The Protective Effects And Pathogensis Of Mycophenolate Mofetil (MMF) On Diabetes In NOD Mice

The Protective Effects and Pathogenesis of MycophenolateMofetil(MMF)on Diabetes in NOD miceObjects: 1. To investigate the protective effects of mycophenolatemofetil(MMF)on insulitis and pancreaticβ-cell apoptosis in NOD mice. 2. To studythe effects of MMF on cyclophosphamide-induced diabetes in NOD mice.Methods: Expriment one: Twenty- two female NOD mice of eight-week agewere divided into the following two groups randomly:The control group and theMMF-treated group. MMF (30mg/kg) was administered daily by intragastricintubations throughout the experimental period (30 days) and the control group micewere received an equivalent amount of 0.9% NaCl intragastrically. Before andduring all experiments, blood glucose of all mice was assessed. Following treatment,five mice of each group were anaesthetized and then were killed to remove pancreasimmediately. The degree of insulitis and the morphological changes of pancreaticβ-cells apoptosis were determined by light microscope and terminal-deoxynucleotidyltransferase mediated nick end labeling assay (TUNEL). The rest mice weresacrificed for the expression of IFN-γand TNF-αmRNA of pancreas by reversepolymerase chain reaction (RT-PCR) when they got diabetes or twenty-week age.Expriment two: Twenty- eight female NOD mice of eight-week age were receivedCTX (300mg/kg) intra-peritoneally. The mice got diabetes (blood glucose >16mmol/l for two days, n=16) were divided into two groups:Diabetes mellitus (DM)group (n=8) and MMF treated group (n=8). MMF (30mg/kg) was administered dailyby intragastric intubations throughout the experimental period (28 days) and the DMgroup received an equivalent amount of 0.9% NaCl intragastrically. During all experiments, blood glucose of all mice was assessed. Following treatment, mice ofeach group were anaesthetized and then were killed to remove pancreas and spleenfor the next spleen mononuclear cell proliferation immediately. The degree ofinsulitis and the distribution of CD₄⁺,CD₈⁺ and CD₄⁺ CD₂₅⁺ T cells in pancreaticislets were determined by light microscope and immunohistology.Result:1. The protective effects of mycophenolate mofeti(l MMF)on insulitisand pancreaticβ-cell apoptosis in NOD mice:The concentration of blood glucose inMMF-treated group were as same as those in the control group before and during theexperiment (p>0.05). While the insulitis scores and severity of insulitis in the MMFtreatedmice(1.34±1.27) were significantly lower than those of the controlgroup(2.36±1.42) (p<0.01); The percentage ofβ-cell apoptosis of the control group(0.158±0.012) were significantly higher than that of MMFgroup(0.083±0.022)(P<0.01). The expression level of IFN-γmRNA in pancreas inMMF group was significantly lower than that in the control group ( P<0.05), whilethe expression level of TNF-αmRNA in pancreas of both groups were the same(p>0.05). 2. The effect of MMF on cyclophosphamide-treated diabetes in NOD mice:The concentration of blood glucose were(20.21±3.22)mmol/l and(19.31±2.79)mmol/l in DM group and MMF-treated group before treatment (p>0.05). But they were gradually decreased after the MMF treatment. At the end of theexperiment, the blood glucose of MMF treated group (11.6±3.80 mmol/L) was lowerthan that of DM group (18.0±2.10 mmol/L) (P<0.01). Based on HE stained tissuesections, DM group elicited severe injury of pancreas, such as decreasing of the isletscells'numbers and diminishing of the diameter of pancreatic islet. The nuclei of isletscells were more shrunken in diabetic mouse compared with MMF group. Theinsulitis scores in diabetic group (2.77±1.35) was much higher than that of MMFtreatedgroup(2.02±1.31) (p<0.01). The spleen mononuclear cell proliferation in MMF group were inhibited significantly compared with DM group (p<0.01).Immunohistochemical analysis: The majority of mononuclear cells participating ininsulitis process in DM group were almost CD₄⁺ T cells, distributed in the center andisperiphery of the islets, while CD₄⁺ CD₂₅⁺ T cells were not detected. In comparisonto diabetic group, pancreatic islets obtained from mice treated with MMF weremainly without infiltrated cells, and only few mild infiltrates of CD₄⁺T cells could befound in a small number of the islets, and the CD₄⁺ CD₂₅⁺ T cells could been found.CD₈⁺ T cells couldn't be seen in the islets of both groups.Conclusions: 1.MMF could ameliorate early pancreatic injury by the inhibitionof insulitis andβ-cell apoptosis in NOD mice. It suggested that MMF could beeffective in delaying and preventing diabetes especially if treatment is started beforethe onset of diabetes. 2. MMF could decrease blood glucose, abatement insulitis byinhibiting the spleen mononuclear cell proliferation and reducing inflammatoryinfiltration of CD₄⁺T cells in islets. It suggested that MMF could be still effective inthe treatment of diabetes if it was given after the onset of diabetes.