Effect Of Qingfei Tonghuo Pill On Multi - Target Of RSV Pneumonia In Young Rats

Purpose:To explore the effective substance basis and biological transmission way of Qingfei Tongluo plaster which based on theory of collaterals in cutaneous regions and determine optimal extraction process.To establish model of respiratory syncytial virus(RSV) pneumonia model of young rats in order to analyze the differences in protein and clear biological marker in toxic heat closed lung syndrome stage.To clarify mechanism of Qingfei Tongluo plaster and its disassembled prescriptions and medicine complementary theory through they intervene pulmonary viral load quantity, MAPK p38/NF-κB signaling pathway and T cell subsets in peripheral of RSV pneumonia in yong rats,and provide the experimental and theoretical basis for the Chinese herb drug development.Material and method:1.To establish the optimum extraction process through to applicate of high performance liquid chromatography(HPLC), use L9(34) orthogonal experimental design, set the content of baicalin emodin, rhein, and dry extract rate as the index, investigate the concentration of ethanol, dosage of ethanol, extraction time, extraction times.2.To investigate transdermal absorption characteristics of the effective substance and transmission mechanism of Qingfei Tongluo plaster through to select modified Franz diffusion apparatus, use rat skin as transdermal barrier, use high performance liquid chromatography(HPLC),determine cumulative transdermal amount of emodin, rhein and baicalin.3.To identificate model through to induce RSV pneumonia model in young wistar rats by nasal inoculation with RSV long strains, to observe general condition of rats and lung tissue pathological,to test virus load by reverse transcription polymerase chain reaction4.To seek biomarkers and intervention targets of toxic heat closed lung syndrome by applicate the two-dimensional fluorescence difference gel electrophoresis combined with matrix assisted laser desorption ionization time of flight mass spectrometry identification technology to analyse differential protein of lung tissue of RSV pneumonia in yong rats.5.To compare pulmonary viral load quantity and MAPK p38/NF-κB signaling pathway between Qingfei Tongluo plaster and its disassembled prescriptions by use real-time quantitative polymerase chain reaction technique and Western Blot technology to test RSV F protein m RNA, p38 MAPK and NF-κB m RNA and protein expression levels of lung tissue of RSV pneumonia in young rats of in different time point.6. To compare T cell subsets and related cytokines in peripheral of RSV pneumonia in yong rats between Qingfei Tongluo plaster and its disassembled prescriptions by use flow cytometry and enzyme linked immunosorbent assay to test CD3+、CD3+CD4+、CD3+CD8+T lymphocyte,IL-4 and IFN-γ.Results:1. The best extraction process of Qingfei Tongluo plaster: A3B1C3D1. The optimum condition was 6 times the amount of 70%,3 times of extraction, 1 hour each time..2. Transdermal absorption characteristics of Qingfei Tongluo plaster:Emodin, rhein, baicalin can transdermal. The cumulative transdermal amount per unit area and the transdermal rate of baicalin is the most. The transdermal absorption of main ingredient were all zero-order kinetics.In the different compatibility tests, the 8h accumulation permeation amount and transdermal rate of each ingredient in the full prescription were the largest,which compared with main formula group have significant difference(p<0.05).3. Pathological changes of lung: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 3 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 no dilatation and congestion,in alveolar can be seen a small number of inflammatory cell infiltration. After 5 days of modeling, the lung tissue of rats in the model group show the alveolar walls vessel had middle dilatation and congestion,in alveolar can be seen a middle number of inflammatory cell infiltration. After 7 days of modeling, the lung tissue of rats in the model group show the alveolar walls vessel had significant dilatation and congestion,in alveolar can be seen a large number of inflammatory cell infiltration.4. Amplification products of RT-PCR: Respiratory syncytial virus was not detected in normal group. Respiratory syncytial virus was detected in control group.The expression of respiratory syncytial virus in the model group increase graduall and were higest after 7 days of modeling(P<0.05).5.The result of 2D-DIGE: More than 1.5 times protein spots between the model group and the normal group were 41. The expression of 4 protein spots were higher in normal group than in the model group. The expression of 37 protein spots were higher in model group than in the normal group.Eight proteins were identified.They include hemopexin, haptoglobin, T-kininogen 1, T-kininogen 2, moesin, ezrin, protein disulfide-isomerase A3, vimentin.6.The results of viral load in lung tissue in the different groups:Respiratory syncytial virus was not detected in normal group. Viral load in the model group was significantly higher than in the normal group at each time point(P<0.05). The viral load of the treatment group was lower than the model group at each time point. The treatment group was lower than the model group,but the full prescription group was significantly lowest(p<0.05).7.The results of MAPK p38 m RNA expression in lung tissue in the different groups: The normal group could be detected small amounts of MAPK p38 m RNA expression. The MAPK p38 m RNA expression in the model group,treatment group was significantly higher than the normal group(P<0.05). The MAPK p38 m RNA expression in the full prescription group was significantly lower than in the model group(P <0.05).The MAPK p38 m RNA expression in the chief and minister group was also lower than in the model group,but there were not significant difference(P<0.05). The MAPK p38 m RNA expression in the full prescription group was significantly lower than in the chief and minister group(P<0.05).8.The results of NF-κB p65 mRNA expression in lung tissue in the different groups: The normal group could be detected small amounts of NF-κB p65 m RNA expression. The NF-κB p65 m RNA expression in the model group,treatment group was significantly higher than the normal group(P<0.05). The NF-κB p65 m RNA expression in the the full prescription group was significantly lower than in the model group(P <0.05).The NF-κB p65 m RNA expression in the chief and minister group was also lower than in the model group,but there were not significant difference(P<0.05). The NF-κB p65 m RNA expression in the full prescription group was significantly lower than in the chief and minister group(P<0.05).9.The results of TSLP m RNA expression in lung tissue in the different groups: The normal group could be detected small amounts of TSLP m RNA protein expression. The TSLP m RNA protein expression in the model group,treatment group was significantly higher than the normal group(P<0.05). The TSLP m RNA protein expression in the the full prescription group was significantly lower than in the model group(P <0.05).The TSLP m RNA protein expression in the chief and minister group was also lower than in the model group,but there were not significant difference(P<0.05). The TSLP m RNA protein expression in the full prescription group was significantly lower than in the chief and minister group(P<0.05).10.The results of MAPK p-p38 protein expression in lung tissue in the different groups: The normal group could be detected small amounts of MAPK p-p38 protein expression. The MAPK p-p38 protein expression in the model group,treatment group was significantly higher than the normal group(P<0.05). The MAPK p-p38 protein expression in the the full prescription group was significantly lower than in the model group(P<0.05).The MAPK p-p38 protein expression in the chief and minister group was also lower than in the model group,but there were not significant difference(P<0.05). The MAPK p-p38 protein expression in the full prescription group was significantly lower than in the chief and minister group(P<0.05).11.The results of NF-κB p65 protein expression in lung tissue in the different groups: The normal group could be detected small amounts of NF-κB p65 protein expression. The NF-κB p65 protein expression in the model group,treatment group was significantly higher than the normal group(P<0.05). The NF-κB p65 protein expression in the the full prescription group was significantly lower than in the model group(P <0.05).The NF-κB p65 protein expression in the chief and minister group was also lower than in the model group,but there were not significant difference(P<0.05). The NF-κB p65 protein expression in the full prescription group was significantly lower than in the chief and minister group(P<0.05).12.The results of TSLP protein expression in lung tissue in the different groups: The normal group could be detected small amounts of TSLP protein expression.The TSLP protein expression in the model group,treatment group was significantly higher than in the normal group(P<0.05). The TSLP protein expression in the full prescription group was significantly lower than in the model group(P <0.05).The TSLP protein expression in the chief and minister group was also lower than in the model group,but there were not significant difference(P<0.05). The TSLP protein expression in the full prescription group was significantly lower than in the chief and minister group(P<0.05).13.The results of T cell subsets in peripheral blood in the different groups: The CD3+、CD3+CD4+、CD3+CD8+T lymphocytes in the model group was significantly lower than the normal group(P<0.05),but CD4+/CD8+T lymphocytes was significantly higher(P<0.05). The CD3+、CD3+CD4+、CD3+CD8+T lymphocytes in the treatment group was significantly higher than the model group(P<0.05),but CD4+/CD8+T lymphocytes was significantly lower. The CD3+、CD3+CD4+、CD3+CD8+T lymphocytes in the full prescription group was significantly higher than in the chief and minister group(P < 0.05),but CD4+/CD8+T lymphocytes was significantly lower.14.The results of IL-4/INF-γ in peripheral blood in the different groups: The IL-4 in the model group was significantly higher than the normal group(P<0.05). The IL-4 in the treatment group was significantly lower than the model group(P<0.05). The IL-4 in the full prescription group was significantly lower than in the chief and minister group(P<0.05). The INF-γ in the model group was significantly lower than the normal group(P<0.05). The INF-γ in the treatment group was significantly higher than the model group(P<0.05). The INF-γ in the full prescription group was significantly higher than in the chief and minister group(P<0.05).Conclusion:1.Emodin, rhein, baicalin is effective substance basis of Qingfei Tongluo plaster. Collaterals in cutaneous regions is biological transmission way. The minister medicine can promote infiltration capacity.2.The fluorescence two-dimensional difference gel electrophoresis and matrix assisted laser desorption ionization time-of-flight mass spectrometry is the method to study the pathogenesis of RSV pneumonia in yong rats and discover biomarkers and new therapeutic targets.3.Qingfei Tongluo plaster can reduce pulmonary viral load, control MAPK p38/NF-κB signaling pathway, regulate and control T cell subsets and related cytokines in peripheral blood of RSV pneumonia in yong rats. The minister medicine can increase efficacy of the chief medicine.