The Function And Apoptosis Mechanism Of Porcine Circovirus Type 2 ORF3 And ORF5 Proteins On PUVEC

Objective:In the present study,we investigated the effect of PCV2 infection on the placenta of Kunming mouse,and further explored the effect of PCV2,stable expression of PCV2 ORF3 or ORF5 proteins on PUVEC functions and apoptotic mechanism.The aim is to explore the effect of PCV2 on placenta development and the potential pathogenic mechanism of PCV2 in pregnancy.Methods:(1)In vivo study,pregnant mice were intraperitoneally injected 0.1 m L PCV2 with 1000 TCID50 on gestation day(GD)15 or the same volume of cell culture medium(control group).Placental tissues were collected on GD19,fixed and embedded with paraffin.Histomorphological changes were detected by Hematoxylin-Eosin(H.E.)staining.The co-localization of PCV2 antigen and CD31(a marker for vascular endothelium)in the placenta was checked by double immunofluorescence labeling method.Cell apoptosis in the placenta was detected by TUNEL assay.The expression of caspase-3 and 9,vascular endothelial growth factor(VEGF)and proliferating cell nuclear antigen(PCNA)were determined by Western Blot(WB)assay.(2)In vitro study,we used porcine umbilical vein endothelial cells(PUVEC)as material,cells were infected with PCV2(10^5.5TCID50),cell proliferation and the protein marker PCNA were detected by CCK8 assay and WB,respectively.Moreover,cell apoptosis and cell cycle were also examined after PCV2infection by flow cytometry.(3)PLVX-PCV2-ORF3 and PLVX-PCV2-ORF5 were stably expressed in PUVEC transducted by a lentivirus system.The effect of stable expression of PCV2 ORF3 or ORF5 protein on PUVEC functions including cell proliferation,migration,invasion and angiogenesis were detected,and the apoptosis-related protein was measured.Results:(1)PCV2 infection caused pathological changes in mouse placenta,with hemorrhage in labyrinth layer,a narrower junctional zone.Cell apoptosis was observed on trophoblastic cells and vascular endothelial cells in placenta.Meanwhile,the expression of VEGF and PCNA was significantly decreased in placenta,while the expressions of caspase-3 and 9 were significantly increased after PCV2 infection.(2)PCV2 infection inhibited proliferation of PUVEC and the expression of PCNA.Cell cycle was blocked in G1 phase and the cell apoptosis was induced after PCV2infection.(3)Stable expression of PCV2 ORF3 or ORF5 proteins inhibited the PUVEC growth,and cell cycle was arrested in the S phase.The abilities of migration,invasion and tube formation of PUVEC with PCV2 ORF3 or ORF5 expression,and the expression of VEGF was decreased.PCV2 ORF3 and ORF5 proteins induce PUVEC apoptosis,the expression of caspase-3,PARP and Bax/Bcl-2 in the stable ORF3-expressing PUVEC cells and caspase-3,PARP and Bax/Bcl-2 except PARP the stable ORF5-expressing were increased,when compared to those in normal PUVEC.Conclusion:PCV2 can cause pathological changes in mouse placenta and infected the vascular endothelial cells in placenta.In vitro study further confirmed that PCV2 can infect the PUVEC,and inhibited the cell proliferation and induced cell apoptosis.PCV2 ORF3 or ORF5 proteins inhibited the functions of PUVEC,including proliferation,migration,invasion and tube formation.Moreover,PCV2ORF3 or ORF5 proteins could induce PUVEC apoptosis,partially via up-regulation the expression of Bax/Bcl-2 and activing the caspase-3 pathway.All the above data can provide theoretical basis for understanding the pathogenesis of reproductive disorders induced by PCV2.