Signal Transduction Pathways Of Apoptosis Induced By Porcine Parvovirus In ST Cells

Porcine parvovirus disease is an infectious disease of reproductive failure in swine. It is caused by porcine parvovirus(PPV) and characterized by abortion of pregnant swine, embryonic death, stillbirth and fetal mummification in swine. PPV infection induced significant cytopathic effect(CPE) in some host cells such as PK-15 cell and ST cell. But the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we detected the role of PPV infection in apoptosis of ST cell. Then we investigated PPV-induced apoptotic pathways and the role of PPV-induced ROS in apoptotic pathways. The results were shown below.1. Effect of PPV infection on ST cell apoptosisThe effect of PPV at various doses on cells viability was firstly determined by MTT assay at different time points. The results showed that PPV infection significantly reduced the ST cells viability and inhibited the growth of ST cells in a time- and dose-dependent manner. PPV-infected ST cells appeared typical apoptosis features, including chromatin condensation and nuclear fragmentation via AO/EB staining assay. In addition, characteristic ladder patterns of chromosomal DNA were detected when ST cells were infected with 1.0 MOI of PPV for 48 h using agarose gel electrophoresis. The apoptotic rate of PPV-infected ST cells was increased with the extension of infection time through flow cytometry analysis compared with that in mock-infected cells.2. Effects of PPV infection on the expression of apoptosis-related proteinsPPV infection activated caspase-9 and caspase-3, while not affecting caspase-8 activity, indicating that PPV-induced apoptosis depended on caspase-3 and caspase-9 activation. Western blot assay showed that PPV infection did not affect the expression of Fas and Fas L in ST cells, while PPV infection up-regulated the expression of Bax, down-regulated the expression of Bcl-2, and promoted the translocation of cytosol Bax to mitochondria and the release of Cyt c from mitochondria to cytoplasm, followed by the cleavage of PARP. In addition, with the translocation of cytosol Bax to mitochondria, the rate of Δψm-depolarized cells was significantly increased.3. Regulation of ROS in PPV-induced cell apoptosisConsistent with the observation under fluorescence microscopy, flow cytometry analysis showed that PPV infection induced the accumulation of ROS in ST cells. In order to explore the accumulation of ROS in PPV-induced ST cell apoptosis, ST cells were treated with ROS scavengers PDTC and NAC prior to infection with PPV. The results showed that PDTC and NAC inhibited the accumulation of ROS induced by PPV, reduced the rate of Δψm-depolarized cells and blocked the down-regulation of Bcl-2, the translocation of Bax and the release of Cyt c, reducing caspase-3 activity and apoptotic rate.This study mainly demonstrated that PPV infection could induce apoptosis of ST cell and shed light on the mechanism of PPV-induced apoptosis of ST cells through mitochondrial signal transduction pathway and the regulation of ROS in the process of apoptosis. These results not only lay the foundations for revealing the molecular pathogenesis of PPV infection, but also provide new ideas for the study of other pathogenic mechanism of porcine disease.