Inhibitory effect of carvedilol on human hepatic stellate cell activation and fibrosis induced by platelet-derived growth factor-BB and related mechanisms of action

Objective To investigate the effect of carvedilol on the migration, invasion and fibrosis of human hepatic stellate cells, as well as related signaling pathways and mechanisms.Methods Human hepatic stellate cell line LX-2 was treated with different concentrations of carvedilol (0, 1, 2, 5, and 10 μmol/L, and platelet-derived growth factor-BB (PDGF-BB) was added to activate the cells.CCK-8 assay was used to measure cell proliferation, wound healing assay was used to measure migration, Transwell chamber assay was used to measure invasion, and Western blot and real-time PCR were used to measure the protein and mRNA expression of fibrosis markers and pathway proteins.The cells were divided into blank control group, PDGF-BB group (only PDGF-BB was added) , and four carvedilol groups (with 1, 2, 5, or 10 μmol/L carvedilol, as well as PDGF-BB) .A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the Dunnett-t test was used for comparison between experimental groups and control group.Results Carvedilol inhibited the proliferation of LX-2 cells in a concentration-dependent manner, with a half-inhibitory concentration of28.42 μmol/L.Compared with the PDGF-BB group, the 10 μmol/L carvedilol group had significantly inhibited migration of LX-2 cells (59.780%±8.898% vs 17.270%±2.668%, t=4.576, P=0.010) .PDGF-BB increased the invasion of LX-2 cells, and carvedilol inhibited the invasion of LX-2 cells in a concentration-dependent manner;the invasion of LX-2 cells was reduced from 157.00%±10.52% to 85.15%±13.50% in the 2 μmol/L carvedilol group (t=4.198, P=0.014) , to 55.67%±9.54% in the 5 μmol/L carvedilol group (t=7.133, P<0.01) , and to 45.37%±10.70% in the 10 μmol/L carvedilol group (t=7.438, P<0.01) .The mRNA expression of type I collagen was reduced from 1.068±0.128 to 0.453±0.085 in the 5 μmol/L carvedilol group (t=3.997, P<0.05) and to 0.151±0.019 in the 10 μmol/L carvedilol group (t=7.091, P<0.01) .The mRNA expression of fibronectin was reduced from 1.285±0.042 to 0.879±0.063 in the 1 μmol/L carvedilol group (t=5.345, P<0.01) , to 0.768±0.010 in the 2 μmol/L carvedilol group (t=4.773, P<0.01) , to 0.742±0.117 in the 5 μmol/L carvedilol group (t=4.385, P=0.012) , and to 0.591±0.049 in the 10 μmol/L carvedilol group (t=10.76, P<0.01) .The expression of fibronectin was reduced from 2.103±0.414 to 0.739±0.132 in the 5 μmol/L carvedilol group (t=3.137, P=0.035) and to 0.600±0.114 in the 10 μmol/L carvedilol group (t=3.499, P=0.025) , and the expression of α-smooth muscle actin was reduced from 1.418±0.241 to 0.543±0.215 (t=2.710, P=0.035) and 0.343±0.118 (t=4.005, P <0.01) , respectively.Y751 phosphorylation was reduced from 2.309±0.181 to 1.278±0.304 in the 2 μmol/L carvedilol group (t=2.912, P=0.044) , to 0.555±0.038 in the 5 μmol/L carvedilol group (t=9.476, P<0.01) , and to 0.175±0.039 in the 10 μmol/L group (t=11.51, P<0.01) .Akt phosphorylation was reduced from 1.106±0.185 to 0.335±0.132 in the 5 μmol/L carvedilol group (t=3.386, P=0.015) and to 0.137±0.110 in the 10 μmol/L carvedilol group (t=4.494, P<0.01) .Conclusion Carvedilol can inhibit the proliferation, migration, invasion, and fibrosis of LX-2 cells induced by PDGF-BB, mainly by blocking the PDGF-BB/PDGFR-β/Akt signaling pathway.