The Effect Of Oxidative Stress On The Expression Of Toll-Like Receptor3Induced By Respiratory Syncytial Virus Infected Human Lung Epithelial Cells

Respiratory syncytial virus （RSV）, an enveloped, non-segmental,negative-stranded RNA virus and a member of the family paramyxoviridae, is the majorcause of serious lower respiratory tract infections in infancy and early childhood andrelate to asthma and lung function abnormalities. Infected with RSV can cause damageto the body by oxidative stress and then causing pathophysiological changes, if so,itmay find some diseases；For example, RSV infected respiratory epithelial cells,inducing cells to produce large amounts of cytokines, chemokines, reactive oxygen andother biological activity medium, leading to the occurrence of infant bronchiolitis,pneumonia, asthma and other diseases.The research of oxidative stress focuses on thestate of oxidative stress in patients in china,study on oxidative stress mediated by Toll-Like Receptor is fewer. Toll-like receptors, type I integral membrane proteins, veryimportant receptors to identify pathogens, are central to early host defense. They detectbacterial, viral and environmental exposures and activate both innate and adaptiveimmune responses. It has been found that TLR3signal transduction pathway is involvedin activating of airway epithelial cells，with secreting a large number of cytokine relatedto inflammatory and immune，which plays an important role in the development andprocess of pneumonia. We study A549cells infected with RSV can induced the generateof oxidative stress in vitro were pretreated with antioxidant and pro-oxidant, the effect of expression of Toll-Like Receptor3. In order to provide basic research for thediagnosis and clinical therapy of RSV infection.Objective: To investigate the effect of oxidative stress on the expression of Toll-likereceptor3（TLR3） induced by respiratory syncytial virus infected human lung epithelialcells （A549） in vitro, and its related effect in the TLR3. Accordingly this study has veryimportant medical significance to prevention and treatment of RSV infection.Method: A549cells infected with RSV in vitro were pretreated with or withoutInhibitors and agonist of oxidative stress, cells and cellular supernatants were harvestedat the selected time points after RSV infection （4,8,12and24, respectively）. Theuninfected A549cells were as normal controls. The expression of TLR3, NF-κB p65,IRF3and SOD gene mRNA were evaluated semiquantitatively by RT-PCR. Theconcentrations of NO, hydroxy radical （OH·）, superoxide dismutase（SOD） in cellularsupernatants were measured according to the kit reference.Results:①I t was found that RSV infection could markedly up-regulate the expressionof TLR3and NF-κB mRNA in a time-dependent manner; down-regulate the expressionof IRF3and SOD mRNA in a time-dependent manner. The changes significantlycompared with the control group. Pretreatment oxidative stress with specific inhibitorNAC, whereas, significantly down-regulate the activation of TLR3and NF-κB; theexpression of IRF3and SOD mRNA are also decrease, but not obviously; Pretreatmentoxidative stress with specific agonist H₂O₂, the expression of TLR3and NF-κB mRNAare more increased obviously, but the expression of IRF-3、SOD mRNA decreasedsignificantly;②It was found that RSV infection could down-regulate the expression ofSOD in a time-dependent manner, but in NAC treatment group the expression of SODdescented less markedly than RSV-infected group；in H₂O₂treatment group the expression of SOD decreased more markedly than RSV-infected group;③In the cellularsupernatants, the levels of NO and OH· rose gradually; in NAC treatment group thelevels of NO and OH· descented gradually; in H₂O₂treatment group the levels of NOand OH· increased more markedly than RSV-infected group. These results indicate thatNAC and H₂O₂inhibited and excited oxidative stress were very important, respectively.Thus affecting the expression of TLR3activation and its major signal molecules at thetranscriptional levels, further influence the changes of downstream inflammatory factorschanges.Conclusions: RSV infected A549cells in vitro can induce oxidative stress, RSVinfection could up-regulate the expression of TLR3.Pretreatment with specific inhibitorNAC, down-regulate the activation of TLR3. Pretreatment with specific agonist H₂O₂,significantly up-regulate the expression of TLR3. Pretreatment oxidative stress withspecific inhibitor and agonist, respectively, the expression of TLR3and its majordownstream signal molecules show significant changes. It illustrated that the effect ofexpression of TLR3is probably regulated by oxidative stress induced by RSV infectionin A549cells.