Effect Of Jinxin Oral Liquid On TLR7Signal Transduction Pathway Induced By RSV

Pneumonia, especially bronchial pneumonia, is a major childhood diseases, which mainly occurs in winter, spring or during climate changes. Seriously threatened children’s health, pneumonia has become the top death cause of the hospitalized children. The World Health Organization list its name as one of the three major global pediatric diseases, and Chinese Ministry of Health as one of the four pediatric diseases. According to statistics, of all the urban hospital pediatric ward inpatients, pneumonia accounted for25%to50%, among which viral pneumonia is a common type, accounting for about50%of the total cases. Respiratory syncytial virus (RSV) is the main pathogen that causes viral pneumonia in children, and western medicine therapy shows little effect in treating RSV pneumonia, while Chinese medicine has its unique advantages in treating viral infectious diseases.Jinxin oral liquid, developed to promote lung, resolve phlegm, detoxicate and remove blood stasis, proves an effective prescription in clinic which treats the viral pneumonia with phlegm-heat closed lung syndrome. Preclinic cases and experimental studies have shown that Jinxin oral liquid significantly inhibits RSV through depressing its membrane fusion, the target of which may lies in the virus’F protein or its receptor; Jinxin also sharply reduces the cytopathic effect (CPE) of Hep-2cell line infected with RSV; moreover, it induces single lymphocyte to produce IL-2, IFN-y to enhance antiviral immune function.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.As a single-stranded negative-strand RNA (ss-RNA) virus, RSV adheres and fuses to the host cell and then ss-RNA is released, which can be recognized by TLR7’s specific module in the inner body membrane, and through a series of signal transduction pathway, downstream cytokines secrete in high level and thus results in systemic inflammatory response. In view of the multiple target effect of Chinese medicine, this experiment starts with the TLR7and its signal transduction pathway, investigates the regulatory role of Jinxin oral liquid on the pathway induced by RSV infection, and researches the correlation to other TLRs pathways.ObjectiveTo study the role of Jinxin oral liquid on TLR7signal transduction pathway induced by RSV, and explore its possible immunological mechanism in treating RSV pneumonia.MethodsExperiments in vitro:1. 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 TLR7, MyD88, NF-κB, IRF7mRNA by real-time PCR assay, and the protein expression of TLR7, MyD88, NF-κB, IRF7by western blot assay, and TLR7expression and distribution in cells by confocal laser scanning technology. And cell supernatants were collected to detect TNF-α, IFN-a levels by ELISA assay.2. TLR7siRNA was used to interfere TLR7expression, and then detect the expression of above substances to observe the effect of Jinxin oral liquid on TLR7signal 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 after72,144hours of the first inoculation, mice lungs were taken to evaluate lung lesions by histologic slide analysis, to detect the levels of TLR7, MyD88, NF-κB, IRF7mRNA by real-time PCR assay, and to examine the protein expression of TLR7, MyD88, NF-κB, IRF7by western blot assay. The broncho alveolar lavage liquids (BALF) were collected to measure TNF-α, IFN-α levels by ELISA assay.ResultsExperiments in vitro:A. Before TLR7siRNA transfection1.24h after RAW264.7cells infected by RSV, expression of TLR7, NF-κB, IRF7mRNA significantly increased (more than2times more than the normal group, P<0.01), the expression of MyD88mRNA can improve, but was not more than two times than the normal group. The serum containing Jinxin oral liquid significantly reduced TLR7, MyD88, NF-κB mRNA expression (P<0.01), but the effect of down-regulating IRF7mRNA not obviously (P>0.05).2.24h after RAW264.7cells infected by RSV, TLR7, MyD88, NF-κB protein expression were significantly higher than that in normal group (P<0.01), and the protein expression of IRF7had no obvious change. The serum containing Jinxin oral liquid down-regulated significantly the expression of TLR7, MyD88, NF-κB protein (P<0.01), and had no effect on IRF7expression.3.24h after RAW264.7cells infected by RSV, in cell supernatants the expression of TNF-a was significantly increased (P<0.01), The serum containing Jinxin oral liquid significantly reduced TNF-a expressionThe serum containing Jinxin oral liquid.But IFN-a was not found.B. After TLR7siRNA transfection1. After RAW264.7cells transfected by TLR7siRNA, TLR7mRNA expression decreased by46.6%, then infected by RSV, TLR7mRNA expression significantly increased (more than two times than siRNA transfection group, P<0.01). And showed that TLR7siRNA transfection interfered TLR7mRNA generation. The expression of MyD88, NF-κB mRNA were also inhibited, but the inhibition rate lower than that of TLR7mRNA (respectively26.9%,27.3%<46.6%), and that may show that MyD88, NF-κB mRNA expression may also be regulated by other factors, especially by other members of TLRs family. IRF7mRNA expression was unaffected after TLR7siRNA transfection.2. The serum containing Jinxin oral liquid significantly reduced TLR7, MyD88, NF-κB mRNA expression (P<0.01) in RAW264.7cells treated by TLR7siRNA transfection and RSV infection, the down-regulation on IRF7mRNA expression was not obvious (P>0.05)3. The expression of TLR7, MyD88, NF-κB protein significantly increased24h after RSV infection (P<0.01), while IRF7protein expression had no significant change. The serum containing Jinxin oral liquid significantly down-regulated TLR7, NF-κB protein expression (P<0.01), but had no obvious regulation on MyD88protein expression (P>0.05), and had no regulating effect on IRF7protein expression.4.24h after TR7siRNA transfection followed with RSV infection, IFN-α failed to check out in the cell supernatant, and TNF-α expression significantly increased (P <0.01). The serum containing Jinxin oral liquid significantly reduced serum TNF-a expression (P<0.01)Experiments in vivo:1. Pulmonary pathological changes in BALB/c mice infected by RSV were mainly including interstitial inflammation, vascular dilatation and congestion, vascular wall edema and thickening, inflammatory cell infiltration in alveolar wall and interstitial lung. Alveolar cavity had no obvious exudation, and the bronchial cavity no significant inflammatory exudation, and bronchial epithelial cells were no significant degeneration and necrosis. With the RSV infection prolonged, the degree of lung lesions gradually worsened. 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.2.72h after RSV infecting BALB/c mice, TLR7, MyD88, NF-κB mRNA expression in lung tissue were significantly increased compared with the normal group (P<0.01), while at144h those expressions decreased. In Jinxin group TLR7, MyD88, NF-κB mRNA expression decreased compared with RSV group (P<0.01)72h and144h after RSV infection, IRF7mRNA expression had no obvious difference compared with normal group (P>0.05). At72h, high-dosage Jinxin oral liquid significantly down-regulated IRF7mRNA expression (P<0.01). At144h, high-dosage and equivalent-dosage Jinxin oral liquid both up-regulated IRF7mRNA expression, but had no statistics difference compared with RSV group (P>0.05)3.72h after RSV infection in BALB/c mice, TLR7、MyD88、NF-κB protein expression were higher than nomal group (P<0.01or P<0.05), and at144h the expression decreased and had no difference compared with normal group (P>0.05). TLR7、MyD88、NF-κB protein expression in Jinxin group at72h decreased significantly compared with RSV group (P<0.01or P<0.05), and at144h those expression decreased but had no obvious difference compared with RSV group (P>0.05)72h after RSV infecting BALB/c mice, IRF7protein expression had no difference compared with normal group, and at144h the expression increased (P>0.05). At72h IRF7protein expression was no difference between in RSV group and Jinxin group. At144h, Jinxin oral liquid significantly increased IRF7protein expression (P<0.01)4.72h after RSV infecting BALB/c mice, TNF-α and IFN-α expression in BALF were significantly higher than that in normal group (P<0.01) while at144h TNF-a and IFN-a expression decreased. At72h, TNF-a and IFN-a expression in Jinxin group significantly increased (P<0.01), and at144h the expression obviously reduced, but had no difference comared with RSV group.Conclusions1. Jinxin oral liquid significantly alleviate the lung inflammation of the RSV infected mice.2. Jinxin oral liquid has an evident down-regulatory effects on TLR7, MyD88, NF-κB mRNA and protein expression in TLR7signal transduction pathway induced by RSV; in the late stage after SRV infection, Jinxin oral liquid can up-regulate IRF7mRNA and protein expression, while in the early stage, no obvious regulation effect can be observed.3. Jinxin oral liquid can increase the levels of TNF-a, IFN-a in the early stage of RSV infection. During the infection prolonged period, it can down-regulate their expressions. The results show that Jinxin oral liquid has a bidirectional dynamic regulation on TNF-a and IFN-a expression.4. Jinxin oral liquid is an effective drug in treating RSV infection, and its effect is realized by adjusting the TLR7signaling pathways induced by RSV, mainly relies on the TLR7/MyD88/NF-κB/TNF-α pathway. In the early stage of RSV infection, the effect on TLR7/MyD88/IRF7/IFN-α pathway is not obvious.5. After TLR7siRNA transfection, the expression of TLR7mRNA and protein were inhibited, and the inhibition rate is higher than those of MyD88, NF-κB mRNA and protein, which shows that the expression of MyD88, NF-κB are also controlled by other TLRs and TLR7signal transduction pathway is one of all pathways. Whether TLR7siRNA transfection or not has no significant effects on IRF7expression.