| Objective:In this study,we explored the mechanism of treatment of interstitial lung disease based on Wenfei Buqi Tongluo Method and Bu Yang Huan Wu Decoction through in vivo experimental methods.The Fas/FasL signal transduction pathway was used as the entry point,and the related apoptosis was studied in the depth of apoptosis.The expression of death factor and inflammatory factor protein was explored.The effect and mechanism of Jiawei Buyang Huanwu Decoction on anti-pulmonary fibrosis in bleomycin-induced rats were explored.It was based on Wenfei Buqi Tongluo Method and Fuyang Huanwu Decoction.Interstitial lung disease provides a basis for pharmacodynamic research and lays the foundation for clinical research.Methods:1.Effect of Jiawei Buyang Huanwu Decoction on pathological morphology of rat model of pulmonary fibrosisThe anti-pulmonary fibrosis experiment in vivo was carried out by animal model method.The general condition of rats was observed dynamically.The degree of alveolitis in lung tissue was evaluated according to the pathological examination method of HE and Masson staining.The effect of Jiawei Buyang Huanwu Decoction on pulmonary fibrosis was discussed.The blank normal group and the bleomycin model were used as the model control group and the positive drug prednisone group.The Jiayang Buyang Huanwu Decoction was divided into high,medium and low dose groups,with 20 rats in each group.The rats with pulmonary fibrosis model were given high,medium and low concentrations of Bu Yang Huan Wu Tang in the dose group,and the traditional Chinese medicine was intervened and compared with the model control group and the positive drug group.2.Study on serum TNF-α,TGF-β and HMGB1 in rats with pulmonary fibrosis by Jiawei Buyang Huanwu DecoctionRats in the bleomycin lung fibrosis model were examined on the 7th,14 th and 28 th day.The expression levels of TNF-α,TGF-β and late inflammatory factor HMGB1 were detected by enzyme-linked immunosorbent assay(ELISA)at each time point.3.Effects of Jiawei Buyang Huanwu Decoction on the expression of Fas and FasL mRNA in lung tissue of rats with pulmonary fibrosisImmunohistochemical technique,real-time PCR and Western blot were used to observe the expression of Fas and FasL mRNA in lung tissue of rats with bleomycin pulmonary fibrosis model.4.Effect of Jiawei Buyang Huanwu Decoction on Caspase-3 in lung tissue of rats with pulmonary fibrosisFlow cytometry and Western blot were used to detect the content of Caspase-3 in rat lung tissue.Results:1.Effect of Jiawei Buyang Huanwu Decoction on pathological morphology of lung tissue in rats with pulmonary fibrosis:(1)HE staining:Positive drug group and Jiawei Buyang Huanwu Decoction group: Compared with the model group,the color,volume,hardness,elasticity and nodular changes of the lungs in each dose group improved,and on the 7th day,the positive drug group and There was significant difference between the high-dose and middle-dose groups of Jiawei Buyang Huanwu Decoction and the model group(P<0.01 or P<0.05).On the 14 th day,there was a significant difference between the positive drug group and the high-dose group of Jiawei Buyang Huanwu Decoction group(P<0.01 or P<0.05).On the 28 th day,there was a significant difference between the high-dose group of Jiawei Buyang Huanwu Decoction and the model group(P<0.01).Compared with the high-dose group of Jiawei Buyang Huanwu Decoction,there was no significant difference in alveolitis scores at each time point(P>0.05).(2)Masson staining: The staining results showed that the collagen fibers were green,the muscle fibers were red,and the red blood cells were orange-red.The 14-day Masson staining showed that the model group had widened alveolar space,increased interstitial fibroblasts,and collagen fibers.Increased formation of diffuse pulmonary fibrosis.Positive drug group and Jiawei Buyang Huanwu Decoction groupshowed significant changes compared with the model group,pulmonary interstitial fibroblasts decreased,alveolar space narrowed,pulmonary fibrosis was alleviated.2.Study on serum TNF-α,TGF-β and HMGB1 in rats with pulmonary fibrosis by Jiawei Buyang Huanwu Decoction:(1)TNF-α: Compared with BLM model group,positive drug group and Jiawei Buyang Huanwu Decoction group The expression of TNF-α was decreased in different degrees.The expression of TNF-α protein in tracheal supernatant and serum of rats in BLM model group was significantly increased(P<0.001).The middle dose group,high dose group and positive drug group were on trachea.The expression of TNF-αprotein in serum and serum was significantly decreased(P<0.01).Compared with the BLM model group,the expression of TNF-α in the middle dose group,high dose group and drug group of Jiawei Buyang Huawu Decoction was used in each sample.There were significant differences in the middle(P<0.01).There was no significant difference in the expression of TNF-α between the high dose group and the positive drug group at different time points(P>0.05).On day 14,the expression of TNF-α in the tracheal supernatant of the low-dose group was significantly lower than that of the BLM model group(P<0.01).(2)TGF-β:TGF-β in the tracheal supernatant and serum of the BLM model group The expression level of protein was significantly increased(P<0.001).Compared with BLM group,the expression of TGF-β protein in tracheal supernatant was significantly decreased in middle dose group,high dose group and positive drug group(P<0.01);Compared with the high-dose group and the positive drug group,the expression of TGF-β was significantly different in each sample(P<0.01).The high-dose group and the positive drug group had TGF-β at different time points and different samples.There was no significant difference in the expression of TGF-β(P>0.05).On the 14 th and 28 th day,the expression of TGF-β in the middle dose group was not significantly different from that in the BLM model group(P>0.05).(3)HMGB1: Compared with the BLM model group,positive drugs and flavored yang at each time point The contents of HMGB1 in rats were significantly decreased in the middle and high dose groups(P<0.001).On the 7th,14 th and 28 th days,there was no significant difference between the high dose group of Bu Yang Huan Wu Tang and thepositive drug group(P>0.05).This shows that the effect of high-dose plus Bu Yang Huan Wu Decoction in inhibiting HMGB1 is consistent with that of the positive drug group;the effect of Jiawei Buyang Huanwu Decoction high-dose group is significantly better than that of the middle and low-dose groups.3.The effect of Jiawei Buyang Huanwu Decoction on the expression of Fas and FasL mRNA in lung tissue of rats with pulmonary fibrosis:(1)Immunosorbent assay: positive drug group and Jiawei Buyang Huanwu Decoction group: Fas protein expression on the 7th day At 14 days and 28 days,Fas positive expression was found in the pulmonary interstitial and alveolar wall,and there was significant difference compared with the model group(P<0.05 or P<0.01).There was no significant difference between the dose groups of Jiawei Buyang Huanwu Decoction and the prednisone group(P>0.05).(2)Western blot: Compared with the model group,the expression of Fas and FasL protein in lung tissue was significantly decreased in the high,middle and low doses of Jiayang Huanwu Decoction.The high-dose group of Jiawei Buyang Huanwu Decoction can effectively inhibit the expression of Fas and FasL proteins in lung tissue,and the effect was not significantly different from that of the positive drug group.(3)qRT-PCR: positive drug group and the Jiawei Buyang Huanwu Decoction group on the 14 th day,the positive drug group and the Jiawei Buyang Huanwu high and middle dose groups were significantly different from the model group(P< 0.001);There was no significant difference between the low-dose group of Jiawei Buyang Huanwu Decoction and the model group(P>0.05).FasL mRNA expression on the 14 th day,the positive drug group and Jiawei Buyang Huanwu Decoction group were significantly different from the model group(P < 0.001);Jiawei Buyang Huanwu Decoction low dose group compared with the model group Significant difference(P>0.05).There was no significant difference between the high-dose group of Jiawei Buyang Huawu Decoction and the positive drug group(P>0.05).4.The effect of Jiawei Buyang Huanwu Decoction on the apoptosis factor Caspase-3 in lung tissue of bleomycin-induced rat:(1)Flow cytometry: Caspase-3 content in lung tissue of each dose group of Jiawei Buyang Huanwu Decoction It was higher than the model controlgroup.Among them,Jiawei Buyang Huanwu Decoction and medium dose group promoted the release of Caspase-3.The content of Caspase-3 in Jiawei Huanwu Decoction high-dose group was significantly different from that in the positive drug group(P<0.05),indicating that the high-dose group of Jiawei Buyang Huanwu Decoction increased the release of Caspase-3 compared with the positive drug group.More obvious.(2)Western blot: The expression of Caspase-3 protein was up-regulated on the 7th,14 th and 28 th day.On the 14 th day of the experiment,the effect of the Chinese medicine intervention group was the most obvious.The expression of Caspase-3 protein was positively correlated with the dose of the medium.Jiawei Buyang Huanwu Decoction significantly inhibited the expression of Caspase-3,and the effect was consistent with the positive drug group.Conclusion:1.Jiawei Buyang Huanwu Decoction significantly reduced the inflammation and pulmonary fibrosis of alveolitis in rats with pulmonary fibrosis,and its effect was similar to glucocorticoid.At present,it is necessary to develop new treatment plans for interstitial lung diseases,and actively carry out research on the intervention mechanism of Bu Yang Huan Wu Tang on interstitial lung diseases.2,Jiawei Buyang Huanwu high dose group inhibited alveolitis and pulmonary fibrosis,reduced the expression of Fas,FasL mRNA,TGF-β,HMGB1 factor,significantly better than the middle and low dose groups.Therefore,the high-dose group of Jiawei Buyang Huanwu Decoction has a good dose-effect relationship for prevention and treatment of pulmonary fibrosis,which may be the most suitable clinical application and prevention dose.3.Jiawei Buyang Huanwu Decoction can improve alveolitis by inhibiting the expression of TNF-α in the serum of rats with pulmonary fibrosis;inhibiting TGF-β,HMGB1,Fas,FasL mRNA,HMGB1 in lung tissue of rats with pulmonary fibrosis The expression,improve the abnormal metabolism of extracellular matrix,promote the release of apoptotic factor Caspase-3,prevent the proliferation and replication of fibroblasts,which may be one of the mechanisms of the application of Bu Yang Huan Wu Decoction inpreventing and treating pulmonary fibrosis.Control the purpose of pulmonary fibrosis. |
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