| Purpose:To expound the theoretical foundation of prescription of Qing Fei Tong Luo plaster in Traditional Chinese Medicine, which is proposed by Prof. Wang Xuefeng, it is " Lung toxin heat syndrome", and the basic theory of application is ‘Sticking Point theoryâ€, which is a kind of external therapy of Traditional Chinese Medicine, as the point application; to use orthogonal design method to quantify the active ingredients of Qing Fei Tong Luo plaster and to evaluate the cream of detection rate of Qing Fei Tong Luo plaster as the evaluation indexes of process, then combining all factors to obtain optimum extraction of Qing Fei Tong Luo plaster; to establish rat pneumonia model induced by respiratory syncytial virus(RSV); to study the regulating role of Qing Fei Tong Luo plaster to RSV induced Toll-like receptor 4(TLR4) signaling pathway and explore the possible immunological mechanisms of treatment of pneumonia induced by RSV; to explore the timeliness relationship and difference effects of TLR4 signaling pathway with intervention of full prescription of Qing Fei Tong Luo plaster and chief and minister drugs of Qing Fei Tong Luo plaster in pneumonia rat model induced by RSV, and provide experimental evidence for the prevention and treatment of RSV pneumonia in Chinese medicine.Material and method:1. Using orthogonal design method to quantify the active ingredients of Qing Fei Tong Luo plaster and to evaluate the cream of detection rate of Qing Fei Tong Luo plaster as the evaluation indexes of process, then combining ethanol concentration, solvent ratio, extraction time and extraction times to sieve and optimize the extraction artworks and to analysis their main active ingredient.2. Using RSV Long strains induced pneumonia model of the young Wister rats throughintranasal inoculation, and observed the difference of rats between the normal group and the model group in the general state, lung tissue pathological section, morphology and lung index, then apply Real time-PCR technology to test the gene levels of viral load in the lung tissue of rats at different time points, then to evaluate the quality of model( Dividing the rats into 4 groups, the normal group was the rats with no drugs and RSV infection, there was no intervention in the normal group; the model group was the rats with RSV infection, but there was no intervention of drugs in the model group; the full prescription group was the rats with RSV infection, and there was intervention of Qing Fei Tong Luo plaster with the whole composition of herbs; the chief and minister group was the rats with RSV infection, and there was intervention of part drugs of Qing Fei Tong Luo plaster composed with Rhubarb and Skullcap herbs). 3. Qing Fei Tong Luo plaster as the external application was applied in the back of young Wister rats(equivalent to the external application on the lung shu points) to intervene pneumonia rats model RSV-induced, and then observe the rats in different groups on the general state, weight and lung index at different time points. 4. Using immunohistochemical and enzyme-linked immunosorbent assay(ELISA) to detect the protein expression and protein content of tumor necrosis factor α(TNF-α) and interleukin 6(IL-6) in lung tissue and serum of pneumonia rats RSV induced, to compare the difference of expression of cytokines TNF-α and IL-6 between the full prescription group and the chief and minister group at different time points. 5. Using immunohistochemistry and Real-time PCR method to detect protein expression and m RNA expression levels of TLR4 and nuclear factor kappa-light-chain-enhancer of activated B-cells p65(NF-k B p65) in the lung tissue of pneumonia rats RSV induced, and discussing the prevention mechanism of Qing Fei Tong Luo plaster on young pneumonia rats RSV induced, meanwhile, to compare the difference of activation level of TLR4 signaling pathway at different time points between the full prescription group and chief and ministergroup.Results:1. Comparison of results of rats weight: the rats weight of the normal group grow into a steady upward trend, the rats weight of model group grow relatively slow, on the 4th day of modeling, comparing the rats weight of the normal group and the rats weight of the model group had significant differences in weight gain(P<0.1), the rats weight both of the full prescription group and the chief and minister group grow steadily, but the increase in rats weight is slow than the normal group on the 5th day of modeling, the rats weight of the full prescription group comparing with the model group showed significantly different(P<0.05); Comparing with the chief and minister group showed not significantly different. On the 7th day of modeling, there was significant difference between the full prescription group and the chief and minister group(P<0.05). 2. Pathological changes in the lung tissue:The lung tissue of rats in the normal group at different times showed no pathological changes in the appearance of the whole lung field. After 5 days of modeling, to observe under light microscope the lung tissue of rats in the model group show damaged epithelial cells, the alveolar walls vessel had dilatation and congestion, the alveolar wall get thickening, interstitial edema, in alveolar can be seen a large number of inflammatory cell infiltration and slurry filled, mainly lymphocytes. Observed under electron microscopy, there showed alveolar wall thickening, necrosis and exfoliation of cells in alveolar, the number of interstitial cells increased, alveolar epithelial type II ciliated epithelial cells broken, epithelial cell degeneration and necrosis. With the prolonging of the infection, the lung lesion gradually worsened. The pulmonary inflammation of rat tissue of both the full prescription group and the chief and minister group reduced in varying degrees, to compare with the model group in pathological scores at each time point showed a significant difference. 3. The semi-quantitative determination results of RSV-RNA in lung tissue atdifferent time points: the lung tissue of rats in the normal group at each time point can not detected any virus nucleic acid of RSV, but the lung tissue of rats in the model group during 4 to 7 days of modeling, RSV-RNA amplified expression can be found and increased simultaneous with the viral load, suggesting that the amount of the virus is proportional to the inflammatory injury level. 4. Lung index test results: lung index of the normal group at different time points were the lowest. The lung index in the model group is higher than in the normal group, and showed significantly different(P <0.01); the lung index in the full prescription group is lower than in the model group, and showed a significant difference(P< 0.01); the lung index in the chief and minister group is lower than in the model group, and showed significantly different(P< 0.05); the lung index in the full prescription group decreased more lower than in the chief and minister group; the full prescription group compared with the normal group, the lung index increased with a significant difference(P <0.05); the chief and minister group compared with the normal group, the lung index increased with a significant difference(P <0.05). 5.The results of TNF-α and IL-6 expression in different groups in lung tissue: the normal group were able to detect small amounts of TNF-α expression. TNF-α expression in model group gradually increased, on the 7th day of modeling, the integrated optical density value of TNF-α was 13.8927±0.5485,and it showed significantly different to compare with the normal group at the same time(P<0.01). At the different time point, TNF-α expression both in the full prescription group and in the chief and minister group were lower than in the model group, but higher than in the normal group(P<0.05), on the 3rd day of modeling, comparing with the model group, these two groups showed no significant different respectively(P>0.05),but on the 5th and 7th day of modeling, comparing with the model group, these two groups showed significantly different respectively(P<0.05). meanwhile, comparison of expression between the full prescription group and the chief and minister group, there was significantlydifferent on the 5th and 7th day of modeling(P<0.05), suggesting that the full prescription group get the best efficacy on the 7th day. The normal group was able to detect small amounts of IL-6 expression. The IL-6 expression in model group gradually increased, on the 7th day of modeling, the integrated optical density value of IL-6 was 33.874±2.1762,and it showed significantly different to compare with the normal group at the same time(P<0.01).At the different time point, the IL-6 expression both in the full prescription group and in the chief and minister group were lower than in the model group, but higher than in the normal group(P<0.05), on the 3rd day of modeling, comparing with the model group, these two groups showed no significant different respectively(P>0.05),but on the 5th and 7th day of modeling, comparing with the model group, these two groups showed significantly different respectively(P<0.05). meanwhile, comparison of expression between the full prescription group and the chief and minister group, there was significantly different on the 5th and 7th day of modeling(P<0.05),suggesting that the full prescription group get the best efficacy on the 7th day. 6.The results of serum TNF-α and IL-6 level in different groups: the normal group were able to detect small amounts of serum TNF-α expression. The serum TNF-α level in model group gradually increased, on the 7th day of modeling, the integrated value of TNF-α was 175.27±9.76,and it showed significantly different to compare with the normal group at the same time(P<0.01).At the different time point, the serum TNF-α expression both in the full prescription group and in the chief and minister group were lower than in the model group,but higher than in the normal group, on the 5th and 7th day of modeling, comparing with the model group, these two groups showed significantly different respectively(P<0.05). meanwhile, comparison of expression between the full prescription group and the chief and minister group, there was significantly different on the 7th day of modeling(P<0.05), suggesting that the full prescription group get the best efficacy on the 7th day. The normal group was able to detect small amounts of serum IL-6 expression.The serum IL-6 level in model group gradually increased, on the 7th day of modeling, the integrated value of IL-6 was 400.48 ± 10.42, and it showed significantly different to compare with the normal group at the same time(P<0.01).At the different time point, the serum IL-6 expression both in the full prescription group and in the chief and minister group were lower than in the model group, but higher than in the normal group, on the 5th and 7th day of modeling, comparing with the model group, these two groups showed significantly different respectively(P<0.05). meanwhile, comparison of expression between the full prescription group and the chief and minister group, there was significantly different on the 7th day of modeling(P<0.05),suggesting that the full prescription group get the best efficacy on the 7th day. 7. The different groups results of TLR4 protein expression in lung tissue: the normal group were able to detect small amounts of TLR4 protein expression,there was no significantly different comparing in different days. The TLR4 protein expression in model group gradually increased, on the 7th day of modeling, TLR4 protein expression reach the strongest; at the same time point, it showed significantly different to compare with the normal group(P<0.01).At the different time point, the protein expression of TLR4 in full prescription group was significantly lower than in the model group, but higher than in the normal group, there were a significant difference(P<0.05); meanwhile, the protein expression in the chief and minister group was lower than in the model group( P < 0.05), but higher than in the normal group( P < 0.05), there was significantly different. Comparison of the protein expression between the full prescription group and the chief and minister group, there was significantly different on the 7th day of modeling(P<0.05). 8. The different groups results of NF-k Bp65 protein expression in lung tissue: the normal group were able to detect small amounts of NF-k Bp65 protein expression,there was no significantly different comparing in different days. The NF-k Bp65 protein expression in model group gradually increased, on the 7th day of modeling, NF-k Bp65 protein expression reach the strongest; at the sametime point, it showed significantly different to compare with the normal group(P<0.01).At the different time point, the protein expression of NF-k Bp65 in full prescription group was significantly lower than in the model group(P<0.05),but higher than in the normal group(P<0.05),there was significantly different; meanwhile, the protein expression in chief and minister group was lower than in the model group(P<0.05), but higher than in the normal group(P<0.05), there was significantly different. The protein expression in the full prescription group was lower than in the chief and minister group, there was significantly different between these two groups on the 7th day of modeling(P<0.05). 9.The results of TLR4 m RNA expression in lung tissue in the different groups: the normal group could be detected small amounts of TLR4 m RNA expression,there was no significantly different comparing in different days.TLR4 m RNA expression in the model group gradually increased, on the 7th day of modeling, the expression reach the peak; TLR4 m RNA in model group was significantly higher than in the normal group(P <0.05).at the different time points, TLR4 m RNA in full prescription group was lower than in the model group(P <0.05), but higher than in the normal group(P <0.05), there was significantly different; meanwhile, the expression in chief and minister group was also lower than in the model group, on the 7th day of modeling, it showed a significant difference(P<0.05). Comparison of TLR4 m RNA expression between the full prescription group and the chief and minister group, there was significantly different on the 7th day of modeling(P<0.05). 10.The results of NF-k Bp65 m RNA expression in lung tissue in the different groups: the normal group could be detected small amounts of NF-k Bp65 m RNA expression,there was no significantly different comparing in different days. NF-k Bp65 m RNA expression in the model group gradually increased, on the 7th day of modeling, the expression reach the peak; NF-k Bp65 m RNA in model group was significantly higher than in the normal group(P <0.05). At the different time points, NF-k Bp65 m RNA in the full prescription group was lower than in the modelgroup(P <0.05), but higher than in the normal group(P <0.05), there was significantly different; meanwhile, the expression in the chief and minister group was also lower than in the model group, on the 7th day of modeling, it showed a significant difference(P < 0.05). Comparison of NF-k Bp65 m RNA expression between the full prescription group and the chief and minister group, there was significantly different on the 7th day of modeling(P<0.05).Conclusion:1.Qing Fei Tong Luo plaster as the external application was applied in the back of young Wister rats(equivalent to the point application on the lung shu points), Qing Fei Tong Luo plaster can significantly increase the rats weight, decrease the lung index, relieve the pulmonary pathological changes at different point reduce pulmonary inflammation in rats with RSV infection. 2. Qing Fei Tong Luo plaster is the effective drug to prevent RSV infection and to treat pneumonia RSV induced, the mechanism of treatment is associated with inhibiting the over-activation of TLR4 signaling pathway. RSV infection induced pneumonia may be associated with the over-activation of TLR4-NF-κB signaling pathway, which can lead to the abnormal secretion of inflammatory cytokines TNF-a and IL-6 in the downstream of the TLR4 signaling pathway; Thereby, TLR4 signaling pathway can be one of the immunological mechanisms for causing serious lung injury in rats. 3. Both the full prescription group and the chief and minister group of Qing Fei Tong Luo plaster can inhibit TLR4 signaling pathway over-activation to varying degrees, which is induced by RSV infection. and Both the full prescription group and the chief and minister group of Qing Fei Tong Luo plastert also can decrease the secretion of downstream cytokines TNF-α and IL-6 of TLR4 signaling pathway. 4.Qing Fei Tong Luo plaster can intervene RSV induced pneumonia, the full prescription drug showed more efficient on inhibiting TLR4 and NF-κB P65 m RNA over expression in the disease process at most of the time points than the chiefand minister composition drugs, it indicates that the full prescription is more effective than the chief and minister composition of plaster. It was associated with the role of the media in the Chinese herb plaster, it should not be ignored.
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