The Role Of 1,25-（OH）₂D₃ In Cathepsin G-mediated Islet Dysfunction In Type 1 Diabetes Mellitus

Background:Type 1 diabetes mellitus（T1DM）is an autoimmune disease with progressive damage of isletβcells under the influence of multiple genetic and environmental factors,the immune damage of isletβcells started before the appearance of hyperglycemia symptoms,and gradually aggravated with the course of the disease.Therefore,it is necessary to explore how to delay the immune damage of islet function in the early stage of T1DM,protect the residual isletβcell function and restore the islet function in the patients with T1DM.We have previously found that Cathepsin G（Cat G）plays an important role in the activation of CD4+T cells in T1DM mice,specific blocking of Cat G can reduce the activity of CD4+T cells in NOD mice,which can reduce the damage of isletβcells caused by inflammatory factors secreted by CD4+T cells.Vitamin D deficiency is common in patients with T1DM,and vitamin D supplementation can reduce the risk of T1DM.Our previous study found that supplementation with 1,25-（OH）₂D₃attenuated proinsulin-induced CD4+T cell activation in T1DM patients,and 1,25-（OH）₂D₃downregulated Cat G in normal humans,Besides Cat G is a protease that plays a dominant role in proinsulin processing and presentation in vitro,and Cat G-specific inhibitors can attenuate the T cell-mediated immune response in T1DM,Therefore we proposed that 1,25-（OH）₂D₃may play a protective role on T1DM by regulating the activity of Cat G and influencing CD4+T cell activation.Object:1.To confirm the harmful effect of Cat G on the isletβcells.2.The apoptosis ofβcells induced by IL-1βand IFN-γcan be influenced by the level of Cat G.3.To clarify that 1,25-（OH）₂D₃can modulate the activity of CD4+T cells by regulating the level of Cat G in T1DM,thus exert the protective effect on the function of isletβcells in T1DM.Method:1.To study the effects of Cat G on the viability,apoptosis and insulin secretion of islet cell line Min-6.Min-6 cells were cultured in vitro with different concentrations of Cat G（0.02μg/ml,0.1μg/ml,0.5μg/ml,2.5μg/ml）.Cell viability was measured by CCK-8,apoptosis was detected by TUNEL staining,insulin content in supernatant was measured by ELISA,and DNA content was measured by Flow cytometry to analyze cell cycle.2.To observe the effect of Cat G on cytokine-induced apoptosis of Min-6 cells.Min-6 cells were transfected with lentiviral vector containing over-or low-expression of Cat G,and the empty vector was used as control group.After 24 hours of co-culture with IL-1β（150 pg/m L）and IFN-γ（5 ng/m L）,the changes of cell apoptosis and insulin secretion in the supernatants were observed.3.To investigate the effect of 1,25-（OH）₂D₃on the disease progression in newly diagnosed NOD mice.The newly diagnosed NOD mice were given Calcitriol（0.05 ml of peanut oil,0.03 g/kg.d,orally administered with peanut oil as a solvent control group）for 4 weeks.The blood glucose,body weight and food intake were monitored,and the glucose level was evaluated by glucose tolerance test.4.To explore the effect of 1,25-（OH）₂D₃on islet function in newly diagnosed NOD mice.The newly diagnosed NOD mice were given Calcitriol（0.05 ml of peanut oil,0.03 g/kg.d,orally administered with peanut oil as a solvent control group）for 4 weeks.The function of mouse isletβcells was evaluated by insulin release test,and then the islets were isolated.The pathological changes of islets and the apoptosis of isletβcells were observed.5.To investigate the role of 1,25-（OH）₂D₃in regulating the immune microenvironment of islets of Langerhans by down-regulating the expression of Cat G.Isletsβcells from 6-week-old NOD mice were primarily isolated with CD4+T cells.After the isletβcells were transfected with Cat G-silenced lentivirus and highly expressed lentivirus,they were co-cultured with CD4+T cells,1,25-（OH）₂D₃intervention for 24 hours.The expression of Cat G in mouse pancreatic monocytes was detected by WB and RT-q PCR,and the levels of TNF-α,IFN-γ,TGF-β1,IL-4and other cytokines in the supernatants were measured before and after1,25-（OH）₂D₃intervention CD4+T cell activation（Th1,Th2,and Treg cell ratios）was measured using Flow cytometry.6.Co-culture of CD4+T cells with 1,25-（OH）₂D₃inhibited the activation of CD4+T cells by down-regulating the expression of Cat G.The transfected isletβcells were co-cultured with CD4+T cells and treated with 1,25-（OH）₂D₃for 24 hours.The Flow cytometry of CD4+T cell activation markers（CD25,CD69）were measured by CCK-8,Flow cytometry and ELISA were used to detect the viability,apoptosis and insulin level of isletβcells co-cultured with CD4+T cells.Result:1.Cat G can directly damage isletβcells by inhibiting cell proliferation,decreasing cell viability,shortening cell cycle,inducing apoptosis and decreasing insulin secretion.2.Overexpression of Cat G can aggravate the apoptosis ofβcells induced by cytokines.3.The intervention of 1,25-（OH）₂D₃could delay the disease progression of T1DM mice.4.1,25-（OH）₂D₃could improve islet function of T1DM mice by inhibiting apoptosis.5.1,25-（OH）₂D₃regulates the islet immune microenvironment by down-regulating the expression of Cat G.6.Overexpression of Cat G may interfere with the protective effect of1,25-（OH）₂D₃on isletβcells and the inhibitory effect on the activation of CD4+T cells.Conclusion:1.Cat G can directly damage isletβcells,and up-regulation of Cat G level can aggravate cytokine-inducedβcell apoptosis.2.1,25-（OH）₂D₃could down-regulate the level of Cat G in NOD mice,inhibit the activation of CD4+T cells,and improve blood glucose and islet function.There were synergistic effects when combined with Cat G inhibitors.3.Overexpression of Cat G may interfere with the protective effect of1,25-（OH）₂D₃on isletβcells and the inhibitory effect on CD4+T cell activation.This study provides a new theoretical and experimental basis for clinical application of 1,25-（OH）₂D₃to protect islet cell function and delay or block the development of T1DM.