口服胰岛素抑制胰岛β细胞凋亡预防NOD鼠糖尿病

Oral administration of insulin inhibits islet β cell apoptosis and prevents diabetes in NOD mice

目的:观察口服胰岛素免疫干预对非肥胖糖尿病(non-obese diabetic,NOD)鼠胰岛炎、β细胞凋亡和糖尿病的影响,并探讨其诱导免疫耐受的机制。方法:86只NOD雌鼠随机分为胰岛素处理组(n=43)和磷酸盐缓冲液(phosphate buffered saline,PBS)对照组(n=43),从4周龄始每周灌胃人普通胰岛素1mg(70μL)2次,12周后改为每周灌胃1次至30周,对照组予等体积的PBS;于12周龄观察胰岛炎和胰岛β细胞凋亡;检测胰岛Fas和FasL的表达;测定血清IL-4和IFN-γ浓度,以及胰岛内I-Aβg7,IL-1β,IFN-γ,Fas,IL-4,TGF-βmRNA和小肠PP(Peyer's Patch)淋巴结IL-4,IFN-γ,TGF-βmRNA的表达水平。结果:NOD鼠口服胰岛素组30周龄和52周龄时发病率为55.6%和70.4%,分别比PBS对照组(85.7%和96.4%)低(P<0.05)。胰岛素组胰岛炎积分比对照组低,但差异无统计学意义(P>0.05)。胰岛素组胰岛Fas抗原表达和β细胞凋亡率均比PBS对照组低(均P<0.05)。胰岛素组胰岛内I-Aβg7,IFN-γ,IL-1β,FasmRNA和PP淋巴结IFN-γmRNA表达均较PBS对照组低(均P<0.05),而IL-4,TGF-βmRNA表达较对照组高(均P<0.05);胰岛素组血清IL-4比PBS组高,IFN-γ比PBS组低(均P<0.05)。结论:口服胰岛素能诱导NOD鼠的免疫耐受而预防糖尿病的发生,但不能阻断胰岛炎的进展。口服胰岛素能诱导调节性T细胞产生,使全身和胰岛局部T细胞由Th1向Th2转型,从而抑制Fas介导的β细胞凋亡而预防糖尿病。

Objective To investigate the effect of oral administration of insulin on insulitis, β cell apoptosis and diabetes in non-obese diabetic （NOD） mice, and to explore the mechanism of immune tolerance induced by insulin. Methods Eighty-six female NOD mice were randomly divided into an insulin group （n =43） and a phosphate buffered saline （PBS） group （n =43）. From 4 weeks of age, the recombinant human insulin （ Humulin R ） 1 mg （ 70 μL ） was administrated in the oral insulin group and 70 μL PBS in the control group respectively, twice per week before 12 weeks of age and then once weekly until 30 weeks. Insulitis and β cell apoptosis of islets were observed at 12 weeks. IL-4 and IFN-γ in the sera were measured by enzyme linked immunosorbent assay （ ELISA）. The expression levels of I-Aβ^g7, IL-4, IFN-γ, IL-1β, Fas and TGF-β mRNA of islets, and IL-4, IFN-γ, TGF-β mRNA of Peyer＇ s patch were measured by reverse transcription-polymer-ase chain reaction （RT-PCR） at 12 weeks. Results The incidences in the insulin group were significantly lower than those in the PBS group （55.6% vs 85.7% at 30 weeks, 70.4% vs 96.4% at 52 weeks, P 〈 0.05 ）. The insulitis scores in the insulin group were lower than those in the PBS group, but there was no statistical significance. Fas expression on islets and apoptotic β cell rates in the insulin group were lower than those in the PBS group （ P 〈 0.05 ）. In the insulin group, serum IL-4 levels were higher, and IFN-γ levels were lower than those in the PBS group （ P 〈 0.05 ） . The levels of I-Aβ^g7, IFN-γ, IL-1β and Fas mRNA transcription in islets and IFN-γ mRNA transcription in Peyer＇s patch were both lower in the insulin group, and IL-4, TGF-β mRNA levels were higher than those in the PBS group （ P 〈 0.05 ）. Conclusion The specific autoantigen insulin may induce the immune tolerance and prevent the diabetes in NOD mice, but it can＇t block the progression of insulitis. Oral administration of insulin can induce the regulatory T cells, and make Th1 to Th2 cytokine shifts in the system and islets, thus preventing the Fas-mediated β-cell apoptosis and diabetes.