芦荟苷通过降低热休克蛋白70的表达增强顺铂诱导的胃癌细胞凋亡  

Aloin enhances apoptosis of gastric cancer cells induced by cisplatin through down-regulating HSP70 expression

作　　者：程振宇 陈雪雷 葛菲 程锦 吴晓明 戚之琳 CHENG Zhen-yu;CHEN Xue-lei;GE Fei;CHENG Jin;WU Xiao-ming;QI Zhi-lin(Department of Biochemistry and Molecular Biology,School of Preclinical Medicine,Anhui Provincial Key Laboratory of Active Biological Macro-molecules,Wannan Medical College,Wuhu 241002,China;School of Clinical Medicine,Wannan Medical College,Wuhu 241002,China;School of Pharmacy,Wannan Medical College,Wuhu 241002,China)

机构地区：[1]皖南医学院基础医学院生物化学与分子生物学教研室,活性生物大分子安徽省重点实验室,安徽芜湖241002 [2]皖南医学院临床医学院,安徽芜湖241002 [3]皖南医学院药学院,安徽芜湖241002

出　　处：《中国病理生理杂志》2022年第11期2021-2027,共7页Chinese Journal of Pathophysiology

基　　金：安徽高校自然科学研究项目重大项目(No.KJ2020ZD54);2021年学科(专业)拔尖人才学术资助项目(No.gxbjZD2021060);活性生物大分子研究安徽省重点实验室项目(No.1306C083008);安徽省大学生创新创业训练计划项目(No.S202110368109);2021年度校大学生科研资助金项(No.WK2021XS07,No.WK2021XS64)。

摘　　要：目的:探讨芦荟苷能否通过降低热休克蛋白70(HSP70)的表达,增强顺铂诱导的胃癌细胞凋亡。方法:胃癌HGC-27细胞分为对照组、200 mg/L芦荟苷组、5 mg/L顺铂组和两者联合组(200 mg/L芦荟苷+5 mg/L顺铂)。DAPI染色和流式细胞术分别检测细胞凋亡形态改变及凋亡率;RT-qPCR检测HSP70 mRNA的表达水平;Western blot检测HSP70和凋亡相关蛋白的表达;免疫沉淀(IP)检测HSP70的泛素化水平。结果:DAPI染色结果显示,与药物单独刺激组相比,联合组细胞核浓缩与核碎裂现象显著增强。流式细胞术分析细胞凋亡率表明,顺铂组细胞凋亡率为16.71%,显著高于对照组的6.95%(P<0.01)。虽然芦荟苷组与对照组相比,凋亡率无显著差异,但是其与顺铂联合作用后,细胞凋亡率为33.74%,显著高于顺铂和芦荟苷单独刺激组(P<0.01)。RT-qPCR显示,芦荟苷能够显著下调顺铂诱导的HSP70 mRNA表达(P<0.01)。Western blot结果表明,联合组cleaved PARP和cleaved caspase-3/-7的表达均高于单独作用组(P<0.05或P<0.01),pro-caspase-3的水平则呈现出相反的变化(P<0.01);芦荟苷组与联合组HSP70的蛋白表达与对照组相比显著下降(P<0.01),而顺铂组其表达则无显著变化。IP结果显示,顺铂可诱导HSP70泛素化,但芦荟苷对HSP70的泛素化水平并无显著影响。结论:芦荟苷通过降低HSP70的表达,增强顺铂诱导的胃癌细胞凋亡。ATM:To investigate the effect of aloin on the apoptosis of gastric cancer cells.METHODS:HGC-27 cells were divided into control group,200 mg/L of aloin group,5 mg/L of cisplatin group and combination group(200 mg/L of aloin and 5 mg/L of cisplatin).Morphological changes of the nucleus were detected by DAPI staining and apoptosis rates were determined using flow cytometry.RT-qPCR was used to determine the expression of HSP70 mRNA,and the levels of apoptosis-related proteins and HSP70 protein were detected by Western blot.Immunecoprecipitation(IP)was used to detect the ubiquitination level of HSP70.RESULTS:DAPI staining showed that nuclear condensation and fragmentation were significantly increased after treatment with combination of aloin and cisplatin.Flow cytometry revealed that the apoptosis rates of gastric cancer cells stimulated by cisplatin were significantly higher than those in control group(P<0.01).Although there was no significant statistic difference in the apoptosis rate between aloin and control groups,the apoptosis rate of combination group was 33.74%,significantly higher than that in aloin group(P<0.01).The result of RT-qPCR showed that aloin decreased cisplatin-induced HSP70 mRNA levels(P<0.01).Western blot showed that the levels of PARP cleavage and cleaved caspase-3/-7 in combination group were higher than those in aloin or cisplatin group(P<0.05 or P<0.01),while the level of pro-caspase-3 showed the opposite change(P<0.01).Aloin and combination treatment both decreased the protein expression of HSP70(P<0.01),but its level was not affected by cisplatin.The results of IP showed that the level of HSP70 ubiquitination was enhanced upon cisplatin stimulation,but was not affected by aloin treatment.CONCLUSION:Aloin enhances cisplatin-induced apoptosis of gastric cancer HGC-27 cells through down-regulating the expression of HSP70.

关 键 词：芦荟苷 顺铂 热休克蛋白70 细胞凋亡 胃癌 

分 类 号：R735.2[医药卫生—肿瘤] R730.23[医药卫生—临床医学]