| Muscovy duck reovirus infection is a high morbidity and mortality acute infectious disease,caused by Muscovy duck reovirus.It mainly endangers waterfowl aged from7 old day to 40 old day.Muscovy duck is the main infected host.The infection of the virus mainly affects the survival rate of muscovy ducks.The epidemic situation is becoming more and more complication.and the disease has caused serious economic losses to the muscovy duck breeding industry.MDRV is often accompanied by other viruses or bacteria,so the immune effect of the conventional vaccine is not satisfactory.Muscovy duck breeding is one of the main industries of animal husbandry in Fujian province.The annual hatching of Muscovy duck and mule duck was to hundreds of millions.However,the MDRV infection of young muscovy ducks has not been eradicated,which will pose a great threat to the Muscovy duck breeding industry development.the pathogenic mechanism of MDRV infection.In addition,studies have shown that MDRV P10.8 protein can induces cell apoptosis,but the mechanism is not clear.Therefore,it is significant to illuminate the mechanism of apoptosis induced by MDRV p10.8 protein for the prevention and treatment of MDRV infection.In this study,young muscovy ducks and DF-1 cells were used as infection models.Transmission electron microscopy(TEM)and flow cytometry(FCM)were used to observe the endoplasmic reticulum(ER)stress and apoptosis level before and after MDRV infection and p10.8 protein transfection.The fluorescence quantitative PCR,Western blot,Co-immunoprecipitation,endoplasmic reticulum stress enhancer and inhibitor were used to detecte the expression levels of mRNA and protein about endoplasmic reticulum stress and apoptosis.The molecular mechanism of apoptosis induced by MDRV p10.8 protein via ERS pathway was clarified in this study.The main contents and results were as follows:1 MDRV endoplasmic reticulum stress induces apoptosisIn this experiment,MDRV-YB strain TCID50=10-5.40)was used to infect 5-day-old muscovy duck and DF-1 cells,liver and DF-1 cells were collected,endoplasmic reticulum(ER)status was investigated by transmission electron microscope,apoptosis level was detected by flow cytometry.Fluorescence quantitative PCR was used to detect the mRNA expression level of XBP1,Bip,CHOP and Caspase3.The expression level of activated XBPls was detected by PCR.The cells were treated with TM or TUDCA at the final concentration of 2ugr/ml and then infected with MDRV.The relationship between MDRV activated endoplasmic reticulum(ER)and apoptosis was analyzed.The results showed that the diameter of endoplasmic reticulum(ER)of liver cells in muscovy ducks increased significantly after infected with MDRV 24 hours(p<0.01).The apoptosis rate of liver cells in MDRV group was significantly higher than that in control group(p<0.01).The expression level of mRNA in XBP1,Bip,CHOP and Caspase3 was significantly up-regulated than control group(p<0.01).The results of PCR showed that the expression of XBPls in the liver of muscovy duck infected with MDRV was significantly up-regulated than the control group(p<0.01).After infected with MDRV,the diameter of endoplasmic reticulum in DF1 cells of MDRV infection group was significantly higher than that of the control group.And the level of apoptosis was significantly higher than that of control group(p<0.01).It was obvious that MDRV infection induced endoplasmic reticulum stress and apoptosis.Endoplasmic reticulum stress agonist chlamycin(TM)was used to treat DF1 infected with MDRV.The results showed that the expression of mRNA in Bip,XBP1,CHOP1 and Caspase3 was significantly up-regulated than the TM and MDRV alone groups(P<0.01),and the activation level of XBPls was also significantly increased(p<0.01).Under the combined action of TUDCA and MDRV,the expression of mRNA in Bip,XBP1,CHOP and Caspase3 was significantly down-regulated than the TM and MDRV alone groups(p<0.01),and the transcription of XBPls was also significantly decreased(p<0.01).It can be seen that MDRV induced apoptosis through endoplasmic reticulum stress.2 p10.8 protein is the key protein that MDRV induces apoptosis through endoplasmic reticulum stress pathway.In this experiment,pCI-flag-p10.8 eukaryotic plasmids were transfected into DF-1 cells for 24h.The diameter of endoplasmic reticulum(ER)was observed by electron microscope and the mRNA expression level of Bip,XBP1,CHOP,Caspase3 were detected by fluorescence quantitative PCR.Flow cytometry was used to detect the level of apoptosis and the expression of activated XBPls was detected by PCR.The cells were treated with TM and TUDCA with final concentration of 2ug/ml and transfected into p10.8.The relationshiop between the endoplasmic reticulum and apoptosis activated by p10.8 protein.XBP1-siRNA was co-transfected with p10.8 at a concentration of lug/ml.To analyze the important role of XBP1 gene in the endoplasmic reticulum stress pathway induced by p10.8 protein.The results showed that the endoplasmic reticulum was enlarged and the diameter was enlarged after transfection of p10.8.Compared with the control group,the expression levels of XBP1,Bip,CHOP and Caspase3 were significantly up-regulated(p<0.01),and the activation level of XBP1s was also significantly increased(p<0.01).The apoptosis rate was as high as 15%,which was significantly different from that of the control group(p<0.01).Endoplasmic reticulum stress agonist chlamycin(TM)was used to treat with DF-1 cell which transfected with p10.8.The results showed that the expression of mRNA in Bip,XBP1,CHOP and Caspase3 genes were significantly up-regulated than the TM and p10.8 alone groups(p<0.01),and the activation level of XBPls was also increased significantly(p<0.01).Under the combined action of TUDCA and p10.8,the expression of mRNA in Bip,XBP1,CHOP and Caspase3 genes were significantly down-regulated than the TM and MDRV alone groups(p<0.01),and the transcription of XBPls was also significantly decreased(p<0.01).It is clear that p10.8 is key protein that induced apoptosis by endoplasmic reticulum stress pathway.Then siXBP1 was co-stained with p 10.8 plasmid,The ratio of endoplasmic reticulum diameter to that of p10.8 group decreased significantly(P<0.01),and the rate of apoptosis also decreased significantly(p<0.01).The expression of XBP1s was also significantly down-regulated(p<0.01),and the expression of XBPls was significantly down-regulated than that of control group(p<0.011).It can be seen that XBP1 plays an important role in p10.8-induced endoplasmic reticulum stress pathway.3 Study on BIP/IRE1XBP1/Caspase3 signaling Pathway of apoptosis induced by p10.8 proteinIn order to further study the molecular mechanism of p10.8 protein inducing apoptosis,the key proteins in BIP/IRE1 XBP 1/Caspase3 signaling pathway were analyzed by Western blot and Co-immunoprecipitation.The results showed that the Bip-IRE1 stable binding protein was dissociated,under the transfection with p10.8 plasmid,autonomous phosphorylation of IRE1 protein and the expression of Bip,XBPls,Cleave-Caspase3 was up-regulated.The protein expression levels of XBP1s and Cleave-Caspase3 were significantly down-regulated than those transfected with P10.8 alone group in treating with IRE1 inhibitor(4μ8C).After co-transfection with siXBP1 and p10.8 plasmid,the expression of key apoptotic protein Cleave-Caspase3 was significantly lower than p10.8 group.lt can be seen that p10.8 protein triggers endoplasmic reticulum stress through IRE1 dissociation and autonomous phosphorylation,which activates apoptosis executive protein Caspase3 and induces apoptosis through the regulation of XBP1 protein.In conclusion,p10.8 is a key protein of MDRV induced apoptosis.It actived the apoptosis mainly through the endoplasmic reticulum stress IRE1/Bip/XBP1 signaling pathway,the caspase chain reaction was activated and the target protein was activated by caspase3 apoptotic executive protein,which promoted cell apoptosis. |
