猪流行性腹泻病毒非结构蛋白NSP15抑制细胞焦亡的分子机制研究  

Molecular mechanism of PEDV non structural protein 15 inhibiting pyroptosis

作　　者：李鑫悦 徐维律 吕倩 傅心雨 石玉华 李丹月 何苏慧 董露 陈楠 师福山 LI Xin-yue;XU Wei-lv;LV Qian;FU Xin-yu;SHI Yu-hua;LI Dan-yue;HE Su-hui;DONG Lu;CHEN Nan;SHI Fu-shan(Department of Animal Medicine,College of Animal Science,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学动物科学学院动物医学系,浙江杭州310058

出　　处：《中国预防兽医学报》2024年第7期731-739,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：浙江省尖兵领雁计划项目(2024C02004);国家自然科学基金区域联合基金项目(U22A20521);国家自然科学基金面上项目(32072817)。

摘　　要：为探究猪流行性腹泻病毒(PEDV)及其NSP15蛋白对细胞焦亡的影响及初步的分子作用机制,本研究将表达pGSDMD-p30的质粒转染猪小肠上皮细胞(IPEC-J2细胞),24 h后将PEDV感染该细胞,于感染后12 h和24 h分别收集细胞上清液和细胞,采用LDH试剂盒测定各时间点细胞上清中乳酸脱氢酶(LDH)的释放比例;采用qPCR检测细胞中GSDMD mRNA的转录水平。结果显示,各时间点,转染表达pGSDMD-p30质粒的IPEC-J2细胞中LDH的释放比例均极显著升高,表明细胞发生焦亡,且与转染p GSDMD-p30质粒的细胞相比,PEDV感染的细胞中LDH的释放比例及GSDMD mRNA的转录水平均极显著降低。将表达pGSDMD-p30的质粒分别与不同剂量的PEDV-NSP15质粒共转染HEK293T细胞(pGSDMD-p30+NSP15组);将表达Caspase-1、pGSDMD和表达NSP15的质粒分别共转染HEK293T和IPEC-J2细胞(Caspase-1+pGSDMD+NSP15组)。上述细胞于24 h后均测定各组细胞上清液中LDH的释放比例。结果显示,与pGSDMD-p30组(共转染表达pGSDMD-p30与空载体的细胞)相比,pGSDMD-p30+NSP15组HEK293T细胞及IPEC-J2细胞中LDH的释放比例均显著降低;与Caspase-1+pGSDMD对照组细胞相比,Caspase-1+pGSDMD+NSP15组细胞中LDH的释放比例极显著降低。上述结果表明,NSP15在不同细胞中均可以抑制GSDMD-p30及GSDMD+Caspase-1两种方式引起的细胞焦亡,提示PEDV可能通过NSP15抑制细胞的焦亡。将自噬抑制剂3-MA和蛋白酶体途径抑制剂MG132分别加入不同剂量的NSP15质粒与MYC-pGSDMD质粒共转染的HEK293T细胞中,6 h后采用western blot检测细胞中GSDMD的表达水平;将上述质粒共转染HEK293T细胞,24 h后通过qPCR检测细胞中GSDMD mRNA的转录水平。结果显示,随着NSP15转染剂量的增加,细胞中GSDMD的表达水平逐渐减少,加入3-MA和MG132并不影响GSDMD的表达水平;相比于共转染空载体和NSP15质粒的对照组,MYC-pGSDMD+NSP15组细胞中GSDMD mRNA的转录水平极显著降低。上述结果表明,PEDV NSP15降解GSDTo investigate the effects of porcine epidemic diarrhea virus(PEDV)and its non-structural protein 15(NSP15)on cell pyroptosis,this study transfected pGSDMD-p30 plasmid into porcine intestinal epithelial cells(IPEC-J2 cells).The cells were infected with PEDV 24 hours after transfection.Cell supernatants and cells were collected at 12 and 24 hours post-infection.The percentage of lactate dehydrogenase(LDH)release was measured to assess pyroptosis,and the transcription level of GSDMD mRNA was analyzed using qPCR.The results showed that LDH release in pGSDMD-p30-transfected IPEC-J2 cells was significantly higher than in the control groups at all time points,indicating pyroptosis occurred.Compared to cells transfected with p GSDMD-p30plasmid,LDH release and GSDMD m RNA transcription levels were considerably reduced in PEDV-infected cells.The p GSDMD-p30 plasmid was co-transfected with varying doses of PEDV-NSP15 plasmid into HEK293T(pGSDMD-p30+NSP15 group);Plasmids expressing Caspase-1,pGSDMD,and PEDV-NSP15 were co-transfected respectively into HEK293T cells and IPEC-J2 cells(Caspase-1+pGSDMD+NSP15 group).LDH release was measured in the cell supernatants 24 hours after transfection.The results showed that co-transfection with PEDV-NSP15 and pGSDMD-p30 significantly reduced LDH release in HEK293T and IPEC-J2 cells compared to the pGSDMD-p30 group.In addition,co-transfection of Caspase-1+pGSDMD+NSP15 also significantly reduced LDH release compared to the Caspase-1+pGSDMD group.These results indicated that NSP15 can inhibit pyroptosis induced in different manners in different cell types.Furthermore,HEK293T cells were co-transfected with NSP15 and MYC-pGSDMD plasmids and the cells were treated respectively with different doses autophagy inhibitor 3-MA and proteasome inhibitor MG132 for 6 hours.Western blot showed that GSDMD expression levels decreased with increasing doses of NSP15,and 3-MA or MG132 did not reverse this effect.Additionally,qPCR results indicated a significant reduction in GSDMD mRNA transcription levels i

关 键 词：猪流行性腹泻病毒 非结构蛋白15 核酸内切酶 细胞焦亡 GSDMD 

分 类 号：S852.65[农业科学—基础兽医学]