The effects of curcumin derivatives C66 on proliferation and activation of rat hepatic stellate cells induced by transforming growth factor-β

Abstract

Objective To investigate the effects of curcumin derivatives (C66) on proliferation and expressions of α-smooth muscle (α-SMA) and Collagen Ⅰ in rat hepatic stellate cells (HSC) induced by transforming growth factor-β (TGF-β) in vitro and the relationship with cannabinoid receptor type 1 (CB1). Methods To determine the optimum time and concentration of C66, HSC-T6 cell line was cultured in vitro and divided into control group and groups with different doses of C66 (1 μmol/L, 2 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmol/L). Cell proliferation was detected by Cell Counting Kit-8 assay. Then, according to the time and concentration of C66 above, cells were divided into 5 groups including control group, TGF-β only group, TGF-β combined with CB1 antagonist group, TGF-β combined with C66 group and TGF-β combined with CB1 antagonist plus C66 group. Quantitative real-time polymerase chain reaction and western blot were used to assess the expressions of α-SMA, Collagen Ⅰ, CB1, JNK and phosphorylation of JNK (p-JNK). The variance homogeneity of multiple samples was compared by LSD method. The variance was compared with Dunnett T3 test. One-way analysis of variance was performed to compare the mean values among the groups. Results The inhibitory effect of C66 on HSC-T6 proliferation was dose and time dependent. The optimum time and concentration were 48h and 10 μmol/L, respectively, with the inhibition rate of 54%. Compared with control group, expressions of α-SMA, collagen Ⅰ and CB1 were significantly elevated in TGF-β group (t=6.188, 3.48 and 20.64, respectively, all P<0.05). TGF-β1 could increase the relative mRNA expressions of CB1, collagen Ⅰ and α-SMA with significant differences (t=4.705, 9.492 and 38.27, respectively, all P<0.05). Compared with control group, p-JNK expression was significantly elevated in TGF-β group (t=9.567, P<0.05). Conclusions C66 could inhibit the proliferation and collagen synthesis in HSC-T6 induced by TGF-β and the effect is strengthened when combined with CB1 antagonist, which may involve JNK phosphorylation. Our study provides a better understanding on the mechanism and a new target for treatment of liver fibrosis. Key words: Curcumin derivative; Hepatic stellate cell; Liver fibrosis; c-Jun N-terminal kinase