| Objective:In this study, successor to the "Treatise on febrile disease" lift drifting drug laws. Binding cell migration modern experimental techniques, to compare the differences of ephedra, Schisandra lift drifting medicinal effects characteristic. In vitro studies explore the effect of Ephedra and Schisandra containing serum on normal rats AM, IM cell migration, comparative analysis of mechanisms leading to differences in cell migration. In vivo studies to observe Ephedra, Schisandra decoction impact on the number of AM, IM cells of pulmonary fibrosis rats, comparative analysis of the mechanisms leading to the number of differences. Through this study, the theory of traditional Chinese medicine and modern science combine to build the lift drifting medicinal effect characteristic evaluation method research to explore new models and ideas.Methods:(1) Theory:Collecting the 113 prescription of the "Treatise on febrile disease", according to the six meridians and eight law attributes to determine prescription lift ups or downs, descripting the use of prescription lift drifting medicinal characteristics in the "Treatise on febrile disease" in detail. Collection the 84 herbs of the "Treatise on febrile disease", collation and analysis the traditional Chinese medicine’s character, taste and medicinal parts to determine the 84 herbs and 43 flavor to the lung medicine liter floating or sedimentation properties, discussed in detail the use of traditional Chinese medicine’s lift drifting medicinal characteristics by Zhang Zhongjing.(2) In vitro study:Experiments using 22 SPF male Wistar rats, body weight (185-215) g, lavage method for extracting AM cells and digested lung tissue for extracting IM cells. Respectively Ephedra containing serum, Schisandra containing serum and blank serum cultured cells 48 hours, to investigate the effects of serum containing to the cells viability by MTT, transwell chamber was used to observe the impact of Ephedra, Schisandra containing serum on AM, IM migration and Western Blot was used to detect cell surface expression of CCR2, CCR5 receptor protein, compare Kvl.3, Kvl.5 channel protein expression differences.(3) In vivo study:Experiments using 74 SPF male Wistar rats, weight (185-215) g, were randomly divided into five groups, divided into sham operation group, model group, hydrocortisone group, ephedra group and Schisandra group. Outside the sham group, the other groups were given bleomycin A5 was established by intratracheal instillation of pulmonary fibrosis in rats. They were treated for 7 and 28 days coverage, cell counting plate method to observe the number of AM and IM, ELISA assay lavage fluid MCP-1, MIP-1α content changes, HE staining of lung tissue, using an optical microscope rats lung tissue biopsy.Results:(1) Theory:In the "Treatise on febrile disease", a large proportion of floating up side was 47.8%, a smaller proportion of the settlement side of 40.7%,11.5% neutral party.43 flavor to the lung of Chinese medicine, Floating up drug was smaller proportion of 25.58%, but the frequency of 41.90 percent, settlement drug was larger proportion of 62.79%, but the frequency of 36.87%.(2) In vitro study: ①Compared the affect of Ephedra, Schisandra containing serum on rat AM, IM cells with the same concentration of serum blank, the OD value was no significant difference (P> 0.05). When the Ephedra, Schisandra containing serum at 20% concentration, the cultured cells are better than other concentrations. So choose a concentration of 20% Ephedra, Schisandra containing serum sample to detect the relevant indicators in this study. ② The number of migrating cells of Ephedra and Schisandra containing serum group was significantly higher than blank serum group, with blank serum group were significantly different (P<0.01). Number of rat AM cells migration of Ephedra containing serum group compared Schisandra containing serum group were significantly increased, with Schisandra containing serum group were significantly different (P<0.01). Number of rat IM cells migration of Schisandra containing serum group compared Ephedra containing serum group were significantly increased, with Ephedra containing serum group were significantly different (P<0.01).③The IOD of AM, IM cell surface CCR2, CCR5 protein expression in Ephedra, Schisandra containing serum group were significantly higher than the blank serum group, there was a significant difference compared with the control serum(P<0.01). The IOD of AM cell surface CCR2, CCR5 protein expression in Ephedra containing serum group was higher than Schisandra containing serum group, with Schisandra containing serum group were significantly different (P<0.01). The IOD of IM cell surface CCR5 protein expression in Schisandra containing serum group was higher than Ephedra containing serum group, with Ephedra containing serum group were different (P<0.05), but IOD of CCR2 expression lower than Ephedra containing serum group, but the difference was not significant.④ The IOD of AM, IM cell surface Kv1.3、Kvl.5 channel protein expression in Ephedra, Schisandra containing serum group were significantly higher than the blank serum group, there was a significant difference compared with the control serum(P<0.01). The IOD of AM cell surface Kv1.3、Kvl.5 channel protein expression in Ephedra containing serum group was higher than Schisandra containing serum group, with Schisandra containing serum group were different (P<0.05). The IOD of IM cell surface Kv1.3、Kv1.5 channel protein expression in Schisandra containing serum group was higher than Ephedra containing serum group, with Ephedra containing serum group were different (P<0.05).(3) In vivo study:①Modeling rats on day 7, the number of AM cells in the model group was significantly higher than the sham group, there was a significant difference compared with the sham group(P<0.01); Ephedra decoction group, Schisandra decoction group" were significantly lower than the number of AM cells in the model group, there was significant difference with the model group(P<0.01). The number of Ephedra decoction group AM cells was below Schisandra decoction group, there are differences (P<0.05). After modeling the first 28 days, the number of AM, model group was still significantly higher than the sham group, there was a significant difference compared with the sham group(P<0.01). The number of AM cells, Ephedra, Schisandra decoction group were significantly lower than the model group, there was significant difference compared with the model group (P<0.01). Modeling rats on day 7, the number of IM cells of the rats increased to varying degrees. After modeling the first 28 days, the number of IM cells in the model group was significantly increased compared with 7 days, and there is a significant difference compared with the sham group(P<0.01). The number of IM cells in each treatment group rats were significantly lower than the model group, there was significant difference with the model group(P<0.01). The number of Schisandra decoction group IM cells was below Ephedra decoction group, there are differences (P<0.05).②The MCP-1 and MIP-1α in model rats’alveolar lavage fluid were significantly higher than the sham group, compared with the sham group had significant difference (P<0.01). The MCP-1 and MIP-la content in each treatment group alveolar lavage fluid was significantly lower than the model group, there was significant difference with the model group(P<0.01).7 days, the MCP-1, MIP-la content in Ephedra decoction group alveolar lavage fluid lower than the Schisandra decoction group. Process description ephedra, Schisandra decoction intervention idiopathic pulmonary fibrosis have a certain relationship with the inhibition of chemokine expression. This shows that Ephedra, Schisandra decoction intervention idiopathic pulmonary fibrosis have a certain relationship with the inhibition of chemokine expression.③In every group of HE staining, we found that sham group rat alveolar structure relatively intact, the phenomenon is no fusion, no obvious defects in the case, did not see the infiltration of inflammatory cells, no thickening of alveolar septa, not found to form a fiber bundle. After 7 days, the model group alveolar found a large number of inflammatory cell infiltration, some alveolar fusion seriously damaged, the formation of inflammatory lesions, occasionally alveolar interstitial thickening, no significant fiber bundle. The alveolar structure of Ephedra, Schisandra decoction group were varying degrees of integration, damaged, showing a small amount of inflammatory cell infiltration in the alveoli, but the extent and scope of infiltration are lighter than the model group.28 days after modeling, model group, there are plenty of formation of fibrous tissue, especially around the bronchial alveolar septal thickening, alveolar structural disorder, alveolar wall damaged, inflammatory infiltration than the 7 days has been reduced; Ephedra decoction group, Schisandra decoction group lung tissue of rats were formed with varying degrees of fiber bundles, but to a lesser degree than the model group.Conclusion:Through literature review analysis the "Treatise on febrile disease" ups and downs of drug law theory, found Zhang Zhongjing attaches great importance to the medicine’s ups and downs, the drug will be too divergent prescriptions converges, the convergence of the drug to be too divergent. It used 84 herbs,43 flavor to the lung, a large proportion of drug settlement, but floating up drug dosage frequency, and this with the lung, "Xuan-Fa-Su-Jiang" function. Can make full use of Chinese herbs lift drifting law, research and treatment of lung diseases, analyze the mechanism and treatment differences practical significance. In vitro experiments exploratory discovery, Ephedra on normal rat AM cell chemotactic migration capability, Schisandra on IM chemotactic migration capability, and its mechanism may be through the stimulation of cells, promotion of cell surface expression of CCR2, CCR5 receptor protein, will signaling molecules passed to the Kv1.3, Kv1.5 ion channel, so that changes in the cytoskeleton, and ultimately promote cell chemotactic migration. In vivo confirmatory discovery, Ephedra, Schisandra for intervention pulmonary fibrosis may be through inhibition of chemokine expression of MCP-1, MIP-la, and the regulation of AM, IM Cell gather in the lungs damaged area and secrete inflammatory factors achieved. Ephedra on the number of AM cells suppressed ability better, Schisandra on the number of IM cells suppressed ability better. During the 7 days alveolitis, Ephedra group less than other groups of rats administered breakage and inflammatory cell infiltration; 28 days stage pulmonary fibrosis, the degree of pulmonary interstitial fibrous connective tissue generated Schisandra group than the other group of administered rats lighter. This effect of Ephedra in the early alveolitis, Schisandra on the late pulmonary fibrosis conclusions coincide. It also verified the Ephedra on AM cell targeting and Schisandra on IM cell targeting. |
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