吡格列酮对非肥胖性糖尿病小鼠糖尿病的预防作用及机理探讨

Preventive effects of pioglitazone on diabetes and relevant mechanisms, experimental study on non-obese diabetic mice

目的 探讨吡格列酮对非肥胖性糖尿病 (NOD)小鼠胰岛炎和糖尿病的影响及其作用机理。方法 4周龄NOD雌性小鼠随机分为 3组 ,分别摄食 0 0 1% (小剂量组 ,2 3只 )和 0 0 4 %(大剂量组 ,2 5只 )的吡格列酮混合饲料和普通营养饲料 (对照组 ,2 5只 )。每周检测尿糖 1次 ,发现阳性后用血糖仪测血糖 ,连续 2次血糖≥ 16 7mmol/L即诊断为糖尿病。各组取 12周龄未患病的小鼠胰腺组织切片HE染色后计算胰岛炎积分 ,取脾后制成脾细胞悬液行细胞培养 72h ,酶联免疫吸附试验法测定血清、脾细胞培养液上清干扰素 γ(IFN γ)和白介素 (IL) 4水平 ;抽提胰腺总RNA后行反转录 聚合酶链反应半定量检测IFN rmRNA水平。结果 (1) 30周龄时 ,对照组、小剂量组和大剂量组糖尿病发病率分别为 80 0 %、6 0 9%和 6 0 0 % (P >0 0 5 ) ;但在下列时点药物组与对照组发病率之间差异有显著意义 (P <0 0 5 ) :小剂量组 :① 10 0d时发病率 0 %对 16 % ;185d时发病率 39%对6 8% ;②大剂量组 :110d时 :0 %对 16 % ;12 0d时 :4 %对 2 4 % ;135d时 :12 %对 36 %。 (2 ) 12周龄时 ,小剂量组、大剂量组和对照组胰岛炎积分分别为 1 0 1± 0 6 8、1 19± 0 84和 1 99± 0 75 (P >0 0 5 ) ;但总药物组和对照组相比差异有显著意义 (

Objective To explore the effects of pioglitazone on insulitis and diabetes and relevant mechanism. Methods Seventy-three female non-obese diabetic (NOD)/Lt mice aged 4 weeks were randomly divided into 3 groups, control group (n=25, fed with regular diet), low dosage pioglitazone group (n=23, pioglitazone of the concentration of 0.01% was added into the feed) and high dosage pioglitazone group (n=25, pioglitazone of the concentration of 0.04% was added into the feed). The mice were killed when diabetes developed or they reached the age of 30 weeks. The body weight and amount of food intake were measured every week and the amount of drug intake was calculated. Urine glucose was checked weekly from week 10 to week 30. When urine glucose was positive and relevant symptoms appeared, blood glucose was measured. The criterion of diagnosis of diabetes was the consecutive blood glucose level ≥16.7 mmol/L for 2 times. At the 12th week 4～7 mice from the 3 groups respectively were killed and their pancreases were removed to be scored on insulitis by HE staining, the spleen cells were cultured. The IL-4 and IFN-r levels in serum and supernatants of spleen cell cultures were measured by ELISA. The pancreatic IFN-r mRNA level was tested using RT-PCR method. Results (1)At the age of 30 weeks, the diabetes incidence rates was 80%(20/25) in the control group, 60.9% (14/23) in the low dose group, and 60% (15/25) in the high dose group (P>0.05). At the following time points the diabetes incidence rates of the 2 treated groups were lower than that of the control group (all P<0.05): ①0% in the low dose group vs 16% : of the control group at the age of 100 days, and 39% vs 68% at the age of 185 days; and ② 0% in the high dose group vs 16% in the control group at the age of 110 days, 4% vs 24% at the age of 120 days, and 12%vs 36% at the age of 135 days. (2)There was no difference in insulitis scores between the control group and low dose or high dose groups at the age of 12 weeks (1.99±0.75 vs 1.01±0.68 and 1.19±0.84, both P>0.05) , however, the score of the combined pioglitazone group (low dose group + high dos group) was significantly higher than that of the control group (1.12±0.75 vs 1.99±0.75, P<0.05). (3)There was no differences in the IL-4/IFN- r ratios in serum and splenocyte culture supernatant and pancreatic IFN-r mRNA levels among the three groups (all P>0.05). Conclusion Pioglitazone, to some extent, lessens the insulitis severity and delays the diabetes onset. Its mechanism may be unrelated to immune deviation of Th1 to a Th2.