Sinapic Acid Inhibits TGF-β1-induced Fibrosis Of LX2 Cells By TGF-β1/Smad Signling Pathway

Objective:Hepatic fibrosis is the pathologic repair of chronic liver injury and the fundamental process of the development of chronic liver disease.Activation of hepatic stellate cells is the core event of liver fibrosis.Activated hepatic stellate cells can migrate to the injury site and secrete extracellular matrix to promote the formation of fibrous scar.As one of the common active ingredients in Chinese herbal medicine,sinapic acid,belongs to phenolic acids and is considered as an important chain breaking antioxidant with good antioxidant,anti-inflammatory,anti-tumor and anti-fibrosis activities.The purpose of the study was to investigate the effect of sinapic acid on liver fibrosis and its potential molecular mechanism,in order to provide theoretical and experimental basis for exploring the effective drugs of liver fibrosis treatment.Methods:1.LX2 cells were the research object.OD values of LX2 cells given different concentrations of sinapic acid were measured by MTS method.Control group,5μmol/L group,10μmol/L group,20μmol/L group,40μmol/L group,80μmol/L group and 100μmol/L group were set.The cell survival rate was calculated by the formula[Cell survival rate=(OD value of administration group/OD value of control group)× 100%].2.RT-qPCR was used to observe the mRNA expression of α-SMA and COL-1 in LX2 cells given 5ng/ml and 10ng/ml TGF-β1 group which compared with the control group.3.Western blot was used to observe the protein expression levels of α-SMA,COL-1 and fibronectin in LX2 cells given 5ng/ml and 1Ong/ml TGF-β1 group which respectively compared with the control group.4.It selected an appropriate concentration of TGF-β1 to stimulate LX2 cells.This was set the model group,then given different concentrations of sinapic acid as the administration group.RT-qPCR was used to observe the mRNA expression of α-SMA and COL-1 in the control group,the model group,the low dose administration group and the high dose administration group.5.The optimum concentration of TGF-β1 was selected,then it was used to stimulate LX2 cells,called the model group.The model group was given different concentrations of sinapic acid as the administration group.Western blot was used to observe the protein expression levels of α-SMA,COL-1 and fibronectin in the control group,the model group,the low dose administration group and the high dose administration group.6.It selected an appropriate concentration of TGF-β1 to stimulate LX2 cells.This was set the model group,then given different concentrations of sinapic acid as the administration group.Western blot was used to observe the protein expression levels of pSmad2 and pSmad3 in TGF-β1/Smad signaling pathway.Results:1.The results of MTS showed that different concentrations of(5,10,20,40,80,100μmol/L)sinapic acid had no toxic effect on LX2 cells.2.Different concentrations of TGF-β1(5,10ng/ml)could promote the mRNA expression of fibrotic-related genes in LX2 cells by RT-qPCR.Compared with the control group,the mRNA expression of fibrotic-related genes type Ⅰ collagen andα-smooth muscle actin were up-regulated(P<0.001).3.Western blot showed that different concentrations of TGF-β1(5,10ng/ml)could stimulate the expression of fibrotic-related protein in LX2 cells,and the expression levels of fibrotic-related protein type I collagen,fibronectin an α-smooth muscle actin were increased,when compared with the control group(P<0.001).4.The results of RT-qPCR showed that compared with the modeling group,the mRNA expressions of fibrosis related genes α-smooth muscle actin were down-regulated in the low dose group(20μmol/L)and high dose group(40μmol/L)(P<0.01);the mRNA expressions of type I collagen were down-regulated in the low dose group(20μmol/L)and high dose group(40μmol/L)(P<0.01,P<0.001).5.By western blot,the expression levels of fibrotic-related protein type Ⅰ collagen and α-smooth muscle actin in the low dose group(20μmol/L)and high dose group(40μmol/L)were less than the modeling group.The protein expression levels of fibronectin in the low-dose group(20μmol/L)was more than its modeling group(P>0.05);its high dose group(40μmol/L)was decreased(P<0.001).6.Compared with the control group by Western blot,the expression levels of TGF-β1/Smad signaling pathway related proteins pSmad2 and pSmad3 increased;compared with the modeling group,the protein expression levels of pSmad2 and pSmad3 in different concentrations(20 and 40μmol/L)were decreased(P<0.01).Conclusion:Sinapic acid can inhibit the expression of fibrosis-related gene and protein in TGF-β1-induced LX2 cell,which to indicate that sinapic acid has an anti-fibrosis effect.The mechanism of sinapic acid against liver fibrosis is related to the inhibition of Smad2 and Smad3 protein phosphorylation in TGF-β1/Smad signaling pathway.