黄芪注射液对肠道病毒71型感染引起的细胞凋亡及自噬的抑制作用  

Inhibitory effect of astragalus membranaceus injection on apoptosis and autophagy induced by enterovirus 71

作　　者：郝晋芳 孙晨曦 杜建平 韩倩 张晓延 HAO Jin-fang;SUN Chen-xi;DU Jian-ping;HAN Qian;ZHANG Xiao-yan(Department of Medical Laboratory,Fenyang college,Shanxi Medical University,Fenyang 032200,Shanxi Province,China)

机构地区：[1]山西医科大学汾阳学院医学检验系,山西汾阳032200 [2]山西医科大学研究生院,山西太原030001

出　　处：《中国生物制品学杂志》2023年第1期53-58,69,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81301426);山西省自然科学基金(201901D111329);吕梁市重点研发计划(2020SHFZ38);临床分子诊断学重点实验室(2020ZDSYS17)。

摘　　要：目的探讨黄芪注射液(astragalus membranaceus,AM)对肠道病毒71型(enterovirus 71,EV71)感染诱导的人胃上皮细胞(GES-1)凋亡及自噬的影响。方法体外培养GES-1细胞,将细胞分为感染组(按MOI=3感染EV71)和对照组(未感染病毒),倒置显微镜观察EV71感染后细胞形态变化;Western blot检测EV71感染后GES-1细胞中病毒结构蛋白VP1的水平;CCK-8法检测EV71感染后GES-1细胞活力;用DAPI进行细胞核染色,观察EV71感染的细胞核凋亡形态变化。再将GES-1细胞分为对照组(未感染病毒)、感染组及终浓度分别为1、2.5、5和10μg/mL的AM干预组,Western blot检测AM干预对EV71感染的GES-1细胞凋亡相关蛋白Caspase-3、PARP及自噬相关蛋白LC3、P62表达的影响;CCK-8法检测AM干预对EV71感染的GES-1细胞活力的影响。结果EV71感染引起了GES-1细胞变圆、皱缩,细胞核固缩,大小不均,细胞中VP1水平明显增高(t=41.56,P<0.01),细胞活力明显降低(t=19.07,P<0.01),细胞中Caspase-3和PARP蛋白均发生了明显的剪切(t分别为35.29和3.648,P分别为<0.01和0.0218),LC3Ⅱ/LC3Ⅰ比例显著增加(t=10.16,P=0.0005),P62蛋白发生了降解(t=68.68,P<0.01);AM干预后,抑制了EV71引起的Caspase-3、PARP、P62蛋白的降解(t分别为52.66、59.60和40.22,P均<0.01)及LC3Ⅱ/LC3Ⅰ比例增加(t=5.521,P=0.0053),并减轻了EV71对GES-1细胞活力的抑制作用(t=4.420,P=0.0115)。结论EV71感染诱导了GES-1细胞凋亡,AM干预通过抑制EV71引起的细胞自噬来抑制EV71诱导的细胞凋亡。Objective To investigate the effect of astragalus membranaceus(AM)injection on apoptosis and autophagy of human gastric epithelial cell line(GES-1)induced by enterovirus 71(EV71).Methods GES-1 cells were cultured in vitro and divided into infected group(EV71 infected at a MOI of 3 and control group(no virus infected).The morpho-logical changes of EV71 infected cells were observed by inverted microscope.The level of VP1 in GES-1 cells infected with EV71 was detected by Western blot;CCK-8 assay was used to detect the viability of GES-1 cells infected with EV71;Nuclear staining with DAPI was used to observe the morphological changes of nuclear apoptosis infected with EV71.GES-1 cells were divided into control group(without virus infection),infection group and AM intervention group with final concentration of 1,2.5,5 and 10μg/mL,respectively.Western blot was used to detect the effect of AM intervention on the expression of apoptosis-related proteins Caspase-3,PARP and autophagy-related proteins LC3 and P62 in GES-1 cells infected with EV71.CCK-8 method was used to detect the effect of AM intervention on the viability of GES-1 cells infected with EV71.Results GES-1 cells were round,shrunken with nuclear pyknosis and uneven size;VP1 level increased(t=41.56,P<0.01),cell viability decreased(t=19.07,P<0.01),Caspase-3 and PARP proteins were cut off(t=35.29 and 3.648,P<0.01 and 0.0218,respectively),LC3Ⅱ/LC3Ⅰratio increased(t=10.16,P=0.0005)and P62 protein was degraded(t=68.68,P<0.01);AM inhibited the degradation of Caspase-3,PARP and P62 proteins induced by EV71(t=52.66,59.60 and 40.22,respectively,each P<0.01)and increased the ratio of LC3Ⅱ/LC3Ⅰ(t=5.521,P=0.0053),and reduced the inhibitory effect of EV71 on the viability of GES-1 cells(t=4.420,P=0.0115).Conclusion EV71 infection induced apoptosis of GES-1 cells and AM intervention inhibited EV71 induced apoptosis by inhibiting EV71 induced autophagy.

关 键 词：肠道病毒71型 黄芪注射液 GES-1细胞 细胞凋亡 细胞自噬 

分 类 号：R310.17[医药卫生—基础医学]