Molecular Mechanism Study On Apoptosis In Vero Cells Induced By Porcine Epidemic Diarrhea Virus

Characterized by diarrhea,vomiting,dehydration and loss of appetite,Porcine epidemic diarrhea(PED)is a highly contagious intestinal infectious disease caused by Porcine epidemic diarrhea virus(PEDV).PEDV has a very high morbidity and mortality rate for piglets,which seriously endangers the healthy development of the pig industry.PEDV is a single-stranded and positive-strand RNA virus belonging to the family Coronaviridae and a member of the alpha coronavirus.Since its first discovery in the UK in 1971,PED has spread to many countries and regions around the world,causing huge economic losses to the pig industry.At present,there is no specific treatment for this disease,and it can only rely on vaccine immunization for preventive control.The pathogenesis of PEDV is still not fully understood and need to be further researched.Therefore,studying the molecular mechanism of PEDV-induced apoptosis is of great significance for finding new antiviral pathways.In this study,we screened the apoptosis signal of PEDV-infected Vero cells and studied the correlation between apoptosis signal and apoptosis,and obtained the following results:1.Indirect immunofluorescence assay,Western blot and flow cytometry were used to screen the changes of different molecular signals in PEDV-infected Vero cells.First,PEDV was used to infect Vero to observe the pathological effects of cells.Indirect immunofluorescence assay was used to observe the proliferation of PEDV.On the basis of confirming that PEDV infection can cause cytopathic effects,different multiplicities of infection were detected by flow cytometry(MOI of 0.1,0.5,1 respectively)The incidence of apoptosis of PEDV-infected cells at 24 h,and the changes of p53 family,ROS content and MAPK family proteins at different infection times were detected.The results showed that with the increase of the dose of infection,the incidence of apoptosis induced by PEDV in Vero cells also increased.Western blot analysis of p53 showed that PEDV significantly activated p-p53(Ser20),significantly increased the expression of CBP(cAMP-responsive element-binding protein)and MDM2(Murine double minute 2 protein).The IFA on p53 showed that,during viral infection,p53 translocated to the nucleus to execute function.Western blot analysis of MAPK family showed that PEDV infection significantly activated p38 MAPK and SAPK/JNK,and increased the expression of p-p38 MAPK and p-SAPK/JNK protein.The results of flow cytometry on ROS content in different PEDV infection time indicated that PEDV can significantly induce the accumulation of ROS,and this cumulative effect is more obvious with the prolonged infection time.2.The relationship between activated ROS,MAPK and p53 family and PEDV-induced apoptosis of Vero cells was confirmed by Western blot and flow cytometry using inhibition test.First,Vero cells were treated with p53-specific inhibitor Pifithrin-?(PFT-?),and then infected with PEDV for 24 h.The apoptosis rate was detected by flow cytometry and the expressions of apoptosis-related proteins such as Bax,Bcl-2 and FasL were detected by Western blot.Next,after treatment of Vero cells with p38 MAPK and JNK inhibitor SB203580 and SP600125,PEDV was infected for 24 h,and the expressions of apoptosis-related proteins such as Bax,Bcl-2 and FasL were detected.Finally,Vero cells were treated with antioxidants NAC and PDTC and then infected with PEDV for 24 h,the incidence of apoptosis was detected by flow cytometry and the relative expression of apoptotic proteins such as Fas,FasL,Bax and Bcl-2 was detected by Western blot.The results showed that PEDV infection significantly increased the expression of pro-apoptotic Bax and decreased the expression of anti-apoptotic protein Bcl-2,and PFT-? could significantly reduce Bax relative expression,increase Bcl-2 relative expression and up-regulate the ratio of Bax/Bcl-2,which reduced the incidence of apoptosis,showed that inhibition of p53 significantly reversed apoptosis;compared with PEDV-infected group,neither SB203580 nor SP600125 treatment could change the expression of Bcl-2,Bax and FasL.The amount of Vero cells induced by PEDV was not reversed in the presence of p38 MAPK and SAPK/JNK,ie,activation of p38 MAPK and SAPK/JNK was not associated with PEDV-induced apoptosis;compared with PEDV-infected group,the rate of apoptosis was significantly reduced in PDTC and NAC treatment group.The ratio of FasL/Fas was not significantly changed in the PEDV-infected group or in the antioxidant treatment,while the ratio of Bax/Bcl-2 was significantly decreased,indicating that PDTC and NAC treatment significantly reversed PEDV induced apoptosis.Further inhibition experiments indicated that the p53 portion is mediated by ROS,ie ROS is upstream of p53.In summary,cytopathic effects appeared during PEDV infection and PEDV induced apoptosis in Vero cells in a dose-dependent manner.Moreover,PEDV infection induced p53 activation,ROS accumulation and activation of p38 MAPK and SAPK/JNK.Activated p53 and ROS were involved in PEDV-induced apoptosis,whereas activation of p38 MAPK and SAPK/JNK was not associated with PEDV-induced apoptosis.