摘要
猪急性腹泻综合症冠状病毒(SADS-CoV)是一种新发现的肠道冠状病毒,引起仔猪严重的临床腹泻和肠道病理损害。为探究甘草提取物的主要成分甘草酸(GLY)抑制SADS-CoV复制的机制,本研究利用不同浓度的GLY对非洲绿猴肾细胞(Vero E6)和猪回肠上皮细胞(IPI-2I)预处理2 h并感染不同浓度的SADS-CoV后,分别经western blot和TCID_(50)检测,结果显示N蛋白的表达量显著减少且病毒滴度下降,表明GLY在体外能够抑制SADS-CoV的感染。利用不同浓度GLY对Vero E6和IPI-2I细胞预处理2 h后,感染灭活的SADS-CoV,western blot结果显示在GLY浓度为0.8 mmol/L时,N蛋白表达量显著减少,表明GLY能够抑制该病毒的侵入。选取0.4 mmol/L GLY处理细胞,再感染SADS-CoV后2 h、6 h、12 h收取细胞样品经western blot检测,结果显示各时间点N蛋白表达量均显著减少,表明GLY对该病毒的复制抑制效果显著。GLY是高迁移率族蛋白B1(HMGB1)的竞争性抑制剂,而HMGB1的受体主要有TLR4和RAGE,因此构建了HMGB1关键性位点(C^(45)S、C^(106)S、C^(45/106)S)突变的质粒和RAGE受体的siRNA,分别转染Vero E6细胞并感染SADS-CoV,收获上清液和细胞样品,利用western blot和TCID_(50)检测。结果显示,N蛋白表达量显著减少且病毒滴度下降,表明GLY在SADS-CoV感染时通过影响HMGB1与TLR4和RAGE的结合而发挥功能。为了进一步探究GLY发挥作用的信号通路,分别将SADS-CoV感染Vero E6和IPI-2I细胞,收获细胞样品,利用western blot检测MAPK信号通路相关蛋白的变化,结果显示p-p38、p-JNK、p-ERK表达量在感染早期和晚期上调,表明SADS-CoV感染激活了MAPK通路。利用不同浓度的GLY和TLR4抑制剂TAK对Vero E6和IPI-2I细胞预处理2 h后感染SADS-CoV,收获蛋白样品,western blot结果显示p-JNK和N蛋白表达量显著减少,其他蛋白无明显变化,表明GLY和TAK调控JNK的磷酸化,而不能调控p38和ERK的磷酸化。利用HMGB1中和抗体预处理Vero E6细胞、将HMGB1的siRNA以及构建的HMGB1关键位点突变的真核表达质粒分别转染Vero E6细胞后感染SADS-CoV,收获上述细胞样品,western blot结果显示p-JNK的表达量均减少,表明HMGB1影响了JNK的磷酸化;利用不同浓度的JNK抑制剂SP600125对Vero E6和IPI-2I预处理2 h,再感染SADS-CoV,利用western blot、TCID_(50)和IFA检测,结果显示N蛋白表达量及病毒滴度均下降且病毒复制减少,表明SP600125抑制了病毒的复制。综上所述,本研究首次证实GLY主要通过HMGB1/TLR4/JNK信号通路抑制SADS-CoV的体外复制,该通路的发现为研究抗SADS-CoV的新型药物提供了数据支持。
Swine acute diarrhea syndrome coronavirus(SADS-CoV),a newly discovered enteric coronavirus,is the etiological agent that causes severe clinical diarrhea and intestinal pathological damage in piglets.In this study,Vero E6 and IPI-2I cells were pretreated with different concentrations of glycyrrhizin(GLY)for 2 hours,and then infected with different concentrations of SADS-CoV,aiming to investigate the inhibitory effect of GLY on SADS-CoV.Western blot and TCID_(50)results revealed a significantly decreased N protein expression and viral titer,indicating that GLY can inhibit the infection of SADS-CoV.Vero E6 and IPI-2I cells were pretreated with different concentrations of GLY for 2 hours and infected with SADS-CoV.Western blot results showed that when the concentration of GLY was 0.8mmol/L,the expression of N protein decreased significantly,indicating that GLY inhibited the invasion of the virus.At first,cells were treated with 0.4mmol/L GLY,and cell samples were collected at 2 hours6 hours and 12 hours after being infected with SADS-CoV for analysis,and the expression of N protein were found to be significantly reduced at all points,indicating that GLY had a significant inhibitory effect on the replication of the virus.GLY is a competitive inhibitor of high mobility group box 1(HMGB1),and the receptors of HMGB1 mainly include TLR4 and RAGE Based on this fact,the mutant plasmid at the key sites of HMGB1(C^(45)S,C^(106)S,C^(45/106)S)and the siRNA of the RAGE receptor were transfected to Vero E6 cells and infected with SADS-CoV,and the cell supernatant and samples were harvested.The western blot and TCID_(50)results showed that the expression of N protein and the virus titer were decreased,suggesting that GLY exerts its function by affecting the binding of HMGB1/TLR4/RAGE during SADS-CoV infection.To further explore the signaling pathway through which GLY functions,Vero E6 and IPI-2I cells were inoculated with SADS-CoV,and cell samples were harvested,western blot was used to detect the changes of MAPK proteins.The results showed that the protein expression levels of p-p38,p-JNK and p-ERK were up-regulated in the early and late stages,indicating that the MAPK pathway was activated by SADS-CoV infection Vero E6 and IPI-2I were pretreated with different concentrations of GLY and TLR4 inhibitor TAK for 2 hours and infected with SADS-CoV.Protein samples were harvested and analysed by western blot which showed a decreased p-JNK and N proteins,while other proteins showed no significant changes.These results indicated that GLY and TAK regulated the phosphorylation of JNK but did not regulate the phosphorylation of p38 and ERK.Also,Vero E6 cells were treated with HMGB1 antibody,the siRNA of HMGB1 and HMGB1 mutants plasmid,and infected with SADS-CoV.Protein samples were harvested,western blot results showed that phosphorylation of JNK decreased,indicating that HMGB1 affected JNK phosphorylation.Finally,Vero E6 and IPI-2I cells were pretreated with different concentrations of JNK inhibitor SP600125 to infect SADS-CoV,western blot,TCID_(50)and IFA results showed that the expression of N protein and virus titer,as well as virus replication were reduced,indicating that SP600125inhibited virus replication.In conclusion,our results revealed that GLY can inhibit in vitro replication of SADS-CoV,mainly through the HMGB1/TLR4/JNK signaling pathway.The discovery of this pathway provides theoretical support for the research of novel anti-SADS-CoV drugs.
作者
冯书风
时洪艳
张记宇
陈建飞
张鑫
张燎原
冯廷帅
景朝阳
季朝阳
石达
冯力
FENG Shu-feng;SHI Hong-yan;ZHANG Ji-yu;CHEN Jian-fei;ZHANG Xin;ZHANG Liao-yuan;FENG Ting-shuai;JING Zhao-yang;JI Zhao-yang;SHI Da;FENG Li(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第10期1076-1083,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
黑龙江省自然科学基金(TD2020C002)。
