Study On The Mechanism Of Qingluo Yin's Intervention In AE-IPF TGF-?₁/Smads Signal Transduction Pathway Based On The Structure-activity Theory Of Lungs

Purpose:Through the experiment of AE-IPF rat model,the traditional Chinese medicine compound Qing Luo Yin prepared by the theory of lung complex structure was used to intervene to explore the regulation of TGF-?₁/Smads signaling by Chinese herbal compound Qing Luo Yin,and to study the intervention of traditional Chinese medicine compound Qingluo Decoction.The mechanism of action of AE-IPF and evaluation of the regulation effect of Chinese herbal compound Qing Luo Yin on AE-IPF provide an effective and reliable theoretical basis for the treatment of AE-IPF with Chinese herbal compound.Materials and Methods:1.Establishment of acute exacerbation model of pulmonary interstitial fibrosis in rats 1.1 Ninety clean-grade male rats were randomly divided into 3 groups with 30 rats in each group,namely blank group,IPF group and AE-IPF group.The experimental rats were fed in animal experiment center for the first time after 3 days of feeding.The experimental rats in IPF group and AE-IPF group were prepared into 10mg/ml injection according to the bleomycin of 7.5mg/kg.According to the body weight of the experimental rats,the rats were injected with the concentration of 0.3ml/100g in the trachea.Seven days after the completion of the first model,the rats in the AE-IPF group were given the second tracheal injection of0.3ml/100g according to their body weight.The acute exacerbation stage model of IPF was established by injecting the rats'trachea for the second time according to the body weight.According to the dynamic pathological changes of pulmonary fibrosis,the samples and indexes were observed at 7 d,14 d and 28 d after modeling.1.2 The general survival state of three groups of rats was observed,and the dry/wet weight ratio,lung coefficient and so on were calculated.1.3 Hydroxyproline content?HYP?in lung tissue of three groups of rats was measured at three time points.2.Study on the mechanism of Chinese herbal compound Qing Luo Yin in inducing AE-IPF TGF-?₁/smads signaling pathway induced by bleomycin in rats2.1 90 clean male rats were randomly divided into 3 groups,30in each group,namely control group,AE-IPF group,Chinese medicine group and hormone group.The lung tissues of 10 experimental rats were taken out at 7 days,14 days and 28 days after the administration of the experimental rats,and the indicators were observed.2.2 The general survival status,dry-to-wet weight ratio,lung coefficient,HE staining,Masson staining,etc.of the three groups of experimental rats were observed.2.3 To detect the content of hydroxyproline in the lung tissue of each group of rats;2.4 Immunohistochemistry,western-blot and RT-PCR were used to detect the expression of TGF-?₁,Smad3,Smad7 protein and mRNA in lung tissue of three groups of rats at three time points,and to explain the intervention experiment of Chinese herbal compound Qingluo Decoction.Mechanism of action of rat AE-IPF TGF-?₁/smads signaling pathway.Results:1.Establishment of acute exacerbation model of pulmonary interstitial fibrosis in rats 1.1 General state:After the first modeling,compared with the control group,the experimental rats in the IPF group and the AE-IPF group were generally in poor condition,and the general status of the experimental rats in the IPF group and the AE-IPF group did not differ;after the second modeling,compared with the control group,and general condition of IPF group AE-IPF group rats worse,rapid breathing significant compared to AE-IPF group rats and IPF group,like grasping the nasal and stick the neck performance,body weight showed a downward trend,and mortality increased significantly.1.2 Pulmonary coefficient:After the second modeling,compared with the IPF group and the control group,the lung coefficient of the experimental rats in the AE-IPF group was significantly increased.1.3 Hydroxyproline content in lung tissue:After the second modeling,the hydroxyproline content in the lung tissue of the AE-IPF group was significantly increased compared with the IPF group and the control group.2.Study on the mechanism of Chinese herbal compound Qing Luo Yin in inducing AE-IPF TGF-?₁/smads signaling pathway induced by bleomycin in rats2.1 Successfully construct a rat AE-IPF animal experimental model,and optimize and explore methods and conditions close to the human body model;2.2 Chinese herbal compound Qing Luo Yin significantly improved the general state of AE-IPF in experimental rats at three time points and reduced mortality.2.3 Chinese herbal compound Qing Luo Yin can significantly reduce the hydroxyproline content in lung tissue at three time points of AE-IPF,reduce the collagen content in lung tissue and reduce the deposition of ECM.2.4 Qing Luo Yin could up-regulate the expression of TGF-?₁,smad3 protein and mRNA in the signal transduction pathway of TGF-?₁/smads in experimental rats with AE-IPF.The expression of smad7 protein and mRNA in the TGF-?₁/smads signal transduction pathway of the experimental rat AE-IPF model was down-regulated,and the effect of the TGF-?₁/smads signal transduction pathway on the lung tissue of the experimental rats was brought into full play,thus the process of controlling the idiopathic pulmonary fibrosis was achieved.Compared with the blank group,the expression levels of TGF-?₁,smad3 protein and mRNA in the lung tissue of the model group were significantly up-regulated,while the expression of smad7 protein and mRNA were significantly decreased?P<0.05?.The results showed that smad3 and smad7 could promote and inhibit the expression of protein and mRNA.Compared with the model group,the expression levels of TGF-?₁,smad3 protein and mRNA in the lung tissue of the experimental rats in the traditional Chinese medicine group and hormone group were decreased in different degrees after the AE-IPF model was induced by Qing Luo Yin bleomycin?P<0 05?,and compared with the model group?P<0 05?.The expression of smad7 protein and mRNA were up-regulated?P<0.05?,which indicated that methylprednisolone and Qing Luo Yin could regulate the promotion and inhibition of smad3and smad7 protein and mRNA in lung tissue of AE-IPF model rats,compared with those of hormone group.There was no significant difference in the expression of TGF-?₁,smad3,smad7 protein and mRNA in the lung tissue of the experimental rats in the Chinese medicine group?P>0 05?.Conclusion:1.The experimental rats were injected with bleomycin twice before and after the tracheal interval,and the AE-IPF model of the experimental rats was successfully induced.It is close to the clinical manifestations and observation indexes of human AE-IPF,and can be used as AE-IPF.The mouse experiment observed the model.2.Chinese herbal compound Qing Luo Yin can effectively improve the general state and clinical manifestations of AE-IPF rats,reduce mortality,reduce collagen content in lung tissue,reduce deposition of ECM,and improve experimental rats.AE-IPF course of disease.Chinese herbal compound Qing Luo Yin delayed the development of AE-IPF in experimental rats by regulating the expression of TGF-?1,smad3,smad7 protein and mRNA in AE-IPF TGF-?1/smads signaling pathway.Anti-pulmonary fibrosis.