Studies On The Mechanism Of Shenks In Treatment Of Pulmonary Fibrosis

Objectives Recently, traditional Chinese medicine, with the characteristics of havingmore targets and being safe for long-term medication, showed some advantages in thetreatment of lung fibrosis. Shen-mai-kai-fei-san (Shenks), a Chinese herb preparation,developed by Yiling Hospital affiliated to Hebei Medical University, has been provedeffective in the therapy of scleroderma-associated lung fibrosis. The aim of the presentstudy is to explore the mechanism of Shenks in treatment of pulmonary fibrosis.Methods For in vivo studies, C57BL/6female mice of7weeks were randomly dividedinto four groups, normal control group, bleomycin group, shenks group, acetylcysteinegroup. Pulmonary fibrosis was induced by a single intratracheal instillation of bleomycin.Shenks and acetylcysteine dissolved in saline (4g/kg and490mg/kg, respectively) wasfeed to the mice daily from the Tenth day after bleomycin instillation. Histopathologicalstatus were assessed by hematoxylin and eosin (HE) and Masson’s trichrome stains; Thegene expression of some extracellular matrix (ECM) proteins, including collagen, andseveral cytokines, such as TGF-β, Ctgf, was examined by real-time quantitative (RT-PCR); collagen content of the lung samples was assessed by Sircol assay; and the proteinlevel of3-nitrotyrosine(3-NT), which is the oxidative damage product of proteins, inmice lung tissues was assessed by western blot. For in vitro studies, NIH/3T3fibroblastswere treated by transforming growth factor-β (TGF-β)(5ng/ml) to investigate the anti-fibrotic role of Shenks. There are three groups in the cell experiments, including normalcontrol group, TGF-β group and TGF-β+Shenks Group. xCELLigence System was usedto determine the optimal concentration of shenks.Real-time quantitative PCR wasperformed to examine the transcript levels of ECM proteins and oxidant/antioxidantrelated genes, and Sircol assay was used to measure the collagen protein secreted to thecell supernatant; while western blot was conducted to determine the protein levels ofCOL I,3-NT and p-Smad3in NIH/3T3fibroblasts. Luciferase activity assay wasperformed to measure the activity of SBE4reporter, which locate on the promoter ofcollagen genes.Results For in vivo study: Compared with normal control group, bleomycin couldinduce the occurrence of fibrosis in the lung tissue. The expression of ECM-related genes, collagen content and the content of protein oxidative damage product in the lung tissuewere significantly increased with bleomycin treatment. Moreover the gene expressionlevels of antioxidant-related were significantly reduced by bleomycin. Shenks oracetylcysteine significantly alleviated pulmonary fibrosis in mice, the correlation indexclose to normal levels. For in vitro study:TGF-β stimulation increased the mRNA level ofcollagen gene,oxidation-related genes, the collagen content and protein oxidativedamage product3-NT in NIH/3T3fibroblasts. The activity of SBE and thephosphorylation level of Smad3was increased as well.The mRNA expression ofextracellular superoxide dismutase (Ec-sod) in NIH/3T3fibroblasts significantly reducedwith TGF-β treatment. With the1mg/ml shenks intervention, the correlation index nearnormal levels.Conclusions Shenks inhibits collagen synthesis in both mouse model and cell model,and we considered that shenks inhibit fibrosis by shifting the oxidant/antioxidantbalance. Moreover, our in vivo study showed that the anti-fibrosis effect of shenkstreatment was better than acetylcysteine treatment. In addition, results of in vitro studydemonstrated that shenks inhibited fibrosis through blockade of TGF-β pathway.