Study On The Pathway Of P38 / JNK MAPK Signaling Pathway Induced By Pneumoconiosis In Pneumocarcinoma In Rats

Purpose: To expound the theoretical foundation of prescription of Qing Fei Tong Luo plaster in Traditional Chinese Medicine, and the basic theory of application is “Sticking Point theory”, which is a kind of external therapy of Traditional Chinese Medicine, as the point application; to establish rat pneumonia model induced by influenza virus(IV); to study the regulating role of Qing Fei Tong Luo plaster to IV induced miotgen—acitvated protein kinases(MAPKs) signaling pathway and explore the possible mechanisms of treatment of pneumonia induced by IV; to explore the timeliness relationship and difference effects of MAPKs signaling pathway with intervention of high,moderate and low doses of Qing Fei Tong Luo plaster in pneumonia rat model induced by IV, and provide experimental evidence for the prevention and treatment of IV pneumonia in Chinese medicine. Through high efficiency liquid chromatography(HPLC) to determine the active component contents of Qing Fei Tong Luo plaster to identify its material foundation of IV infected pneumonia treatment.Material and method:1. Using influenza A virus FM1 to infect younger Wistar mice to mice pneumonia, observed the general status、the differences of lung tissue、morphology, observe the differenences between normol groug and model group,applied RT-PCR method to detect IV-RNA viral load to accredit the animal model.2. Qing Fei Tong Luo plaster as the external application was applied in the back of young Wistar rats(equivalent to the external application on the lung shu points) to intervene pneumonia rats model IV-induced, and then observe the rats in different groups on the general state, weight and lung index at different time points.3. Using immunohistochemistry and Real-time PCR method to detect protein expression and m RNA expression levels of mitogen-activated protein kinase p38(p38)、c-jun n-terminal kinase(JNK) and mitogen-activated protein kinase kinase 4(MKK4) in the lung tissue of pneumonia rats IV induced, and discussing the prevention mechanism of Qing Fei Tong Luo plaster on young pneumonia rats IV induced, meanwhile, to compare the difference of activation level of MAPKs signaling pathway at different time points in the high,moderate and low doses of Qing Fei Tong Luo plaster.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 2th 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<0.05), the rats weight of the treatment groups group grow steadily, but the increase in rats weight is slow than the normal group on the 3th 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 low dose group, high and moderate doses of Qing Fei Tong Luo plaster groups showed significantly different(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 IV-RNA in lung tissue at different time points: the lung tissue of rats in the normal group at each time point can not detected any virus nucleic acid of IV, but the lung tissue of rats in the model group during 4 to 7 days of modeling, IV-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<0.05); the lung index in the treatment groups are lower than in the model group, and showed a significant difference(P<0.05); the lung index in the high dose group decreased more lower than in the moderate and low dose groups; the high dose group compared with the normal group, the lung index increased with a significant difference(P<0.05); the moderate and low dose groups compared with the normal group, the lung index increased with a significant difference(P<0.05).5. The results of P38 expression in different groups in lung tissue: the normal group were able to detect small amounts of P38 expression. P38 expression in model group gradually increased, on the 7th day of modeling, the integrated optical density value of P38 was 18.720±1.254,and it showed significantly different to compare with the normal group at the same time(P<0.05). Comparing with the model group, these high dose group showed significantly different respectively on 3th、5th and 7th day(P<0.05).The P38 expression in high dose group was higher than normol group and lower than model group(P<0.05),which was statistical significance compared with moderate group on 7th day(P<0.05).6. The results of JNK expression in different groups in lung tissue: the normal group were able to detect small amounts of JNK expression. JNK expression in model group gradually increased, on the 7th day of modeling, the integrated optical density value of JNK was 35.839±4.929,and it showed significantly different to compare with the normal group at the same time(P<0.05). Comparing with the model group, these high dose group showed significantly different respectively on 3th、5th and 7th day(P<0.05).The JNK expression in high dose group was higher than normol group and lower than model group(P<0.05).7. The results of MKK4 expression in different groups in lung tissue: the normal group were able to detect small amounts of MKK4 expression. MKK4 expression in model group gradually increased, on the 7th day of modeling, the integrated optical density value of MKK4 was 20.806±1.662,and it showed significantly different to compare with the normal group at the same time(P<0.05). Comparing with the model group, these high dose group showed significantly different respectively on 3th、5th and 7th day(P<0.05).The MKK4 expression in high dose group was higher than normol group and lower than model group(P<0.05),which was statistical significance compared with moderate group on 7th day(P<0.05).8. Correlation analysis of P38 and MKK4 protein level expression in different groups in lung tissue:Linear correlation analysis method was used to proved the expression of P38 and MKK4 correlation r close to 1. Variation of P38 and MKK4 expression were relevant(P<0.05) in different groups except the Fuxiong cream high dose group on 3th day.9. Correlation analysis of JNK and MKK4 protein level expression in different groups in lung tissue: Linear correlation analysis method was used to proved the expression of JNK and MKK4 correlation r close to 1. Variation of JNK and MKK4 expression were relevant(P<0.05) in different groups except the Fuxiong cream moderate dose group on 3th day,normol group on 5th day and 7th day.10. The results of P38 m RNA expression in lung tissue in the different groups: the normal group could be detected small amounts of P38 m RNA expression,there was no significantly different comparing in different days. P38 m RNA expression in the model group gradually increased, on the 7th day of modeling, the expression reach the peak; P38 m RNA in model group was significantly higher than in the normal group(P<0.05). Comparing with the model group, these high dose group showed significantly decreased in different times,which was higher than normol group(P<0.05). Comparing with the moderate group, these high dose group showed significantly different respectively on 3th and 7th day(P<0.05).11. The results of JNK m RNA expression in lung tissue in the different groups: the normal group could be detected small amounts of JNK m RNA expression,there was no significantly different comparing in different days. JNK m RNA expression in the model group gradually increased, on the 7th day of modeling, the expression reach the peak; JNK m RNA in model group was significantly higher than in the normal group(P<0.05). Comparing with the model group, these high dose group showed significantly decreased in different times,which was higher than normol group(P<0.05). Comparing with the moderate group, these high dose group showed significantly different respectively on 7th day(P<0.05)).12. The results of MKK4 m RNA expression in lung tissue in the different groups: the normal group could be detected small amounts of MKK4 m RNA expression,there was no significantly different comparing in different days. MKK4 m RNA expression in the model group gradually increased, on the 7th day of modeling, the expression reach the peak; MKK4 m RNA in model group was significantly higher than in the normal group(P < 0.05). Comparing with the model group, these high dose group showed significantly decreased in different times,which was higher than normol group(P<0.05).13. Correlation analysis of P38 and MKK4 m RNA level expression in different groups in lung tissue:Linear correlation analysis method was used to proved the expression of P38 and MKK4 correlation r was close to 1. Variation of P38 and MKK4 m RNA expression were relevant(P<0.05) in different groups except the Fuxiong cream low dose group on 3th day、model group、Fuxiong cream high dose group and moderate dose group.14. Correlation analysis of JNK and MKK4 m RNA level expression in different groups in lung tissue: Linear correlation analysis method was used to proved the expression of JNK and MKK4 correlation r was close to 1. Variation of JNK and MKK4 m RNA expression were relevant(P<0.05) in different groups except the Fuxiong cream moderate dose group、normol group and model group on 3th day,model group、Fuxiong cream moderate dose group on 7th day.Conclusion:1.IV induced Wistar rat pneumonia model could be successfully established by the method on intranasal inoculation 15LD50 Influenza A virus 0.1ml through ether anesthesia by the observation on the general condition of rats, the lung tissue pathology and real-time fluorescence quantitative method in the detection of lung tissue IV load.2. Qing Fei Tong Luo plaster can significantly improve the general status in rats, nflammation of lung tissue damage, lung tissue pathology and Lung index. Prevention and cure function of Qing Fei Tong Luo plaster of IV infection induced pneumonia rats may be associated with the over-activation of P38/JNK MAPK signaling pathway, the high and moderate dose drug showed more efficient on inhibiting P38、JNK、MKK4 over expression in the disease process than the low dose drugs on 3th、5th and 7th day.3. Established the determination method of 6 kinds of active ingredients in Qing Fei Tong Luo plaster by HPLC, the method is simple, sensitive and accurate with a high reproducibility.HPLC could become a mature technology to content determination and quality control.