【作者】 刘毅；

【导师】 王文健； 沈自尹；

【作者基本信息】 复旦大学 ， 中西医结合临床， 2007， 博士

【摘要】 背景胰岛素抵抗（IR）是代谢综合征（Metabolic Syndrome，MS）众多组分疾病共同的病理生理基础，给机体带来严重危害。增加胰岛素敏感性、改善IR对于MS的防治具有非常重要的意义。TZD类药物罗格列酮是目前治疗IR的代表药物，作为胰岛素增敏剂，它通过激活PPARγ，进而促进脂肪细胞分化、增加脂肪酸代谢、降低血浆FFA水平；通过激活胰岛素信号转导通路PI-3K／PKB途径，增加外周组织GLUT4的表达而参与葡萄糖生成、转运和利用。但罗格列酮最终使脂肪积聚增多引起体重和体脂增加，并且有头痛、水肿等不良反应，而肥胖与IR有高度相关性，这样就造成了治疗上的矛盾。因此，寻求既能改善IR又能减轻或避免TZD类药物弊端的胰岛素增敏剂成为目前研究的热点。黄连是中医治疗消渴病的常用药物，研究表明其有效成分小檗碱具有改善IR及糖脂代谢的作用，我们在以前的研究工作中也发现，Ber可增加脂肪细胞葡萄糖摄取和利用，抑制前脂肪细胞向成熟脂肪细胞的分化，其作用途径完全不同于PPARγ激动剂罗格列酮，提示Ber在发挥胰岛素增敏剂作用的同时不会引起体脂的聚积而造成体重增加，可能更适合于治疗肥胖和IR相关的代谢性疾病，这将为IR的防治提供新思路，但其确切机制有待于深入研究。目的建立IR脂肪细胞模型，观察小檗碱对体外培养的3T3-L1前脂肪细胞增殖分化和成熟脂肪凋亡的作用，以及对脂肪细胞因子脂联素和内脂素表达的影响，为深入探讨小檗碱改善胰岛素抵抗的细胞分子机制提供实验依据。方法在高糖高胰岛素的培养环境下，以³H-葡萄糖摄取试验为标准建立IR脂肪细胞模型；XTT比色法建立3T3-L1前脂肪细胞“OD₅₇₀值——细胞数目”曲线，观察Ber不同浓度和作用不同时间后对3T3-L1前脂肪细胞增殖的影响；流式细胞术（Annexin V-FITC/PI双染）观察Ber对成熟脂肪细胞凋亡的影响；Real-time PCR和Western blotting分别检测前脂肪细胞分化过程中关键转录因子PPARγ、C/EBPa mRNA和蛋白的表达；酶联免疫吸附检测法（ELISA）检测不同浓度、作用不同时间的Ber对IR脂肪细胞培养上清中APN的含量；Real time PCR检测APN、Visfatin mRNA的表达，Western blotting检测AdipoR1、AdipoR2和Visfatin蛋白水平的表达。结果高糖、高胰岛素、高糖+高胰岛素分别使脂肪细胞³H-葡萄糖摄取率下降53.65％、47.69％、61.40％，与正常对照组比较有显著差异（P＜0.01）。1～80μM Ber作用48小时后对前脂肪细胞增殖均有明显的抑制作用（与空白对照组比较P＜0.05或P＜0.01），且呈量效关系；高浓度Ber（20～80μM）对细胞增殖的抑制作用较强，24小时即有明显差异（P＜0.05），10μM以下的Ber作用24小时对前脂肪细胞增殖无明显的影响（P＞0.05）；前脂肪细胞经Ber干预后，分化相关基因PPARγ、C／EBPa mRNA和蛋白的表达下调，胞浆中脂滴明显减少（与空白对照组及罗格列酮干预组比较P＜0.05或P＜0.01）；分化成熟的脂肪细胞经Ber作用24小时后出现明显凋亡，在一定浓度范围内存在量效关系（与模型对照组比较P＜0.05）。0.1～10μM Ber逐渐上调脂肪细胞Visfatin mRNA的表达，10μM作用最明显（是空白对照组的4.96倍）；10μM Ber作用3小时后Visfatin mRNA表达开始增强，至作用12小时时表达增强最明显（是作用0小时的4.57倍）；5、10、20μM的Ber分别使Visfatin蛋白表达增加1.13、2.46、2.34倍，与正常对照组比较有显著差异（P＜0.05）。Ber作用3小时后脂肪细胞APN的分泌即开始增加（与正常对照组比较P＜0.01），且呈一定时效关系；IR脂肪细胞APN分泌减少，经Ber干预后则分泌增加（与模型组对照比较P＜0.05，与罗格列酮干预组比较P＞0.05）；10μM Ber可上调脂肪细胞APN mRNA表达，作用18小时时效应最明显（是正常对照组的6.20倍）；高糖高胰岛素可下调APN mRNA表达，Ber干预后可以使降低的APN mRNA表达增加（与模型对照组比较P＜0.05）。IR脂肪细胞AdipoR1蛋白表达减少（与正常对照组比较P＜0.05），AdipoR2变化不明显（与模型对照组比较P＞0.05）；Ber和Ros均能增加AdipoR1蛋白的表达（分别是模型对照组的1.64、1.92倍）；Ros能明显增加AdipoR2蛋白表达（是模型对照组2.58倍）；Ber虽能增加AdipoR2蛋白表达，但与模型对照组比较无统计学差异（P＞0.05）。结论Ber能抑制体外培养的3T3-L1前脂肪细胞增殖，显著诱导成熟脂肪细胞的凋亡；能抑制前脂肪细胞分化，减少细胞内脂质积聚，抑制脂肪细胞分化关键转录因子PPARγ、C／EBPαmRNA和蛋白的表达；促进Adiponectin、Visfatin mRNA和蛋白的表达，增加AdipoR1蛋白的表达。Ber影响脂肪细胞增殖、分化、凋亡以及脂肪细胞因子的效应可能是其增加胰岛素的敏感性，改善IR的作用机制之一。更多还原

【Abstract】 BackgroundInsulin resistance (IR) is considered as a core component in the pathophysiology of the metabolic syndrome (MS) which represents a cluster of abnormalities such as central obesity, hyperglycemia, hypertension, dyslipidemia. It is regard as a principal risk factor for increased cardiovascular disease, the leading cause of morbidity and mortality. The worldwide prevalence of MS is increasing, leading to serious public health concerns. However, the underlying mechanisms of MS are incompletely understood. Many studies show that it is very important and indispensable to enhance insulin sensitivity for prevention and treatment of MS. Currently, thiazolidinediones (TZD), the agonists of peroxisome proliferator-activated receptors gamma (PPARγ) with insulin sensibilization and antidiabetic action, is most widely used and recommended for diabetes, but there are some side-effects caused by TZD, such as increasing body weight and body fat volume, headache, extremity edema. Accordingly, to find new insulin-sensitizing agents without TZD’s side-effect becomes the highlight. Berberine (Ber), an alkaloid originally isolated from Huanglian (Copitis chinensis), shows the beneficial pharmacological actions of decreasing blood glucose, regulating lipid metabolic disorders, increasing insulin sensitivity, consequently, it has been extensively used as a perspective drug for prevention and treatment of type 2 diabetes, obesity and MS.ObjectiveIn order to explore the molecular mechanisms of Berberine on treatment of insulin resistance, 3T3-L1 preadipocytes were cultured with high glucose and high insulin as a insulin resistance cellular model. Then we observed the effects of Berberine on cell proliferation, differentiation, apoptosis and some adipocytines expression in 3T3-L1 preadipocytes and adipocytes.MethodsInsulin resistance cellular model determined by ~3H-glucose uptake rates was induced with high concentration of insulin and glucose medium. To observe the effects of various concentrations of Berberine and different incubation time on the proliferation in 3T3-L1 preadipocytes, the curve of cellular counts and OD570 were set up by XTT methods. Effects of Berberine on apoptosis rates in fully differentiated 3T3-L1 adipocytes were detected by Flow cytometry (Annexin V-FITC/PI, double dye). The expressions of peroxisome proliferation activated receptorγ(PPARγ) and CAAT/enhancer binding protein (C/EBPa) mRNA and protein during cell differentiation were determined by real time PCR and Western blotting respectively. Adiponectin contents in supernatant were detected by ELISA; the expression of adiponectin and visfatin mRNA were assayed by real-time PCR; Western blotting were used to determine the expressions of adiponectin receptor 1, adiponectin receptor 2 and visfatin.ResultsThe rate of ~3H-glucose uptake declined 53.65 %、47.69%、61.40% in the three cell groups cultured with in the high glucose medium, high insulin medium and high glucose + high insulin medium respectively (P<0.05 or 0.01 vs. control group).Berberine with various concentrations in the experiment (10～80μM) could affect 3T3-L1 preadipocytes proliferation and it showed a dose-dependent relationship. 3T3-L1 preadipocytes treated with 10μM Berberine had little lipid droplets in the cytoplasm which were less than cells treated with Rosiglitazone (Ros). Berberine significantly decreased the mRNA and protein expression of PPARγand C/EBPa (P <0.05 or 0.01 vs. control group) in the course of 3T3-LI preadipocyte differentiation.Berberine can induce apoptosis in fully differentiated adipocyte in dose-dependent manner (P<0.05 vs. control group).0.1～10μM Berberine could increase visfatin mRNA level dose-dependently, especially 10μM in which visfatin mRNA level was 4.96 fold as much as control group. After intervention of 10μM Berberine for 3 hours, the visfatin mRNA level began to increase and reached a peak after 12 hours intervention (4.57 fold as much as 0 hours intervention group). Berberine with 5, 10, 20μM could increase visfatin protein expression level by 1.13, 2.46, 2.34 folds respectively (P<0.05 or 0.01 vs. control group).After Berberine intervention, adiponectin content in cultural supernatant and adiponectin mRNA expression increased in time-dependent manner, and its mRNA expression reached a pick after intervention for 18 hours (6.20 fold vs. 0 hours intervention group). The protein secretion and mRNA expression of adiponectin in insulin resistance celluar were decreased, but Berberine could upregulate them (P< 0.05 vs. control group).In insulin resistance celluar, the expression of adiponection receptor 1 was decreased (P < 0.05 vs. control group) while adiponection receptor 2 had no significant changes (P>0.05 vs. control group). Berberine as well as Rosiglitazone can increase protein expression of adiponectin receptor 1 (1.64 and 1.92 folds vs. control group respectively). Rosiglitazone increased protein expression of adiponectin receptor 2 significantly (2.58 folds vs. control group) while Berberine had no notable changes (P>0.05 vs. control group).ConclusionsIn vitro, Berberine can inhibit proliferation and differentiation of 3T3-L1 preadipocyte, induce adipocyte apoptosis, decrease mRNA and protein expression of PPARγand C/EBPa, increase mRNA and protein expression of adiponectin and visfatin, up-regulate adiponectin receptor 1 expression. These beneficial effects may be one of its mechanisms for amelioration of insulin resistance.更多还原