普伐他汀对非肥胖糖尿病小鼠糖尿病预防作用的机制

Effects of pravastatin in prevention of diabetes and mechanism thereof： experiment with non-obese diabetic mice

目的探讨普伐他汀对非肥胖糖尿病（NOD）小鼠糖尿病的预防作用及机制。方法3^-14周龄NOD雌鼠分为4组，对照组（摄普通饲料，30只），小剂量组（普伐他汀1mg·kg^-1·d^-1，29只），中剂量组（普伐他汀10mg·kg^-1·d^-1，29只）和大剂量组（普伐他汀40mg·kg^-1·d^-1，30只），观察糖尿病发病至30周龄。各组另取8只12周龄未患病NOD鼠，胰腺HE染色观察胰岛炎；制备脾细胞悬液后，流式细胞仪检测脾细胞中CD4＋CD25＋调节性T细胞数量；氚-胸腺嘧啶核苷掺入法检测脾细胞对特异性抗原的刺激增殖反应；ELISA法测特异性抗原刺激后脾细胞培养上清干扰素γ（IFN-γ）和白细胞介素4（IL-4）水平；RT-PCR检测脾脏IFN-γ、IL-4mRNA的表达水平。结果30周龄时，大剂量普伐他汀组NOD鼠糖尿病的发病较对照组明显降低（P〈0．01）。12周龄时，大剂量普伐他汀组胰岛炎严重程度低于对照组（P〈0．001）；大剂量普伐他汀组脾细胞培养上清中IFN-γ水平（42±20）pg／ml，脾脏IFN-γmRNA表达水平（0．24±0．10）pg／ml均低于对照组（157±32）pg／ml，（0．81±0．18）ps／ml，均P=0．000），IL-4水平（91±22）pg／ml，脾脏IL-4mRNA表达水平（0.39±0．18）pg／ml均高于对照组[（44±20）pg／ml，P=0．000；（0．20±0．08）pg／ml，P=0．002）]；小、中、大剂量普伐他汀组和对照组特异性抗原增殖指数分别为3．85±0．35、3．53±0．82、3．32±0．44、3．70±0．62，各组间差异无统计学意义（均P〉0．05）；小、中、大剂量普伐他汀组和对照组脾细胞中CD4＋CD25＋调节性T细胞数量分别为（9．6±2．6）％、（10．2±2．4）％、（8．9±2．7）％、（10．0±2．4）％，各组间差异无统计学意义（均P〉0．05）。结论早期使用大剂量普伐他汀进行干预，可以通过下调Th1细胞因子INF-γ，上调Th2细胞因子IL-4，促使免疫平衡向Th2方向偏移，从而减轻NOD鼠胰岛炎，预防NOD鼠糖尿病的发生。

Objective To explore the effects of pravastatin in prevention of diabetes and mechanisms thereof. Methods 1183 to 4-week-old female non-obese diabetic （NOD） mice were randomly divided into 4 groups： control group （ n = 30）, fed with regular diet, low-dose pravastatin group （ n = 29 ）, fed with 1 mg·kg^-1·d^-1 pravastatin via the diet, medium-dose pravastatin group （n =29） fed with 10 mg·kg^-1·d^-1 pravastatin via the diet, and high-dose pravastatin group （n = 30 ）, fed with 40 mg ·kg^-1·d^-1 pravastatin via the diet. The mice were followed up till they were 30-week old. Urine glucose was measured every week. Eight 12-week old mice without onset of DM from each group were killed with the pancreas taken out to undergo insulitis scoring via microscopy. Another 8 12-week old mice without onset of DM from each group were killed with their spleens taken out. Suspension of splenocytes was made, put into a 96-well plate, and stimulated by rGAD65 ; and [ ^3H]-thymidine was incorporated. The stimulation index （SI） of the splenocytes was calculated. Flow cytometry was used to observe the population of CD4＋ CD25＋ regulatory T cells. ELISA was used to detect the interferon （IFN）-γ and interleukin （IL）-4 levels in the supernatants of splenocytes. RT-PCR was used to detect the mRNA expression of IFN-γand IL-4 in the spleen. Results At 30 weeks of age, the incidence rate of DM onset of the high-dose pravastatin group was significantly lower than that of the control group （ P = 0. 003 ）, and the incidence rates of DM onset of the medium and low dose groups were not significantly different from that of the control group （ both P 〉 0.05 ）. The severity of insulitis at 12 weeks of age of the high-dose pravastatin group was significantly lower than that of the controls group （P 〈 0.001 ）. There were no significant difference in the SI level and in the percentage of CD4＋ CD25＋ regulatory T cells among the four groups （ all P 〉 0.05 ）. The IFN-γ level in the supernatant of splenocytes of the high-dose pravastatin group was （42 ±20） pg/mi, significantly lower than that of the control group [ （ 157 ± 32 ） pg/mi, P = 0. 000 ]. The IFN-γ mRNA expression level in the spleen of the high-dose pravastatin group was 0. 24 ±0. 10, significantly lower than that of the control group （0.81± 0. 18, P = 0. 000）. The IL-4 level in the supernatants of splenocytes of the high-dose pravastatin group was （91 ± 22） pg/m, significantly higher than that of the control group [ （44 ± 20） pg/ml, P =0.000） ]. The IL-4 mRNA expression level in spleen of the high-dose pravastatin group was 0.39 ± 0. 18, significantly higher than that of the control group （0.20 ± 0.08, P = 0. 002 ）. However, there were not significant differences in the IFN-γ and IL-4 levels in the supernatant and the IFN-γ and IL-4 mRNA expression in spleen among the medium dose, low dose, and control groups （ all P 〉 0.05 ）. Condusion High-dose pravastatin therapy at early time decreases the IFN-γlevel and increases the IL-4 level, shifts the immune response to the direction of Th2, and subsequently lessens insulitis and prevents DM.