Mechanisms Of Baicalein-inhibited Lung Fibroblast Differentiation

Background:Fibroblast foci of pulmonary fibroblasts and pulmonary myofibroblasts differentiated from pulmonary fibroblasts are is the main pathological feature of pulmonary fibrosis,and is also drug target for treatment of pulmonary fibrosis.At present,pirfenidone and nintedanib have been used to treat pulmonary fibrosis,which attenuate lung fibrosis by repressing lung fibroblast differentiation.Our laboratory reported that baicalein could alleviate pulmonary fibrosis in bleomycin-induced animal fibrosis,which might be related to down-regulation of microRNA-21(miR-21)expression.However,the molecular mechanism of baicalein on pulmonary fibrosis has yet to be elucidated.In this paper,the molecular mechanism of baicalein will be investigated from the perspective of lung fibroblast differentiation.Method:1.Baicalein represses TGF β1-induced lung fibroblast differentiation through the inhibition of miR-21(1)To screen the safe dose of baicalein on lung fibroblast by CCK8 method;(2)The experiment of the effects of baicalein is divided into control group,transforming growth factor β1(TGF β1)group,vehicle group and different doses of baicalein group.Western Blot is used to detect of the levels of a-smooth muscle actin(a-SMA)and extracellular matrix protein(ECM),such as type I collagen(collagen I)and fibronectin(Fn)in human lung fibroblast cell line MRC-5 cell;(3)Cell immunostaining method was used to analyze the formation of cell myofilament in lung fibroblasts;(4)Real-time quantitative PCR was used to determine the expression of miR-21 in lung fibroblasts;(5)Overexpression of miR-21 in MRC-5 cells was used to confirm the role of miR-21 is involved in the effect of baicalein-inhibited lung fibroblasts differentiation through detecting the changes in a-SMA protein levels and collagen contraction gel assay including lung fibroblasts;(6)To determine the activity of one of miR-21 transcription factor---API by dual luciferase reporter gene assay,and to assay the STAT3 activity,another transcription factor of miR-21 by Western Blot;(7)To measure the levels of downstream targets of miR-21,Spryl and Smurf2 protein,after baicalein treatment on MRC-5 cell by using Western blot.(8)By down-regulation of Spryl protein expression,to explore whether baicalein inhibits lung fibroblasts differentiation through Spryl.2.Quantitative proteomic analysis of Baicalein-inhibited Type Ⅰ collagen production in human lung fibroblast(1)Western blot was used to determine the effects of baicalein on type Ⅰ collagen production in the MRC-5 cell;(2)Applying SILAC quantitative proteomic technique to find key molecules involved in the effects of baicalein-inhibited type Ⅰ collagen production in MRC-5 cell;(3)Western blot was used to detect the levels of connective tissue growth factor(CTGF)protein expression in MRC-5 cell after baicalein treatment;(4)To determine the CTGF mRNA levels in MRC-5 cells after baicalein treatment by real-time quantitative PCR;(5)By overexpression of CTGF in MRC-5 cells to investigate whether baicalein inhibits type I collagen production through CTGF;(6)To detect the levels of CTGF transcription factors,SP1,Smad3/p-Smad3,and Smad2/p-Smad2 in MRC-5 cells after baicalein treatment by western blot.Results:1.Baicalein represses TGF β1-induced lung fibroblast differentiation through the inhibition of miR-21(1)The CCK8 results showed that 1 μM and 10 μM baicalein had no obvious effect on MRC-5 cell proliferation;(2)10μM baicalein significantly reduced the levels of α-SMA,type Ⅰ collagen and fibronectin in TGF β1-stimulated MRC-5 cells,while 1 μM baicalein did not affect the levels of a-SMA protein.But 1 μM baicalein could decrease the levels of type Ⅰ collagen and fibronectin in a time-dependent manner.(3)Cellular immunofluorescence staining showed that 10 μM baicalein significantly inhibited the formation of cell myofilament during lung fibroblast differentiation;(4)10 pM baicalein could significantly down-regulate the expression level of miR-21 during TGF β1-stimulated lung fibroblast differentiation;(5)MiR-21 mimics could significantly antagonize the effect of baicalein-inhibited of lung fibroblast differentiation;(6)Baicalein significantly inhibited the activation of miR-21 transcription factor,STAT3,but it had no significant effect on the activity of another miR-21 transcription factor,AP-1.(7)Baicalein could up-regulate the levels of one of miR-21 downstream targets,Spryl protein,which is,but it has no obvious effect on the levels of another miR-21 downstream target,Smurf2 protein;downregulation of Spryl protein expression by Spryl siRNA could significantly antagonize the effect of baicalein inhibition of lung fibroblast differentiation;2.Quantitative Proteomic Analysis of Baicalein-Inhibited Type Ⅰ Collagen Production in Human Lung Fibroblast(1)10 μM baicalein significantly decreased the levels of type Ⅰ collagen in TGFβ1-stimulated MRC-5 cells;(2)Proteins with significant differences in protein levels between baicalein group and TGF β1 group were obtained by searching protein database.We found that Connective Tissue Growth Factor(CTGF)was one of the most down-regulated proteins,and the result of searching matescape database also showed that CTGF was associated with type I collagen production.(3)Western blot results showed that baicalein significantly down-regulated CTGF protein levels,and qPCR results demonstrated that baicalein also remarkably reduced CTGF mRNA levels;(4)Overexpression of CTGF could significantly antagonize the effect of baicalein-inhibited type Ⅰ collagen production in the TGF ββ1 stimulated MRC-5 cells;(5)Baicalein could down-regulate the levels of CTGF transcription factors,p-Smad2 in MRC-5 cells,but it had no significant effect on the expression level of SP1 and p-Smad3 protein.Conclusion:(1)Baicalein could inhibit the differentiation of lung fibroblasts through the STAT3/miR-21/Spryl signaling pathway;(2)Baicalein could reduce the production of type Ⅰ collagen through Smad2/CTGF pathway in lung fibroblasts differentiation;...