活化T细胞核因子在吡格列酮预防非肥胖糖尿病小鼠糖尿病中作用机制的探讨

The mechanism study of NFAT in pioglitazone prevent diabetes of NOD mice

目的探讨吡格列酮预防非肥胖糖尿病(NOD)小鼠糖尿病的机制及活化T细胞核因子(NFAT)的作用。方法 (1)将4周龄NOD雌鼠随机分为吡格列酮组(摄食含0.02%吡格列酮的混合饲料)及对照组(普通营养饲料),各21只。观察30周龄的累积糖尿病发病率。(2)各组取12周龄未发病NOD鼠(n=15)胰腺苏木素-伊红(HE)染色观察胰岛炎的严重程度;RTPCR半定量检测脾脏白细胞介素4(IL-4)、γ干扰素(IFN-γ)和核因子过氧化物酶体增殖物活化受体γ(PPARγ)mRNA表达水平;酶联免疫吸附试验(ELISA)法测定血清IL-4和IFN-γ水平及脾脏PPARγ、活化T细胞核因子c1(NFATc1)活性。结果 (1)15周龄时,吡格列酮组及对照组发病率分别为4.76%和33.33%(P<0.05);30周龄时,吡格列酮组及对照组发病率分别为57.14%和76.19%(P>0.05)。(2)12周龄时,吡格列酮组胰岛炎指数(1.79±0.75)低于对照组(2.38±0.66)。(3)吡格列酮组脾脏IFN-γmRNA相对光密度值(0.16±0.07)显著低于对照组(0.53±0.26);而PPARγmRNA表达水平(0.91)则高于对照组(0.25)。(4)12周龄NOD鼠吡格列酮组血清IFN-γ水平(561.05±78.61)pg/mL显著低于对照组(666.43±28.42)pg/mL。(5)12周龄吡格列酮组NOD鼠脾细胞PPARγ活性(0.05±0.01)高于对照组(0.02±0.01),NFATc1活性(0.23±0.04)低于对照组(0.33±0.04)。结论吡格列酮活化PPARγ,降低NFATc1活性,减少IFN-γmRNA,血清IFN-γ下降,辅助性T(Th)细胞向辅助性T细胞1型(Th1)方向分化减少,减轻NOD鼠胰岛炎,降低糖尿病的发生。

Objective To investigate the mechanism of pioglitazone preventing diabetes and the role of nuclear factor of attired T cells （NFAT） on non-obese diabetic（NOD） mice. Methods （1）Female NOD mice at 4 weeks of age were randomly divided into pioglitazone group（n= 21） and control group（n= 21）. The accumulative diabetes incidence was followed-up to 30 weeks of age in each group of NOD mice. （2）Pancreas were removed from NOD mice at 12 weeks of age in each group（n= 15） to score insulitis se- verity by routine HE staining. IL-4,IFN-γ, and peroxisome proliferator-activated receptor γ（PPARγ） mRNA levels in spleens were tested by RT PCR. IL-4 and IFN-γ levels in sera,the activity of PPARy and NFATcl nuclear protein in spleens were measured by enzyme linked immunosorbent assay （ELISA）. Results （1） At 15 weeks of age,the diabetes incidence was 4.76 % in pioglitazone group,and 33.33G in control group（P〈0.05）. At 30 weeks of age,the diabetes incidence was 57.14% in pioglitazone group,and 76.19% in control group（P〉0.05）. （2） At 12 weeks of age,the insulitis score in pioglitazone group was lower than that in control groupE（1.79t0.75） vs. （2.38±0.66）,P〈0. 051. （3） IFN-γ mRNA level in pioglitazone group was lower than that in control group[（0.16±0.07） vs. （0.53±0.26） ,P〈0.05] ,and PPAR3＇ mRNA level in pioglitazone group was higher than that in control group（0.91 vs. 0. 25,P〈0.05）. （4）IFN-γ, level in pioglitazone group was lower than that in control group [-（561.05±78.61）pg/ mL vs. （666.43±28.42）pg/mL,P〈0. 051. （5）At 12 weeks of age,the spleen PPAR＇/nuclear protein activity in pioglitazone group was higher than that in control group （0. 05±0. 01） vs. （0.02±0.01） ,P〈0.05）],and NFATcl nuclear protein activity was low- er than that in control groupr（0.23±0.04） vs. （0.33±0.04） ,P〈0. 051. Conclusion Pioglitazone could activate PPARy nuclear protein,inhibit activity of NFATcl nuclear protein,downregulate IFN-7,diminish Th cells deviating to Thl, and sequently prevents insulitis and diabetes onset in NOD mice.