Protective Effects Of Gentiopicroside Against Pulmonary Fibrosis

Objectives:To investigate the protective effects of gentiopicroside(GPS)against Bleomycin(BLM)-induced pulmonary fibrosis in mice,confirm the anti-pulmonary firosis effect in vitro,and preliminarily explore the mechanism of action.Methods:1.Pulmonary fibrosis was induced in mice by single tratracheal instillation of bleomycin,and GPS was injected intraperitoneally once a day for consecutive 28 days.Then all mice were sacrificed to collect BALF and lung tissue samples.The pulmonary coefficient was calculated.2.Hemotoxylin and eosin(HE)and masson’s trichrome staining were used for pathological examination.3.The protein concentration of BALF were detected by BCA Protein Assay Kit4.The content of hydroxyproline in lung tissue were measured by Hydroxyproline Assy Kit.5.RAW264.7,A549 and MRC-5 cells were cultured in vitro and the cell growth curves were assessed by MTT.6.The toxicity of different concentrations of GPS on RAW264.7,A549 and MRC-5 cells were assessed by MTT and IC50 values of GPS were calculated.7.In GPS BLM-induced A549 cells incubated with GPS,the expression of apoptosis-related proteins Bax and Bcl-2 were detected by immunocytochemistry(ICC).The apoptotic rate of A549 cells were measured by flow cytometry.8.In TGF-β1-induced A549 cells(which differentiates to firoblasts by EMT)incubated with GPS,the expression of E-cadherin and a-SMA were detected by immunocytochemistry(ICC).9.In TGF-β1-induced MRC-5 cells(which differentiates to firoblasts)incubated with GPS,the expression of a-SMA were detected by immunocytochemistry(ICC).10.Immunocytochemistry(ICC)and Western blotting were employed to examine the protein of NF-κB-p65 in LPS-induced RAW264.7 cells incubated with GPS.Results:1.Different concentration of GPS reduced the lung coefficient of mice with BLM-induced pulmonary fibrosis.2.Histopathological evalution:in NS group,the lung structure including alveolar space and alveolar septum were normal,no inflammatory cells were found in alveolar space and lung interstitium;in model group,there were inflitration of a large number of inflammatory cells,thickness of the alveolar walls,the collapse of alveoli,pulmonary consolidation and interstitial fibro-plastic proliferation;compared with the model group,the pathological changes were relieved obviously in DXMS and GPS therapy groups.3.Compared with the model group,the protein concentration and the ratio of inflammatory cells in BALF of mice were reduced obviously in DXMS and GPS therapy groups.4.Compared with BLM model group,lung HYP content of mice in both of DXMS and GPS therapy groups were decreased(P<0.05).5.The RAW264.7、A549 and MRC-5cells were well-grown.According to the growth curve,the cells in the experiment were in the logarithmic phase.Therefore,0.8 ×105 cells/mL was selected as the seed plate concentration.6.The IC50 values of GPS to RAW264.7,A549 and MRC-5 were over 2mg/ml.7.The expression of apoptosis-related proteins Bax were decreased and Bcl-2 were increased in BLM-induced A549 cells in both of DXMS and the high dose GPS therapy groups.The apoptosis rate of BLM-induced A549 cells were detected by flow cytometry.Compared with blank group,the apoptosis rate were increased in negative control group(P<0.05).Compared with negative control group,the apoptosis rate were decreased in differentiation concentration of GPS groups.8.Effects of GPS on EMT induced by TGF-0 1 in A549:Compared with the blank control group,the expression of E-caherin protein was decreased in the negative control group(P<0.05);compared with the negative control group,the expression of E-caherin protein in high and middle dose group of GPS group and the DXMS group was significantly increased(P<0.05).Compared with the blank control group,the a-SMA protein levels in the negative control group were increased significantly(P<0.05);Compared with the negative control group,the a-SMA protein levels in the DXMS group and differentiation concentration of GPS groups were decreased(P<0.05).9.The result of immunocytochemistry(ICC):Compared with blank control group,the expression of NF-κB-p65 protein in negative control group was increased(P<0.01);Compared with the negative control group,the expression of NF-κB-p65 protein in DXMS group and GPS groups were decreased(P<0.01).The result of Western blotting:Compared with blank control group,the expression of NF-κB-p65 protein in the negative control group was increased(P<0.05);Compared with the negative control group,the expression of NF-κB-p65 protein in the DXMS group and GPS groups were decreased(P<0.05).Conclusions:1.GPS can alleviate the pathological changes of BLM-induced pulmonary fibrosis in mice,and reduce the proliferation of collagen fibers,GPS has the effects of anti-pulmonary firosis.2.GPS can reduce the lung coefficient of mice,and reduce the HYP content,GPS has anti-inflammatory effects.3.GPS can down-regulate Bax expression and up-regulate Bcl-2 expression in BLM-induced A549 cells;GPS can inhibit the apoptosis of alveolar epithelial cells induced by BLM.4.GPS can significantly prevent EMT in alveolar epithelial cells A549 induced by TGF-β1.5.GPS can down-regulate the expression of NF-κB-p65 protein in LPS-induced RAW264.7 cells.