辅助降糖颗粒与二甲双胍联合用药对ZDF大鼠胰岛素抵抗的作用及机制研究

发布时间：2018-06-25 02:49

本文选题：胰岛素抵抗 + microRNA　；参考：《北京中医药大学》2017年博士论文

【摘要】：目的:探讨辅助降糖颗粒与二甲双胍联合用药对ZDF大鼠胰岛素抵抗的作用及机制,为中西药联合应用提供科学依据,为临床用药提供指导。方法:采用随机对照设计方法,动物实验选取自发性2型糖尿病动物模型ZDF(fa/fa)大鼠28只,pumina#5008饲料诱导性喂养4周,不同次随机血糖≥11.1 mmol/L纳入实验。按体重、随机血糖随机分为:模型组、二甲双胍组、辅助降糖颗粒组(降糖颗粒组)、辅助降糖颗粒联合二甲双胍组(联合组),每组7只;另设7只同周龄正常ZDF(fa/+)大鼠为正常组,连续干预6周。实验过程中观察记录大鼠一般情况、体重、空腹血糖,第6周分别进行OGTT、ITT实验;6周后大鼠麻醉、取血,分别冻存和固定肝脏,检测血清中IR相关指标FBG、Fins和HOMA-IR,血脂指标TG、TC和FFA,肝肾功能指标AST、Scr和BUN,氧化应激和炎性细胞因子MDA、SOD、CAT和TNF-α,以及细胞因子Adiponectin、Resistin;检测肝脏糖原含量,行HE、PAS染色观察肝脏病理形态变化;高通量测序检测肝脏microRNAs表达,NCBI、The Gene ontology、KEGG、TIGR、miRBase数据库对差异microRNAs进行生物信息学分析,预测靶通路;Western blot 检测 IRS1ser307/ser612/ser1101/Tyr989、PI3K、Akt、GSK-3β 磷酸化水平以及 Real time-PCR检测肝脏InsR、G-6-P、PEPCKmRNA表达,来验证microRNAs生物信息学分析结果。细胞实验采用雄性SD大鼠50只,适应性喂养1周,按体重随机分为:正常组、二甲双胍组、辅助降糖颗粒组(降糖颗粒组)、辅助降糖颗粒联合二甲双胍组(联合组),每组10只,每日早晚各给药1次,连续给药7次后麻醉取血获得含药血清。H4IIE肝细胞常规复苏、培养,胰岛素浓度为10-6mol/L的培养基诱导36 h建立肝细胞胰岛素抵抗模型,含药血清进行干预后,CCK-8法检测细胞活性;Westernblot和Real time-PCR检测胰岛素信号通路关键靶点基因、蛋白表达,验证动物实验结果。结果:1.血清学检测:①在改善IR和糖脂代谢方面,与正常组大鼠比较,模型组大鼠血清FBG、Fins和HOMA-IR明显升高(P0.01或P0.05),OGTT、ITT试验峰值延后(P0.01或P0.05),体重、肝重、肝重/体重以及TG、TC和FFA显著升高(P0.01或P0.05),而肝糖原含量显著降低(P0.01);与模型组比较,联合组显著降低大鼠FBG、Fins、HOMA-IR、体重、肝重、肝重/体重以及TG、TC和FFA(P0.01或P0.05),显著升高肝糖原含量(P0.01),而且效果优于二甲双胍组和降糖颗粒组(P0.01或P0.05)。②在改善氧化应激和细胞因子方面,与正常组大鼠比较,模型组大鼠血清 MDA、Resistin、TNF-α 显著升高(P0.01 或 P0.05),而 SOD、CAT、Adiponectin 水平显著降低(P0.01 或 P0.05);联合组大鼠血清 MDA、Resistin、TNF-a显著降低(P0.01 或P0.05),SOD、CAT、Adiponectin 水平显著升高(P0.01 或 P0.05),其中调节MDA、SOD、CAT、Adiponectin、TNF-α的效果优于二甲双胍组、降糖颗粒组(P0.01或P0.05)。③在肝肾功能方面,各组间AST无差异;模型组Scr、BUN较正常组显著升高(P0.01),而二甲双胍组、降糖颗粒组、联合组较模型组显著降低(P0.01),联合组与二甲双胍组比较Scr显著降低(P0.05)。2.形态学检测:HE染色显示各药物干预组肝细胞较模型组排列整齐、脂肪样变性减轻、炎细胞浸润减少;PAS染色显示各药物干预组较模型组胞质中紫红色糖原颗粒分布增多,联合组更为明显。3.microRNAs生物信息学分析:药物干预6周后,进行大鼠肝脏microRNA测序及差异基因趋势分析发现,与正常组、模型组比较,二甲双胍组rno-miR-182、rno-miR-33-3p、rno-miR-33-5p、rno-miR-3553、rno-miR-96-5p发生差异性趋势变化,降糖颗粒组rno-miR-122-3p、rno-miR-33-3p、rno-miR-33-5p、rno-miR-22-3p 发生差异性趋势变化,联合组 rno-miR-122-3p、rno-miR-33-3p、rno-miR-33-5p、rno-miR-742-3p、rno-miR-22-3p发生差异性趋势变化(P0.01或P0.05);对差异趋势显著的microRNA进一步行靶基因预测、功能分析以及Pathway分析显示,与正常组、模型组比较,二甲双胍组、降糖颗粒组、联合组均显著性影响胰岛素信号传导通路,但联合组显著性较二甲双胍组、降糖颗粒组更强。4.Western blot和Real time-PCR检测:与模型组比较,联合组大鼠肝脏IRS1ser307/ser612/ser1101 蛋白磷酸化水平显著降低(P0.01 或 P0.05),IRS1Tyr989、PI3K、Akt、GSK-3β蛋白磷酸化水平显著升高(P0.01或P0.05),InsRmRNA表达显著升高(P0.01),G-6-P、PEPCKmRNA表达显著降低(P0.01);而与二甲双胍组、降糖颗粒组比较,联合组调控以上蛋白和基因表达的效果显著,存在差异性(P0.01 或 P0.05)。5.肝细胞检测结果:对胰岛素抵抗肝细胞活性检测显示,模型组含药血清干预后细胞活性较正常组显著降低(P0.01);二甲双胍组、降糖颗粒组、联合组较模型组显著升高(P0.01);联合组较二甲双胍组、降糖颗粒组显著升高(P0.01或P0.05)。Western blot和Real time-PCR检测显示,联合组含药血清干预后细胞较模型组IRS1ser307/ser612/ser1101蛋白磷酸化水平显著降低(P0.01 或 P0.05),IRS1Tyr989、PI3K、Akt、GSK-3β蛋白磷酸化水平显著升高(P0.01或P0.05),InsRmRNA表达显著升高(P0.01),G-6-P、PEPCK mRNA表达显著降低(P0.01);而与二甲双胍组、降糖颗粒组比较,联合组调控以上蛋白和基因表达的效果显著,存在差异性(P0.01 或 P0.05)。结论:辅助降糖颗粒联合二甲双胍明显改善糖尿病大鼠IR以及糖脂代谢紊乱,促进糖原合成,效果优于二甲双胍、辅助降糖颗粒单独应用,其机制可能是通过调节microRNA差异表达,来调控胰岛素信号传导通路中IRS1、PI3K、Akt、GSK-3磷酸化及InsR、G-6-P、PEPCK基因表达,发挥改善IR的作用;此外,辅助降糖颗粒联合二甲双胍还具有抗氧化,调节细胞因子Adiponectin、Resistin、TNF-α的作用,这可能也是其作用机制之一。
[Abstract]:Objective: To explore the effect and mechanism of the combination of auxiliary hypoglycemic granule and metformin on insulin resistance in ZDF rats, provide scientific basis for the combination of Chinese and Western medicine and provide guidance for clinical use. Methods: a randomized controlled design method was used and 28 fa/fa rats of spontaneous type 2 diabetes were selected and 28 rats were selected, pumina#5008 Feed inducible feeding for 4 weeks, different random blood glucose more than 11.1 mmol/L were included in the experiment. According to weight, random blood sugar was randomly divided into model group, metformin group, auxiliary hypoglycemic group (hypoglycemic granule group), auxiliary hypoglycemic granule combined with metformin group (combined group), 7 rats in each group, and 7 normal ZDF (fa/+) rats of the same week age as the normal group, continuous dry During the 6 weeks, the general condition, body weight, fasting blood glucose, OGTT, ITT experiment were recorded in sixth weeks. After 6 weeks, rats were anesthetized, and blood was taken to freeze and fix the liver respectively. The serum IR related indexes, FBG, Fins and HOMA-IR, TG, TC and FFA, liver and kidney function indicators AST, Scr and BUN, oxidative stress and inflammatory cell cause were detected. MDA, SOD, CAT and TNF- alpha, as well as cytokines Adiponectin, Resistin; detection of liver glycogen content, HE, PAS staining to observe the liver pathological changes; high throughput sequencing detection of liver microRNAs expression, NCBI, The Gene. T test IRS1ser307/ser612/ser1101/Tyr989, PI3K, Akt, GSK-3 beta phosphorylation level and Real time-PCR to detect liver InsR, G-6-P, PEPCKmRNA expression to verify the results of microRNAs bioinformatics analysis. Cell experiments used 50 male SD rats for 1 weeks, and were randomly divided into normal group, metformin group and auxiliary hypoglycemic group according to body weight. Granule group (Jiangtang granule group), auxiliary hypoglycemic granule combined with metformin group (combined group), 10 rats in each group were given 1 times a day and morning and evening. After 7 times of continuous administration, blood was taken to obtain serum.H4IIE liver cell routine resuscitation and culture. The insulin concentration was induced by 10-6mol/L in 36 h to establish the liver cell insulin resistance model. The cell activity was detected by CCK-8 method, and the key target genes of insulin signaling pathway were detected by Westernblot and Real time-PCR, and the protein expression was detected. Results: 1. serological test: 1. In improving IR and glycolipid metabolism, the serum FBG, Fins and HOMA-IR in the model group were significantly higher than those in the normal group (P0.01). Or P0.05), OGTT, ITT test peak delay (P0.01 or P0.05), weight, liver weight, liver weight / weight and TG, TC and FFA significantly increased (P0.01 or P0.05), while liver glycogen content decreased significantly (P0.01). Compared with the model group, the combined group significantly reduced the rats' FBG, weight, liver weight, liver weight / weight, and significantly increased Liver glycogen content (P0.01), and the effect is better than metformin group and hypoglycemic group (P0.01 or P0.05). (2) in improving oxidative stress and cytokines, compared with normal rats, the serum MDA, Resistin, TNF- alpha in the model group increased significantly (P0.01 or P0.05), but SOD, CAT, Adiponectin level decreased significantly (P0.01 or P0.05). The serum MDA, Resistin, and TNF-a decreased significantly (P0.01 or P0.05), SOD, CAT, Adiponectin level increased significantly (P0.01 or P0.05). The effect of regulation MDA, SOD, and Adiponectin was better than that of metformin group and hypoglycemic granule group. Significantly increased (P0.01), and metformin group, hypoglycemic group, combined group was significantly lower than the model group (P0.01), the combination group and metformin group compared with the metformin group significantly lower (P0.05).2. morphological detection: HE staining showed that the liver cells in the drug intervention group were arranged in a whole group, the adipose degeneration and inflammatory cell infiltration decreased; PAS staining showed that each group was Scr. In the drug intervention group, the distribution of the purple red glycogen granules in the cytoplasm of the model group increased, and the combined group was more obviously.3.microRNAs bioinformatics analysis. After 6 weeks of drug intervention, the microRNA sequencing and differential gene trend analysis of rat liver were found, compared with the normal group, the model group, and the group of two metformin groups rno-miR-182, rno-miR-33-3p, rno-miR-33-5p, rno-. The difference trend of miR-3553, rno-miR-96-5p, rno-miR-122-3p, rno-miR-33-3p, rno-miR-33-5p, rno-miR-22-3p in the group of hypoglycemic granula was changed, and the trend changes of rno-miR-122-3p, rno-miR-33-3p, rno-miR-33-5p, rno-miR-742-3p, and rno-miR-22-3p in the combination group were different. IcroRNA further target gene prediction, functional analysis and Pathway analysis showed that compared with the normal group, the model group, the metformin group, the hypoglycemic granule group and the combined group significantly affected the insulin signaling pathway, but the combined group was significantly more.4.Western blot and Real time-PCR in the metformin group and the hypoglycemic granule group: and the model of the model. The level of phosphorylation of IRS1ser307/ser612/ser1101 protein in the liver of the combined group was significantly lower (P0.01 or P0.05), IRS1Tyr989, PI3K, Akt, the level of phosphorylation of GSK-3 beta protein was significantly increased (P0.01 or P0.05), InsRmRNA expression increased significantly (P0.01), G-6-P, and the expression of InsRmRNA was significantly lower than that in the metformin group, and the ratio of hypoglycemic granule group was compared with the group of metformin. Compared with the combination group, the effect of the above protein and gene expression was significant, and there was a difference (P0.01 or P0.05).5. hepatocyte detection results: the activity of insulin resistant liver cells showed that the cell activity of the model group was significantly lower than that of the normal group (P0.01) after the intervention of the drug serum, and the metformin group and the combination group were more significant than the model group. Higher (P0.01); compared with metformin group, the level of.Western blot and Real time-PCR in the group of hypoglycemic granule group (P0.01 or P0.05) showed that the level of IRS1ser307/ser612/ser1101 protein phosphorylation in the combined group was significantly lower than that of the model group (P0.01 or P0.05), IRS1Tyr989, PI3K, and beta protein phosphorylation level. Significantly increased (P0.01 or P0.05), InsRmRNA expression significantly increased (P0.01), G-6-P, PEPCK mRNA expression significantly decreased (P0.01), and compared with the metformin group, hypoglycemic group, the combined control of the protein and gene expression of the effect is significant, there is a difference (P0.01 or P0.05). Conclusion: auxiliary hypoglycemic Granules Combined with metformin obviously improved sugar. Urinary disease rats IR and glycolipid metabolic disorders, promote glycogen synthesis, the effect is better than metformin, the effect is better than metformin, auxiliary hypoglycemic granules alone, the mechanism may be by regulating the differential expression of microRNA, to regulate the insulin signal transduction pathway IRS1, PI3K, Akt, GSK-3 phosphorylation and InsR, G-6-P, PEPCK gene expression, play the role of improving IR; in addition, supplemented by the role of IR; in addition, auxiliary The combination of "Jiangtang Granule" and metformin also has antioxidant effect, which regulates cytokine Adiponectin, Resistin and TNF- alpha. This may be one of its mechanisms.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R965

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