Study On The Mechanism About The Inhibitory Effects Of Tanshinone â…¡ On Pulmonary Fibrosis

Idiopathic Pulmonary Fibrosis(IPF) is a serious disease which threatens human health and life; its morbidity and mortality increased in recent yeas. The pathogenesis of IPF remains unclear; and it is lack of effective treatment. Evidences showed there had no effective drugs which could heal IPF except lung transplantation according to 2011 Guide. But studies suggested that some drugs might be beneficial to IPF, which implies it could be important to look for effective drugs to treat and control the progress of pulmonary fibrosis.Tanshinone II A(Tan II A) is a bioactive constituent extracted from the root of salvia miltiorrhiza Bunge, a Chinese herbal medicine. Tan II A is used in the treatment of cardiovascular disease in clinic. It is not only a specific molecular structure, but also the highest levels of many tanshinone compositions, the most stable and effective active ingredient, with most of the drug effect of salvia miltiorrhiza. A removal of oxygen free radicals, anti-inflammatory, anti-tumor activities, cardioprotective and neuroprotective effects of Tan II A have been reported in many earlier studies. Studies have shown that Tan II A can relieve the bleomycin-induced pulmonary fibrosis in rats. The excessive infiltrations of alveolar macrophages contribute to inflammation of lung fibrosis. Our study showed that lung expression of CD68, a specific mark of alveolar macrophage, was decreased after treating with Tan II A in bleomycin-induced pulmonary fibrosis which suggested Tan II A can inhibit inflammatory response. Rats model of pulmonary fibrosis reduced by bleomycin were used to confirm Tan II A can inhibit lung inflammation and alleviate fibrosis of lung by inhibiting collagen deposition.The pathological character of IPF was the accumulation and proliferation of undetermined mesenchymal cell including fibroblast and myofibroblast. The latest studies suggested IPF was induced by alveolar epithelial injury, EMT and formation of fibroblast foci. EMT was considered the main source of fibroblasts.EMT manifested as cells obtained characters of fibroblast as decreased of E-cadherin and increased of mesenchymal cells markers such as vimentin, fibronection and alpha smooth muscle actin. EMT was closely associated with pulmonary fibrosis, which had become the focous in the pathogenesis of pulmonary fibrosis.Studies showed that EMT of breast cancer cell lines as MCF-7 and HCC1973, and liver cancer cell were blocked and by Tan II A. Our previous work demonstrated that Tan II A could inhibit lung fibrosis, which suggested Tan II A might inhibit EMT to alleviate fibrosis. However, few studies have been carried about the molecular mechanism of Tan II A inhibiting EMT in pulmonary fibrosis. Lung expression of E-cadherin decreased and expression of Fibronection and Vimnetin increased in rats with pulmonary fibrosis; but these factors expressions could be reversed after treated by Tan II A, which confirmed the inhibitory effect of Tan II A on EMT. So, further study should be continued to explore the mechanism of Tan II A inhibitory effect on EMT.Transforming growth factor-β(TGF-β) is a kind of multifunctional cytokine by affecting cell proliferation, differentiation, apoptosis, extracellular matrix(ECM) formation to adjust the morphogenesis and differentiation of the organization. TGF-β was considered as recognized fibrosis factor and induced transformation of EMT, including renal tubular epithelial cells, corneal epithelium cells and alveolar epithelial cells. TGF-β/Smad is an important signaling pathway, participating in the EMT of pulmonary fibrosis. Smad is downstream factor of TGF-β. Studies have shown that TGF-β can be induced EMT through activating Smad-2/3 and promote the formation of pulmonary fibrosis. Renal interstitial fibrosis could be relieved through inhibiting the TGF-β1/Smads signaling pathway in renal interstitial fibroblasts after dealing with Tan II A. Besides, inflammations in pulmonary fibrosis and lung expression of TGF-β1 decreased by Tan II A management. But mechanism about the effect of Tan II A on TGF-β depended EMT need to be further study. Therefore, in the present study, we hypothesized that Tan ⅡA could mitigate TGF-β-related EMT process during IPF progress and investigated the potential molecular mechanisms in vivo and in vitro.Caveolin-1 is a iconic protein of caveola, a small uncoated pit in the cell membrane, which plays important role in maintaining the structure and function of caveola. Caveolin scaffolding domain(CSD), interacting with other signaling factors in Caveolin-1, can down-regulate activity of many signaling factors. Caveolin-1 were expressed in many differentiated cells, especially in alveolar type I epithelial cells,smooth muscle cell, fibroblasts, adipose cells, endothelial cells and other terminally differentiated cells, which implied Caveolin-1 might be a kind of negative regulate factor. In recent years, studies showed that expression of Caveolin-1 decreased in lung of pulmonary fibrosis; meanwhile, mice of Caveolin-1 gene being knock-out showed lung changes as pulmonary fibrosis; besides, Caveolin-1 expression in epithelial cell decreased in lung after dealing with TGF-β1. These results demonstrated that Caveolin-1 was related with pulmonary fibrosis. Caveolin-1 together with type I, ⅡTGF-β1 receptor and Smad2 located in cellular region with rich plasma membrane. Therefore, Caveolin-1 can inhibit TGF-β/Smad signaling pathway by preventing TGF-β to combine with its receptor. In the study regarding inhibitory effect of Tan II A on the TGF-β induced EMT, we discovered Tan II A could suppress BLM-induced activity of TGF-β/Smad signaling pathway, alveolar macrophages increased, collagen deposition and EMT; in vitro, Tan ⅡA was confirmed to inhibit the TGF-β/Smad pathway in A549 and TGF-β induced EMT. But the mechanism of Tan ⅡA inhibiting TGF-β/Smad and TGF-β induced EMT remains unclear. Caveolin-1, the upstream factor of TGF-β, decreased in pulmonary fibrosis and it inhibited TGF-β/Smad pathway to remit lung fibrosis. Since both caveolin-1 and Tan ⅡA had the effect of inhibiting TGF-β signaling pathways in pulmonary fibrosis, the effect of Tan ⅡA might be related with caveolin-1. Experiment in vitro was performed to test whether Tan ⅡA could regulate TGF-β/Smad induced EMT by caveolin-1. We aim to explore the effect of Tan ⅡA on treatment for pulmonary fibrosis and its mechanism to provide more theoretical basis for new drugs development.Part I Tanshinone IIA inhibited rat pulmonary fibrosis induced by bleomycinObjective: To study the effect of Tan ⅡA on lung morphology, type I collagen and hydroxyproline content to definite protective role of Tan ⅡA on pulmonary fibrosis.Methods: Sprague-Dawley(SD) rats, 8 weeks, weighing about 250 g, were divided into four groups: Control group(intratracheal instillation of normal sterile saline), BLM group(intratracheal instillation of saline containing BLM, 5 mg/kg), TanⅡA group(rats received daily injection of Tan ⅡA) and BLM + Tan ⅡA group(BLM-induced rats received daily injection of Tan ⅡA). Rats from each group were sacrificed on day 28 after the initial instillation. Lung tissues were obtained to examinewet/dry weight ratio, to observe the pathological changes by HE and Masson’s staining, to measure the concentration of type Ⅰcollagen, hydroxyproline by ELISA and to detect expression of CD68, the lung macrophage marker, by immunohistochemistry.Results: The wet/dry weight ratio of BLM + Tan ⅡA group was significantly lower than that in the BLM group. BLM instillation induced severe pulmonary lesions in rats, including thickened interstitium and excessive infiltration of inflammatory cells as compared with control group. As expected, these pathological alterations were significantly attenuated by intraperitoneal injection of Tan ⅡA compared with BLM group. Similar inhibitory effect of Tan ⅡA on BLM-stimulated aberrant collagen deposition in the rat lung was also observed. Also, BLM-induced up-regulation of collagen Ⅰand hydroxyproline and CD68 was markedly suppressed by Tan ⅡA administration.Conclusion:(1) Tan ⅡA alleviated lung inflammatory response in BLM-induced pulmonary fibrosis by inhibiting the infiltration of lung alveolar macrophages.(2) Tan ⅡA reduced ECM deposition by inhibiting lung expression of type Ⅰcollagen and hydroxyproline to relieve pulmonary fibrosis.Part II Study on the mechanism of tanshinone ⅡA inhibiting TGF-β depended EMTObjective: To study the impact of Tan ⅡA on EMT and TGF-β/Smad signaling pathway in rats lung tissue with pulmonary fibrosis induced by BLM and A549 cells stimulated by TGF-β1 to discuss the mechanism of tanshinone ⅡA alleviating pulmonary fibrosis.Methods: SD rats, 8 weeks, weighing about 250 g, were divided into four groups: Control group, BLM group, TanⅡA group and BLM + Tan ⅡA group. Rats from each group were sacrificed to obtain lung tissues on day 28 after the initial instillation. Cells were divided into four groups: Control group(treated with 10 μM DMSO for 2 h), Tan ⅡA group(treated with 10 μM Tan ⅡA for 2 h), TGF-β1 group(stimulated with 10ng/ml recombinant TGF-β1for 48 h) and TGF-β1+ Tan ⅡA group(treated with 10μM Tan ⅡA for 2 h, followed by stimulation with 10 ng/m L recombinant TGF-β1 for 48 h). E-cadherin was examinated by immunofluorescence; TGF-β1 was detected by real-time PCR; TGF-β1、Smad2、p-Smad2、Smad3、p-Smad3、 E-cadherin、Fibronectin and vimentin were detected by western blot.Results: Immunofluorescence staining assay was used to detect the expression of epithelial marker, E-cadherin. Lung tissues from rats treated with BLM showed less positive staining cells of E-cadherin when compare with these from normal control or Tan ⅡA-treated control rats. This pulmonary reduction of E-cadherin could be partially reversed by Tan ⅡA injection. Western blot confirmed the immunofluorescence staining results. To the contrast, the expression levels of mesenchymal markers, fibronectin and vimentin, increased in BLM-treated rats and almost decreased to normal levels after Tan ⅡA treatment. Western blot method and real-time PCR showed BLM group, TGF-β1 groups compared with control group, respectively, E-cadherin expression decreased obviously, Fibronectin and vimentin protein expression significantly increased, TGF-β1 RNA and protein expression, p-Smad2 and p-Smad3 expression were higher. Tan ⅡA interventional therapy group, all the above factor expression reversed. Tan ⅡA have no effect on the expression of Smad2 and Smad3, but it affects Smad2 and Smad3 level of phosphorylation.Conclusion: Tan ⅡA ameliorated pulmonary fibrosis, probably through inhibition of TGF-β/Smad pathway-dependent EMT process. 。Conclusion:(1) Tan ⅡA alleviated BLM-induced pulmonary fibrosis by inhibiting lung EMT and activity of TGF-β/Smad signaling pathway.(2) Tan ⅡA relieved fibrosis by inhibiting TGF-β depended EMT in A549 cells stimulated by TGF-β1.Part III Tanshinone ⅡA suppressed TGF-β1 induced EMT in A549 cells by up-regulating caveolin-1Objective: To test whether Tan ⅡA could regulate TGF-β/Smad induced EMT by caveolin-1 to to provide more theoretical basis for new drugs development.Methods: High transfection efficiency of caveolin- 1 sh RNA expression vector was built for cell transfection. Cells are divided into 7 groups: Control group(A), Tanshinone ⅡA group(B)(10μM Tan ⅡA, 2h), Caveolin-1si RNA+ Tan ⅡA group(C)(transfection caveolin-1si RNA, 24h; 10μM tanshinone ⅡA, 2h), TGF-β1 group(D)(10ng/ml TGF-β1, 48h), TGF-β1+ caveolin-1si RNA group(E)(transfection caveolin-1si RNA, 24 h, then 10ng/ml TGF-β1, 48h), Tan ⅡA + TGF-β1 group(F)(10μM Tan ⅡA, 2h, and then 10ng/ml TGF-β1, 48h) and caveolin-1si RNA+ Tan ⅡA + TGF-β1 group(G)(transfection caveolin-1si RNA, 24 h, then 10μM Tan ⅡA, 2h, then with 10ng/ml TGF-β1, 48h). Caveolin-1, TGF-β1, E-cadherin, Fibronectin and vimentin expressions were detected by RT-PCR and western blot. p-Smad2 and p-Smad3 were detected by western blot.Results: TGF-β1 significantly reduced caveolin-1 expression compared with control group and Tan ⅡA treated control cells. Tan ⅡA pre-treatment restored caveolin-1 expression in A549 cells. Expression of Fibronectin and vimentin increased in TGF-β1-stimilated cells and decreased by pretreatment with Tan ⅡA. Factors changes as caveolin-1, TGF-β1, p-Smad2, p-Smad3, E-cadherin, Fibronection, Vimenin have no difference in A549 with or without Tan ⅡA. In transfection Caveolin-1sh RNA group, the expressions of TGF-β1, p-Smad2 and p-Smad3 increased; protein expression of E-cadherin decreased; fibronection and vimenin increased. In TGF-β1+caveolin-1sh RNA group, expression of TGF-β1, p-Smad2, p-Smad3, fibronection and vimentin increased significantly and E-cadherin decreased significantly, compared with TGF-β1 group. In caveolin-1sh RNA+ Tan ⅡA + TGF-β1 group, expression of TGF-β1, p-Smad2, p-Smad3, fibronection and vimentin increased significantly and E-cadherin decreased significantly, compared with Tan ⅡA +TGF-β1 group.Conclusion:(1) The expression of Caveolin-1 is increased by Tan ⅡA in A549 cells stimulated by TGF-β1.(2)Tanshinone ⅡA suppressed TGF-β1 induced EMT in A549 cells by up-regulating caveolin-1.