Effect of overexpression of apoptosis-stimulating protein 2 of p53 on activation and apoptosis of hepatic stellate cells induced by transforming growth factor-β1 and its mechanism

Abstract

Objective: To study the effect of apoptosis-stimulating protein 2 of p53 (ASPP2) on the activation and apoptosis of hepatic stellate cells induced by transforming growth factor-β1 (TGF - β1), and to explore the role of autophagy in this process. Methods: Mouse hepatic stellate cells were primarily isolated and cultured with green fluorescent protein (GFP) expressing empty vector adenovirus (Ad-GFP) and ASPP2 expressing adenovirus (Ad-ASPP2) for 12 h by transfection kit, and then treated with TGF-β1 (10ng/ml) for 24 h. The experiments were grouped as follows: control group: green fluorescent protein (GFP) expressing empty vector adeno (Ad-GFP); experimental group 1: transfected with Ad-GFP and added with TGF-β1; experimental group 2: transfected with Ad-ASPP2 and induced by TGF-β1. Western blot and quantitative fluorescence PCR were used to detect the expression of ASPP2, α-smooth muscle actin (SMA). At the same time, autophagy was determined by microtubule-associated protein 1 light chain 3-β (LC3). Autophagy and apoptosis of MHSc were observed by immunocytochemistry and RNA interference (RNAi). Multiple pairwise-comparisons between the mean of groups was performed by one-way ANOVA. Results: The relative expression of α-SMA mRNA in mHSC of TGF-β1 + Ad-GFP group (16.83 ± 2.41) was significantly higher than Ad-GFP group (3.62 ± 0.56) (P < 0.05), while the relative expression of α-SMA mRNA (4.22 ± 0.48) in TGF-β1 + Ad-GFP group was significantly lower than TGF-β1 + Ad-GFP group (P < 0.05). The expression of α-SMA protein in each group was consistent with mRNA expression. The proportion of mHSC autophagy in TGF-β1 + Ad-GFP group (80%) was significantly higher than Ad-GFP group (35%); however, there was no statistically significant difference between the two groups. The proportion of mHSC autophagy in TGF-β1 + Ad-ASPP2 group was 42%, which was significantly lower than TGF-β1 + Ad-GFP group, but the apoptotic rate was significantly increased. Cells were simultaneously treated with autophagy inhibitors 3-MA and TGF-β1. The level of autophagy was not statistically significantly different from that of TGF-β1 + Ad-ASPP2 group, but the apoptotic rate was increased. In addition, the RNAi group added with ASPP2 had increased autophagy (LC3-II/LC3-I) than control RNAi group, and the rate of apoptosis was significantly decreased. Conclusion: Overexpression of ASPP2 can alleviate the activation of mHSC and promote the apoptosis of HSC by inhibiting autophagy, so as to alleviate liver fibrosis.