艾塞那肽诱导自噬流受损引起大鼠胰腺损伤机制的实验研究

Exenatide induce the impairment of autophagy flux to damage rat pancreatic tissue

目的探讨艾塞那肽作用10周后，大鼠胰腺腺泡细胞内自噬的变化情况。方法50只SD雄性大鼠中按随机数字表法随机选取30只予以高糖高脂饲料喂养2个月后，再腹腔注射链脲佐菌素（35mg／kg）以诱导糖尿病模型，并将26例糖尿病造模成功的大鼠随机分为数量相当的2组，即糖尿病模型实验组13只和糖尿病模型对照组13只。另外20只大鼠常规饲养2个月后，按随机数字表法随机等分为正常大鼠对照组和正常大鼠实验组。之后。2实验组大鼠皮下注射艾塞那肽5μg／kg·次，2次/d，每日清晨8点和下午6点在餐前1h给药，2对照组大鼠在同样时间予以等量生理盐水皮下注射。10周后取胰腺组织标本。免疫组化检测胰腺组织中GLP-1R的表达，western blot检测胰腺组织中LC3-Ⅰ、LC3-Ⅱ和p62的表达并计算LC3-Ⅱ／Ⅰ比值和p62的相对表达量，每例胰腺标本均行HE染色以了解病变情况。结果正常大鼠实验组有6只出现胰腺腺叶结构破坏、胰腺腺泡细胞萎缩和细胞间隔增宽等病理改变．糖尿病模型实验组11只大鼠有7只出现上述病理改变。2实验组GLP-1R表达高于2对照组，差异有统计学意义（P〈0．05）。2实验组LC3-Ⅱ／Ⅰ比值和p62／β-actin比值大于各组相应对照组，差异均有统计学意义（P〈0．05）。结论艾塞那肽长期作用于正常大鼠和糖尿病模型大鼠可上调胰腺腺泡细胞上GLP-1R的表达并诱导胰腺腺泡细胞自噬流受损．p62蛋白积累，而引起胰腺损伤。

Objective To explore the alteration and effect of autophagy in pancreas tissue of rat injected by exenatide. Methods Diabetes model rats were induced by two-month high-sugar and high-fat diet and streptozotocin injection （35 mg/kg） in normal rats. 50 SD male rats were divided into four groups according to the principle of complete random design, namely normal control group（n=10）, normal exenatide-injected group （n=10）, diabetes-model control group （n=lS） and diabetes-model exenatide-injeeted group （n=15）. Rats in exenatide-injected groups were subcutaneously injected with exenatide respectively in 5μg/kg dose each time, twice a day, at 8 a.m. and 6 p.m. Animal weights were weighted weekly and the dose of exenatide was adjusted according to current weight. Rats in the two control groups were injected with the corresponding amount of saline. After 10 weeks of treatment, all rats were killed and pancreatic tissues were disposed. Immunohistochemistry was used to measure the expression of GLP-1R in pancreatic tissues. Western blot was used to test the expressions of LC3-Ⅰ,LC3-Ⅱ and p62 in pancreatic tissues, and LC3-Ⅱ/Ⅰ ratio and p62 were compared between any two groups. All specimens were stained with hematoxylin-eosin （HE）. The data were expressed as means ± standard deviation and were analyzed by unpaired Student t test using SPSS 18.0 statistics software. P value 〈0.05 was considered to be statistically significant for all tests. Results The pancreatic tissues from 13 rats （6 from the normal exenatide-injected group and 7 from the diabetes-model exenatide-injected group） appeared pathological changes such as gland structure damage, pancreatic cells atrophy and cells compartment broadening. The expressions of GLP-1R, LC3-Ⅱ and p62, and LC3B-Ⅱ/Ⅰ ratio in the two exenatide-injected groups were higher than those in the respective control group, and the differences had statistical significance （P〈0.05）. Conclusions Long-term sub- cutaneous injection of exenatide can upregulate the expression of GLP-1R in rat pancreatic acinar cells and may induce the impaiment of autophagy flux in rat pancreatic cell.