氨磷汀通过上调PKM2表达增强肾近端小管上皮细胞抵抗顺铂毒性  

Amifostine enhances the resistance of renal proximal tubular epithelial cells to cisplatin by upregulating PKM2

作　　者：薛嵘[1] 马金刚 李莹屏[1] 张健 高佩娟 黄文辉[1] 杨晓军[2] 李小丽[1] XUE Rong;MA Jingang;LI Yingping;ZHANG Jian;GAO Peijuan;HUANG Wenhui;YANG Xiaojun;LI Xiaoli(Department of Nephrology,Gansu Provincial Hospital,Lanzhou,Gansu Province,730000;Department of General Surgery,Gansu Provincial Hospital,Lanzhou,Gansu Province,730000;Institute for Drug and Instrument Control of Xining Joint logistics Support Center,Lanzhou,Gansu Province,730050,China)

机构地区：[1]甘肃省人民医院肾内科,兰州730000 [2]甘肃省人民医院普外科,兰州730000 [3]西宁联勤保障中心药品仪器监督检验站,兰州730050

出　　处：《陆军军医大学学报》2023年第5期433-440,共8页Journal of Army Medical University

基　　金：国家自然科学基金地区科学基金项目(81660398);甘肃省自然科学基金(21JR7RA625,22JR11RA256)。

摘　　要：目的探究氨磷汀缓解顺铂肾毒性的分子机制。方法在肾近端小管上皮细胞系HK-2及原代细胞中,采用CCK-8细胞活性实验检测氨磷汀对顺铂细胞毒性的抑制作用。通过在线数据库tabula-muris分析肾脏组织各细胞亚群PKM2的表达。通过流式细胞术检测细胞凋亡比例。通过荧光定量PCR和蛋白免疫印迹实验检测细胞中PKM2基因表达。通过小RNA干扰技术敲低细胞中PKM2基因表达。结果氨磷汀显著增强HK-2细胞和原代肾近端小管上皮细胞抵抗顺铂的毒性。在顺铂处理的细胞中,随着氨磷汀浓度的增加,细胞的活力从0.37增加到0.45至0.77(P<0.001)。另外,随着氨磷汀浓度的增加,HK-2细胞中PKM2基因表达依次增加,在1μmol/L浓度时增加到8.76倍(P<0.001)。在HK-2细胞中干扰PKM2基因表达,氨磷汀对顺铂的细胞毒性的保护作用显著受到抑制。顺铂诱导的细胞凋亡比例、标志蛋白Cleaved-PARP和Cleaved-caspase3的表达被氨磷汀显著抑制。单细胞数据分析的结果表明,相对于肾脏组织其他细胞亚群,PKM2在肾近端小管上皮细胞中表达最低。结论氨磷汀通过上调肾近端小管上皮细胞PKM2的表达抑制顺铂诱导的细胞凋亡,从而缓解顺铂的细胞毒性。ObjectiveTo explore the molecular mechanism of amifostine in relieving cisplatin nephrotoxicity.MethodsCCK-8 assay was used to detect the inhibitory effect of amifostine on cisplatin cytotoxicity in HK-2 and primary renal proximal tubular epithelial cells.The expression of PKM2 was analyzed by online database tabula muris.The proportion of apoptotic cells was detected by flow cytometry.The expression of PKM2 gene was detected by fluorescence quantitative PCR and Western blotting.PKM2 gene expression was knocked down by small RNA interference.ResultsAmifostine significantly enhanced the resistance of renal proximal tubular epithelial cells to cisplatin toxicity.In cisplatin treated cells,with the increase of the concentration of amifostine,the cell viability was increased from 0.37 to 0.45 to 0.77(P<0.001).In addition,with the increase of amifostine concentration,PKM2 gene expression in HK-2 cells was increased in turn.The expression of PKM2 gene was increased to 8.76 times higher at 1μmol/L of amifostine(P<0.001).PKM2 gene expression in HK-2 cells was increased with the increase of amifostine concentration.When PKM2 gene expression was depleted in HK-2 cells,and the protective effect of amifostine on cisplatin cytotoxicity was significantly inhibited.The proportion of apoptotic cells was reduced and the levels of apoptotic markers Cleaved-PARP and Cleaved-Caspase3 induced by cisplatin were significantly inhibited by amifostine.Single-cell data analysis indicated that the expression of PKM2 in renal proximal tubular epithelial cells was the lowest compared with other cell subsets in renal tissue.ConclusionAmifostine inhibits cell apoptosis induced by cisplatin by up-regulating PKM2 in renal proximal tubular epithelial cells,and thus alleviates the cytotoxicity of cisplatin.

关 键 词：氨磷汀 肾近端小管上皮细胞 PKM2 顺铂 毒性 

分 类 号：R322.61[医药卫生—人体解剖和组织胚胎学] R966[医药卫生—基础医学] R979.19