| In this study,primary chicken renal tubular epithelial cells were used as the research object to establish a NIBV infection model,aiming to explore the relationship between MDA5 signaling pathway and NIBV-induced renal tubular epithelial cell damage as well as the regulatory mechanism of cepharanthine.Hy-line brown chicks aged 1-7 days were selected to establish the primary chicken renal tubular epithelial cell culture model by enzyme digestion.The cells were inoculated with 1 MOI SX9 NIBV and collected at 12 hpi,24 hpi,36 hpi and 48 hpi to detect the effect of NIBV on pyroptosis and MDA5 signaling pathway of renal tubular epithelial cells.The peak point of NIBV stimulation on the cells was selected for 36 h.After challenge,20 μM MDA5 signaling pathway TRAF6 inhibitor C25-140 was used to verify whether NIBV regulated primary chicken renal tubular epithelial cells pyroptosis through MDA5 signaling pathway.And to investigate whether it can treat renal infectious bronchitis through this mechanism by using 0.5 μM cepharanthine.The collected cells were tested for the following indicators: CCK8 was used to detect cell viability,quantitative fluorescent PCR and western blotting were used to detect MDA5 signaling pathway related genes and proteins,NIBV viral load and N protein expression level,LDH activity of supernatant was detected by enzyme label,pyroptosis level was observed by fluorescence microscope,pyroptosis rate was detected by flow cytometry.The expression of NLRP3 protein was observed by confocal laser.The results are as follows:1.Compared with Con,the activity of LDH increased significantly after NIBV infection(P<0.01),and there was a certain aging relationship..The NIBV group showed a time-dependent increase in viral load.The pyroptosis rate increased significantly in the flow results,and the number of double dye fluorescence points increased significantly in the fluorescence results(P<0.01).Gene and protein experiments showed that NIBV could promote the expression of MDA5,MAVS,TRAF6,TAK1,IKKα,IKKβ,NF-κB P65,NF-κB P50,NLRP3,Caspase-1,IL-18,and IL-1β.These results indicated that NIBV could activate MDA5 signaling pathway and induce Pyroptosis.2.After co-treatment with 20μM C25-140,compared with NIBV group,LDH activity and NLRP3 red fluorescence point of N+C25-140 group were significantly decreased(P<0.05),and pyroptosis rate and pyroptosis level were significantly decreased by flow and fluorescence results(P<0.01).The MDA5 signaling pathway and the above related genes and proteins were down-regulated,and the viral load was significantly decreased(P<0.01),suggesting that NIBV could regulate the pyroptosis of chicken renal tubule cells through MDA5/NF-κB signaling pathway.3.Compared with the NIBV group,the LDH activity of NIBV+CEP group was significantly decreased(P<0.01),the immunofluorescence fluorescence point of NLRP3 was significantly reduced to the normal level,the pyroptosis rate and pyroptosis level of cells were significantly decreased by flow cytometry and fluorescence structure(P<0.001).The m RNA expression levels of MDA5 signaling pathway and the above related genes and proteins were down-regulated(P<0.05 or P<0.01),and the viral load results showed that the viral copy number was significantly decreased(P<0.05).The expression of N protein was significantly decreased(P<0.05).These results suggest that cepharanthine can alleviate the pyroptosis induced by NIBV based on MDA5/NF-κB/NLRP3 signaling pathway.In conclusion,NIBV infection induces pyroptosis regulated by MDA5/NF-κB signaling pathway,and cepharanthine can alleviate NIBV-induced pyroptosis of renal tubular epithelial cells by inhibiting MDA5/NF-κB/NLRP3 signaling pathway. |
