代综方干预骨骼肌细胞胰岛素抵抗及其作用机制研究

发布时间：2018-06-10 10:17

本文选题：代综方 + 骨骼肌　；参考：《中国中医科学院》2017年博士论文

【摘要】：代谢综合征(metabolic syndrome,MS)是指多种代谢异常聚集于同一个体的病理状态,可直接增加心血管疾病、2型糖尿病和全因死亡的风险。目前其患病率迅猛增加,已成为影响全世界的一个重大公共卫生问题。在美国,20岁以上的成年人中MS的患病率已由34%上升到44.2%;2009年国家健康与营养调查发现,我国成人MS的患病率已达到18.2%;老年人群中MS患病粗率达25.5%。胰岛素抵抗是指代谢器官或组织对胰岛素的敏感性低于正常的一种病理生理状态,是MS腹型肥胖、高血糖、高血压、血脂异常等代谢紊乱的共同病理基础。骨骼肌是机体葡萄糖摄取和利用的主要场所,也是发生胰岛素抵抗的重要场所。骨骼肌胰岛素抵抗主要表现为胰岛素刺激的葡萄糖摄取和利用能力下降,是导致全身胰岛素抵抗及MS等代谢性疾病发病的原因之一。因此,调节骨骼肌葡萄糖代谢,改善骨骼肌胰岛素抵抗对防治MS的发生发展具有重要意义。导师刘喜明教授认为,MS以腹型肥胖为核心组分,是由脂质在腹部过度堆积所致,病位在中焦脾胃,根据“脾主散精”、“脾主肌肉四肢”理论,脂质在腹部堆积,脾不散精,精微异常,游溢四肢是导致骨骼肌胰岛素抵抗的重要病因。代综方是导师基于二十余年临床经验并结合现代临床药理研究所创制的治疗MS痰热互结证的基本方,主要由黄连、姜半夏、瓜萎皮等药物组成。本课题组在国家“十二五”重大新药创制课题“代综方干预代谢综合征早期糖脂代谢紊乱的新药临床前研究”中已开展了代综方的药学、药效学、毒理学、临床及基础试验研究,证实代综方可减轻MS患者体重、改善糖脂代谢紊乱;促进脂肪细胞葡萄糖消耗和胞内甘油三酯分解,改善脂质合成与分解的动态平衡;下调C/EBP-α、PPAR-γ、FAS、ACC的基因表达抑制3T3-L1前脂肪细胞的分化;抑制油酸所致的肝细胞内脂质堆积。本研究将中医理论与骨骼肌胰岛素抵抗相结合,为代综方治疗MS提供进一步的证据支持。目的1.研究代综方含药血清对C2C12骨骼肌细胞葡萄糖代谢的影响,并从GLUT1、GLUT4的膜蛋白表达探讨其作用机制。2.研究代综方含药血清对棕榈酸诱导的C2C12骨骼肌细胞胰岛素抵抗的影响,并从胰岛素相关PI3K/AKT信号通路、AMPK通路及炎症方面探讨其作用机制。3.研究代综方对自发性2型糖尿病KKAy小鼠糖脂代谢紊乱的影响。方法1.制备代综方含药血清及空白血清;采用MTT法检测1%、5%、10%及20%代综方含药血清干预24h后C2C12细胞的活性,确立含药血清浓度。2.以高、中、低剂量代综方含药血清(10%、5%、2.5%)分别干预C2C12细胞8h、16h及24h后,采用葡萄糖氧化酶法检测葡萄糖消耗;以高剂量代综方含药血清干预C2C12细胞24h后,2-NBDG法检测基础状态及胰岛素刺激状态下的C2C12细胞葡萄糖摄取。3.以高剂量代综方含药血清干预C2C12细胞24h后,采用Western blot检测基础状态及胰岛素刺激状态下的GLUT1、GLUT4膜蛋白表达。4.以0.5mM棕榈酸孵育C2C12骨骼肌细胞24h,制造骨骼肌细胞胰岛素抵抗模型;采用2-NBDG法检测胰岛素抵抗C2C12细胞葡萄糖摄取;采用甘油三酯酶法测定胰岛素抵抗C2C12细胞内甘油三酯浓度。5.采用RT-PCR检测胰岛素抵抗C2C12细胞PI3K/AKT通路关键分子IRS-1、P13K、AKT、GLUT4的基因表达;检测炎症因子IL-6、TNF-α、NF-κB的基因表达。6.采用Western blot检测胰岛素抵抗C2C12细胞PI3K/AKT通路AKT、GSK-3β磷酸化蛋白及GLUT4膜蛋白表达,检测AMPK通路AMPK、ACC磷酸化蛋白表达。7.采用DCFH-DA荧光探针孵育胰岛素抵抗C2C12细胞20min,分别应用荧光显微镜、流式细胞仪检测DCF荧光强度,根据其荧光强度判断ROS水平。8.采用NF-κB激活-核转运检测试剂盒检测胰岛素抵抗C2C12细胞NF-κB核转运。9.选用8周龄KKAy小鼠高脂饲料喂养,筛选空腹血糖13.9mmol/L的小鼠60只,按体重随机分为5组:模型对照组(Con)、二甲双胍组(Met,200mg/kg/天)、代综方高剂量组(DZFh,2000mg/kg/天)、代综方中剂量组(DZFm,1500mg/kg/天)、代综方低剂量组(DZFl,1000mg/kg/天);另取8周龄C57BL/6J小鼠8只作为正常对照组(Nor)。每日上午灌胃给药一次,为期8周,模型对照组与正常对照组给予相同剂量的无菌水。每周检测小鼠进食量、体重、空腹血糖,第8周检测小鼠糖耐量。实验结束后取材,检测血糖、血脂4项、血清胰岛素并计算胰岛素敏感指数(QUICKI)。10.每组随机选取4只小鼠,分离骨骼肌,常规石蜡包埋切片,免疫组化检测小鼠骨骼肌GLUT4蛋白表达。结果1.与正常对照组相比,空白血清组及不同浓度代综方含药血清组细胞存活率无明显差异(P0.05)。2.与空白血清组比较,代综方含药血清干预8h后,高、中剂量组C2C12细胞葡萄糖消耗即显著增加(P0.05),增加幅度分别为22.54%、18.75%,干预16h、24h后,高、中、低剂量组C2C12细胞葡萄糖消耗均显著增加(P0.05),三组含药血清相比,高剂量组作用效果最佳;以高剂量代综方含药血清干预C2C12细胞24h后检测葡萄糖摄取,与空白血清组比较,基础状态下,代综方含药血清高剂量组荧光强度增加19.88%,胰岛素刺激状态下,荧光强度增加9.54%,差异均有统计学意义(P=0.041,P=0.030)。3.以高剂量代综方含药血清干预C2C12细胞24h,基础状态下,可显著增加 C2C12 细胞 GLUT1、GLUT4 膜蛋白表达(P=0.008,P=0.004),与空白血清组相比,分别增加30.98%、59.07%;胰岛素刺激状态下,可显著增加GLUT1膜蛋白表达(P=0.000),与空白血清组相比,增加42.26%。4.棕榈酸孵育C2C12细胞24h后,胰岛素刺激下的葡萄糖摄取量下降30.72%,产生胰岛素抵抗;代综方含药血清干预胰岛素抵抗C2C12细胞24h后,葡萄糖摄取量增加,其中高、中剂量组葡萄糖摄取量分别增加20.62%、13.27%,差异显著(P=0.001,P=0.039);棕榈酸孵育 C2C12 细胞24h后,细胞内甘油三酯浓度显著增加(P=0.000),高剂量代综方干预后,甘油三酯浓度显著降低(P=0.004)。5.高剂量代综方含药血清干预胰岛素抵抗C2C12细胞24h后,可上调AKT mRNA、GLUT4mRNA 的表达(P=0.042,P=0.041);增加 p-AKT、p-GSK-3β蛋白表达(P=0.002,P=0.000),增加GLUT4膜蛋白表达(P=0.001)。6.高剂量代综方含药血清干预胰岛素抵抗C2C12细胞24h后,可增加p-AMPK及 p-ACC 蛋白表达(P=0.006,P=0.012)。7.高剂量代综方含药血清干预胰岛素抵抗C2C12细胞24h后,可降低IL-6mRNA、TNF-αmRNA 表达(P=0.003,P=0.013),降低 ROS 水平(P0.05),降低 NF-κBmRNA 表达及 NF-κB 核转运(P=0.002)。8.代综方各剂量组KKAy小鼠的体重与模型组相比虽无明显差异,但体重增长呈下降趋势;给药过程中,与模型组相比,代综方各剂量组KKAy小鼠空腹血糖呈下降趋势,给药6周时,代综方高剂量组血糖下降15.19%(P0.05),给药8周时,代综方中、高剂量组血糖分别下降17.46%、15.29%(P0.05);与模型组相比,代综方高剂量组KKAy小鼠的糖耐量异常有明显缓解(P0.05);与模型组相比,代综方高剂量组TC水平降低11.33%(P0.05),代综方高剂量及中剂量组TG水平分别下降28.78%、18.80%(P0.05),代综方各剂量组HDL-C水平无明显差异(P0.05);代综方高剂量组LDL-C的水平降低25.20%(P0.05);代综方高、中剂量组空腹胰岛素水平明显下降(P0.05),QUICKI指数显著增加(p0.05)。9.与模型组相比,代综方高剂量组骨骼肌GLUT4蛋白表达显著增加(P=0.003)。结论1.代综方含药血清浓度在20%以内时,对C2C12细胞活性无明显影响;代综方含药血清可增加骨骼肌细胞葡萄糖消耗和摄取,其作用机制可能与增加GLUT1、GLUT4膜蛋白表达相关。2.代综方含药血清可改善C2C12骨骼肌细胞胰岛素抵抗,其作用机制可能与上调PI3K/AKT通路中关键分子AKT的基因表达,增加AKT蛋白磷酸化,进而促进GLUT4基因表达及膜转位并降低糖原合成激酶GSK3活性;激活AMPK信号通路;抑制骨骼肌细胞炎症有关。3.代综方可改善KKAy小鼠糖脂代谢紊乱,增加胰岛素敏感性,其作用机制可能与增加骨骼肌GLUT4蛋白表达相关。
[Abstract]:Metabolic syndrome (MS) refers to the pathological condition of various metabolic abnormalities that accumulate in the same individual. It can directly increase the risk of cardiovascular disease, type 2 diabetes and all causes of death. At present, the prevalence rate has increased rapidly. It has become a major public health problem affecting the whole world. In the United States, among adults over 20 years old, MS The prevalence rate has risen from 34% to 44.2%. In 2009, the national health and nutrition survey found that the prevalence rate of adult MS in China has reached 18.2%, and the MS prevalence of 25.5%. insulin resistance in the elderly is a pathological condition of metabolic organ or tissue sensitivity to insulin lower than normal, and it is MS abdominal obesity, hyperglycemia, and hypertension. Skeletal muscle is the main site of glucose uptake and utilization, and it is also an important place for insulin resistance. The insulin resistance of skeletal muscle is mainly manifested by insulin stimulation of glucose uptake and utilization ability, which leads to systemic insulin resistance and MS metabolic diseases. One of the causes of the disease is that it is of great significance to regulate the glucose metabolism of skeletal muscle and improve the insulin resistance of skeletal muscles to prevent and control the occurrence and development of MS. Professor Liu Ximing of the tutor believes that MS is the core component of abdominal obesity, which is caused by excessive accumulation of lipids in the abdomen, the position of the disease in the middle of the spleen and the stomach, and "spleen main muscle", "spleen main muscle" Extremities "theory, lipid accumulation in the abdomen, spleen inseminal essence, subtle abnormalities, and overflowing extremities is an important cause of insulin resistance in skeletal muscle. The agent is based on twenty years of clinical experience combined with modern clinical pharmacology research to treat MS phlegm heat exchange syndrome, mainly by Coptis chinensis, Jiang Banxia, melon wilt skin and other drugs. The research group has carried out the pharmacology, pharmacodynamics, toxicology, clinical and basic test of the new drug in the national "12th Five-Year" major new drug creation subject, "the new drug in the preclinical study of the early glycolipid metabolic disorder of metabolic syndrome." it has been proved that the agent can reduce the weight of MS patients and improve the disorder of glycolipid metabolism. Promote glucose consumption and intracellular triglyceride decomposition in adipocytes and improve the dynamic balance of lipid synthesis and decomposition; down regulation of C/EBP- alpha, PPAR- gamma, FAS, ACC gene expression inhibits the differentiation of 3T3-L1 preadipocytes and inhibits the accumulation of lipid in liver cells induced by oleic acid. To provide further evidence support for the treatment of MS. Objective 1. to study the effect of the serum on the glucose metabolism of C2C12 skeletal muscle cells, and to explore the effect of.2. on the insulin resistance of the skeletal muscle cells induced by palmitic acid, and from the insulin phase, from the expression of the membrane protein expression of GLUT1 and GLUT4. The effect of PI3K/AKT signaling pathway, AMPK pathway and inflammation on the effect of.3. on the glucose and lipid metabolism disorder of spontaneous type 2 diabetes KKAy mice. Method 1. to prepare the serum and blank sera of the synthesizer, and to detect the activity of C2C12 cells after the intervention of 24h in 1%, 5%, 10% and 20% generation with MTT. Serum concentration of.2. in high, middle and low doses (10%, 5%, 2.5%) interfered with C2C12 cells 8h, 16h and 24h respectively. Glucose oxidase method was used to detect glucose consumption, and C2C12 cell 24h was intervened by high dose heald serum containing serum, and 2-NBDG assay was used to detect C2C12 cell glucose uptake under the condition of insulin stimulation. After taking.3. to intervene the C2C12 cell 24h with high dose of the drug containing serum, Western blot was used to detect the basic state and GLUT1 in the state of insulin stimulation. The GLUT4 membrane protein expressed.4. with 0.5mM palmitic acid incubating C2C12 skeletal muscle cells 24h, producing insulin resistance model of skeletal muscle cells, and using 2-NBDG method to detect insulin resistance to Staphylococcus aureus cells. Glucose uptake and determination of triglyceride concentration in insulin resistance C2C12 cells by triglyceride method.5. using RT-PCR to detect the gene expression of IRS-1, P13K, AKT, GLUT4, the key molecules of the PI3K/AKT pathway of insulin resistance C2C12 cells, and the detection of inflammatory factors IL-6, TNF- alpha. Cell PI3K/AKT pathway AKT, GSK-3 beta phosphorylated protein and GLUT4 membrane protein expression, AMPK pathway AMPK, ACC phosphorylated protein expression.7. use DCFH-DA fluorescence probe to incubate insulin resistance C2C12 cell 20min. Fluorescence microscopy, flow cytometry, respectively, detect DCF fluorescence intensity. Activation nuclear transfer detection kit was used to detect the NF- kappa B nuclear transport of insulin resistance C2C12 cells, and 60 mice were fed with high fat diet of KKAy mice of 8 weeks old, and 60 mice were selected for screening empty fasting blood glucose 13.9mmol/L. They were randomly divided into 5 groups according to weight: model control group (Con), metformin group (Met, 200mg/ kg/ days), and the generation of high dose group (DZFh, 2000mg/kg/ days), and the generation side. Medium dose group (DZFm, 1500mg/kg/ days), lower dose group (DZFl, 1000mg/kg/ days), and 8 C57BL/6J mice of 8 weeks age as normal control group (Nor). Every morning gavage was given for 8 weeks. The model control group was given the same dose of aseptic water with the normal control group. The mice intake, body weight, fasting blood glucose and eighth week test were detected weekly. After the experiment, the blood glucose, blood lipid 4 items, the serum insulin and the insulin sensitivity index (QUICKI).10. were selected and 4 mice were randomly selected to separate the skeletal muscle, the routine paraffin embedded section and the immunohistochemical detection of GLUT4 protein in the skeletal muscle of mice were detected. Results 1. compared with the normal control group, the blank sera group and the normal control group were no more than the normal control group. Compared with the blank sera group, there was no significant difference in the cell survival rate of the serum group with the same concentration (P0.05).2. and the blank sera group. After the intervention of 8h, the glucose consumption of the C2C12 cells in the middle dose group increased significantly (P0.05), the increase was 22.54%, 18.75%, and after 24h, the glucose consumption in the high, middle and low dose groups of C2C12 cells was all after 24h. Significantly increased (P0.05), compared with the three groups of serum containing drugs, the high dose group had the best effect, and the glucose uptake was detected after the intervention of the C2C12 cell 24h in the high dose heald prescription serum. Compared with the blank sera group, the fluorescence intensity increased by 19.88% in the high dose group containing the heald prescription serum, and the fluorescence intensity increased by 9.5 under the state of insulin stimulation. 4%, the difference was statistically significant (P=0.041, P=0.030).3. was used to interfere with 24h in C2C12 cells with high dose of agent serum, which could significantly increase the GLUT1, GLUT4 membrane protein expression (P=0.008, P=0.004) in C2C12 cells, and increased by 30.98%, 59.07% respectively compared with the blank sera group, and the GLUT1 membrane protein could be significantly increased under the condition of insulin stimulation. Expression (P=0.000), compared with the blank sera group, after increasing 42.26%.4. palmitic acid to incubate C2C12 cell 24h, the glucose uptake under insulin stimulation decreased by 30.72% and produced insulin resistance. The glucose uptake increased after the intervention of the insulin resistance C2C12 cell 24h by the heald serum containing drug serum, and the glucose uptake in the medium dose group increased respectively. Adding 20.62%, 13.27%, the difference was significant (P=0.001, P=0.039); after the palmitic acid incubated 24h, the intracellular triglyceride concentration increased significantly (P=0.000). The concentration of triglyceride decreased significantly (P=0.004) (P=0.000), and the high dose of the triglyceride concentration was significantly decreased (P=0.004), and the high dose of the.5. in the high dose heald serum intervened the 24h of the insulin resistance of C2C12 cells, and the AKT mRNA could be up-regulated. GLUT4mRNA Expression (P=0.042, P=0.041); increase p-AKT, p-GSK-3 beta protein expression (P=0.002, P=0.000), increase GLUT4 membrane protein expression (P=0.001) and.6. high dose heald prescription containing serum intervention of insulin resistance C2C12 cell 24h, can increase the expression of p-AMPK and protein expression. After 24h, IL-6mRNA, TNF- alpha mRNA expression (P=0.003, P=0.013), ROS level (P0.05), NF- kappa BmRNA expression and NF- kappa B nuclear transfer were reduced, although there was no significant difference between the model group and the model group, the weight gain decreased. The fasting blood glucose of KKAy mice in each dose group decreased, and the blood glucose decreased by 15.19% (P0.05) in the high dose group at 6 weeks. The glucose tolerance of the high dose group decreased by 17.46% and 15.29% (P0.05) in the high dose group at 8 weeks. Compared with the model group, the glucose tolerance abnormality in the high dose group of KKAy mice was significantly relieved (P0.05). Compared with the model group, the high dose group of the high dose group was compared with the model group. The level of TC in the high dose group was reduced by 11.33% (P0.05), and the level of TG in the high and middle dose groups of the synthesizer decreased by 28.78%, 18.80% (P0.05), and there was no significant difference in HDL-C level in each dose group of the generation (P0.05); the level of LDL-C in the high dose group was decreased by 25.20% (P0.05), and the level of fasting insulin in the medium dose group was significantly lower (P0.05). The QUICKI index increased significantly (P0.05).9. compared with the model group, the expression of GLUT4 protein in the skeletal muscle of the high dose group was significantly increased (P=0.003). Conclusion the serum concentration of the 1. generation heald prescription was less than 20%, and the serum glucose consumption and intake of skeletal muscle cells could be increased. It can improve the insulin resistance of C2C12 skeletal muscle cells with the increase of GLUT1 and GLUT4 membrane protein expression, which may improve the gene expression of the key molecule AKT in the PI3K/AKT pathway, increase the phosphorylation of the AKT protein, and then promote the expression of GLUT4 gene and the translocation of the membrane and reduce the GSK3 activity of the glycogen kinase, and stimulate the activity of the glycogen kinase. Living AMPK signaling pathway, inhibiting the inflammation of skeletal muscle cells related to.3. generation can improve the metabolic disorder of glucose and lipid in KKAy mice and increase the sensitivity of insulin. The mechanism may be related to the increase of GLUT4 protein expression in skeletal muscle.
【学位授予单位】：中国中医科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R259

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