The Effect Of 1,25?OH?₂D₃ On Inflammation Responses Of INS-1 Cells And Zebrafish Diabetes Mellitus

Objective:To investigate the effects of 1,25?OH?₂D₃ on the inflammatory responses of INS-1 cells and zebrafish model of diabetes mellitus and the molecular mechanisms.Methods:1.INS-1 beta cells model of diabetes mellitus and treatment.To culture pancreatic INS-1 beta cells and establish a model of diabetes mellitus?DM?with 5m M Streptozotocin?STZ?.INS-1 cells were then divided into five groups as the following:Normal control group: 1640 completely normal culture medium;DM cell model group: STZ-induced INS-1 DM cell culture;1,25?OH?₂D₃ intervention group: cells were treated with 10-7mmol/L 1,25?OH?₂D₃ for 24h;NF-kappa B inhibitor intervention group: cells were treated with 5umol/L BAY11-7082 for 24h;NLRP3-si RNA group: cells were treated with liposome transfection and interfere with m RNA of NLRP3 of INS-1 cells.ELISA was used to detect the serection of insulin and inflammatory cytokines?TNF-alpha,IL-1?and IL-18?,the mitochondrial reactive oxygen species?ROS?was detected by DCF,RT-PCR,Western blot and substrates analysis were used to detect the expression of m RNA or protein levels of caspase-1,NF-?B p65 and NLRP3 in each group.2.Zebrafish model of diabetes mellitus and treatment 60 zebrafish of adult male were used in this study.15 zebrafish were randomly selected as a normal control group,the rest 45 zebrafish were treated with intraperitoneal injection of 350 mg/Kg STZ combined with overfeeding to establish model of type 2 diabetes mellitus?the blood glucose levels of zebrafish remained above 310 mg/dl?.The diabetes model of zebrafish was randomly divided into 3 groups as the following.Diabetes group;1,25?OH?₂D₃ intervention group: diabetes model of zebrafish were treated with10ul/ml 1,25?OH?₂D₃ for 6h every other day;BAY11-7082 intervention group :diabetes model of zebrafish were treated with 10ul/ml BAY11-7082 intervention for6 h every other day.The level of fasting blood glucose in each group was monitored every week,after three weeks of intervention,ELISA kits was used to detect serum inflammatory cytokines?TNF alpha,IL-18,IL-1??,mitochondrial ROS levels was detected by DCF,the expression of proteins related to inflammation in renal were detected by Western blot.Results:1.The effects of 1,25?OH?₂D₃ on the secretion and inflammatory responses of INS-1 cells.?1?Compared with the control group,1,25?OH?₂D₃ of different doses could promote the proliferation activity of INS-1 cells and increase the secretion of insulin?P<0.01?.Among them,10-7m M was the most significant?P<0.01?.?2?Compared with the DM group,the insulin secretion of 1,25?OH?₂D₃,BAY11-7082,and NLRP3-si RNA group was significantly increased?P<0.05?,and the improvement of NLRP3-si RNA group was most obvious?P<0.01?.?3?Compared with the DM group,the m RNA level of NLRP3 in 1,25?OH?₂D₃,BAY 11-7082,and NLRP3-si RNA intervention group were significantly decreased?P<0.01?.?4?ROS content in supernatant of cells of DM group that was significantly increased compared with control group?P < 0.01?,and 1,25?OH?₂D₃,BAY11-7082,and NLRP3-si RNA intervention could significantly reduce ROS level?P < 0.01?,there was no significant difference between NLRP3-si RNA group and the normal control group?P > 0.05?.?5?Both caspase-1 activity and p65 m RNA level of NF-kappa B were increased in cells of DM group,and both them were significantly decreased after theintervention.?6?The level of TNF alpha,IL – 18 and IL-1?was increased in cells of DM group?P < 0.05 or P < 0.01?,and each of them was decreased after the intervention with 1,25?OH?₂D₃,BAY11-7082 or NLRP3-si RNA.The decrease of IL-18 was most significant.The effects of 1,25?OH?₂D₃ on zebrafish model of type 2 diabetes mellitus..?1?Compared with normal control group,blood glucose of zebrafish treated with300 mg/Kg,350 mg/Kg or 400mg/Kg STZ was significantly increased,and the higher blood glucose level was most obvious in zebrafish treated with 350 mg/Kg STZ.?2?The tail fin of the DM zebrafish model had a regeneration disorder,and the regeneration rate was the lowest in 3 weeks.?3?Compared with normal control group,the fasting blood glucose of the DM model was significantly increased,and decreased significantly after intervention,and the blood glucose tended to be normal as the time of intervention prolonged.?4?The ROS content of zebrafish of DM group was much higher than that in the control group?P<0.05?.Compared with the DM group,ROS content was significantly decreased in zebrafish of 1,25?OH?₂D₃ intervention group?P<0.05?.?5?The contents of inflammatory correlation proteins NLPR3 and NF-kappa B p65 as well as IL-1?level were significantly increased in DM group?P<0.05?,and decreased after 1,25?OH?₂D₃ or BAY intervention.Conclusions:1.The expression of inflammatory cytokines and proteins were significantly increased in diabetes mellitus model,which indicated that diabetes is closely related to inflammatory response.2.1,25?OH?₂D₃ can improve the insulin secretion of INS-1 cells,while reduce the expression level of NF-?B,NLRP3,and inflammatory cytokines.3.1,25?OH?₂D₃ could reduce the level of inflammatory responses in diabetic zebrafish and relieve symptoms of diabetes.4.The signaling pathway of inflammatory response,ROS-NLRP3-NF-?B is closely related to the development of diabetes,and 1,25?OH?₂D₃ might affect this pathway.