The Investigation Of COX-2 Expression Induced By Respiratory Syncytial Virus In Vivo And In Vitro And Its Partial Mechanism

ObjectiveTo investigate the expression changes of COX-2 in human lung epithelial cells (A549 cells) and the lung tissue of BALB/c mice infected with respiratory syncytial virus (RSV), and to explore the partial mechanism of COX-2 expression and the possible relationship with the inflammatory response to RSV infection.Methods①A549 cells were infected with 1×10~6 PFU/ml RSV in vitro,then collected cells and cellular supernatants in the infection course of 4h, 8h, 16h, and 24h. The expression of cyclooxygenase 2 (COX-2) was evaluated by semiquantitative RT-PCR, immunocytochemistry and Western-blot methods, respectively. The expression of NF-κB p65 activity was detected by Western-blot using nuclear protein. The level of PGE2 in cellular supernatants was measured by ELISA assay. The viral titers in cellular supernatants were measured by plaque forming unit (PFU) assay. Additionally, BALB/c mice were infected with 1×106PFU/ml RSV intranasally. After infection of 24h, 48h and 96h, the expression of lung COX-2 protein was measured with immunohistochemical method (SP assay) and Western-blot methods, respectively. HE stain was performed to detect inflammatory level of lung tissues. The uninfected A549 cells and BALB/c mice were as normal controls. COX-2 protein in lung tissues and A549 cells were assessed quantitatively by biological image analysis system under the immunohistochemical method.②Furthermore, the cells infected with RSV were treated with 50μmol/L pyrrolidine dithiocarbamate (PDTC) to inhibit the activation of NF-κB. The indices of COX-2 were detected and compared with the group that did not use PDTC inhibitor. And the expression of COX-2 was also measured by semiquantitative RT-PCR and Western-blot methods, respectively.③The specific inhibitor of COX-2, 100μmol/L Nimesulide, was used in the experiment. The levels of PGE2 and viral titers in cellular supernatants were evaluated to compare with the groups that did not use Nimesulide inhibitor.Results①The expression of COX-2 mRNA and protein in normal A549 cells were very weak, whereas the amounts of COX-2 mRNA and protein in A549 cells infected with RSV were increased in a time-dependent manner. RSV infection promoted COX-2 mRNA and protein expression, which were much higher than that of uninfected group (P < 0.05). The expression of COX-2 mRNA and protein in lung tissues of RSV-uninfected mice were very weak, but the expression of COX-2 mRNA and protein in lung tissues of infected mice were increased in a time-dependent manner compared with that of normal group (P<0.05). HE staining showed pulmonary inflammation in RSV-infected mice was heavier along with the infection progress. Furthermore, the levels of COX-2 mRNA and protein were consistent with the degree of mouse pulmonary inflammation. The amount of NF-κB p65 was increased since 8 hours of postinfection (P<0.01). In the cellular supernatants, the levels of PGE2 increased gradually. The virus titers were increased significantly in a time-depandent manner.②PDTC inhibitor could significantly down-regulate the activation of NF-κB, and the expression of COX-2 mRNA and protein were also decreased since 8 hours after infection. The variations of PDTC group were significant compared with the RSV group (P<0.05).③The PGE2 contents in the cellular supernatants were significantly reduced when Nimesulide inhibited the COX-2 activation (P<0.05). But the titers of RSV were not changed significantly (P>0.05).ConclusionsRSV infection could induce A549 cells and BALB/c mouse lung tissues to express high-level of COX-2 mRNA and protein. The levels of PGE2 increased gradually in a time-depandent manner. COX-2 may be involved in the lung inflammatory process caused by RSV infection. The activation of NF-κB may play an important role in the regulation of COX-2 expression. The level of PGE2 was associated with the activation of COX-2, which might not influent the viral titers in the infection process.