| Objective:To study the ideas and herbal components in traditional Chinese medicine for the treatment of pulmonary fibrosis.To explore the effects of osthole on treating bleomycin-induced pulmonary fibrosis in mice and its mechanisms from the point of TGF-?1/Rac-1 mediated oxidative stress.Methods:To study the recordation of Atrophic Lung Disease in ancient traditional Chinese medicine(TCM)literatures and discuss the connotation of pulmonary fibrosis in TCM.To analyze the pathogenesis,treatment principles and medications of Atrophic Lung Disease in TCM.Enlightened by the clinical experience and theory of Prof.Zhang Wei,treating pulmonary fibrosis with Warming Kidney Yang were discussed.Finally,common herbs in treating pulmonary fibrosis such as Qianhu(Radix Peucedani),Danggui(Angelica Sinensis),Baizhi(Radix Angelicae),Buguzhi(Fructus Psoraleae)and Shechuangzi(Fructus Cnidii)were investigated,and from which osthole was targeted as an effective monomer in anti-pulmonary fibrosis.Then in vivo and in vitro experiments were done to certify the hypothesis.In vivo: C57BL/6J strain mice were randomly divided into control group,BLM group,BLM+OST group and OST group.Pulmonary fibrosis animal models in mice were established by intratracheal injection of bleomycin(3.5mg/kg).From the operation day,in mice of BLM+OST group and OST group,osthole(40?mg/kg)? was administered by gavage?daily;and in mice of control group and BLM group,0.5% CMC-Na was administered by gavage?everyday.28 days later,mice were sacrificed and lungs were harvested.Lung index was recorded.Inferior lobe of right lung was fixed for HE,Masson to measure inflammation,fibrosis.The rest lungs were kept in liquid nitrogen.After homogenate,concentrations of hydroxyproline(HYP),MDA,T-SOD,T-AOC were measured by biochemical analysis.And with ELISA kit or Western blot,level of TGF-?1 and Rac1 in lungs of the mice were detected.In vitro: In LDH assay,MLFs were cultured with four different concentration of OST(0.1?mol/L to 50?mol/L)for 48 h.In other assays,cells were devided into five groups: control,TGF-?1,TGF-?1+OST(1?mol/L),TGF-?1+OST(10?mol/L),TGF-?1+OST(50?mol/L).LDH assay was used to test the toxicity of osthole to mouse lung fibroblasts.CCK-8 assay,cell counting and 3H-proline uptake assay were used to assess the proliferation,collagen synthesis of MLFs.Western blot and immunofluorescence assay were applied to determine the expression of ?-SMA,Rac-1,collagens I and collagen III.And DCFH-DA assay were to test the level of ROS in the cells.Results:Pulmonary fibrosis belongs to Atrophic Lung Disease in TCM,with the pathogenesis of deficiency in origin and enrichment in symptom.In treatment,excepting dispelling pathogenic factors,it is also important to build up healthful vital energy,especially warming Kidney Yang.Shechuangzi(Fructus Cnidii)could warm Kidney Yang.It contains osthole,which has significant effect in anti-pulmonary fibrosis.In vivo:(1)OST could improve bleomycin-induced body weight loss and rised lung index;(2)OST could alleviate bleomycin-induced alveolitis;(3)OST could alleviate bleomycin-induced interstitial fibrosis,and OST also decreased the content of HYP in the fibrotic lung tissue;(4)ELISA and Western blot showed,OST could significantly down-regulate bleomycin-induced expression of protein TGF-?1 and Rac-1;(5)Results of biochemical analysis showed,OST could obviously decrease the content of MDA and increase the activity of total-SOD and total-AOC.In vitro:(1)0.1-50?M osthole showed no toxicity to MLFs;(2)OST could concentration dependently inhibit TGF-?1-induced MLFs proliferation,collagen synthesis;(3)OST could concentration dependently inhibit TGF-?1 induced?-SMA expression and phenotype transformation in MLFs;(4)OST could concentration dependently inhibit TGF-?1-induced Rac-1 expression and ROS generation in MLFs.Conclusion:Osthole could significantly attenuates bleomycin-induced pulmonary fibrosis in mice,with the mechanism of down-regulating TGF-?1/Rac-1 in lung fibroblasts and inhibiting oxidative stress in lung. |
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