The Effect And Mechanism Of The Expression Of Nitric Oxide In Respiratory Syncytial Virus-Infected Human Lung Epithelial Cells

ObjectiveTo investigate the effect of expression of nitric oxide (NO) on human lung epithelial cells (A549) infected with respiratory syncytial virus (RSV) in vitro, and its related mechanism in the course of RSV infection.Methods①A549 cells infected with RSV in vitro were used to collect cells and cellular supernatants. The expression of inducible nitric oxide synthase (iNOS) mRNA and protein were evaluated by semiquantitative RT-PCR and immuocytochemistry, respectively. The protein expression of NF-κB p65 activity was detected by Western-Blot using nuclear protein. The concentrations of NO, hydroxy radical (OH·), superoxide anion (O2.￣) and malondialdehyde (MDA) in cellular supernatants were measured according to the kit reference. The virus titers in cellular supernatants were evaluated by plaque forming unit (PFU) assay.②Furthermore, the cells infected with RSV were treated with 50μmol/L pyrrolidine dithiocarbamate (PDTC) to inhibit the activation of NF-κB. The indices of iNOS were detected and compared with the groups that did not use PDTC inhibitor.③The specific inhibitor of iNOS, 1mmol/L Aminoguanidine (AG), were used in the experiment. The levels of NO, OH, O2., MDA and PFU were evaluated to compare with the groups that did not use AG inhibitor. Results①RSV infection could markedly up-regulated the expression of iNOS mRNA and protein in A549 cells since 4 hours after infection, and induced the activation of NF-κB since 8 hours postinfection. In the cellular supernatants, the levels of OH·and O2.￣rose gradually. Meanwhile, the levels of MDA and PFU increased obviously in a time-dependent manner. The changes of each index varied significantly compared with the control group.②PDTC inhibitor could significantly down-regulate the activation of NF-κB, and the expression of iNOS mRNA and protein were also decreased since 4 hours after infection. The changes of PDTC group were significant compared with the RSV group.③The NO content in the cellular supernatants could significantly decrease when AG inhibited the iNOS expression, the levels of OH·, O2.￣and MDA were also decreased accordingly when AG inhibitor was used in the infection. But the titer of virus was increased, which was about 1.5 times as many the RSV group 24h after RSV infection.ConclusionsRSV infection could induce high-level NO, which is mainly synthesized by iNOS. The activation of NF-κB may play an important role in the expression of iNOS. The NO may associate with the level of free radical, which could decrease the titer of virus in the infection, but lead to injure normal cells.