摘要
目的:探讨p38 MAPK在顺铂诱导大鼠近端肾小管上皮细胞(RPTC)凋亡中的作用。方法:首先采用Western blot实验检测0、5、10和20μmol/L顺铂处理24 h对细胞凋亡的影响,确定最佳处理剂量;而后采用20μmol/L顺铂联合50 mg/L p38 MAPK抑制剂SB203580刺激RPTC,实验分为对照组、顺铂组及顺铂+SB203580(加入SB203580处理RPTC 1 h后再给予顺铂处理24 h)。采用相差荧光显微镜观察和流式细胞术分析顺铂处理后RPTC的凋亡情况;采用Ac-DEVD-AFC试剂盒检测RPTC裂解液中的caspase活性;Western blot实验检测p38、磷酸化p38、cleaved PARP和cleaved caspase-3等的蛋白水平;pH计检测顺铂处理后RPTC外环境pH值改变。结果:20μmol/L顺铂处理RPTC 24 h,可以明显诱导细胞凋亡;顺铂处理15 min后RPTC中p38 MAPK开始磷酸化并达到高峰。顺铂处理后12.08%的RPTC呈凋亡形态,具有增强的caspase活性,并且cleaved PARP和cleaved caspase-3水平明显升高(P<0.05);p38 MAPK抑制剂SB203580可抑制p38的磷酸化,降低RPTC的凋亡率和caspase活性,并减少cleaved PARP和cleaved caspase-3的蛋白水平。同时,SB203580可逆转顺铂诱导的RPTC培养基pH值的改变。结论:p38 MAPK的磷酸化在顺铂诱导的RPTC凋亡中发挥作用。顺铂诱导RPTC凋亡后,可改变细胞外酸性环境,并可被p38 MAPK抑制剂SB203580所抑制。
AIM:To investigate the role of p38 MAPK in cisplatin-induced rat renal proximal tubular cell(RPTC)apoptosis.METHODS:To determine the optimal concentration of cisplatin to induce RPTC apoptosis,the cells were treated with 0,5,10 and 20μmol/L cisplatin for 24 h,and then the cell lysates were collected for Western blot analysis of cleaved PARP,p38 and phosphor ylated p38(p-p38).To determine the role of p38 MAPK in cisplatin-induced RPTC apoptosis,the cells were divided into control group,cisplatin group(the cells were treated with cisplatin for 24 h)and cisplatin+p38 MAPK inhibitor group(the cells were treated with p38 MAPK inhibitor SB203580 for 1 h,and then treated with cisplatin for another 24 h).The morphological changes of apoptotic cells were observed under phase-contrast fluorescence microscope.The apoptotic rate of the cells were analyzed by flow cytometry.The caspase activity of RPTC lysates was examined using Ac-DEVD-AFC kit.The protein levels of p-p38,p38,cleaved PARP and cleaved caspase-3 were determined by Western blot.The pH value of extracellular environment of the cells was measured by pH meter.RESULTS:Cisplatin at 20μmol/L obviously induced apoptosis of RPTC.The p38 MAPK was phosphorylated and its phosphorylation peaked at 15 min after cisplatin treatment.The apoptotic rate of RPTC was 12.08%after cisplatin induction.Cisplatin treatment also enhanced caspase activity,and increased cleavage of PARP and caspase-3 proteins(P<0.05).The p38 MAPK inhibitor SB203580 effectively inhibited the phosphorylation of p38 MAPK,down-regulated the RPTC apoptosis rate and caspase activity,and reduced the cleavage of PARP and caspase-3 proteins.The pH value change in RPTC culture medium was also inverted by SB203580.CONCLUSION:The phosphorylation of p38 MAPK is involved in cisplatin-induced apoptosis of RPTC.The apoptosis induced by cisplatin results in the change of acidic extracellular environment,which is inhibited by p38 MAPK inhibitor SB203580.
作者
娄强
张芳
李淑莲
LOU Qiang;ZHANG Fang;LI Shu-lian(National and Local Joint Laboratory for Antibody Drug Development Techniques,Basic Medical School of Henan University,Kaifeng 475004,China;The First Affiliated Hospital of Henan University,Kaifeng 475000,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2018年第9期1678-1683,共6页
Chinese Journal of Pathophysiology
基金
河南省科技发展计划项目(No.152102310302)
河南省教育厅科学技术研究重点项目(No.14B310015)
河南大学科研基金资助项目(No.zzjj20140053)