Study On Jinxin Oral Liquid On Regulating TLR4Activated By RSV

Respiratory syncytial virus(RSV) is one of the most important virus pathogens which lead to infantile viral lower respiratory infection in the world.However,there is no pointed medicin resistance to RSV at present,The development of related vaccine has yet to mature.With the overall concept and syndrome differentiation and treatment as its diagnostic guidance,Chinese medicine withstand RSV by clearing lung、eliminating phlegm、relieving a cough and it always achieve good clinical efficacy. Toll like receptors(TLRs),an important pattern recognition receptors(PRRs),can identify different microbial pathogen associated molecular patterns(PAMPs).TLRs plays an important role in the process of RSV infection and becomes the new trend in this research field.Preliminary research results of "the effect and mechanism of Jinxin Oral Liquid on TLRs signal transduction pathway induced with RSV infection(81072840)" a National Natural Science Foundation project led by professor Wang Shou-chuan,have shown that Jinxin oral liquid can regulate the expression levels of TLR3,TLR4and downstream signaling molecules induced with RSV infection;Jin Xin oral liquid has obvious effect to restrain RSV,its acting site may be F protein of the virus or its receptor, these study offer a new way for the treatment of infant RSV pneumonia.In view of the multiple target effect of Chinese medicine, this experiment starts with the TLR4and ts signal transduction pathway,and using RNA interference (RNAi)technology,investigates the regulatory role of Jinxin oral liquid on the pathway induced by RSV infection,and researches the correlation to other TLRs pathways.1ObjectiveTo study the role of Jinxin oral liquid on TLR4signal transduction pathway induced by RSV, and explore its possible immunological mechanism in treating RSV pneumonia.2MethodsExperiments in vitro:The serum containing Jinxin oral liquid was applied to intervene RAW264.7cells infected with RSV,and after24hours, the cells were collected to detect the levels of TLR4and TAK1.Meanwhile,TLR4siRNA was used to interfere TLR4expression, to observe the effect of Jinxin oral liquid on mRNA and protein expression of TLR4and TAK1after interference,and then detect the expression of above substances to observe the effect of Jinxin oral liquid on TLR4signal transduction pathway and the correlation with other TLRs pathways.Experiments in vivo:Jinxin oral liquid in different dosage was applied to treat BALB/c mice inoculated with RSV,and after24hours of the first inoculation,mice lungs were taken to evaluate lung lesions by histologic slide analysis,to examine the protein expression of TLR4by western blot assay. 3Result3.1Experiments in vitro3.1.1RAW264.7cells which transfected by TLR4siRNA interfered the expression of TLR4.After transfection TLR4mRNA expression decreased by0.456,it shows that After the transfection,the model of the RAW264.7cell to interfere TLR4mRNA is successful. RSV group and ribavirin injection can cut the expression of TLR4mRNA, mRNA expression in the serum containing Jinxin oral liquid is ascending and it is different compared with ribavirin group(P<0.05),Jinxin oral liquid can raise the expression of TLR4mRNA which induced by RSV.3.1.2After RAW264.7cells transfected by TLR4siRNA,TAK1mRNA expression also decreased.Infected with RSV, TAK1mRNA expression decreased by0.632times.Both Jinxin oral liquid and ribavirin group can up-regulate TAK1mRNA expression compared with transfection group(P<0.01).The expression of Jinxin oral liquid and ribavirin group is higher than RSV group(P<0.01),but Jin Xin oral liquid is slightly higher than that of ribavirin group (P <0.05).3.1.3RAW264.7cells which transfected by TLR4siRNA interfered the expression of TLR4,After transfection,TLR4protein expression exist a downward trend.RSV group’s protein expression is different with transfection group(P<0.01).Both the group of serum containing Jinxin oral liquid and ribavirin group can up-regulate TLR4protein expression compared with transfection group and Jinxin oral liquid is better than ribavirin(P<0.05).3.1.4After transfection, RAW264.7cells which transfected by TLR4siRNA exist a obvious change in TAK1protein, transfection group’s protein expression is lower than normal group (P <0.01),but the protein expression of RSV group is reduced.Both the group of serum containing Jinxin oral liquid and ribavirin group can down-regulate TAK1protein expression,but ribavirin group have a more apparent down-regulation,there is a statistically significant between them (P <0.05).3.2Experiments in vivo3.2.1Pulmonary pathological result in BALB/c mice24h after RSV infected show that lung inflammation in mice treated by Jinxin oral liquid alleviate to some extent.The pathological scores of treatment group showed statistically significant differences at each time point compared with RSV infection group(P<0.05).3.2.2Jinxin Oral Liquid’influence on the Expression of lung sample TLR4and TAK1of BALB/c mice Infected with RSV:After24hours of infected with RSV,the expression of TLR4and TAK1protein were up-regulated observably than normal group (P<0.01),the expression of TLR4and TAK1protein in the group of Jinxin Oral Liquid were down-regulated compared with the RSV group(P<0.05),the effect is better than ribavirin group(P<0.01);Meanwhile,according to Pearson correlation coefficient1=0.980,P=0.000<0.01,we consider that the expression present a positive correlation between TLR4and TAK1.4Conclusion4.1Jinxin oral liquid significantly alleviate the lung inflammation of the RSV infected mice. And its role in early stage of disease is slightly better than that of ribavirin.4.2Jinxin oral liquid can down-regulate the levels of TAK1in the early stage of RSV infection.TAKl is a significant cohesion protein in signaling pathways which mediate by TLR4.4.3Jinxin Oral Liquid can inhibit the protein expression of TLR4/TAK1in signal transducing pathway of TLR4,the expression present a positive correlation between TLR4and TAK1.4.4After TLR4siRNA transfection,the expression of TLR4mRNA and protein were inhibited, simultaneously,the expression of TAK1mRNA and protein were inhibited too,which shows that the expression of TAK1are controlled by TLR4, Meanwhile TLR4mRNA and protein expression in oral group Jinxin been raised after transfection proved TLR4pathway to other signaling pathways also exist some impact,but this does not explain that the TAK1is the only factor the TLR4signaling pathways.4.5TLR4’signal transducing pathway in the antiviral mechanism of action of Jinxin Oral Liquid may be realized through TAKl,that is TLR7/MyD88/IRF7/IFN-a pathway.