Study On Jinxin Oral Liquid And Resveratrol On Regulating TLR3and Downstream Signaling Transduction Pathway Activated By RSV Infection

Pneumonia, one of the most common diseases in pediatrics, is harmful to children's health. It has been one of three important global pediatric diseases by WHO and also been one of four imprtant diseases by the Ministry of health of PRC. Viral pneumonia is accounted for about half of the children's pneumonia, and respiratory syncytial virusis (RSV) is the most commom pathogen in viral pneumonia, which can cause infant capillary bronchitis, bronchial pneumonia, interstitial pneumonia and so on.It is not fully understood, so far, the pathogenesis about RSV infection, and there are nearly no safe and effective vaccines and treatments in the medical market. As it is know to all that Chinese medicine has its unique advantage in treating viral infections, and Jinxin oral liquid, which have the function of opening lung reducing phlegm and detoxification is an efficacy prescription in treating viral pneumonia with the syndrome of obstruction of the lung by phlegm heat. Many scientific research have also shown that it has some certain anti-RSV activity. Resveratrol, which is reported having the effect of anti-inflammatory and anti-viral, is one of the effective components of Polygonum cuspidatum in Jinxin oral liquid.Toll-like receptors (TLRs), the proteins belone to type I across membrane, are very important pattern recognition receptors (PRRs), which could identify different Pathogens associated molecule pattens expressed by infectious agents. RSV, an enveloped, negative-stranded RNA virus, can induces the synthesis of dsRNA during its replication and transcription, so TLR3can produce a marked effect during the infection by recognizing the dsRNA, and then the signal transduction pathways are activatded accompanyed by the expression of cytokine, finally cause systematic inflammatory response. In view of multiple targets of traditional Chinese medicine, this experiment are developed from the Toll like receptor3and its signal pathway, in order to discuss the antiviral mechanism of Jinxin oral liquid and its effective component resveratrol.Objective:Study on Jinxin oral liquid and resveratrol on regulating TLR3and its downstream signaling transduction pathway activated by RSV, in order to demostrate the possible immunological mechanism in treating RSV pneumonia.Methords:In vitro experiment:RAW264.7cell line was infected with RSV, and then treated with the drug serum of Jinxin oral liquid and resveratrol.24hours later, detected the level of TLR3, TRIF,TRAF6mRNA using Realtime RT-PCR assey; observe the expression of TLR3protein using laser confocal microscopy by immunofluorescence (IF) assay; collected the supernatant and measured the level of interleukin6(IL-6),interleukin1β (IL-1β),tumor necrosis factor alpha (TNF-α) and interferon β (IFN-β)In vivo experiment:BALB/c mice were inoculated with RSV, and treated with Jinxin oral liquid in different dosage and resveratrol, delivery method. After24,72and144hours of the first inoculated with RSV, killed the mice and collected the lung tissue of each mouse, evaluate pulmonary lesions by Histological; detected the level of TLR3,TRIF,TRAF6mRNA using Realtime RT-PCR assey; observe the expression of TLR3protein using western blot assey; and collected the bronchoalveolar lavage fluid, measured the level of IL-6,IL-1β,TNF-α,IFN-β.Results:In vitro experiment:1. The expression of TLR3mRNA in RAW264.7was up-regulated significantly compared with the control group (P<0.01) after24h of infection with RSV, but it was typically down-regulated in Jinxin oral liquid group and resveratrol groups with different degree compared with RSV infected groups (P<0.01). The expression of TRIF and TRAF6mRNA in RAW264.7were slightly higher compared with the control group after24h of infection with RSV, but no statistical difference (P>0.05), and there were no statistical difference among Jinxin oral liquid group, resveratrol groups and RSV infected groups also with the expression of TRIF and TRAF6mRNA (P>0.05)2. The expression of TLR3protein in RAW264.7was up-regulated significantly compared with the control group (P<0.01) after24h of infection with RSV, but it was typically down-regulated in Jinxin oral liquid group and resveratrol groups in different degree compared with RSV infected groups (P<0.01)3. There was no expression of IL-1P in the supernatant after24h of infection with RSV. But the expression of IL-6and TNF-a were up-regulated significantly compared with the control group (P<0.01), but IL-6(P<0.05) and TNF-α (P<0.01) were typically down-regulated in Jinxin oral liquid group and resveratrol groups in different degree compared with RSV infected groups. The expression of IFN-β was up-regulated compared with the control group (P<0.01), and it was typically up-regulated in Jinxin oral liquid group and resveratrol groups in different degree compared with RSV infected groups.In vivo experiment:1. The pulmonary pathological changes of BALB/c mice that infected RSV were mainly pulmonary interstitial inflammation, dilatated and congested for the vascular of alveolar wall, and with inflammatory cell infiltration in interstitia; The alveolar cavity was clear, without obvious exudation; and there were no apprent degeneration and necrosis in bronchial epithelial cells, and without inflammatory exudate in the lumen. Pulmonary lesions was lighe after24h of infection with RSV, and with the extention of infection time, the lesions were gradually increased. The pulmonary inflammation degree were relieved in Jinxin oral liquid and resveratrol groups and the pathological score of treatment group showed statistically significant difference at each time point compared with the model groups.2. The expression of TLR3mRNA in the lung tissue was up-regulated significantly compared with the control group (P<0.01) after24h of infection with RSV, and it was declined gradually with the extention of infection time. The expression of TLR3mRNA was typically down-regulated in Jinxin oral liquid and resveratrol groups with different degree compared with RSV infected group (P<0.01), but it was slightly higher in Jinxin group with equivelent dosage than RSV infected groups with the extention of infection time, and the high dosage group of Jinxin oral liquid and resveratrol group had no difference with model groups on TLR3mRNA expression.3. The expression of TRIF and TRAF6mRNA in lung tissue of BALB/c mice had no statistical difference (P>0.05) compared with the control group after24h,72h and144h of infection with RSV, and there were no statistical difference among Jinxin oral liquid group, resveratrol groups and RSV infected groups also with the expression of TRIF and TRAF6mRNA (P>0.05)4. The expression of TLR3protein in the lung tissue was up-regulated compared with the control group (P<0.01) after24h of infection with RSV, and it reached to the peak at72h after infection (P<0.01), then declined gradually with the extention of infection time. The expression of TLR3protein was typically down-regulated in Jinxin oral liquid and resveratrol group at24h and72h after infection, but there was no difference at144h (P>0.05)5. The expression of IL-lβ,IL-6and TNF-α were up-regulated significantly compared with the control group (P<0.01) in BALF at24h after infection with RSV, and then declined gradually with the extention of infection time. They were typically down-regulated in Jinxin oral liquid and resveratrol group in different degree compared with RSV infected groups at24h after the infection. With the extension of infection time, the expression of IL-1β,IL-6and TNF-α in Jinxin oral liquid group with equivelent dosage were up-regulated compared with RSV group. There was no difference between control and model group on the expression of IFN-β (P>0.05) at24h after infection with RSV. But with the extention of infection time, the expression of IFN-P was up-regulated gradually in RSV group. The expression of IFN-β were up-regulated typically in Jinxin oral liquid and resveratrol group in different degree at24h after infection, but there was no difference between them with the extention of infection time (P>0.05)Conclusion:1. Jinxin oral liquid and resveratrol can typically improve the lung lesions after the infection with RSV in BALB/c mouse.2. Jinxin oral liquid and resveratrol can inhibit the activation of TLR3expression by RSV at the early stage from nucleic acid and protein levels significantly.3. Jinxin oral liquid and resveratrol have no direct effect on regulating the expression of the critical molecular TRIFn TRAF6mRNA in TLR3signaling pathways.4. Jinxin oral liquid and resveratrol can obviously control the over-expression of the inflammatory cytokines like IL-1β,IL-6and TNF-α mediated by TLR3signaling pathways at the early stage of RSV infection. Along with the extension of time, Jin Xin oral liquid can increase the expression of that inflammatory cytokines moderately, with the effect of two-way regulation, expect of resveratrol. Jinxin oral liquid and resveratrol can induce the expression of IFN beta rapidly after RSV infection, to play certain antiviral effect coordinated with the body.5. Jinxin oral liquid and resveratrol are the effective drugs to cure RSV infection, and the mechanism may be the regulation of TLR3and its signal transduction pathway activated by RSV.