| Toll-like receptors (TLRs), type I across membrane proteins, very important receptors in innate immune responses in the recent studies, which could identify difference pathogen-associated molecular patterns expressed by infectious agents, play critical roles in activating the innate immune response and mediating the inflammatory reaction, now they are considered as the new targets of the drugs to cure the acute and chronic diseases. Recent studies indicate that Toll-like receptor 3(TLR3) is the specific receptor for double-stranded RNA (dsRNA). Respiratory syncytial virus (RSV) is the major cause of serious lower respiratory tract infections in infancy and early childhood and is also becoming widely recognized as an important pathogen in the elderly and in immunosuppressed adults. The nucleic acid of RSV is single- stranded RNA (ssRNA), but the virus induces the synthesis of dsRNA during its replication and transcription, so the TLR3 can produce a marked effect during the infection by recognizes the dsRNA of RSV. After the combination between TLR3 and dsRNA, the nuclear factor kappa B (NF-κB) would be activated through MyD88-dependent or MyD88-independent pathway, which can up-regulates the proinflammatory factor, chemotatic factor and collaborative stimulating factor, caused systematic inflammatory response. Therefore, the discovery of TLRs may give a significant revolution in the pneumonia caused by the infection of RSV, and it will provide a new method for the research about the mechanism of RSV-induced inflammatory and immune reaction. Up to now, its mechanism is unknown since it was so complex that there is no satisfactory medicine or vaccine. Accordingly, it has very important medical significance to deep research the mechanism of RSV infection.Studies showed that melatonin (MT) had the effects on anti-inflammation, regulating the immune response and great anti-oxidation, cutting down the inflammatory injury significantly and down-regulating the iNOS through inhibiting the activation of NF-кB. Melatonin may produce an effect through combining with the melatonin receptor directly in the immune cells. Some previous data showed that MT could reduce the inflammatory injury caused by bacterial endotoxin. But whether melatonin could give a regulatory effect on RSV–induced inflammatory and immune response, or its target and mechanism in the TLR3 signaling pathway, these questions are unknown and will be a focus in our present work. We here study the TLR3 activated signaling pathway involvement of the mechanism of RSV-induced inflammatory and immune response, in vitro and in vivo, and the effect of intervention and target of pharmacological profile of MT in RSV-caused inflammation through observation the MT's influence on TLR3 signaling pathway.Objective:To investigate the expression changes of TLR3 and its downstream signaling molecules, and the relationships between TLR3 signal transduction pathway and its mediated inflammatory and immune response to RSV. Using MT as an intervention, to study the potential target of MT through compared with the change of TLR3 and its downstream signaling molecules. Meanwhile, observe the expression of membrane melatonin receptor and nuclear receptor, explain which receptor can combine with the melatonin and take effect.Methods1. The TLR3-mediated inflammatory and immune response to RSV in vitroRAW264.7 cells infected with RSV in vitro were used to collect cells and cell culture supernatants. Semiquantitative RT-PCR was used to evaluate the expression of TLR3, MyD88, TNF-αand iNOS mRNA. The protein expression of TLR3 and NF-κB p65 activity was detected by Western-Blot using total protein and nuclear protein, respectively. The expression levels of TNF-αprotein were measured by ELISA assay in cell culture supernatant.2. The TLR3-mediated inflammatory and immune response to RSV in vivo The Kung Ming mice inoculated intranasally with RSV were used to collect lung and mouse serum. Semiquantitative RT-PCR was used to evaluate the expression of TLR3, MyD88, TNF-αand iNOS mRNA. The protein expression of TLR3 and NF-κB p65 activity was detected by Western-Blot using total protein and nuclear protein, respectively. The expression level of TNF-αprotein in mouse serum was measured by ELISA assay.3. The effect of melatonin on TLR3-mediated inflammatory and immune response to RSV in vitroThe concentration of MT at 10-7, 10-6, and 10-5M was given as an intervention in three levels before the infection of RSV in RAW264.7 macrophages, respectively. The same works were done to compare with the groups which not used MT, and also the analysis of membrane melatonin receptor(MT1,MT2) and nuclear receptor(RORα) mRNA expression were tested at same time.4. The effect of melatonin on TLR3-mediated inflammatory and immune response to RSV in vivoThe concentration of MT at 5mg·kg-1, 10mg·kg-1, and 20mg·kg-1 was given as an intervention in three levels before the infection of RSV in Kung Ming mice, respectively, and lasting seven days. The same works were done to compare with the groups which not used MT, and also the analysis of melatonin membrane receptor(MT1,MT2) and nuclear receptor(RORα) mRNA expression were tested at same time.Results:1. The results of TLR3-mediated inflammatory and immune response to RSV in vitro RSV infection could up-regulate the transcriptional levels of TLR3, TNF-αand iNOS mRNA since 4 hours in RAW264.7 cells. RSV stimulation also induced the TLR3 protein and the activation of NF-κB p65 since 8 hours, and enhanced TNF-αproduction in cell culture supernatants. The changes of each index varied significantly and in a time-dependent manner, which compared with the control group. But the MyD88 mRNA had no evident change.2. The results of TLR3-mediated inflammatory and immune response to RSV in vivo RSV infection can up-regulates the transcription of TLR3 since 4 hours in mouse lungs. In accordance with the expression of TLR3, RSV stimulation induced the activation of NF-κB p65. The up-expression of TNF-αmRNA was detected since 48 hours, and the iNOS mRNA was detected since 72 hours. The TNF-αin the mouse serum was also increased after 48 hours. The changes were significant compared with the control group. But the MyD88 mRNA had no evident change, and the protein of TLR3 was not detected within 24 hours.3. The results of effect of melatonin on TLR3-mediated inflammatory and immune response to RSV in vitroMelatonin could down-regulate the elevated transcriptional levels of TNF-αmRNA which induced by RSV infection, and the down-regulation had a dose-dependent manner. Result showed that 10-7M concentration of MT did not have an evident effect on down regulation the transcription of iNOS mRNA, but 10-6M and 10-5M of MT could do since 4 hours. The effect of melatonin on down regulation the activation of NF-κB was similar to that of iNOS mRNA. There was no change in the protein of TLR3 and the MyD88 mRNA treated with MT. The membrane melatonin receptors MT1 and MT2 were not detected, but nuclear receptor RORαwas opposite and the level in each group was uniformity. TNF-αproduction in cell culture supernatant could decrease treated with MT in 10-6M and 10-5M since 8 hours, and the effect of 10-5M on down regulation TNF-αproduction was obvious, but there is no down-regulation role at the level of 10-7M.4. The results of effect of melatonin on TLR3-mediated inflammatory and immune response to RSV in vivoMT could down-regulate the transcriptional levels of TNF-αand iNOS mRNA in lung following 72h of RSV infection, but the down-regulation of NF-κB was detected at 4h of large dose and 8h of middle dose melatonin, respectively. Moreover, MT cut down the production of TNF-αin the mouse serum at the dose of 10mg·kg-1 since 48 hours of RSV infection, 20 mg·kg-1 per mouse can greatly reduce the TNF-αproduction. But MT had no effect on the expression of TLR3, MyD88 and RORα. Also we did not detect the MT1 and MT2 receptor expression. Conclusion:1. RSV infection significantly up-regulates the transcriptional levels of TLR3, iNOS, and TNF-αmRNA. Meanwhile, RSV stimulation induces the activation of NF-κB, and enhances TNF-αproduction not only in cell culture supernatants but also in mouse serum. The inflammatory and immune reaction induced by virus is related with the TLR3 signal transduction pathway, and TLR3 might take effect in the inflammatory and immune response to RSV and activate the NF-κB through MyD88-independent or partial-dependent pathway.2. Melatonin at a range of levels and dose could down-regulate the activation of NF-κB and the transcription of iNOS and TNF-αmRNA, decrease the TNF-αproduction both in cell culture supernatants and mouse serum caused by RSV infection. But there were no evident effects of melatonin on suppressing the expression levels of TLR3 mRNA and protein,as well as MyD88 and RORαmRNAexpression both in RAW264.7 cells and mouse lung.3. The inhibitory effects of melatonin on RSV-induced increasing NF-κB, TNF-αand iNOS expression have a relationship with RSV-infected times and melatonin′s dose.4. The effect of melatonin on the anti-RSV infection probably is that melatonin may be combining with its nuclear receptor and suppress the activation of NF-κB.5. Some of melatonin′s effects on the immune and inflammatory response caused by RSV infection probably do not inhibit the TLR3 or MyD88 signaling molecules.
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