The Mechanism Of Porcine Placental Trophoblast Cells Necroptosis Induced By Porcine Parvovirus

Porcine parvovirus（PPV）often causes reproductive disorders in primigravid sows and brings huge economic losses to the pig industry.Porcine placental trophoblast cells（PPTCs）are an important part of the porcine placental barrier,play an important role in the formation of porcine placenta and embryonic development,and are also the main target cells of PPV attack.Necroptosis is a form of non-apoptotic cell death,which is characterized by the rupture of target cell membrane and the release of cell contents,which induces severe inflammatory response in local tissues.At present,studies have shown that a variety of viruses such as IAV can cause necroptosis after infecting host cells.Previous studies in our laboratory have found that PPTCs undergo non-apoptotic cell death in the late stage of PPV infection.However,whether these non-apoptotic cell death are necroptosis and the specific mechanism are not clear so far.Therefore,the aim of this study is to investigate whether PPTCs infected with PPV can cause necroptosis by inhibiting cell apoptosis caused by PPV infection,observing cell morphology,measuring the release of Lactate dehydrogenase（LDH）and detecting the expression levels of key proteins of necroptosis.Furthermore,a series of PPTCs cell lines with the deletion of host cell key regulatory genes in the process of necroptosis were constructed.Finally,these cell lines were used to study the role and mechanism of ZBP1/Caspase8/RIP3/MLKL pathway proteins in the process of PPTCs necroptosis induced by PPV.The following results were obtained.1.PPV infection induced necroptosis in PPTCsPPTCs were pretreated with apoptosis inhibitor Ac-DEVD-CHO for 1 h and then infected with 1 MOI PPV.Light microscopy showed swelling,rupture and ill-defined lesions at 48 h and 72 h after infection.The cells at 72 h of infection showed typical programmed necrosis characteristics such as swelling,vacuolization,irregular organelles and membrane rupture.The LDH release assay showed that the LDH release of PPTCs at 24 h of PPV infection was significantly higher than that at 0 h of infection（P<0.05）.LDH release from PPTCs was significantly increased at 48 h and 72 h after infection（P<0.01）.western blotting showed that the expression of p-MLKL in the cells infected with PPV at 24 hours was significantly higher than that at 0 hours（P<0.05）,and the expression of p-MLKL at 48 and 72 hours was extremely higher than that at 0 hours（P<0.01）.The subcellular localization of MLKL detected by laser confocal microscopy showed that MLKL was located in the cytoplasm of PPTCs in the MOCK group,and was located in the cell membrane at 72 h after PPV infection.The results of RIP3/Caspase8 detected by western blotting showed that the expression of RIP3in the cells infected with PPV at 24 h was significantly higher than that at 0 h（P<0.05）,and the expression of RIP3 at 48 h and 72 h was extremely significantly higher than that at 0 h（P<0.01）.However,there was no significant difference in the expression of Caspase8 at 24 h,48 h and 72 h after PPV infection（P>0.05）.The expression level of ZBP1 in the cells was detected by western blotting.The results showed that the expression level of ZBP1 in the cells infected with PPV for 24 h was significantly higher than that at 0 h（P<0.05）,and the expression level of ZBP1 in the cells infected with PPV for 48 h and 72 h was extremely significantly higher than that at 0 h（P<0.01）.PPTCs were pretreated with zbp1 si RNA for12 h and then infected with 1 MOI PPV.The results of LDH assay at 72 h showed that the release of LDH in the si-zbp1 group was significantly lower than that in the si-NC group（P<0.05）.2.The PPTCs^zbp1-/-,PPTCs^caspase8-/-,PPTCs^rip3-/-,PPTCs^mlkl-/-gene knockout cell lines were successfully constructedThe lentivirus plasmids targeting zbp1（352,368,403 sites）,caspase8（219,715,721sites）,rip3（300,376,481 sites）and mlkl（126,520,543 sites）were successfully constructed.The constructed lentiviral plasmid was transfected into 293T cells,and the cell supernatant was collected 72 hours later.After 48 hours of infection,PPTCs were screened by M199medium containing 10μg/m L puromycin.The corresponding target proteins were not detected in the PPTCs knockout cells targeting the 403 site of zbp1 gene,219 site of caspase8 gene,481 site of rip3 gene and 126 site of mlkl gene.Sequencing results showed that there were base deletions near the target sites.The successful knockout cell lines were named403PPTCs^zbp1-/-,219PPTCs^caspase8-/-,481PPTCs^rip3-/-and 126PPTCs^mlkl-/-.3.PPV mediated necroptosis of PPTCs through the ZBP1-RIP3-MLKL pathwayAfter pretreatment with Ac-DEVD-CHO,an apoptosis inhibitor,for 1 h,wild-type PPTCs,403PPTCs^zbp1-/-,219PPTCs^caspase8-/-,481PPTCs^rip3-/-and 126PPTCs^mlkl-/-were infected at 1 MOI PPV.LDH release assay showed that there was no significant difference in LDH release between the four gene deletion cells and the wild type cells at 24 hours after PPV infection（P>0.05）.LDH release of 403PPTCs^zbp1-/-,481PPTCs^rip3-/-and 126PPTCs^mlkl-/-was significantly decreased at 48 and 72 h after infection（P<0.01）.There was no significant difference in LDH release between 219PPTCs^caspase8-/-cells（P>0.05）.The results of detecting ZBP1,RIP3,Caspase8 and p-MLKL in these cells showed that in zbp1 null cells,the expression levels of RIP3,Caspase8 and p-MLKL at 24,48 and 72 hours after PPV infection were not significantly different from those at 0 hour（P>0.05）.In caspase8 null cells,the expression levels of ZBP1,RIP3 and p-MLKL were significantly increased at 24 h of PPV infection compared with those at 0 h of PPV infection（P<0.05）,and the expression levels of the three proteins were extremely significantly increased at 48 h and 72 h of PPV infection（P<0.01）.In rip3 null cells,the expression levels of Caspase8 and p-MLKL were not significantly different at 24 h,48 h and 72 h of PPV infection compared with those at 0 h of PPV infection（P>0.05）,but the expression level of ZBP1 was significantly increased at 24h of PPV infection（P<0.05）.The expression level of ZBP1 was significantly increased at 48and 72 h after infection（P<0.01）.In mlkl null cells,the expression levels of ZBP1 and RIP3were significantly increased at 24 h of PPV infection compared with those at 0 h（P<0.05）,and the expression levels of ZBP1 and RIP3 were extremely significantly increased at 48 h and 72 h of PPV infection（P<0.01）.There was no significant difference in the expression level of Caspase8 between the two groups（P>0.05）.This study found that PPV infection could induce necroptosis of PPTCs,and403PPTCs^zbp1-/-,219PPTCs^caspase8-/-,481PPTCs^rip3-/-,126PPTCs^mlkl-/-cell lines were successfully constructed.Further studies found that,PPV induces necroptosis of PPTCs through the ZBP1-RIP3-MLKL pathway.The results of this study provide a theoretical basis for further elucidating the pathogenic mechanism of PPV.