Antophagy inhibition can decrease AR42J cell apoptosis induced by exendin-4

Abstract

Objective To explore whether exendin-4 inhibits AR42J cells and its mechanism. Methods AR42J cells were treated with exendin-4 under multiple concentrations(1, 5, 10 pmol/L) at 24, 48, 72, 96, 120 h to assess its cell viability by MTT assay and got the IC-50 and time points. Then checking whether exendin-4 could induce the AR42Jcells apoptosis by setting normal control (NC) group, exendin-4 (Ex-4) group and Ex-4+ z-VAD-fmk (apoptosis inhibitor) group, and exploring whether 3-MA which is autophagy inhibitor could inhibit the AR42J cells apoptosis induced by exendin-4 by setting NC group, Ex-4 group and Ex-4+ 3-MA group. Cell viability was analyzed by MTT and the cells apoptosis was detected by flow cytometry and the protein levels of caspase-3, LC3 and p62 were studied by Western blot. Results Concentration of 10 pmol/L exendin-4 and and time point 72 h were selected for the further study . z-VAD-fmk pretreatment can significantly inhibit the cell viability of exendin-4 by (81.2±3.3)% vs. (49.4±3.0)% (P<0.05). Flow cytometry showed that exendin-4 could induce the AR42J cells apoptosis by (28.2±1.4)% vs. (3.6±0.8)%, and increased the caspase-3 level by Western blot, which both can be reversed by (79.1±2.3)% vs. (49.8±2.5)% (P<0.05) when the cells were treated for 72 h, as was apoptosis ratio by (14.5±2.1)% vs. (29.2±3.2)%. Western blot showed that exendin-4 can upregulate protein levels of LC3B-II, p62和caspase-3 and 3-MA, and pretreatment can inhibit the upregulation of LC3B-II and caspase-3 but further increased the upregulation of p62 induced by exendin-4. Conclusions Exendin-4 can induce AR42J cells apoptosis and 3-MA pretreating can inhibit exendin-4 cytotoxicity through downregulating autophagy. So autophagy inhibitor 3-MA could potentially extenuate the cytotoxicity of exendin-4 in pancreatic acinar cells. Key words: Exendin-4; Pancreatic acinar cell; Apoptosis; Autophagy inhibitor, 3-MA