摘要
肠道病毒-A71型(EV-A71)重症感染患儿多表现为过激的炎症反应,病毒感染引起的细胞焦亡可能是机体炎症发生的重要原因之一。本文旨在探究姜黄素对EV-A71病毒感染引起的细胞焦亡与细胞损伤的保护作用及可能的机制。首先,观察了姜黄素对EV-A71引起的细胞毒性的影响。CCK8检测结果显示,EV-A71感染降低细胞的增殖活力;LDH测定表明,病毒增加细胞培养上清中LDH的释放,造成了细胞的损伤;DAPI核染色及Dil细胞膜染色后观察到,EV-A71感染引起了细胞的形态变化和数量减少。姜黄素可以逆转病毒引起的上述变化,提示姜黄素对病毒感染的细胞毒性具有保护作用。细胞焦亡发生时,可促进炎症因子IL-1β的成熟、产生和释放。我们观察了EV-A71及姜黄素干预对细胞IL-1β产生的影响。Western印迹结果显示,病毒感染细胞内IL-1β的活化增加。ELISA检测结果显示,EV-A71病毒感染引起细胞上清中IL-1β的分泌水平增加;qPCR测定结果显示,EV-A71病毒感染细胞中IL-1β的转录水平上调;而姜黄素干预可抑制染毒细胞IL-1β的活化和分泌。Western印迹检测细胞内焦亡相关分子的变化。结果显示,EV-A71感染可诱导细胞发生焦亡,并呈时间和剂量依赖性,分子NOD样受体热蛋白结构域相关蛋白3(NLRP3)、GSDMD、胱天蛋白酶1(caspase-1)参与EV-A71诱导细胞发生的焦亡;姜黄素干预可以有效抑制EV-A71诱导的细胞细胞焦亡。另外,Western印迹检测显示,姜黄素可以减少细胞内EV-A71结构蛋白VP1的水平,通过检测细胞上清中病毒的CCID50发现,姜黄素可以降低细胞上清中病毒的滴度。最后,对姜黄素抗焦亡机制进行了探索,发现EV-A71感染诱导了细胞自噬并激活了p38/NLRP3通路,姜黄素能抑制自噬标志蛋白LC3及自噬底物p62的降解,并抑制p38/NLRP3的激活。总之,本研究明确了姜黄素通过对自噬溶酶体阶段及p38/NLRP3通路的抑制,有效减轻了EV-A71诱导的细胞焦亡,为姜黄素在抗EV-A71感染的临床应用提供参考。
The severe individuals with enterovirus A71(EV-A71) infection usually present excessive inflammatory response.Pyroptosis can trigger the host inflammatory response.The inflammatory response was usually due to pyroptosis elicited by EV-A71.This study was performed to explore the protective effect of curcumin on cell pyroptosis and cell damage caused by EV-A71 infection.Firstly,the protective effect of curcumin against the EV-A71 induced cytotoxicity was observed.Gastric epithelial cells(GES-1) were infected with EV-A71 of different multiplicity of infection(MOI).CCK8 test results showed that EV-A71 infection decreased the proliferation of cells.LDH test showed that the virus increased the release of LDH in cell culture supernatant and caused cell damage.After staining with DAPI and Dil,it was observed that EV-A71 infection caused cell morphological changes and decline of the number of cells.The encouraging results that curcumin could reverse the above changes caused by the virus suggest that curcumin has a protective effect on the cytotoxicity of viral infection.Pyroptosis always accompany with the maturation,generation and extracellular release of the inflammasome-dependent cytokines interleukin IL-1β,which contributes to inflammatory pathology.The increased secretion level of IL-1β in cell supernatant had been measured by ELISA;the upregulated transcriptional level of IL-1β had been detected by RT-qPCR;the increased level of IL-1βhad been tested by Western blotting in EV-A71 infected cells,and these upregulation had been significantly suppressed by the treatment with curcumin.Furthermore,the changes of pyroptosis related molecules in EV-A71 infected cells were detected by Western blot assays.The results showed that EV-A71 infection could induce pyroptosis in a time and dose dependent manner,and the molecules NOD-like receptor thermal protein domain associated protein 3(NLRP3),gasdermin D(GSDMD),and caspase-1 participated in this process.Curcumin intervention can effectively inhibit EV-A71 induced pyroptosis.In addition,Western-blot and CCID50 assay were performed to detect the replication level of EV-A71,respectively.Curcumin could reduce the level of EV-A71 structural protein VP1 in cells and the viral titer in cell supernatant.Finally,we explored the mode of action of anti-pyroptosis mechanism of curcumin and found that EV-A71 infection induced autophagy and activated the p38/NLRP3 pathway.Curcumin showed similar effects with the autophagy inhibitor 3-MA and chloroquine that could inhibit the degradation of autophagy marker LC3 and autophagy substrate p62,and inhibit the activation of p38/NLRP3.In conclusion,this study confirmed that curcumin can effectively inhibit the occurrence of EV-A71 induced cell pyroptosis though the suppression on autophagy lysosome stage and p38/NLRP3 signaling pathway.These findings provide a guide for future research on antiviral therapy for EV-A71 infection.
作者
张晓延
张鑫艳
杜建平
ZHANG Xiao-Yan;ZHANG Xin-Yan;DU Jian-Ping(Department of Medical Laboratory,Fenyang College of Shanxi Medical University,Fenyang 032200,Shanxi,China;Clinical Laboratory,Sinopharm Tongmei General Hospital,Datong 037003,Shanxi,China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2023年第1期142-153,共12页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金项目(No.81301426)
山西省自然科学基金(No.201901D111329)
吕梁市临床分子诊断学重点实验室(No.2020ZDSYS17)资助。
