The Impact Of Graptopetalum On The Models Of Pulmonary Fibrosis Cell In Vitro Induced By TGF-β1

Objective:To observation of Graptopetalum solution(Graptopetalum solution,GP)on transforming growth factor beta 1(transforming growth factor beta-1,TGF-beta 1)mediated for human alveolar epithelial cell line A549 cells and human fibroblast cell line HLF cells of lung pulmonary fibrosis cell model in vitro,and radiation-induced pulmonary fibrosis mouse model in vivo,to explore whether Graptopetalum solution has anti pulmonary fibrosis effect.Methods:The first part of this experiment,use TGF-betal(5 ng/mL)stimulated human alveolar epithelial cell line A549 cells to construct pulmonary fibrosis model in vitro,Analyze E calcium proteins,collagen I and fibronectin mRNA and their protein expression to identify the model by morphological observation,fluorescence quantitative PCR,Western blot and immunohistochemistry.The experiment group of A549 cells were treated with GP(1250 μg/mL)and the positive control group were treated with dexamethasone(120 μg/mL).The intervention time were 48h and 72h,respectively.The proliferation of A549 cells in each group was detected by MTT,the apoptosis of A549 cells in each group was detected by flow cytometry,fluorescence quantitative PCR,Western blot and immunohistochemistry were applied to detect E calcium protein,I collagen and fibronectin mRNA and their protein expression.Finally,fluorescence quantitative PCR,Western blot and immunohistochemistry were applied to detect Smad on signaling pathway related protein Smad2,Smad4 and Smad7 mRNA and their protein expression.The second part of this experiment,use TGF-betal(5 ng/mL)stimulated human fibroblast cell line HLF cells to construct pulmonary fibrosis model in vitro,Analyze a-smooth muscle actin,collagen I and fibronectin mRNA and their protein expression to identify the model by fluorescence quantitative PCR,Western blot and immunohistochemistry.The experiment group of HLF cells were treated with GP(650 μg/mL)and the positive control group were treated with dexamethasone(120 μg/mL).The intervention time were 48h and 72h,respectively.The proliferation of HLF cells in each group was detected by MTT,the apoptosis of HLF cells in each group was detected by flow cytometry,fluorescence quantitative PCR,Western blot and immunohistochemistry were applied to detect a-smooth muscle actin,I collagen and fibronectin mRNA and their protein expression.Finally,fluorescence quantitative PCR,Western blot and immunohistochemistry were applied to detect Smad on signaling pathway related protein Smad2,Smad4 and Smad7 mRNA and their protein expression.Results:A549 cells were stimulated by TGF-betal(5 ng/mL)after 7 days,the cell gap broadening,morphology become like spindle shape;compared with routinely cultured A549 cells,E calcium protein mRNA and its protein expression were significantly reduced(P<0.05 or P<0.01),and the expression of collagen I and fibronectin mRNA expression were significantly increased(P<0.05 or P<0.01).The GP and DXM could inhibit the proliferation of A549 model(IC50=1249.617 μg/mL and 118.713 μg/mL),and to promote its apoptosis(P<0.05 or P<0.01,compared with model group),up-regulate the level of E calcium protein mRNA and protein expression(P<0.05 or P<0.01,compared with non-intervention group),down-regulate the level of collagen I and fibronectin protein mRNA and protein(P<0.05 or P<0.01 compared with non-intervention group);The GP could up-regulate the expression of E calcium protein and down-regulate the expression of collagen I and fibronectin protein,but not as good as DXM,there was no significant difference(P>0.05)between the two groups mRNA and its protein expression after the intervention of 72h.The GP and DXM could down-regulate the level of Smad2 and Smad4 protein mRNA and protein expression(P<0.01,compared with non-intervention group),up-regulate the level of Smad7 protein mRNA and protein(P<0.01 compared with non-intervention group);The GP could down-regulate the expression of Smad2 and Smad4 protein and up-regulate the expression of Smad7 protein,but not as good as DXM,there was no significant difference(P>0.05)between the two groups protein expression after the intervention of 72h.HLF cells were stimulated by TGF-betal(5 ng/mL)after 7 days,compared with routinely cultured HLF cells,α-smooth muscle actin,collagen I and fibronectin mRNA and its protein expression were significantly increased(P<0.05 or P<0.01).The GP and DXM could inhibit the proliferation of HLF model(IC50=647.620 μg/mL and 117.939 μg/mL),and to promote its apoptosis(P<0.01,compared with model group),down-regulate the level of a-smooth muscle actin,collagen I and fibronectin mRNA and protein expression(P<0.05 or P<0.01,compared with non-intervention group),The GP could down-regulate the expression of α-smooth muscle actin,collagen I and fibronectin protein,but not as good as DXM,but in addition to the GP on the regulation of fibronectin protein,there was no significant difference(P>0.05)between the two groups mRNA and its protein expression after the intervention of 72h.The GP and DXM could down-regulate the level of Smad2 and Smad4 protein mRNA and protein expression(P<0.05 or P<0.01,compared with non-intervention group),up-regulate the level of Smad7 protein mRNA and protein(P<0.05 or P<0.01,compared with non-intervention group);The GP could down-regulate the expression of Smad2 and Smad4 protein and up-regulate the expression of Smad7 protein,but not as good as DXM,there was no significant difference(P>0.05)between the two groups protein expression after the intervention of 72h.Conclusions:(1)The TGF-beta 1 mediated human alveolar epithelial A549 cells mesenchymal transition and cell proliferation,but also mediated human lung fibroblast cell line HLF cells into muscle fibroblast conversion and cell proliferation showed that two types of cells the pulmonary fibrosis model was constructed successfully in vitro.(2)GP can inhibit the proliferation of human type II alveolar epithelial cell line A549,promote the apoptosis and inhibit the epithelial mesenchymal transition(EMT)of the cells induced by TGF-beta 1.(3)GP can inhibit the proliferation and activation of human lung fibroblast cell line HLF induced by TGF-beta 1,and promote cell apoptosis.(4)GP can reduce the Smad2 and Smad4 mRNA and protein expression,and increased the Smad7 mRNA and protein expression,suggesting that GP intervene the process of lung fibrosis may by regulating TGF-betal/Smad pathway.