高脂饮食肥胖大鼠胰岛细胞凋亡与Mcl-1表达及意义

目的 通过建立高脂饮食诱导肥胖大鼠模型,研究肥胖大鼠胰岛细胞中线粒体途径抗凋亡蛋白髓样细胞白血病因子-1(Mcl-1)表达情况及其与β细胞凋亡的相关性,探究肥胖大鼠胰岛细胞损伤可能的机制。方法 SPF级3周龄雄性SD大鼠72只,随机分为普通饲料组和高脂饲料组 。在高脂喂养8、16、20、24周末筛选出符合肥胖标准的大鼠设为肥胖组,普通饲料组设为对照组。ELISA检测血清,游离脂肪酸(FFA)水平, 通过透射电镜观察胰岛β细胞微观结构改变,免疫组化SP法检测胰岛组织中Mcl-1蛋白的表达情况,并通过Real-Time PCR检测胰腺组织中Mcl-1的mRNA表达水平,原位末端标记法(TUNEL法)检测两组大鼠的胰岛细胞凋亡率。结果 1)高脂喂养20周,肥胖组大鼠血清FFA水平显著高于对照组(P<0.01);2)电镜结果显示,16周末肥胖组大鼠胰岛β细胞结构与对照组相比,出现凋亡样改变,且随时间推移有所加重;3)高脂喂养20周,肥胖组大鼠抗凋亡相关蛋白Mcl-1表达较对照组明显降低(P<0.05);4)RT-PCR结果显示, 高脂喂养16周,抗凋亡相关蛋白Mcl-1的mRNA水平明显降低(P<0.05);5)高脂喂养20周,与对照组大鼠相比,肥胖组凋亡率显著增加(P<0.01);6)大鼠胰岛细胞凋亡率与血清FFA水平呈正相关(r＝0.536,P<0.01),与Mcl-1基因的表达水平呈负相关(r＝-0.776,P<0.01)。结论 高脂饮食肥胖大鼠胰岛β细胞形态结构异常, 线粒体途径抗凋亡蛋白Mcl-1表达降低,胰岛细胞凋亡率与Mcl-1表达呈负相关。提示肥胖大鼠存在胰岛β细胞损伤,其机制与线粒体凋亡途径有关。

Expression and significance of Mcl-1 in islet cell apoptosis of rats with high-fat diet

Objective To analyze the expression level of myeloid cell leukemia-1(Mcl-1) and its correlation with cell apoptosis through the establishment of a high-fat diet induced obese rats model, in order to research islet cells injure mechanism. Methods A total of 72 male Sprague-Dawley rats with specific pathogen free (SPF) weaned were randomly assigned to two groups by 1∶2.Finally 24 rats were in normal diet group and 48 rats in high fat diet group.The rats were killed at the end of the 8,16,20,24 weeks of high-fat diet.After that, the obese rats were sieved out and prepared for following experiment.The obese rats were set up as obese group and the normal diet rats were control group.The serum level of free fatty acids was tested by ELISA to analyze the lipid metabolism in obese rats.The microstructure of pancreas was observed by electron microscope.The protein expression of Mcl-1 was examined by immunohistochemical method, and the mRNA level of Mcl-1 was detected by real-time PCR to explore the possible mechanism of obesity β-cell damage in rats.TUNEL method was used to detect islet cell apoptosis situation of the two groups. Results 1) The serum level of free fatty acids of obese rats was significantly higher than that of the control group after high-fat diet for 20 weeks (P<0.01).2) Electron microscope showed that beta-cells in obese group were hyperplastic, and some appeared apoptosis.3) The protein expression level of Mcl-1 was significantly lower in obese group after high-fat diet for 20 weeks (P<0.05).4) Compared with control group, the mRNA level of Mcl-1 was significantly lower in obese group after high-fat diet for 16 weeks (P<0.05).5) The apoptosis rate of the obese group increased significantly than after high-fat diet for 20 weeks (P<0.01).6) The islet cell apoptosis rate of rats was positively correlated with serum FFA(r＝0.536,P<0.01), and negatively correlated with Mcl-1 gene expression(r＝-0.776,P<0.01). Conclusions Islet β-cell morphological abnormalities existed in obese rats, and the expression level of Mcl-1 gene decreases in obese rats.Moreover, rats′ islet cell apoptosis rate was negatively correlated with Mcl-1 gene expression.It is indicated that islet β-cell is damaged in obese rats, and the mechanism might be associated with mitochondrial apoptosis pathway.