The Effect Of 1,25（OH）₂D₃ On Inflammation And Autophagy Of TypeⅡDiabetes Mouse Model

Objective:1. To investigate the correlation between diabetes occurrence and vitamin D;2. To investigate the effect of 1,25（OH）₂D₃ on inflammation and autophagy of type Ⅱ diabetes mouse model and explore the underlying molecular mechanisms;Methods:1. High-fat diet combined with STZ（streptozotocin, STZ） induced diabetes model of C57BL/6 mouse, spontaneous type Ⅱ diabetes（C57BL /Ks J- db/db） and normal C57BL/6 mice were used to investigate the underlying relationship between serum 25-hydroxyvitamin D₃（25（OH）D₃） content and glucose metabolism（10 mice in each group）. Sugar metabolism index（FBG, AUC, HOMA-IR） and physiological index（weight, drinking water） were detected; the effect of 1,25（OH）₂D₃ of different concentration on diabetes occurrence of mice model were detected.2. Type Ⅱ diabetic mice were randomly divided into 3 groups with drug intervention, respectively. 1,25（OH）₂D₃ group: mice were injected with 5 μg/kg 1,25（OH）₂D₃ at alternate days; RAPA（rapamycin） group: mice were injected with 0.25 mg/kg RAPA at alternate days. BAY group: mice were injected with 20 mg/kg BAY11-7082（inhibitor of NLRP3 inflammasome） every day. Each group consisted of 9 mice. After three weeks treatment, the changes in glucose metabolism（FBG,AUC,HOMA-IR） were detected; levels of cytokines（TNF-α, IL-18, IL-1β, INF-γ） in serum were assayed using ELISA kits; reactive oxygen species（ROS） of mitochondrial changes in different tissues（liver, pancreas, kidneys, spleen） were tested by DCF method; Glomerular and islets analysis were performed under electron microscope and light microscope; Immunohistochemistry and western blot were used to detect the expression of Beclin-1, LC3, NLRP3, NF-κB p65 protein in pancreas and kidneys.Results:（1） The connection between diabetes and vitamin D.1. Compared with the normal group, serum 25（OH）D₃ was significantly decreased in diabetes mice model（P<0.05）.2. Early 1,25（OH）₂D₃ intervention could reduce diabetes occurrence of mice model.3. Weight, drinking volume and fasting glucose of diabetes mice were increased significantly（P<0.05）. Moreover, their glucose metabolism were abnormal.（2） Effect of 1,25（OH）₂D₃ ontype Ⅱ diabetes mice model.1. Compared with Diabetes group, the glucose metabolism of diabetes mice intervened with 1,25（OH）₂D₃, RAPA or BAY improved significantly（P<0.05）.2. Compared with Diabetes group, the concentration of TNF-α, IL-1β, INF-γ in 1,25（OH）₂D₃, RAPA and BAY intervention group were decreased significantly（P<0.05）.3. Compared with Diabetes group, mitochondrial ROS of liver, pancreas and kidney in mice intervened with 1,25（OH）₂D₃ was significantly decreased（P<0.05）4. Diabetes mice displayed serious pathological changes in pancreas and glomerulus. While 1,25（OH）₂D₃ intervention improved lesions of islets and glomerulus, and autophagosomes could be found in β cells（P<0.05）.5. Compared with Diabetes group, the expression of LC3, Beclin-1 in 1,25（OH）₂D₃ group were decreased（P<0.05）, while the level of NLRP3 and NF-κB p65 were increased in pancreas and kidney（P<0.05）.Conclusions:1. The concentration of 25（OH）D₃ of typeⅡdiabetes mice model was low, and supplementation of 1,25（OH）₂D₃ could prevent the onset of diabetes.2. It was demonstrated the signaling pathway between autophagy and inflammation, NLRP3/IL-1β/NF-κB was related to the development of typeⅡdiabetes, 1,25（OH）₂D₃ could suppress this pathway by promoting autophagy in β cells, and indicated a novel strategy for diabetes treatment.