| Objective: Pulmonary fibrosis is a chronic disease that seriously threatens human health and its morbidity rate has been rising in recent years..Glucocorticoids and immunosuppressants were used which had side effects and poor treatment.Chinese medicine has shown good prospects in the treatment of pulmonary fibrosis recently.In the previous experiment,mKG obtained on the basis of ancient prescriptions shows inhibition of pulmonary fibrosis.The purpose of this experiment is to investigate the therapeutic effect of mKG on BLM-induced pulmonary fibrosis,and to explore its mechanism.Method:1 Animals70 SPF male BALB/c mice were randomly divided into 7 groups: normal control group(NS),BLM model group(BLM),dexamethasone group(DEX),Pirfenidone group;mKG high-dose group(mKG-H),mKG middle-dose group(mKG-M),mKG low-dose group(mKG-L).The pulmonary fibrosis mice model was established by tracheal injection of bleomycin(BLM,2mg/kg).Then mice in NS and BLM model group were intragastrically treated with 0.9% saline,while other groups were intragastrically treated with different medicine.All mice were sacrificed on 21 th day.2 Record the body weight and lung coefficient of mice.Evaluate the extent of inflammation and fibrosis by the HE staining and Masson staining.3 Determine the expression level of IL-6,IL-17 in lung tissues of mice by RT-PCR assay.And evaluate the anti-inflammary effects of mKG on BLM-induced pulmonary fibrosis mice.4 Evaluate the effects of mKG on the deposition of collagen through the immunohistochemical staining of col-Ⅰ,col-Ⅲ and the expression level of HYP in the lung tissue of mice which were determined by ELISA assay.5 Evaluate the effects of mKG on the TGF-β1/Smad3 path in mice through the expression level o Smad2,Smad3,Smad7,TGF-β1,α-SMA in the lung tissue of mice which were determined by ELISA,RT-PCR and Western blot assay.Results:1 The body weight of mice in BLM model group is significantly lower than mice in NS group.The lung coefficient of mice in BLM group is significantly higher than mice in NS group.According to the HE staining and Masson staining,there are serious inflammation and collagen deposition in the lung tissue of mice in BLM model group.Inflammation score and fibrosis score of mice in BLM group is significantly higher than mice in NS group(P<0.01).The BLM-induced pulmonary fibrosis model of mice was successfully established.2 The mice in Pirfenidone,mKG-H,mKG-M groups are significantly heavier than mice in BLM group(P<0.01).The lung coefficient of mice in Pirfenidone,mKG-H groups were significantly lower than those of mice in BLM group(P<0.01).3 According to the HE staining and Masson staining,there are serious inflammation and fibrosis in the lung tissue of mice in BLM model group,while both inflammation and fibrosis in the lung tissue of mice in Pirfenifone,mKG-H and mKG-M groups are significantly lighter(P<0.01).Compared to the lung tissue of mice in BLM model group,the inflammation in the lung tissue of mice in DEX group are lighter to some extent(P<0.05).Inflammation and fibrosis are not significantly decreased in the lung tissue of mice in mKG-L group.4 The mRNA expression of IL-6,IL-17 were detected by RT-PCR assay.The mRNA expression of IL-6,IL-17 significantly increased in BLM model group compared with the NS group(P<0.01).When compared to the BLM group,the mRNA expression of IL-6,IL-17 decreased significantly in Pirfenidone,mKG-H and mKG-M groups(P<0.01).The mRNA expression of IL-6,IL-17 decreased in mKG-L group and DEX group to some extent(P<0.05).5 The expression of HYP were detected by ELISA assay.The expression of HYP were significant lower in the lung tissue of mice in Pirfenifone and mKG-H groups compared with those in BLM group(P<0.01).The expression of HYP were lower in the lung tissue of mice in mKG-M and DEX groups(P<0.05).According to the immunohistochemical staining of col-Ⅰ and col-Ⅲ,col-Ⅰ and col-Ⅲ increased significantly in the lung tissue of mice in BLM group compared with the NS group(P<0.01),while both col-Ⅰ and col-Ⅲ in the lung tissue of mice in Pirfenifone and mKG-H groups decreased significantly(P<0.01).Compared to the lung tissue of mice in BLM model group,col-Ⅰ and col-Ⅲ in the lung tissue of mice in mKG-M and DEX groups decreased to some extent(P<0.05).col-Ⅰ and col-Ⅲ didn’t show significant difference between lung tissues of mice in mKG-L group and BLM group.6 The mRNA expression of Smad2,Smad3,Smad7 were detected by RT-PCR assay.The mRNA expression of Smad2,Smad3 all significantly increased in BLM model group compared with the NS group(P<0.01).When compared to the BLM group,the mRNA expression of Smad2,Smad3 all decreased significantly in both Pirfenidone and mKG-H groups(P<0.01),and the mRNA expression of Smad3 in mKG-M group were significantly lower(P<0.01).The expression of Smad7 didn’t show difference.7 The expression of TGF-β1、α-SMA,P-Smad3,Smad3 were detected by Western blot assay.The expression of TGF-β1、α-SMA,P-Smad3 showed significant increment in BLM model group compared with NS group(P<0.01).When compared to the BLM model group,the expression of TGF-β1,α-SMA,P-Smad3 decreased significantly in DEX,Pirfenidone,mKG-H,mKG-M and mKG-L groups(P<0.01).The expression of Smad3 didn’t show significant difference.The expression of TGF-β1which were detected by ELISA assay were significant lower in the lung tissue of mice in Pirfenifone and mKG-H groups compared with those in BLM group(P<0.01).We also found that the expression of TGF-β1 in DEX,mKG-M and mKG-L groups were lower than those in BLM model group(P<0.05).Conclusion:1 The mKG could effectually inhibit pulmonary inflammation of pulmonary fibrosis mouse.2 The mKG has the effect of inhibition on pulmonary fibrosis of pulmonary fibrosis mouse.3 We proposed the mechanism of therapy of mKG accoring to the experimental data: mKG inhibit the expression of TGF-β1,and then down-regulate the expression of Smad2 and Smad3 through the TGF-β1/Smad3 path without the activation of Smad7. |
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