二氢杨梅素通过抑制PPARγ磷酸化改善胰岛素抵抗的作用及机制研究

发布时间：2018-07-25 10:46

【摘要】：糖尿病是严重危害人类健康的慢性非传染性疾病,中国的2型糖尿病发病率呈现出逐年上升的趋势,成为糖尿病患病大国,糖尿病的防治研究任重道远。胰岛素抵抗是2型糖尿病发病的重要机制之一,改善胰岛素抵抗是临床治疗糖尿病的主要策略。植物化学物具有抗炎、抗氧化、抗癌等多种生物学活性,在疾病的防治中有着重要应用价值。其中类黄酮化合物因其具有降血糖、低毒副作用的功效而备受关注,但其具体机制尚不明了。过氧化物酶体增殖物激活受(peroxisome proliferator-activated receptor,PPAR)-γ参与调节机体糖脂代谢,是临床研发抗糖尿病药物的重要靶点。其中胰岛素增敏剂噻唑烷二酮类(Thiazolidinedione,TZD)药物如罗格列酮(Rosiglitazone,ROSI)虽具有良好降糖效果,但其最为PPARγ的一种完全激动剂可导致成脂增加和肥胖。研究发现类黄酮化合物能通过部分激活PPARγ增强胰岛素敏感性的同时,减少成脂、肥胖和水肿等诸多罗格列酮药物的副作用。二氢杨梅素(Dihydromyricetin,DHM)作为一种类黄酮化合物,具有抗炎、抗氧化、抗酒精性中毒和抗肿瘤等多种生物学活性,也有研究报道其具有降血糖作用,但具体机制还有待进一步阐明。研究发现,PPARγ的磷酸化与胰岛素抵抗的发生密切相关。抑制PPARγ273位点丝氨酸的磷酸化是PPARγ的配体发挥降糖作用的主要机制。细胞外信号调节蛋白激酶(Extracellular Regulating Kinase,ERK)和细胞周期蛋白依赖性激酶(Cyclin-dependent Kinase,CDK5)都能介导PPARγ273位点的磷酸化,导致肥胖相关的胰岛素抵抗的发生。抑制MEK/ERK通路能显著提高动物胰岛素敏感性。多种类黄酮化合物均被证实能抑制MEK/ERK信号通路促进肿瘤细胞凋亡。基于结构-效应关系理论推理和本课题组前期计算机模拟分子对接发现DHM能直接与PPARγ的配体结合区结合,我们推测类黄酮化合物DHM可能通过调控MEK/ERK,抑制PPARγ273位点的磷酸化,进而提高胰岛素敏感性,有效降低血糖。实验方法:本研究包括体内动物实验和离体细胞实验两部分。以Zucker Diabetic Fatty(ZDF)糖尿病模型大鼠和3T3-L1脂肪细胞为研究对象。1.实验分组:健康对照组ZL大鼠,ZDF对照组,DHM(50mg/kg)组,DHM(100mg/kg)组,DHM(200mg/kg)组,罗格列酮(4mg/kg)组;每日灌胃,共8周。隔日记录体重,每周测量进食量和空腹血糖、胰岛素、胰高血糖素水平,第0、4、8周测量血脂水平、脂联素和FGF21水平,实验第7周进行口服葡萄糖耐量实验(OGTT),第8周进行胰岛素耐量实验(ITT)。2.干预第7周,活体小动物CT成像分析大鼠体成分组成。3.实验结束后取肝脏、胰腺、肾脏、脂肪组织进行油红O染色、组织化学染色或免疫组织化学染色。Western Blot测定ZDF大鼠脂肪组织PPARγ蛋白表达及磷酸化。4.采用地塞米松诱导建立3T3-L1脂肪细胞的胰岛素抵抗模型,分析DHM对细胞成脂分化、糖摄取能力的影响。ELISA检测脂肪因子的分泌。5.Western Blot测定脂肪细胞PPARγ磷酸化水平及其上游调控激酶ERK/CDK5的蛋白表达水平。使用PPARγ抑制剂GW9662阻断PPARγ活性,观察其对DHM对脂肪细胞糖摄取和脂联素分泌的影响;用MEK抑制剂PD98059阻断ERK活性,对比研究DHM和MEK抑制剂的作用效应。主要实验结果:1.DHM降低ZDF大鼠空腹血糖,提高胰岛素敏感性。中、高剂量(100mg/kg和200mg/kg)DHM组大鼠空腹血糖显著低于ZDF对照组,低剂量(50 mg/kg)DHM维持大鼠空腹血糖低于10mM到实验第7周。第7周口服葡萄糖耐量实验(Oral Glucose Tolerance Test,OGTT)显示,3个剂量DHM组口服葡萄糖30min后血糖值均显著低于ZDF对照组;第8周胰岛素耐量实验(Insulin Tolerance Test,ITT)显示,中、高剂量(100mg/kg和200mg/kg)DHM组在注射胰岛素30min后,血糖显著低于ZDF对照组。2.DHM改善糖尿病大鼠血脂水平,不引起动物体重增加。DHM显著降低血清TG和LDL-C水平,升高HDL-C水平。干预第8周末,DHM组大鼠体重增加量比罗格列酮组降低了56%~74%。3.DHM对糖尿病大鼠的肝脏、胰腺和肾脏有保护作用。DHM降低肝细胞脂质沉积、维持肝小叶正常形态、减缓肝脂肪样变。DHM增加胰岛体积,维持胰岛形态完整性,提高β细胞的胰岛素含量。DHM显著降低肾间质炎细胞浸润,减少肾小球系膜基质增生。4.DHM降低糖尿病大鼠的脂肪组织含量,减小脂肪细胞体积,增加脂联素分泌。体成分组成显示,与罗格列酮相比DHM显著降低大鼠总脂肪和内脏脂肪含量。脂肪组织油红O染色显示,ZDF对照大鼠皮下、内脏脂肪细胞体积显著增大,DHM降低脂肪细胞体积,其皮下、内脏脂肪细胞大小与ZL健康对照组无显著差异。第0,4,8周分别进行血清脂联素水平检测,ZDF对照组脂联素水平进行性下降,而DHM显著升高血清脂联素水平,与ZL健康对照组无明显差异。5.体内和体外实验Western Blot结果均显示,DHM抑制脂肪组织和细胞中PPARγ273位点丝氨酸磷酸化,而且DHM抑制PPARγ磷酸化能力优于罗格列酮。此外,DHM还显著降低调节PPARγ磷酸化的激酶ERK和CDK5的活性。6.在地塞米松建立的3T3-L1脂肪细胞胰岛素抵抗模型中,DHM剂量依赖性的显著提高细胞的糖摄取能力,增加脂肪细胞分泌脂联素和FGF21水平。PPARγ抑制剂GW9662阻断了DHM提高脂肪细胞糖摄取和增加分泌脂联素、FGF21的能力。DHM表现出与MEK抑制剂PD98059相同的提高脂肪细胞糖摄取和促分泌脂联素、FGF21的作用,且两者联用具有协同效应。结论:1.DHM能降低ZDF糖尿病大鼠空腹血糖,减轻胰岛素抵抗,改善血脂水平。此外,DHM减少肝脏脂质沉积,增加胰腺组织胰岛的体积和胰岛素含量,缓解肾间质炎细胞浸润和肾小球系膜基质增生。研究同时发现,长期使用DHM并不引起动物体重的过多增加。2.体内实验中,DHM降低糖尿病大鼠体脂含量,缩小脂肪细胞体积,增加脂肪细胞脂联素水平。体外实验中利用地塞米松建立3T3-L1脂肪细胞胰岛素抵抗模型,发现DHM提高胰岛素抵抗的3T3-L1脂肪细胞糖摄取能力,促进脂肪细胞分泌脂联素和FGF21。3.通过调节MEK/ERK信号通路抑制PPARγSer273磷酸化是DHM降低胰岛素抵抗的主要作用机制,且DHM和MEK抑制剂协同作用,提高脂肪细胞胰岛素敏感性。综上所述,本研究进一步揭示了二氢杨梅素(DHM)改善胰岛素抵抗的分子作用机制,首次提出抑制PPARγ273位点丝氨酸的磷酸化是DHM的作用机制,为将藤茶或其提取物DHM应用于临床糖尿病的防治提供了重要的科学依据。
[Abstract]:Diabetes is a chronic non communicable disease which seriously endangers human health. The incidence of type 2 diabetes in China is increasing year by year. It has become a major diabetes country. The study of diabetes prevention is a long way to go. Insulin resistance is one of the important mechanisms of the onset of type 2 diabetes. Improving insulin resistance is a clinical treatment for diabetes. Major strategies. Phytochemicals have many biological activities, such as anti-inflammatory, antioxidant and anticancer, which have important application value in the prevention and treatment of diseases. Among them, the flavonoids have attracted much attention because of their hypoglycemic and low toxic and side effects, but the specific mechanisms are still unknown. Peroxisome proliferators are activated by (peroxisome prolif). Erator-activated receptor, PPAR) - gamma is an important target in the clinical research and development of antidiabetic drugs. The insulin sensitizer (Thiazolidinedione, TZD), such as Rosiglitazone, ROSI, has a good hypoglycemic effect, but a complete agonist of the most PPAR gamma can lead to a complete agonist of PPAR gamma. The study found that flavonoids can reduce the side effects of many rosiglitazone drugs, such as lipid, obesity, and edema, by partially activating PPAR gamma. Two Dihydromyricetin (DHM), as a kind of flavonoid compound, has anti-inflammatory, antioxidant, anti alcohol poisoning, and swelling resistance. Many biological activities, such as tumor, have also been reported to have hypoglycemic effect, but the specific mechanism remains to be further elucidated. Studies have found that the phosphorylation of PPAR gamma is closely related to the occurrence of insulin resistance. The inhibition of the phosphorylation of serine at the PPAR gamma 273 site is the main mechanism of PPAR gamma ligand playing the hypoglycemic effect. Protein kinase (Extracellular Regulating Kinase, ERK) and cyclin dependent kinase (Cyclin-dependent Kinase, CDK5) can mediate phosphorylation of PPAR gamma 273 site, leading to the occurrence of obesity related insulin resistance. Inhibition of the MEK/ERK pathway can significantly increase the insulin sensitivity of animals. Inhibition of MEK/ERK signaling pathway to promote apoptosis of tumor cells. Based on the theoretical reasoning of structural effect relationship and the docking of DHM to the ligand binding area of PPAR gamma, we speculate that the flavonoid compound DHM may improve the islet by regulating MEK/ERK, inhibiting the phosphorylation of the PPAR gamma 273 site and thus improving the islets of the islets. Zucker Diabetic Fatty (ZDF) diabetic model rats and 3T3-L1 adipocytes were divided into groups of.1. experimental groups: healthy control group ZL rats, ZDF pairs, DHM (50mg/kg), DHM (100mg/kg) group, 3T3-L1 group, The group of rosiglitazone (4mg/kg) group was administered daily for 8 weeks. The body weight was recorded every other day, daily intake of food and fasting blood glucose, insulin, glucagon level, serum lipid levels, adiponectin and FGF21 levels, oral glucose tolerance test (OGTT) for the first seventh weeks, and eighth weeks of insulin tolerance test (ITT).2. intervention for seventh weeks were performed. Liver, pancreas, kidney, and adipose tissue were stained with oil and red O after the CT imaging analysis of the body composition of the rat body. The expression of PPAR gamma protein in the adipose tissue of ZDF rats was determined by histochemical staining or immunohistochemical staining with.Western Blot, and the phosphorylated.4. was induced by dexamethasone to establish the insulin of 3T3-L1 adipocytes. Resistance model, analysis of the effect of DHM on the differentiation of cells and the effect of sugar uptake..ELISA detected the secretion of adipokine by.5.Western Blot and the level of PPAR gamma phosphorylation in adipocytes and the protein expression level of the upstream regulated kinase ERK/CDK5. The PPAR gamma inhibitor GW9662 was used to block the PPAR gamma activity and to observe the uptake of sugar in DHM to the fat cells and the glucose uptake by DHM. The effect of adiponectin secretion; blocking ERK activity with MEK inhibitor PD98059 and comparing the effect of DHM and MEK inhibitors. Main experimental results: 1.DHM reduced fasting blood glucose in ZDF rats and increased insulin sensitivity. In the high dose (100mg/kg and 200mg/kg) DHM group, the fasting blood sugar of the rats was significantly lower than that in the ZDF control group, and the low dose (50 mg/kg) was maintained. The fasting blood glucose in rats was lower than 10mM to seventh weeks. The oral glucose tolerance test (Oral Glucose Tolerance Test, OGTT) at seventh weeks showed that the blood glucose values of the 3 DHM groups after oral glucose 30min were significantly lower than those in the ZDF control group; eighth weeks of insulin tolerance test (Insulin Tolerance Test) showed that the high dose (Insulin Tolerance Test) group was in the high dose group. After the injection of insulin 30min, the blood glucose was significantly lower than that of the ZDF control group.2.DHM to improve the blood lipid level of the diabetic rats, and no increase of the body weight.DHM significantly decreased the level of serum TG and LDL-C and increased the level of HDL-C. At the end of the eighth weekend, the weight gain of DHM group was lower than that of the rosiglitazone group, and the liver and pancreas of diabetic rats were lower than that of the rosiglitazone group. The protective effect of.DHM and kidney can reduce the lipid deposition of liver cells, maintain the normal form of hepatic lobule, slow down the liver fat like changes, increase the islet volume, maintain the integrity of the islet, improve the insulin content of beta cells, reduce the infiltration of renal interstitial inflammatory cells, reduce the glomerular mesangial matrix hyperplasia and reduce the fat of the diabetic rats by reducing the.4.DHM of the glomerular mesangial matrix, and reducing the fat of the diabetic rats. Tissue content reduced fat cell volume and increased adiponectin secretion. Body composition showed that DHM significantly reduced total fat and visceral fat content in rats compared with rosiglitazone. Fat tissue oil red O staining showed that ZDF control rats were subcutaneous, visceral adipocyte volume increased significantly, DHM decreased fat cell volume, and subcutaneous, visceral fat was thin. There was no significant difference between the cell size and the ZL health control group. The serum adiponectin level was detected at week 0,4,8, and the level of adiponectin in the ZDF control group decreased, while the DHM significantly increased the serum adiponectin level, and there was no significant difference between the.5. body and the ZL healthy control group. The results of Western Blot in both in vivo and in vitro showed that DHM inhibited adipose tissue and cells in the cells. PPAR gamma 273 site serine phosphorylation, and the ability of DHM to inhibit PPAR gamma phosphorylation is superior to rosiglitazone. In addition, DHM also significantly reduces the activity.6. regulating PPAR gamma phosphorylation of kinase ERK and CDK5 in the insulin resistance model established by dexamethasone in the insulin resistance model of dexamethasone. The dose dependence of DHM increases the sugar uptake and increase of cells significantly. Adipocytes secrete adiponectin and FGF21 level.PPAR gamma inhibitor GW9662 block DHM increase sugar uptake and increase the secretion of adiponectin. FGF21's ability.DHM shows the same as MEK inhibitor PD98059 to increase fat cell sugar uptake and promote division of secretin, FGF21, and both have synergistic effect. Conclusion: 1.DHM can be used. Reducing the fasting blood glucose, reducing insulin resistance and improving blood lipid levels in ZDF diabetic rats. In addition, DHM reduces liver lipid deposition, increases the volume and insulin content of pancreatic islets, alleviates the infiltration of renal interstitial inflammation and glomerular mesangial matrix hyperplasia. The study also shows that long-term use of DHM does not cause excessive increase in animal weight. In the.2. experiment, DHM reduced the body fat content of diabetic rats, reduced the volume of adipocyte and increased adipocyte adiponectin level. In vitro, the insulin resistance model of 3T3-L1 adipocytes was established by dexamethasone. It was found that DHM increased the glucose uptake ability of 3T3-L1 adipocytes in insulin resistance, and promoted adipocytes to secrete adiponectin and FG. F21.3. by regulating the MEK/ERK signaling pathway to inhibit PPAR gamma Ser273 phosphorylation is the main mechanism of DHM to reduce insulin resistance, and DHM and MEK inhibitors synergistically to increase the insulin sensitivity of adipocytes. To sum up, this study further revealed the molecular mechanism of two hydrogen myricetin (DHM) to improve insulin resistance, for the first time The inhibition of the phosphorylation of serine at PPAR gamma 273 site is the mechanism of DHM, which provides an important scientific basis for the application of rattan tea or its extract DHM in the prevention and control of clinical diabetes.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5

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