| WHO estimates that more than 220 million people worldwide have suffered with diabetes, and the number is likely to double by 2030 if without intervention. The state of diabetes in China is also alarming, with the highest population of diabetes in the world. Over time, diabetes can damage the heart, blood vessels, eyes, kidneys and nerves, causing chronic diseases and even death. Vitamin D has been shown to be potential in biological actions. 1,25-dihydroxyvitamin D (1,25-(OH)2D3) is the physiologically active metabolite of vitamin D, when combined with its receptor (VDR) in the target tissues, it may modulate inflammatory response, cell maturation and cell differentiation. More recently, animal and human studies have suggested that vitamin D is a potential modifier of the diabetes risk, but the mechanism is not yet clear. Moreover, conclusions from previous investigations are paradoxical. The present study is undertaken to explore the role of vitamin D in prevention and treatment of diabetes and its underlying mechanisms.Objective: To evaluate the relationship between the biological characteristics in vitamin D receptor (VDR) gene knockout mice and their diabetes occurrence, to study the prevention and treatment effects of 1,25-(OH)2D3 in a mouse model of type 1 diabetes, and to explore the underlying mechanism of 1,25-(OH)2 D3 effects on type 1 diabetes in order to provide theoretical and experimental data for the discovery of new drugs for diabetes treatment.Methods: Mice were generated by breeding heterozygotes to produce offspring with three genotypes. Basic breeding data of the mice, including age of maturity and length of pregnancy were monitored. Age-matched wild type (VDR+/+), heterozygotes (VDR+/-) and homozygote (VDR-/-) mice were genotyped by PCR, and weight and histology of liver, kidney, spleen as well as blood biochemical parameters were measured. Alloxan were injected through tail vein into the VDR-/- and VDR+/+ mice. Blood glucose and urine glucose were monitored periodically until four weeks after the last alloxan administration to determine the diabetes occurrence. C57BL/6J mice were used to establish the model of type 1 diabetes with multiple low doses of streptozotocin, which were randomly divided into diabetic group, 1,25-(OH)2D3-treated diabetic group and rapamycin-treated diabetic group. For the latter two groups, 1,25-(OH)2D3 and rapamycin were injected intraperitoneally for two weeks. Mice metabolic parameters including body weight, food and water intake and blood glucose were monitored periodically. Pathological changes of mice pancreas and kidney were observed using haematoxylin-eosin staining, and the expressions of VDR were measured by immunohistochemistry assay. The effects of 1,25-(OH)2D3 and rapamycin on autophagy and apoptosis were observed by electron microscopy. Th1/Th2 lymphocyte composition and Regulatory T cells (Treg) were analyzed by flow cytometer. The MIN6 cells were cultured to logarithmic growth phase, and then divided into STZ group, and 1,25-(OH) 2D3 + STZ group (co-cultured with 1,25-(OH) 2D3 in advance, and then with STZ). The secretion of insulin was measured by ELISA assay, cell viability by MTT essay and apoptosis by flow cytometry after annexin-V/propidium iodide labeling. The expressions of Beclin-1 and Bcl-2 protein were detected by Western blot analysis.Results:1. The average gestation period of VDR+/- mice was 22.5 days, with the proportion of VDR-/- in the offspring of 5% (8/165). There was no significant difference in body weight and organs weight between the VDR-/- mice and other groups. However, inflammatory cells infiltration and vacuolar degeneration could be observed in livers, kidneys and spleens of the VDR-/- mice. WBC, ALT, TG, CK and LDH increased, while serum calcium and phosphorus declined. Blood glucose of all VDR-/- mice was above the threshold, and the incidence of diabetes was 100% in one week after injection of alloxan. While for the the wild-type mice, only 50% occurred diabetes until 3 weeks.2. One week after the injection of STZ, blood glucoses in all mice without 1,25-(OH)2 D3 treatment exceeded 16.7 mmol/L. For those mice treated with 1,25-(OH)2 D3 beforehand, only 66.7% were induced to diabetes. The body weight of diabetic mice was declined, food and water intakes and random blood glucose were continuously increased. Moreover, inflammatory cells infiltration could be observed in the kidneys and pancreases. After treatment with 1,25-(OH) 2D3 for 14 days, the blood glucose of type 1 diabetic mice was decreased, inflammation in pancreas and kidney was markedly relieved, and the expression of VDR increased.3. The pathological changes in pancreases, kidneys, spleens and thymuses of diabetic mice were evident under electron microscope, with swelling of mitochondria, distension of rough endoplasmic reticulum and chromatin margination. In 1,25-(OH)2 D3 -treated diabetic group, the apoptotic lymphocytes in spleens and thymuses were incremental, and the ultrastructure abnormality in pancreases and kidneys was improved. Autophagic vacuoles were visible in pancreatic acinar cells and duct cells. The expression of Beclin-1 was increased, while Caspase-3 was reduced. The treatment of 1,25-(OH)2 D3 could decrease the frequencies of Th1 and Th1/Th2 ratio in mouse model of type 1 diabetes. On the other hand, 1,25-(OH)2 D3 could increase Treg cells. The symptoms and organ lesions in rapamycin-treated diabetic mice were even more aggravated. Data collecting from experiments about MIN6 cells demonstrated that 1,25-(OH)2 D3 pre-treatment could promote both insulin secretion and the expression of Beclin-1 and Bcl-2, and decrease the rate of apoptosis in MIN6 cells.Conclusion1. VDR showed no significant effects on mice growth and development, but its absence could result in inflammation, metabolic abnormalities, and increase the incidence of diabetes significantly.2. 1,25-(OH)2D3 pretreatment could reduce the incidence of diabetes in model mice, and play therapeutic effect on diabetes, indicating a potential role as an alternative drug in prevention and treatment of diabetes.3. The possible mechanisms of 1,25-(OH)2D3 involved in prevention and treatment of diabetes might be realized by the following ways: (1)1,25-(OH)2D3 could induce autophagy in pancreatic cells to clear the aging organelles so as to repair and increase insulin secretion; (2)1,25-(OH)2D3 could promote the apoptosis of lymphocytes to reduce their immune attack on pancreatic islets. (3)1,25-(OH)2D3 could restore the immune balance of Th1/Th2 cells and up-regulate Treg cells; (4)1,25-(OH)2D3 could inhibit the inflammation response and relieve the symptoms of diabetes. |
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