The Effects And Mechanisms Of Glaucocalyxin A-inhibited Lung Fibroblast Differentiation

Background:Lung fibroblasts overactivate and differentiate to lung myofibroblasts,accompanied with abnormal deposition of extracellular matrix,which eventually induced pulmonary fibrosis.Previous studies have found that Glaucocalyxin A(GLA)could alleviate bleomycin-induced pulmonary fibrosis in mice,but its mechanisms remain unclear.This work will explore the effects and mechanisms of GLA-inhibited pulmonary fibrosis from the point of lung fibroblast differentiation.Method:(1)To establish the TGF-β1-induced lung fibroblast transformation model,the groups were divided into control group,model group(transforming growth factor-β1 group),GLA group,and Western blot was used to determine the expression levels of α-Smooth muscle actin(α-SMA)in these groups;(2)Cell immunostaining method was used to analyze the formation of cell myofilament in lung fibroblasts;(3)Make use of collagen gel shrinkage test to determine the experimental group containing lung fibroblast collagen gel shrinkage;(4)Effect of Western Blot of GLA on the expression level of matrix protein Fibronectin in the transformation of lung fibroblasts induced by TGF-β1;(5)Effect of Western Blot of GLA on the expression level of Phosphorylation of Smad2、Smad3、GSK3β、ERK1/2 in the transformation of lung fibroblasts induced by TGF-β1;(6)Application of SILAC quantitative proteomics analysis of GLA inhibition of intracellular protein expression during transformation of lung fibroblasts.Results:(1)2μM and 4 μM GLA could significantly reduce TGF-β1-induced α-SMA protein expression levels,and was dose-dependent;(2)After 24h and 48h,α-SMA protein expression level could be reduced by GLA and the time-dependent expression of α-SMA protein could be reduced by acting on TGF-β1-induced lung fibroblasts;(3)Cellular immunofluorescence staining showed that 4 μM GLA inhibited the formation of cell myofilament during lung fibroblast differentiation;(4)Collagen gel shrinkage test results showed that GLA significantly reduced TGF-β1-induced lung fibroblast collagen shrinkage capacity;(5)GLA significantly down-regulated the expression level of matrix protein Fibronectin in TGF-β1-stimulated MRC-5 cells;(6)GLA significantly reduced TGF-β1-induced MRC-5 cells Smad2 phosphorylation expression levels,but did not affect the expression level of Smad3 phosphorylation;(7)GLA significantly inhibited TGF-β1-induced MRC-5 cells the phosphorylation expression level of GSK3β,but had no effect on the phosphorylation expression level of ERK1/2;(8)Using SILAC quantitative proteomic analysis of GLA inhibition of lung fibroblast transformation process changes in protein,we found that might be associated with lung fibroblast transformation of 19 protein expression levels down,while 6 protein expression levels were upregulated.Conclusion:GLA could significantly inhibit the transformation of lung fibroblasts,and associated with reduced expression levels of matrix protein Fibronectin,it might inhibit Smad2 activity and GSK3β phosphorylation regulation related by GLA-inhibited,while proteomics results also provided clues to clarify GLA molecular mechanisms.