The Role Of Oxidative Stress In The Process Of Apoptosis Induced By NS1 Protein Of H9N2 Avian Influenza Virus In DF-1 Cells

NS1 of H9N2 avian influenza virus(AIV) is a non-structural protein of the virion and a multifunctional regulatory factor. What is well known is that NS1 of H9N2 AIV can suppress immune responses in influenza virus-infected hosts and induces apoptosis in several cultured cells. Our previous studies have confirmed that NS1 of H9N2 AIV induces apoptosis in DF-1 cells. In order to explore whether the NS1 protein of H9N2 influenza virus can cause oxidative stress, plasmid pEGFP-NS1 was transfected into DF-1 cells for different times. The accumulation of ROS were observed with fluorescent microscope and assayed with flow cytometry. The changes of redox signalings were detected via the measurement of SOD and catalase activities, as well as the expression of PRDX3 and TRX. By comparing apoptosis changes of the plasmid pEGFP-NS1 transfected DF-1 cells before and after the treatment with pyrrolidinedithiocarbamic(PDTC) and N-acetyl-L-cysteine(NAC) which using flow cytometry analysis, we have demonstrated that ROS participate in NS1 of H9N2 AIV-activated apoptosis. To investigate the roles and regulation of ROS in the NS1 of H9N2 AIV-activated apoptosis, some apoptosis associated protein and factors including caspase-3, caspase-8, caspase-9, Bax and bcl-2 were detected by Western Blotting. Results suggest that ROS might play a key role in the NS1 of H9N2 AIV- induced apoptosis and NS1 of H9N2 AIV pathogenesis. The results of the study were as follows:1. The NS1 protein of H9N2 avian influenza virus can induce the accumulation of ROS and oxidative stress in DF-1 cells. We observed the accumulation of ROS-positive red fluorescent. The result of measurement of ROS by flow cytometry further confirmed the accumulation of ROS. The detection of SOD and catalase activities showed that the activities of them increased with the extension of time after pEGFP-NS1 transfection. The relative fluorescence quantitative PCR detection of PRDX3 and TRX showed down-regulaton of mRNA expression of PRDX3 and TRX with different degree at 24 hours after pEGFP-NS1 transfection.2. Oxidative stress is involved in the process of apoptosis in DF-1 induced by NS1 of H9N2 AIV. In the present study, the DF-1 cells were divided into five groups: non-transfection, mock-vehicle groups, plasmid pEGFP-NS1 transfection, plasmid pEGFP-NS1 transfectiion with PTDC/NAC pretreatment. The rate of apoptosis were 2.4%,4.0%,15.8%,12.8% and 6.8%, respectively. The rates of antioxidant pretreatment groups were significantly lower than that of the experiment group. The result of caspases activities detection showed that the activities of caspase-3 and caspase-9 of antioxidant pretreatment groups were significantly lower than that of experiment, while the activity of caspase-8 has no obvious change. Consistent with the activities, the expression of activitied caspases showed similar trend. The expression of Bax in antioxidant pretreatment groups was lower than that in experiment group. Contrast with Bax, the expression of bcl-2 in antioxidant pretreatment groups was higher than that in experiment group.In summary, this study confirmed that the NS1 protein of H9N2 AIV can cause the accumulation of ROS and oxidative stress in DF-1 cells, and oxidative stress participate in the process of apoptosis induced by the NS1 protein of H9N2 AIV. The oxidative stress may mediate apoptosis through intrinsic apoptosis pathway.