Protective Effects Of Berberine On Lipapoptosis In Pancreatic βTC3 Cells Induced By Free Fatty Acid

Background:Berberine, the most effective components of Rhizoma Coptidis, is a kind of isoquinoline alkaloid, extracted from the traditional C hinese medicine Rhizoma Coptidis and Cortex Phellodendri. Many studies and observations have demonstrated that the berberine has a lot of biological activity, including regulating gut microbiota, hypoglycemic activity, lipid- lowering activity, improving insulin sensitivity, promoting insulin secretion, inhibiting cell apoptosis and weight reduction. At present, berberine is commonly used for treating type 2 diabetes, obesity and metabolic syndrome, and has drawn greater attention in the mechanisms of its hypoglycemic activity. The intervention effects of berberine on beta cells in high FFAs condition and its potential molecular mechanisms were unclear.PTEN(Phosphatase and tensin homolog deleted in chromosome10), is a phosphatase activity of tumor suppressor genes, plays a key role in the occurrence of many tumors. Studies have shown that PTEN not only plays a role in cell apoptosis, proliferation and migration, also participated in the PI3K-AKT signaling pathways. Recent studies have demonstrated that PTEN could inhibit the AKT signaling pathways activated in the islet beta cells exposed to free fatty acids, and suppressing the expression of PTEN gene markedly improved beta cell function under lipid stress.Therefore, the role of berberine in the beta cell apoptosis induced by free fatty acids was investigated in the present research, and whether PTEN /AKT signaling was involved.Objective: To evaluate the protective effect of berberine on pancreatic βTC3 cells lipoapoptosis induced by free fatty acids, investigated the role of PTEN/AKT signaling in berberine involved beta cell protection.Methods: Experiment 1:The proliferation and apopto sis of βTC3 cells were determined by MTT assay and Flow cytometry analysis when exposed to FFAs with or without berberine. Experiment 2:The basal and glucose stimulated insulin secretion capability of βTC3 cells were evaluated when exposed to FFAs with or without berberine by Radioimmunoassay. Experiment 3: The expression of PTEN, AKT, p-AKT and apoptosis related proteins Bax, Bcl-2 and Active-Caspase3 were detected by western blotting in βTC3 cells when exposed to FFAs with or without berberine.Results:1. The results obtained from MTT indicated that berberine in concentration of 0.001 to 1 μmol/L can promote the proliferation of pancreatic βTC3 cells as compared to the untreated controls(P<0.05). The cell survival rate decreased obviously in the groups treated with 10, 50, 100 and 200 μmol/L berberine, indicating a high concentration of berberine has a distinct cytotoxic effect in pancreatic βTC3 cells in a dose-dependent manner(P<0.01). Cell viability decreased after exposing to the free fatty acids in the concentration of 0.2 to 1.0 mmol/L for 24 to 72 hours. Il ustrating the lipotoxicity was in a time and dose dependent manner. 2. Flow cytometry analysis suggested that apoptosis of βTC3 cells were significantly increased after exposing to palmitate(p<0.05). Berberine substantially inhibited the lipoapoptosis and augmented the proliferation of beta cells. In addition, berberine in the concentration of 0.1 μmol/L has the best protection effect(P<0.01). 3. Berberine substantially facilitated the basal and glucose stimulated insulin secretion of beta cells in high FFAs condition. 4. Western blot revealed that the phosphorylation of AKT and Bcl-2 were markedly decreased under lipid stress but were elevated when treated with berberine. Moreover, FFAs could up-regulate the expression levels of PTEN, Bax, and Active-Caspase3, but down-regulate the expression levels of p-AKT and Bcl-2 in beta cells, which were canceled by the addition of berberine.Conclusion: Berberine observably inhibited the apoptosis, elevated proliferation and the basal and glucose stimulated insulin secretion of beta cells in high FFAs condition. Furthermore, the protective action of berberine was probability mediated by activation of PTEN/AKT, which was accompanied by the down-regulation of Bax, Active-Caspase3 and up-regulation of p-AKT and Bcl-2 expressions.