二甲双胍通过抑制RORγt对2型糖尿病大鼠IL-17的影响

发布时间：2018-03-28 02:25

  本文选题：IL-17　切入点：RORγt　出处：《郑州大学》2017年硕士论文

【摘要】：1研究背景2型糖尿病作为一种慢性代谢性疾病,其发病机制复杂,涉及环境与遗传两大方面,胰岛素分泌不足及胰岛素抵抗是其发病的关键因素。相关研究表明,2型糖尿病不仅是一种代谢性疾病,也是一种亚临床炎症疾病,其发病机制与慢性低度炎症密切相关,因此提出2型糖尿病的炎症学说。大量研究证明,炎性因子、促炎性因子与T2DM密切相关,但其发病机制尚未完全阐明。近年来研究发现,IL-17与2型糖尿病、1型糖尿病、高血压及代谢综合征均有一定的相关性。IL-17是一种促炎细胞因子,具有强大的招募中性粒细胞、促进多种细胞释放炎性因子、促进细胞增值及抑制肿瘤生长等多种生物学作用,可导致炎症反应、自身免疫性疾病等发生和发展。而IL-17也是一种重要的炎症介质,它可以诱导其他炎症细胞如IL-6、TNF-α等的表达,产生致炎作用。大量研究证实,无论体外还是体内,Th17分化IL-17细胞都需要RORγt的参与,证实RORγt是Th17细胞分化的关键正向转录调节因子。在活化T细胞中RORγt高表达可产生大量的IL-17,并且RORγt基因敲除小鼠体内TH17细胞和IL-17明显减少。二甲双胍于20世纪进入临床应用,大量循症医学证据显示其有良好的降糖作用,且安全性及效价比均高,目前许多权威的糖尿病指南把二甲双胍推荐为治疗2型糖尿病的一线首选药物。近年来一些研究显示其除了降糖作用外同时具有抗炎作用,然而其抗炎作用机制仍未完全阐明。2材料和方法2.1分组及饲喂方法SPF级SD雄性大鼠50只,体重为89.62±7.42g。适应性喂养7天后,随机分为2组,一组20只,一组30只,分别饲喂普通饲料、高脂高糖饲料,在第8周末时腹腔注射STZ,使用STZ+高脂高糖饲样联合造模,以30mg/kg STZ腹腔注射造模,3天后测空腹血糖,将空腹血糖大于11mmol/L视作为造模成功。将造模组随机分为3组,每组10只,一组继续饲喂高脂高糖(M组),一组饲喂高脂高糖+二甲双胍(M+MET组),一组饲喂高脂高糖+胰岛素(M+INS);普通饲料组随机分为2组,每组各10只,一组饲喂普通饲养(C),一组饲喂普通饲料+二甲双胍(C+MET)。MET按200mg/Kg剂量灌胃,不给予MET的大鼠使用生理盐水灌胃。整个实验共持续12周。2.2观察指标(1)全自动血标本分析仪检测血糖浓度及胰岛素含量,计算胰岛素抵抗指数;(2)ELISA法检测血清中IL-17的表达水平;(3)Western blotting法检测RORγt蛋白的表达水平;(4)RT-PCR法检测RORγt mRNA基因的表达;(5)免疫组化观察脾脏组织中RORγt及IL-17阳性细胞因子的表达。3结果3.1 HOMA-IR各组大鼠HOMA-IR水平之间的差异有统计学意义(F=163.923,P=0.000)。M组、M+INS组和M+MET组的HOMA-IR均比C组高,差异有统计学意义(P0.05)。M+INS组、M+MET组的HOMA-IR均比M组低,差异有统计学意义(P0.05)。3.2血清IL-17的表达水平各组大鼠血清平均IL-17水平之间的差异有统计学意义(F=59.778,P=0.000)。进一步两两比较,M组、M+INS组、M+MET组的IL-17水平均高于C组,差异有统计学意义(P0.05)。M+MET组的IL-17水平低于M组,差异有统计学意义(P0.05)。C+MET组的IL-17水平低于M+MET组,差异有统计学意义(P0.05)。M+INS组的IL-17水平高于M+MET组,差异有统计学意义(P0.05)。C组的IL-17水平与C+MET组相比无统计学意义(P0.05)。M组的IL-17水平与M+INS组相比无统计学意义(P0.05)。3.3大鼠HOMA-IR与IL-17水平的相关性分析M组,HOMA-IR与血清IL-17水平呈正相关,r=0.718,P=0.019;M+MET组,HOMA-IR与血清IL-17水平呈正相关,r=0.706,P=0.022。3.4各组大鼠脾脏组织RORγt mRNA的表达各组大鼠脾脏组织平均RORγt mRNA水平之间的差异有统计学意义(F=910.176,P=0.000)。进一步两两比较,M组、M+INS组、M+MET组的RORγt mRNA水平均高于C组,差异有统计学意义(P0.05)。M+MET组的RORγt mRNA水平低于M组,差异有统计学意义(P0.05)。C+MET组的RORγt mRNA水平低于M+MET组,差异有统计学意义(P0.05)。M+INS组的RORγt mRNA水平高于M+MET组,差异有统计学意义(P0.05)。C组的RORγt mRNA水平与C+MET组相比无统计学意义(P0.05)。M组的RORγt mRNA水平与M+INS组相比无统计学意义(P0.05)。3.5各组大鼠脾脏组织RORγt蛋白的表达各组大鼠脾脏组织平均RORγt蛋白表达水平之间的差异有统计学意义(F=505.668,P=0.000)。进一步两两比较,M组、M+INS组、M+MET组的RORγt蛋白水平均高于C组,差异有统计学意义(P0.05)。M+MET组的RORγt蛋白水平低于M组,差异有统计学意义(P0.05)。C+MET组的RORγt蛋白水平低于M+MET组,差异有统计学意义(P0.05)。M+INS组的RORγt蛋白水平高于M+MET组,差异有统计学意义(P0.05)。C组的RORγt蛋白水平与C+MET组相比无统计学意义(P0.05)。M组的RORγt蛋白水平与M+INS组相比无统计学意义(P0.05)。3.6各组大鼠脾脏组织IL-17与RORγt的免疫组化表达水平M组较M+INS组、M+MET组、C组、C+MET组的脾脏组织中IL-17及RORγt细胞阳性比均高,差异有统计学意义(P0.05)。4结论(1)2型糖尿病大鼠的血清中IL-17及脾脏组织RORγt的表达水平升高。(2)炎症因子IL-17表达增高可能加重2型糖尿病大鼠的胰岛素抵抗。(3)二甲双胍可通过抑制脾脏RORγt水平使2型糖尿病大鼠IL-17表达减少。
[Abstract]:1 research background of type 2 diabetes mellitus is a chronic metabolic disease, its pathogenesis is complex, involving environmental and genetic aspects of the two, the lack of insulin secretion and insulin resistance is a key factor in its pathogenesis. Related studies have shown that type 2 diabetes is a metabolic disease, is also a kind of subclinical inflammatory disease, the and the pathogenesis of chronic low-grade inflammation is closely related, therefore proposed the type 2 diabetes inflammation theory. Many studies have demonstrated that inflammatory cytokines, inflammatory cytokines are closely related with T2DM, but its pathogenesis has not been fully elucidated. Recent studies have found that IL-17 patients with type 2 diabetes mellitus, type 1 diabetes, hypertension and metabolic syndrome.IL-17 have a certain correlation between syndrome is a proinflammatory cytokine, has a strong recruitment of neutrophils, promote various cells to release inflammatory cytokines, promote cell proliferation and inhibit tumor growth and other biological The role, can cause inflammation, autoimmune disease and development. IL-17 is also an important medium of inflammation, it can induce other inflammatory cells such as IL-6, expression of TNF-, to produce proinflammatory effects. A number of studies have demonstrated that both in vitro and in vivo, Th17 IL-17 cells to ROR gamma the involvement of T, confirmed that the ROR gamma t is a key positive transcription regulatory factor. The differentiation of Th17 cells in the activation of T cells in ROR gamma t high expression can produce a large number of IL-17, ROR and gamma T gene knockout mice TH17 cells and IL-17 decreased significantly. The clinical application of metformin in twentieth Century, a large number of evidence-based medical evidence it has good hypoglycemic effect, and the safety and potency ratio are high, at present many authoritative diabetes guidelines to metformin recommended as the preferred first-line drugs for the treatment of type 2 diabetes. In recent years, some studies show that in addition to its hypoglycemic effect At the same time have anti-inflammatory effects with its anti-inflammatory effect, however the mechanism is not yet fully elucidated.2 material and method 2.1 grouping and feeding method of SPF 50 SD male rats, weight 89.62 + 7.42g. for adaptive feeding for 7 days, were randomly divided into 2 groups, a group of 20, a group of 30 rats were fed normal diet. High fat and high glucose diet, in eighth weeks after intraperitoneal injection of STZ, use the STZ+ high fat and high sugar feeding combined with 30mg/kg like model, intraperitoneal injection of STZ rats, after 3 days of fasting blood glucose, fasting blood glucose greater than 11mmol/L as the successful model. The rats were randomly divided into 3 groups, 10 rats in each group. Continue feeding high fat and high glucose (M group), a group fed with high fat and high glucose + metformin (M+MET group), a group fed with high fat and high glucose + insulin (M+INS); normal diet group were randomly divided into 2 groups, 10 rats in each group, one group was fed with normal chow (C), a group fed with normal diet metformin + (C+MET).MET by 20 0mg/Kg dose administered, not given MET rats using saline. The whole experiment lasted 12 weeks.2.2 observation index (1) full automatic blood analyzer to detect the concentration of blood glucose and insulin levels, insulin resistance index; (2) IL-17 in serum were measured by ELISA expression level; (3) expression the level of detection of ROR gamma t protein Western blotting method; (4) to detect the expression of ROR gene of t mRNA gamma RT-PCR method; (5) immunohistochemistry to observe the expression of.3 in spleen tissue results ROR gamma T and IL-17 positive cell factor differences in the level of HOMA-IR 3.1 HOMA-IR rats between the statistically significant (F=163.923. P=0.000).M group, M+INS group and M+MET group HOMA-IR were higher than C group, the difference was statistically significant (P0.05) in.M+INS group, M+MET group, HOMA-IR was lower than M group, the difference was statistically significant (P0.05) between the serum expression level of rats in each group the average IL-17 level of serum.3.2 IL-17 There was a significant difference (F=59.778, P=0.000). A further 22 compared with M group, M+INS group, M+MET group, IL-17 levels were higher than C group, the difference was statistically significant (P0.05).M+MET levels of IL-17 were lower than those of M group, the difference was statistically significant (P0.05).C+MET levels of IL-17 were lower than group M+MET, there are statistically significant difference (P0.05) of.M+INS group IL-17 was higher than M+MET group, the difference was statistically significant (P0.05).C group IL-17 levels compared with the C+MET group had no statistical significance (P0.05) in.M group compared with M+INS group IL-17 level had no statistical significance (P0.05) of M group between.3.3 and IL-17 levels in HOMA-IR rats and, HOMA-IR was positively related with the serum level of IL-17, r=0.718, P=0.019; group M+MET, HOMA-IR was positively associated with serum levels of IL-17, r=0.706, P=0.022.3.4 between groups of rat spleen tissue ROR mRNA expression of gamma t spleen tissue of rats in each group were averaged ROR gamma t mRNA level There was a significant difference (F=910.176, P=0.000). A further 22 compared with M group, M+INS group, M+MET group, ROR gamma t mRNA levels were higher than C group, the difference was statistically significant (P0.05) group.M+MET ROR y t mRNA level lower than the M group, the difference was statistically significant (P0.05).C+ MET group ROR y t level of mRNA was lower than that in M+MET group, the difference was statistically significant (P0.05) group.M+INS ROR y t mRNA level is higher than that of M+MET group, the difference was statistically significant (P0.05) group.C ROR y t mRNA level compared with the C+MET group had no statistical significance (P0.05) group.M ROR mRNA y t level and M+INS group no significant difference (P0.05) there was significant difference between the expression level of.3.5 group rat spleen tissue ROR protein expression of gamma t spleen tissue of rats in each group were averaged ROR gamma t protein (F=505.668, P=0.000). A further 22 compared with M group, M+INS group, M+MET group, ROR gamma t protein levels were significantly higher than that of C group, difference There was a significant correlation (P0.05) group.M+MET ROR gamma t protein level is lower than the M group, the difference was statistically significant (P0.05) group.C+MET ROR gamma t protein level is lower than the M+MET group, the difference was statistically significant (P0.05) group.M+INS ROR gamma t protein level is higher than that of M+MET group, the difference was statistically significant (P0.05.C) group of ROR gamma t protein levels compared with the C+MET group had no statistical significance (P0.05) group.M ROR gamma t protein levels compared with the M+INS group had no statistical significance (P0.05) group were immunized.3.6 rat spleen tissue IL-17 and ROR gamma t expression level in M group compared with M+INS group, M+ MET group. Group C, IL-17 and ROR gamma t C+MET cells in the spleen tissue were higher than positive, the difference was statistically significant (P0.05) conclusion.4 (1) increased the expression level of IL-17 in serum and spleen tissue of rats with type 2 diabetes in ROR gamma t. (2) increased the expression of inflammatory factor IL-17 may add weight 2 the insulin resistance in diabetic rats (3) metformin can reduce the expression of IL-17 in type 2 diabetic rats by inhibiting the level of ROR gamma t in the spleen.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
，

本文编号：1674356