Study On The Interference Mechanism Of TH5 / TH2 / Smads Signal Transduction Pathway And TH1 / TH2 Intervention In Patients With IPF TGF - β

Purpose: Idiopathic pulmonary fibrosis(IPF) is the treatment problem of the modern medicine whit highly the incidence and mortality rate. Traditional Chinese medicine(TCM) has some advantages in the treatment of IPF. The study based on lung collaterals theory and its physiological structure and physiological function systemize clinical experience for many years, category the IPF into lung collaterals disease. We believe the pathogenesis of IPF which the acute phase is " lung heat blood stasis", remission of pathogenesis is "lung collaterals week and blood stasis”, and according the “Pungent dredging collaterals ”,“insect drugs dredging collaterals” and “dredging and supplement collaterals weaken”, form the standard description. Through the animal experiment, observe the traditional Chinese medicine compound granule(ginseng dragon decoction) intervene IPF TGF- beta 1 / Smads signal transduction pathways and TH1 / TH2 cytokines imbalances from multiple perspectives and targets in different time points. Explain the delay mechanism of idiopathic pulmonary fibrosis and provide a scientific basis for prevention and treatment of IPF, and lay the basis of new drug development.Material and method: 1.Animal in vivo experiments 1.1 Adopt the method of endotracheal injection with bleomycin in making animal model of idiopathic pulmonary fibrosis in rats 1.2 According to male and female and body weight,120 rats were randomly divided into six groups, each group were only 20. The six groups are named blank control group, model control group, western medicine group control group(prednisone acetate tablets group), and dragon decoction granules high dose group(Chinese traditional medicine high does group), and dragon decoctiongranules medium dose group(Chinese traditional medicine medium dose group), and dragon decoction granules low dose group(Chinese traditional medicine medium dose group).According to dynamic evolution law of pathogenesis of IPF, taking samples and measurement the index in 7d, 14 d and 28 d respectively. 1.3 The influence of TCM compound granules in the IPF rats’ model lung tissue form and lung coefficient Observe the rats model form and lung coefficient in different time points analysis and comparison between groups in the overall state and the dynamic evolution; By HE staining, MASSON staining with pathological semi-quantitative analysis, observe the dynamic changes of the pathological tissue morphology between groups of rats, evaluate the model rats of IPF and the effects of traditional Chinese medicine compound. 1.4 The influence of TCM compound granules of IPF model to the hydroxyproline content in model rats. Through the determination of hydroxyproline content in alkaline solution at different time points between groups, by observing the variations of hydroxyproline content in rats, evaluate the success of IPF model preparation and the effect of traditional Chinese medicine compound granules to the collagen fiber formation and the effect of collagen deposition. 1.5 The mechanism research of TCM compound granules coordinating IPF model rats’ THI/TH2 cytokine imbalance By ELISA, immunohistochemistry, Western blot and rt-pcr, reveal serum IL-4 at different time points and dynamic expression of INF- gamma expression and the INF IL- 4- gamma protein in lung tissue and m RNA from multi-level and multi-angle, clarify the mechanism of TCM compound granules coordinate the IPF model rats THI/TH2 imbalance 1.6 The study of TCM compound granules intervening IPF model rat TGF- beta 1 /Smads signal transduction pathwayBy immunohistochemistry, Western blot and rt-pcr, revealed TGF- beta 1, TGF beta RII, Smad3, Smad7 protein and m RNA expression of dynamic in the lung tissue at different time points, clarify the mechanism of TCM compound granules adjusting and regulation the IPF model rat TGF- beta 1 / Smads mechanism of signaling pathways. 2. Animal in vitro experiments 2.1 By trypsin digestion method primitive extraction and cultivate lung fibroblasts; 2.2 lung fibroblasts were divided into 5 groups, named blank groups, blank group which with the normal serum lung fibroblasts + serum of blank group, Model group with lung fibroblasts of model group rats serum + serum of blank group, Inhibitor groups with giving TGF- beta 1 inhibitors LY210976 the lung fibroblasts,Western medicine group with giving western medicine medicated serum intervention,TCM group with traditional Chinese medicine compound granules containing serum intervention model group rats lung fibroblasts, and respectively determine the index in 24 h, 48 h, 72 h; 2.3 Immunofluorescence method to detect the expression of vimentin and positioning, determine the success of lung fibroblasts appraisal; 2.4 By MTT colorimetry to detect cell proliferation, explore the proliferation effect of medicine serum of lung fibroblasts containing TCM compound granules; 2.5 The study of TCM compound granules containing medicine serum intervening lung fibroblasts TGF- beta 1 / Smads signal transduction pathway By immunohistochemistry, Western blot and rt-pcr, revealed TGF- beta 1, TGF beta RII, Smad3, Smad7 protein and m RNA expression of dynamic in the lung tissue at different time points, clarify the mechanism of TCM compound granules adjusting and regulation the lung fibroblasts TGF- beta 1 / Smads mechanism of signaling pathways.Results:1. Animal in vivo experiments 1.1 successful replication of IPF in rats in vivo animal models, and made the conditions optimization of model-making 1.2 TCM compound granules obviously improve the general condition of rats, effectively reduce the pulmonary coefficient and significantly improve IPF overall state of rats. 1.3 TCM compound granules can significantly reduce the degree of inflammation in the rats lung tissue and the degree of fibrosis by observing the pathological organization morphology, the compound Chinese medicine granules can effectively restrain the early alveolar inflammation, improve the deposition of lung fibrosis in the middle-late stage, especially in the medium dose of Chinese medicine compound granules. Its effect in inhibit inflammation in early stage is superior to prednisone(P < 0.01 or P < 0.05); 1.4 TCM compound granules can effectively reduce the content of hydroxyproline(HYP) from the aspects of biochemical and reduce the degree of IPF, which means the traditional Chinese medicine compound granules can inhibit HYP, reduce the collagen content in the lung tissue and the deposition of ECM, and alleviate the pulmonary fibrosis. Compared with western medicine group, hydroxyproline in the lung tissue at 7d reduced significantly(P < 0.05). 1.5 TCM compound granules can inhibit the rat serum IL- 4 expression, promote the INF- gamma expression in serum, can inhibit the expression of IL- 4 protein and m RNA of IL – 4 in lung tissue and promote INF- gamma protein and INF-gamma m RNA expression, to mediate TH1 / TH2 balance system and delay the process of pulmonary fibrosis; By drug intervention, compared with model control group, western medicine and Chinese medicine each dose groups model serum INF- gamma levels were significantly higher(P < 0.01 or P < 0.05), IL- 4 levels were significantly decreased(P < 0.01 or P < 0.05), suggesting the drug can promoteINF- gamma, and inhibit IL- 4. By immunohistochemistry, western blot and rt-pcr method, IL- 4, the protein and m RNA of INF- gamma test results in lung tissue, and serological test results are consistent, Chinese medicine medium dose effect is slightly better than western medicine group. 1.6 TCM compound granules can inhibit the TGF- beta 1, Tp RII, high Smad3 protein and m RNA expression which play a important role in TGF- 1.6 beta 1 / Smads signal pathway, promote the Smad7 protein and m RNA expression level which play a negative in pathways,and adjust the function of TGF –β1/ Smads signal,delay the process of pulmonary fibrosis, and the medium dose effect is best. Compared with the blank control group, the TGF –β1,TGF –βII and Smad3 in the lung tissue of rats in model control group and m RNA expression of significantly raised(P < 0.01 or P < 0.05), while Smad7 protein and m RNA expression significantly declined(P < 0.01 or P < 0.05), confirmed that the relationship between Smad3 and Smad7 competitive inhibition; After drug treatment, compared with model control group, the expression of TGF –β1,TGF –βII, m RNA and Smad3 in the dosage groups are declined to some extent(P < 0.01 or P < 0.05), at the same time the Smad7 protein and m RNA expression has raised(P < 0.01 or P < 0.05), suggesting Chinese medicine compound granules group comparing prednisone tablet have adjusted the relationship between Smad3 and Smad7 competitive inhibition role; Compared with western medicine group, lung tissue TGF –β1,TGF –βII, Smad3,Smad7 and m RNA expression in Chinese medicine medium dose groups level have no statistical difference. 2. Animals experiments in vitro 2.1 successful extraction and culture the rat lung fibroblasts and grope conditions of the extraction and cultured fibroblasts; 2.2 TCM compound granules can effectively restrain the drug-containing serum lung fibroblasts proliferation ability;2. 3 Chinese medicine compound granules containing serum can effectively restrain TGF –β1,Tp RII, Smad3 protein and m RNA expression which plays a important role TGF –β 1 / Smads signal pathway in the lung fibroblasts, promote Smad7 protein and m RNA expression level which play a regulation role in TGF-the role of beta 1 / Smads signal pathway, delay the process of pulmonary fibrosis.The result shows: the model group Fb endogenous TGF –β1,TGF –βII, Smad3 protein and m RNA expression level significantly higher than the blank group(P < 0.01 or P < 0.05), and the expression level of Smad7 is significantly lower than the blank group(P < 0.01 or P < 0.05), and proves that the model successfully simulated in vivo in the process of pulmonary fibrosis Fb activation and raised TGF –β1,TGF –βII, Smad3 protein, cut down the Smad7 protein and m RNA expression. The TGF-β1 Fb protein levels in inhibitor group were no statistical difference in 48 h, 72 h, illustrating TGF-β1 inhibitor is by inhibiting the biological activity and can’t reduce its content. Compared with model group, the TGF –β1,TGF –βII, Smad3 protein and m RNA expression levels of western medicine and Chinese medicine group Fb were significantly declined(P < 0.01 or P < 0.05), while Smad7 protein and m RNA level raised(P < 0.01 or P < 0.05), inhibitors of Fb Smad7 protein and m RNA in three test time levels were significantly higher(P < 0.01 or P < 0.05), TGF –βII, Smad3 protein and m RNA expression have delined in some degree(P < 0.01 or P < 0.05), and the influence of TGF –β1 protein and m RNA is not obvious. It is suggested two kinds of medicated serum could cut in Fb TGF –β1, TGF –βII, Smad3 protein and m RNA expression level, raise Smad7 protein and m RNA level, relieve fibrosis. Compared with inhibitor group, western medicine and Chinese medicine group in Fb TGF –β1, TGF –βII m RNA,Smad3 and m RNA, smad7 and m RNA in each test time had statistical significance(P < 0.01 or P < 0.05), while the TGF –β1 and TGF –βII protein expression was no statistically significant difference, suggesting the effect of TGF- beta 1 m RNA, TGF beta RIIm RNA, Smad3 and m RNA, samd7 and m RNA of the western medicine and Chinese medicine group within the Fb is obvious.Conclusion: 1. Through the animal experiment, the traditional Chinese medicine compound granules could adjust the TH1 / TH2 cytokines balance and regulating the TGF-β1 / Smads signal pathway, so as to delay the process of pulmonary fibrosis; multi-angle, targets 2. By animal experiment in vitro, traditional Chinese medicine compound granules containing medicine serum can effectively inhibit lung fibroblasts proliferation, regulating the TGF-β1 / Smads signal pathway, play a important role of anti fibrosis.