牛病毒性腹泻病毒核心蛋白C激活NLRP3炎症小体诱发细胞焦亡的分子机制研究

Molecular mechanisms of bovine viral diarrhea virus core protein activating NLRP3 inflammasome to induce pyroptosis

为探究牛病毒性腹泻病毒(BVDV)核心蛋白C激活NLRP3炎症小体诱发细胞焦亡的分子机制。本研究构建BVDV C蛋白真核表达质粒pCMV-C,经PCR和测序鉴定正确后转染牛睾丸原代(BT)细胞,48 h采用间接免疫荧光试验(IFA)和western blot检测C蛋白的表达;采用CCK-8法检测转染pCMV-C(过表达C蛋白)的BT细胞活性。结果显示,该BT细胞出现特异性绿色荧光,且在23 ku处出现特异性条带,阴性对照细胞(转染pCMV的细胞,后同)未见绿色荧光也无该条带,表明C蛋白在BT细胞中获得了表达。细胞活性检测结果显示,过表达C蛋白的BT细胞活性极显著低于阴性对照组细胞(P<0.001)。将荧光素酶报告质粒pNF-κB-Luc、内参质粒pRL-TK和pCMV-C共转染BT细胞,并设置相应的阳性和阴性对照,48 h后采用双荧光素酶报告系统检测各组细胞上清中NF-κB的相对荧光素酶活性。结果显示,共转染3种质粒的BT细胞及阳性对照细胞中NF-κB相对荧光素酶活性极显著高于空白对照组和阴性对照组(共转染pCMV、pNF-κB-Luc和pRL-TK的细胞)(P<0.01)。通过荧光定量RT-PCR(qRT-PCR)分别检测过表达C蛋白的BT细胞中NLRP3炎症小体组分[Caspase-1前体(pro-Caspase-1)、凋亡相关斑点样蛋白(ASC)、NLRP3及IL-1β的前体(pro-IL-1β)]mRNA的转录水平;采用western blot检测该BT细胞中NLRP3炎症小体组分(pro-Caspas-1、ASC、NLRP3、pro-IL-1β和IL-1β的表达水平并通过western blot分析pro-Caspase-1和pro IL-1β的切割结果。结果显示,过表达C蛋白的BT细胞中NLRP3和ASC mRNA转录水平(P<0.01)、pro-Caspase-1和pro-IL-1βmRNA转录水平(P<0.001)均极显著高于阴性对照组细胞;该BT细胞中NLRP3、pro-Caspase-1、Caspase-1P20、ASC、pro-IL-1β和IL-1β蛋白表达水平均明显高于阴性对照组细胞。转录和蛋白表达水平均表明BT细胞发生了炎症反应,且pro-Caspase-1被切割成为有活性的Caspase-1和Caspase-1p20,pro-IL-1β被切割成有活性的IL-1β。将pCMV-C和pCMV-ASC、pCMV-C和pCMV-NLRP3分别共转染293T细胞,48 h后通过激光共聚焦显微镜观察C蛋白与NLRP3和ASC的共定位。结果显示,C蛋白分别与NLRP3和ASC在细胞质中存在共定位。为进一步探究C蛋白激活NLRP3炎症小体对细胞焦亡的影响,通过qRT-PCR和western blot检测过表达C蛋白的BT细胞中死亡调节蛋白GSDMD mRNA的转录、蛋白表达水平及切割情况。通过扫描电镜观察转染pCMV-C 12 h和24 h的BT细胞,并采用间接ELISA检测转染pCMV-C 24 h BT细胞上清中IL-1β的含量。qRT-PCR结果显示,GSDMD mRNA转录水平极显著高于空白对照组和阴性对照组细胞(P<0.001);GSDMD蛋白的表达水平也明显高于两个对照组,且该蛋白被切割成有活性的GSDMD-N和GSDMD-C。电镜观察结果显示,与阴性对照细胞相比,随着C蛋白表达时间的延长,BT细胞膜逐渐破裂,细胞膜均已形成渗透孔,并有细胞内容物的释放,且细胞上清中IL-1β的含量极显著高于阴性对照组细胞(P<0.01)。上述研究表明,C蛋白能够显著引起NLRP3炎症小体活化进而激活pro-Caspase-1形成活化的Caspase-1,后者一方面切割GSDMD,形成有活性的GSDMD-N,并在BT细胞膜形成孔洞,释放内容物,诱导BT细胞发生细胞焦亡,另一方面活化的Caspase-1切割pro-IL-1β,形成有活性的IL-1β,并释放到BT细胞外,致细胞上清中IL-1β含量极显著升高。本研究为进一步阐明BVDV的致病机制以及制定BVD的防控策略提供参考。

The molecular mechanism underlying the activation of the NLRP3 inflammasome and induction of cell pyroptosis by the core protein C of bovine viral diarrhea virus(BVDV)was investigated in this study.The BVDV C protein eukaryotic expression plasmid pCMV-C was constructed and transfected into bovine testicular(BT)primary cells after PCR and sequencing validation.The expression of the C protein was detected by indirect immunofluorescence assay(IFA)and western blot at 48 hours post-transfection.The cell viability of BT cells transfected with pCMV-C(overexpression of C protein)was measured using the CCK-8 assay.The results showed that BT cells overexpressing C protein exhibited specific green fluorescence and a specific band at 23ku,which was absent in negative control cells(cells that transfected with the pCMV plasmid).This indicated successful expression of the C protein in BT cells.The cell viability assay revealed that the overexpression of C protein significantly reduced the activity of BT cells compared to the negative control group(P<0.001).The reporter plasmids pNF-κB-Luc and pRL-TK,along with pCMV-C,were co-transfected into BT cells to measure the relative luciferase activity of NF-κB in the cell culture supernatant using a dual-luciferase reporter system.The results showed that the relative luciferase activity of NF-κB in BT cells co-transfected with the three plasmids and the positive control cells were significantly higher than the blank control group and the negative control group(cells co-transfected with pCMV,pNF-κB-Luc,and pRL-TK)(P<0.01).The mRNA transcription levels of NLRP3 inflammasome components(pro-Caspase-1,apoptosis-associated speck-like protein containing a CARD domain(ASC),NLRP3)and pro-interleukin-1β(pro-IL-1β)in BT cells overexpressing C protein were measured using fluorescence quantitative PCR(qRT-PCR).The expressions of NLRP3 inflammasome components(pro-Caspase-1,ASC,and NLRP3)and IL-1βin these BT cells were detected using western blot,and the cleavage of pro-Caspase-1 and pro-IL-1βwas analyzed.The results showed that the mRNA transcription levels of NLRP3,ASC,pro-Caspase-1,and pro-IL-1βin BT cells overexpressing C protein were significantly higher than those in the negative control group(P<0.01 or P<0.001).The protein expression levels of NLRP3,pro-Caspase-1,Caspase-1p20,ASC,pro-IL-1β,and IL-1βin these BT cells were significantly higher than those in the negative control group.These results indicated the occurrence of inflammatory response in BT cells,and pro-Caspase-1 was cleaved to active Caspase-1 and Caspase-1p20,while pro-IL-1βwas cleaved in active IL-1β.Cellular co-localization of the C protein,NLRP3 and ASC were observed by laser confocal microscopy,the results showed that C protein co-localized with NLRP3 and ASC in cytoplasm,respectively.To further investigate the effect of C protein-mediated NLRP3 inflammasome activation on cell pyroptosis,the mRNA transcription,protein expression levels,and cleavage of gasdermin D(GSDMD)in BT cells overexpressing C protein were measured by qRT-PCR and western blot analysis.The morphological changes of BT cells transfected with pCMV-C were observed by scanning electron microscopy(SEM),and the secretion of IL-1βin the supernatant of BT cells transfected with pCMV-C was detected using indirect ELISA.The results of qRT-PCR showed that the transcription level of GSDMD mRNA in BT cells overexpressing C protein was significantly higher than that in the blank control group and the negative control group(P<0.001).The protein expression level of GSDMD was also significantly higher than that of the two control groups,and the protein was cleaved into active GSDMD-N and GSDMD-C.Electron microscopy observations showed that compared with the negative control cells,the BT cell membrane gradually ruptured with the prolonged expression of C protein,permeable pores were formed in the cell membrane,and cellular contents were released.Moreover,the IL-1βcontent in the cell supernatant was significantly higher than that of the negative control group(P<0.01).The above study indicated that C protein could significantly activate NLRP3 inflammasome and subsequently activate pro-Caspase-1 to form activated Caspase-1.On the one hand,the latter cleaved GSDMD to form active GSDMD-N and formed pores in the BT cell membrane to release the contents and induce pyroptosis in BT cells.On the other hand,the activated Caspase-1 cleaved pro-IL-1βto form active IL-1β,which was released into the extracellular space,resulting in a significant increase in IL-1βcontent in the cell supernatant.This research provides a reference for further elucidating the pathogenic mechanism of BVDV and developing prevention and control strategies for BVD.