Objective: To investigate the effect of high fat on the expression of high-mobility group box 1 (HMGB1),α-smooth muscle actin (α-SMA), and matrix metalloproteinase-2 (MMP-2) in rat hepatic stellate cells (HSC-T6 cells).
Methods: HSC-T6 cells were cultured in vitro and treated with palmitic acid (PA) at a concentration of 300μmol/L for 24 hours, and the HSC-T6 cells in the control group were treated with bovine serum albumin (BSA) of the same volume for 24 hours; Western blot was used to measure the expression ofα-SMA and MMP-2. The HSC-T6 cells in the dose-effect group were treated with PA at concentrations of 0, 100, 200, 300, 500, and 1000μmol/L for 24 hours; the HSC-T6 cells in the time-effect group were treated with PA for 0, 4, 8, 12, 24, and 48 hours; in the control group, PA was replaced by BSA of the same volume; Western blot was used to measure the expression of HMGB1,α-SMA, and MMP-2. The HSC-T6 cells were treated with recombinant HMGB1 (rHMGB1) at concentrations of 0, 50, 100, 200, and 500 ng/ml for 48 hours, and in the control group, PA was replaced by phosphate buffer of the same volume. Western blot was used to measure the expression ofα-SMA and MMP-2. The HSC-T6 cells in the PA group were treated with PA at a concentration of 300μmol/L for 24 hours; the HSC-T6 cells in the PA+HMGB1-siRNA group were treated with PA at a concentration of 300μmol/L for 24 hours after HMGB1 in HSC-T6 cells was down-regulated; in the blank control group, PA was replaced by BSA of the same volume. Western blot was used to measure the expression of HMGB1,α-SMA, and MMP-2. One-way analysis of variance was applied for continuous data, and the t-test was applied for comparison between two groups.
Results: (1) The expression ofα-SMA and MMP-2 increased significantly after HSC-T6 cells were treated with PA at a concentration of 300μmol/L for 24 hours (P< 0.05). (2) Compared with the HSC-T6 cells in the untreated group (0μmol/L), the HSC-T6 cells treated with different concentrations of PA showed significant increases in the expression of HMGB1 and MMP-2 (P< 0.01), as well as a significant increase in the expression ofα-SMA at concentrations of 200, 300, 500, and 1000μmol/L (P< 0.01); the HSC-T6 cells treated with PA at a concentration of 300μmol/L for different periods of time showed varying degrees of increase in the expression of HMGB1,α-SMA, and MMP-2, with significant increases at 16, 24, and 48 hours (P< 0.01). (3) Compared with the HSC-T6 cells in the untreated group (0 ng/ml), the HSC-T6 cells treated with rHMGB1 at concentrations of 100, 200, and 500 ng/ml for 48 hours showed significant increases in the expression ofα-SMA and MMP-2 (P< 0.05). (4) Compared with the HSC-T6 cells in the PA group, the HSC-T6 cells treated with PA+HMGB1-siRNA for 24 hours showed significant reductions in the expression of HMGB1,α-SMA, and MMP-2 (P< 0.05).
Conclusions: High fat can increase the expression ofα-SMA and MMP-2 through up-regulating the expression of HMGB1 in HSC-T6, and thus lead to the development of liver fibrosis.