Effects Of Aucubin On Human Hepatic Stellate Cells Activation And Deposition Of Extracellular Matrix Induced By TGFβ1

Objective: To evaluate effects of aucubin on the activation and deposition of extracellular matrix in human hepatic stellate cells induced by TGFβ1, providing theoretical bases for the mechanism of aucubin on liver injury protection.Methods:Human hepatic stellate cells (LX-2) were cultured in vitro and divided into9groups:Control group, DMSO group (1%。 DMSO), TGFβ1model group (5ng/mL TGFβ1), p38MAPK inhibitor group (5ng/mL TGFβ1+10μM SB203580), low concentration Aucubin group (5ng/mL TGFβ1+1μM aucubin group), middle concentration aucubin group (5ng/mL TGFβ1+10μM aucubin group), high concentration aucubin group (5ng/mL TGFβ1+100μM aucubin group), P-glucosidase group (5ng/mL TGFβ1+10U/mL β-glucosidase), aucubigenin group (5ng/mL TGFβ1+100μM aucubin+10U/mL β-glucosidase). LX-2were incubated in a medium containing10%FBS for36hours, using an inverted microscope to oberve the change of cell morphology; Real time-PCR and Western blot were used to detect the mRNA and protein expression of LX-2activation markers a-smooth muscle actin (a-SMA) and reflect the extracellular matrix indicators collagen I (Col I) and collagen Ⅲ(Col Ⅲ).Results:The control cells displayed typical pebbles sample of LX-2cells.5ng/mL TGFβ1treatment of LX-2cells resulted in distinct morphological changes.5ng/mL TGFβ1treated cells showed evidence of fusiform strip sample but inhibited partly in the presence of p38MAPK inhibitor and aucubin.Compared with the control group,5ng/mL TGF β1significantly increased the mRNA level of a-SMA (1.81±0.13vs0.99±0.02, P<0.001), Col Ⅰ (5.36±1.44vs1.00±0.00, P<0.001) and Col Ⅲ (2.51± 0.28vs1.02±0.02,P<0.001).Compared with TGFβ1group,SB203580significantly reduced the mRNA level of α-SMA(0.98±0.05vs1.81±0.13,P<0.001),Col Ⅰ(2.34±1.04vs5.36±1.44,P=0.005)and ColⅢ (0.72±0.03vs2.51±0.28,P<0.001)and the difference is statistically significant.Compared with5ng/mL TGFβ1group,low,middle and high concentration of aucubin has ability to lower mRNA level of Col Ⅰ(5.29±0.89,5.03±0.49,5.12±0.80vs5.36±2.5,P=0.946,P=0.732,P=0.787)and Col Ⅲ(2.32±0.22,2.09±0.37,2.35±0.48vs2.51±0.28,P=0.528,P=0.207,P=0.637),but the difference was not statistically significant；high concentrations of aucubin significantly reduced the mRNA level of α-SMA(1.46±0.09vs1.81±0.13,P=0.015),and the difference was statistically significant；low and middle concentration of aucubin had tendency to lower mRNA level of α-SMA(1.61±0.08,1.62±0.05vs1.81±0.13,P=0.139,P=0.170),but the difference was not statistically significant.Compared with TGFβ1group,the mRNA levels of α-SMA(1.88±0.03vs1.81±0.13,P=0.578)in β-glucosidase group increased slightly, Col I(3.65±0.70vs5.36±1.44,P=0.088)and Col Ⅲ(2.09±0.19vs2.51±0.28,P=0.203)decreased,but the difference was not statistically significant.Compared with TGFβ1group,aucubigenin group decreased the mRNA levels of Col Ⅰ(4.81±2.3vs5.36±2.5,P=0.573),Col Ⅲ (2.19±0.27vs2.51±0.28,P=0.363)and α-SMA(1.61±0.16vs1.81±0.13,P=0.139),but the difference was not statistically significant.Compared with the control group,5ng/mL TGFβ1significantly increased the protein levels of α-SMA(3.12±0.93vs1.00±0.00,P<0.001),Col Ⅰ(3.51±0.32vs1.00±0.00,P<0.001)and Col Ⅲ(7.54±1.65vs1.00±0.00,P<0.001).Compared with TGFβ1group,SB203580significantly reduced the protein levels of α-SMA(0.99±0.14vs3.12±0.93,P<0.001),Col Ⅰ(1.28±0.35vs3.51±0.32,P<0.001)and Col Ⅲ (1.60±0.23vs7.54±1.65, P<0.001), and the difference was statistically significant.Compared with TGFβ1group, low, middle and high concentration of aucubin can lower the protein levels of a-SMA (0.91±0.16,1.10±0.24,1.18±0.42vs3.12±0.93, P<0.001, P<0.001, P<0.001), and the difference was statistically significant; The low and middle concentration of aucubin has ability to lower Col Ⅰ (3.22±0.24,3.28±0.33vs3.51±0.32, P=0.162, P=0.271) and Col Ⅲ (7.35±0.97,7.00±0.77vs7.54±1.65, P=0.674, P=0.240), but the difference was not statistically significant.Compared with TGFβ1group, the protein levels of a-SMA (3.52±1.11vs3.12±0.93, P=0.208) increased slightly inβ-glucosidase group, and the protein levels of Col Ⅰ (3.39±1.26vs3.51±0.32, P=0.721) and Col Ⅲ (7.17±0.83vs7.54±1.65, P=0.418) decreased, but the difference was not statistically significant. Compared with TGFβ1group aucubigenin group decreased the protein levels of Col Ⅰ (2.18±0.37vs3.51±0.32, P<0.001), Col Ⅲ (6.03±0.56vs7.54±1.65, P=0.002) and α-SMA (0.80±0.36vs3.12±0.93, P<0.001), and difference was statistically significant. Compared with high concentration of aucubin group, aucubigenin group decreased the protein levels of Col Ⅰ (2.18±0.37vs2.95±0.45, P=0.009), and difference was statistically significant.Conclusion:Aucubin can inhibit TGβ1-induced LX-2activation and deposition of extracellular matrix;Aucubigenin can inhibit TGFβ1-induced LX-2activation and deposition of extracellular matrix, maybe has more biological activity than Aucubin.