrease the expression of p62(SQSTM1)in HK-2 cells.Moreover,compared with TP group,the ratios of LC3 II/LC3 I and cleaved caspase 9/caspase 9 decreased,the expression of p62(SQSTM1)increased,and the cell proliferation inhibition and pro-apoptotic effect were weakened in 3-MA+TP group.On the contrary,compared with the TP group,the ratios of LC3 II/LC3 I and cleaved caspase 9/caspase 9 increased,the expression of p62(SQSTM1)decreased,and the cell proliferation inhibition and pro-apoptotic effect were enhanced in Rap+TP group.Conclusions TP could induce autophagy and mitochondria mediated apoptosis in HK-2 cells.Inhibition of autophagy could reduce TP-induced HK-2 cell apoptosis.Therefore,inhibition of autophagy may be an effective intervention strategy to alleviate TP-induced nephrotoxicity.

关 键 词：雷公藤甲素 HK-2细胞 凋亡 自噬 肾毒性 3-甲基腺嘌呤 雷帕霉素 

分 类 号：R284.1[医药卫生—中药学] R322.61[医药卫生—中医学] R329.24R329.25R392