| Diabetes mellitus(DM) is a kind of metabolic diseases, which is caused by a variety of factors and characterized by hyperglycemia. One of the main reasons of DM is the decrease of insulin secretion due to the dysfunction and loss of pancreatic islet beta cells. 1, 25-(OH)2D3(Vit D3) is the active form of vitamin D, and it binds to vitamin D receptor(VDR) to play its role. Autophagy is an evolutionarily conserved process, its housekeeping function is to degrade cytosolic long-lived proteins and organelles, It has important biological significance for the maintenance of cellular homeostasis and cell survival.Objective:1. To explore the protective effect of Vit D3 on INS-1 cells damaged by STZ.2. To investigate the role of Vit D3 on the secretion of islet cells and the regulation of autophagy.Methods:INS-1 cells were cultured in dishes, and divided into normal control group and different concentrations of Vit D3 treatment groups(10-6 mmol/L, 10-7 mmol/L, 10-8 mmol/L, 10-9 mmol/L). The cells were incubated with Vit D3 for 24 hours, cell viability was measured by CCK8 method, and insulin secretion was assayed using Elisa kit, to find the optimal concentration of Vit D3 for INS-1 cells. Cells were divided into normal control, STZ treatment group, Vit D3 control group, Vit D3 protection group(Vit D3 + STZ) and Vit D3 repair group(STZ + Vit D3). Cell viability and insulin secretion of STZ-damaged INS-1 cells were measured by CCK8 and Elisa reapectively, Culture media were collected, MDA and T-AOC were measured using Elisa assay. The total protein and RNA were extracted by RIPA and Trizol, Western blot and RT-PCR were used for dection of protein and m RNA level of autophagy. Ultrathin sections were performed, autophagy and the changes of ultrastructure were observed under transmission electron microscope.Results:1. The influence of Vit D3 on insulin secretion of INS-1 cellsWe found that 10-7m M concentration of Vit D3 could significantly promote insulin secretion of INS-1 cells in comparison with normal control(P<0.01). Furthermore, cell viability increased under the same concentration of Vit D3(P<0.01). There were no significant differences in other concentrations of Vit D3 treatment groups(P>0.01).2. Effects of Vit D3 on INS-1 cells damaged by STZCell viability, insulin secretion and T-AOC of cells in STZ group were decreased(P<0.001), and while MDA were significantly increased(P<0.001), Cell viability, insulin secretion and T-AOC of cells both in Vit D3 protection and in Vit D3 repair group were also decreased(P<0.01), and MDA were increased(P<0.01). However, when compared with cells in STZ treatment group, cell viability and insulin secretion and T-AOC were increased(P<0.01), and MDA were decreased(P<0.01).3. Effects of Vit D3 on autophagy of STZ-damaged INS-1 cellsThe results of RT-PCR showed that the expressions of autophagy-related genes Beclin 1 and LC3 in Vit D3 group, Vit D3 protection and repair groups were increased significantly(P<0.01), results of Western blot also showed the same trend. Under transmission electron microscope, cells of normal control showed integrity cell morphology, organelles arranged in an orderly manner, and insulin secretion granules could be observed. The morphological structure of cells in Vit D3 group were the same as normal control.While the cells in STZ treatment group were severely injured, the expansion of rough endoplasmic reticulum, the edema, rupture of crista and the reduction quantity of mitochondrial were observed. And the chromosomes were aggregation, and some cells appeared apoptosis characteristics, there was few insulin secretion granules. Cells both in Vit D3 protection and in repair group were light-injured and autophgosomes with double membranes double membranes structure could be found.Conclusion:1 Vit D3 could reduce the damage of INS-1 cells induced by STZ and promote the secretion of insulin.2 Vit D3 could promote the expression of autophagy-related protein Beclin1 and LC3 in INS-1 cells with STZ treatment, and promote the formation of autophagosomes. Data suggested that Vit D3 could help cells to eliminate the organelles damaged by STZ through inducing autophagy and to achieve the protective effect on INS-1 cells. |
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