| Porcine Parvovirus(PPV),a member of the genus Parvovirus of the family Parvoviridae,is an important pathogen causing reproductive disorders in sows.It can be transmitted vertically from the sows to fetuses through the placental barrier,and can infect a variety of primary and secondary pig cells,among which porcine placental trophoblast cells(PTCs)are its main target cells.PTCs is an important part of the barrier of pig placenta,which plays an important physiological and immune protection role in placenta formation and fetal development.Previous studies in this laboratory have confirmed that PPV infection of PTCs can cause autophagy and apoptosis.However,the relationship between autophagy and apoptosis of PTCs caused by PPV and whether autophagic cell death exists in PTCs infected by PPV is still unknown.In this paper,we firstly identified the relationship between autophagy and apoptosis induced by PPV infection in PTCs,and further examined whether the autophagic cell death caused by PPV infection in PTCs and identified the characteristics of the autophagic cell death.Finally,the mechanism of autophagic PTC death induced by PPV was investigated.The research results are as follows.1.PTCs were infected with 1 MOI PPV,and DNA Ladder results showed that after 36 h of PPV infection,PTCs chromosome DNA began to fragment,and fragments of cell chromosome DNA could be detected in 48 h.p.i.,60 h.p.i.,72 h.p.i.Flow cytometry showed that,the apoptosis rate at 12 h.p.i.,24 h.p.i.and 36 h.p.i.was significantly increased compared with infection at 0 h(p < 0.01).Caspase-3 and caspase-9 activity in PPV infected 12 h.p.i.cells were significantly increased compared with infection at 0 h(p< 0.05)and continued to increase at 24 h.p.i.and 36 h.p.i.after infection(p < 0.01),indicating that PPV infection can induce PTCs apoptosis.PTCs were pretreated with autophagy agonist rapamycin(RAPA,100 n M)or autophagy inhibitor 3-methyladenine(3-MA,5 m M),then infected by 1 MOI PPV.The results of cell apoptosis detection showed that inhibition of autophagy with 3-MA had no significant effects on the activity levels of caspase-3 and caspase-9 in PTCs infected by PPV(p > 0.05),and the rate of apoptosis was significantly increased(p < 0.05).And RAPA-induced autophagy significantly reduced the activity levels of caspase-3 and caspase-9 in PPV infected PTCs(p < 0.01),and significantly decreased the ratio of apoptosis(p < 0.05).The results showedthat autophagy induction could inhibit PPV-induced apoptosis,and autophagy inhibition promoted PPV-induced apoptosis2.PTCs were infected with 1 MOI PPV,flow cytometry showed that the cell mortality was 31.23%±1.98% with PPV infection for 24 h.With Caspase specific inhibitor 10 m M z VAD-fmk pretreatment,still 10.71%±1.12% of cells died,suggesting the existence of other forms of cell death mediated by non-apoptotic pathways in PPV-infected cells.PTCs were pretreated with 5 m M 3-MA or 100 n M RAPA in the presence of z VAD-FMK.3-MA inhibition of autophagy reduced the cell mortality caused by PPV infection(p < 0.05);The cell mortality increased with RAPA pre-treatment(p < 0.05);Specific si ATG5 inhibition of autophagy reduced the cell mortality caused by PPV infection(p < 0.05).These results suggest that PPV infection induces autophagic death of PTCs.Flow cytometry showed that1 MOI PPV infection significantly reduced the mean fluorescence intensity of lysosome tracker DND-26(DND26 MFI)with PTCs infection for 24 h.z VAD-fmk pretreated PTCs could not change the DND26 MFI reduction caused by PPV infection,but in the presence of z VAD-fmk,3-MA pretreatment effectively prevented the DND26 MFI reduction.Western blotting results showed that PPV infection promoted the release of cathepsin D and cathepsin L from lysosomes to cytoplasm,and 3-MA could reduce the cathepsin D and L in cytoplasm of PPV infected cells(p < 0.05),while z VAD-FMK could not.These results suggest that PPV infection can induce autophagic death of PTCs characterized by lysosomal damage.3.PTCs were infected with 1 MOI PPV,further testing cell mortality and lysosomal membrane permeability change within 72 h of PPV infection.Flow cytometry showed that in the presence of z VAD–fmk,1 MOI PPV infected PTCs,with the extension of PPV infection time,the cell mortality continued to increase,the cell death rate at 24 h.p.i.,48 h.p.i.and 72 h.p.i.was significantly increased compared with infection at 0 h(p < 0.01).And DND26 MFI continued to decrease in line with the cell mortality,suggesting that the rate of autophagic cell death,characterized by lysosomal damage caused by PPV infection,increased with the duration of infection.Compared with only the PPV infection,100 n M RAPA pretreatment increased cell mortality and reduced DND26 MFI,especially in 72 h.p.i.But PTCs incomplete autophagy induced by 100 n M RAPA&100 n M BAF had no significant effect on the cell mortality and DND26 MFI with PPV infection.In line with DND-26 MFI,in 72 h.p.i.,cathepsin D and cathepsin L were released from lysosomes into cytoplasm.Cathepsin D and cathepsin L in the PPV infection group pretreated with 100 n M RAPA&100 n M BAF were significantly decreased(p < 0.05).These results suggest thatcomplete autophagy increases the rate of autophagic death in PTCs characterized by lysosomal damage in the later stages of PPV infection.In this study,it was found that induction of PTCs autophagy could inhibit PPV-induced apoptosis,and inhibition of PTCs autophagy promoted PPV-induced apoptosis.It was found that PPV infection caused autophagic cell death of PTCs characterized by lysosomal damage.Further studies found that complete autophagy of cells promoted PPV-induced autophagic cell death of PTCs,while incomplete autophagy of cells had no significant effect on autophagic cell death of PTCs.The results elucidated the relationship between autophagy and apoptosis induced by PPV in PTCs and the influence of autophagy in different stage on autophagic cell death induced by PPV in PTCs,providing a theoretical basis for further revealing the pathogenesis of PPV. |
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