小檗碱通过Sirt1改善3T3-L1脂肪细胞糖代谢的机制研究

发布时间：2017-12-27 13:22

  本文关键词：小檗碱通过Sirt1改善3T3-L1脂肪细胞糖代谢的机制研究　出处：《南京中医药大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 小檗碱 Sirt1 3T3-L1脂肪细胞 胰岛素抵抗 糖代谢

【摘要】：小檗碱(BBR)是常用清热解毒药黄连的主要活性成分,能显著降低糖尿病患者和动物血糖,减轻体重,改善糖耐量和胰岛素抵抗。体内沉默信息调节因子1(Sirt1)是一种去乙酰化酶,在抗炎和调节敏感性脂肪组织糖脂代谢相关信号通路中与BBR作用靶点和生物学效应基本一致。因此,本文以3T3-L1脂肪细胞葡萄糖代谢紊乱为切入点,体外干扰Sirt1表达,探明BBR改善糖代谢是否依赖于Sirt1,为BBR的作用机制提供新的线索。目的:体外培养3T3-L1脂肪细胞,探明小檗碱对脂肪细胞Sirt1蛋白表达和糖代谢及其相关通路蛋白表达的影响是否依赖于Sirt1的介导。方法:1)Western Blot和荧光定量PCR检测不同浓度(1、5、10μMol/L)小檗碱对3T3-L1脂肪细胞中Sirt1表达的影响;2)将分化成熟的3T3-L1脂肪细胞用Sirt1抑制剂Ex527或慢病毒1v-Sirt1干扰Sirt1蛋白表达后分为四组,对照组,BBR组,Ex527(或1v-Sirt1)组,Ex527(或1v-Sirt1)+BBR组,作用24h后更换含1nM胰岛素和0.2%BSA的无酚红低糖培养基作用1Oh,收取细胞上清液,采用葡萄糖氧化酶法检测细胞的上清液中葡萄糖含量,以无酚红高糖培养基中糖含量相减,即为各组细胞葡萄糖消耗量;3)按2)将分化成熟的3T3-L1脂肪细胞分为四组,提取蛋白采用Western Blot检测脂肪细胞中LKB1ser428、AMPK-αThr172、ACCser79的磷酸化蛋白表达,免疫沉淀检测PGC-1α的乙酰化表达;4)将诱导成熟的3T3-L1脂肪细胞分为六组,其中三组用Ex527预处理1h,同时加入TNF-α或合并加入5μMol/LBBR,分别为对照组,TNF-α组,TNF-α+BBR组,Ex527 组,Ex527+TNF-α 组,Ex527+TNF-α+BBR 组,作用 24 后,弃上清,用含有 1nmol/L Insulin 的 0.2%BSA DMEM 处理 15min 后加入 30μM 2-NBDG 和 1 μg/mlDAP2荧光探针的PBS 37℃孵育2h,用高内涵细胞成像仪拍照并计算脂肪细胞平均荧光强度;5)按4)将诱导成熟的3T3-L1脂肪细胞分为六组,分别为对照组,TNF-α组,TNF-α+BBR组,Ex527组,Ex527+TNF-α组,Ex527+TNF-α+BBR组,作用24h,Western Blot检测AKTser 473与IRS-1Y612磷酸化表达。结果:1)BBR可显著上调3T3-L1脂肪细胞中Sirt1蛋白和基因的表达,且5μMol/LBBR作用最为显著(P0.05);2)BBR与正常组相比,BBR明显增加葡萄糖消耗(P0.05),但在Sirt1被Ex527抑制或被1v-Sirt1敲减后,BBR增加葡萄糖消耗这一作用明显减弱;3)BBR 明显增加 LKB1ser428、AMPK-αThr172、ACCser79 的磷酸化表达,并降低 PGC-1乙酰化水平,但在Sirt1被抑制或敲减后,BBR这一作用被减弱;4)与正常组比较,TNF-α刺激后葡萄糖吸收减弱(P0.05),BBR处理后,葡萄糖摄取增加50%(P0.05),然而Sirt1被抑制后,BBR增加葡萄糖摄取作用减弱(P0.05);5)BBR有效恢复炎症因子TNF-α抑制下AKTser473活性损伤,而Sirt1被抑制或敲减后,BBR恢复AKT活性的作用减弱,然而BBR对IRS-1Y612的影响未见明显变化。结论:小檗碱依赖于Sirt1激活AMPK/AKT通路改善3T3-L1脂肪细胞糖代谢。
[Abstract]:Berberine (BBR) is a main active ingredient of Coptis chinensis, a commonly used antipyretic and detoxifying drug. It can significantly reduce blood glucose, weight loss, glucose tolerance and insulin resistance in diabetics and animals. Silencing factor 1 (Sirt1) is a kind of deacetylase, which is basically consistent with BBR target and biological effect in anti-inflammatory and regulating signaling pathways related to glucose and lipid metabolism in sensitive adipose tissue. Therefore, this article takes 3T3-L1 adipocyte glucose metabolism disorder as the breakthrough point, interferes with Sirt1 expression in vitro, and finds out whether BBR improves glucose metabolism and whether it depends on Sirt1. It provides new clues for the mechanism of BBR. Objective: To investigate the effect of berberine on Sirt1 protein expression, glucose metabolism and its related protein expression in cultured adipocytes in vitro, and whether it depends on Sirt1 mediated 3T3-L1. Methods: 1) Western Blot and fluorescence quantitative PCR to detect different concentrations (1, 5, 10 Mol/L) of berberine on expression of Sirt1 3T3-L1 in fat cells; 2) differentiated 3T3-L1 adipocytes expressed by Sirt1 inhibitor Ex527 or lentiviral 1v-Sirt1 interference Sirt1 protein after divided into four groups, control group, BBR group Ex527 (or 1v-Sirt1) group, Ex527 (or 1v-Sirt1) +BBR group, 24h after replacement with 1nM insulin and 0.2%BSA phenol red free low glucose medium 1Oh, collected the cell supernatant were measured by glucose oxidase method cell supernatant Portuguese glucose content in phenol red free sugar content in high glucose medium subtraction medium, i.e. for each cell glucose consumption; 3) 2) according to the differentiated 3T3-L1 adipocytes were divided into four groups, the expression of phosphorylated Western protein in fat cells, LKB1ser428 Blot detection AMPK-, ACCser79 alpha Thr172 extracted by protein, immune precipitation The expression of starch detection PGC-1 alpha acetylation; 4) induced mature 3T3-L1 adipocytes were divided into six groups, three of which were pretreated with Ex527 1H, while adding TNF- and adding alpha or 5 Mol/LBBR, respectively in the control group, TNF- group, TNF- group, +BBR alpha, alpha, alpha Ex527 group, Ex527+TNF- group. Ex527+TNF- +BBR group, 24, Kami Kiyo, 1nmol/L Insulin 0.2%BSA with DMEM 15min after adding 30 M 2-NBDG and 1 g/mlDAP2 fluorescent probe PBS 37 C 2H incubation with high content imaging camera and calculate the average fluorescence intensity of fat cells; 5) by 4) will induce mature 3T3-L1 cells were divided into six groups, including control group, TNF- group, TNF- group, +BBR alpha, alpha Ex527+TNF- alpha, Ex527 group, Ex527+TNF- group, +BBR group, 24h alpha, Western, AKTser 473 and IRS-1Y612 Blot detected the expression of phosphorylated. Results: 1) BBR Sirt1 protein and gene expression of 3T3-L1 was significantly up-regulated in fat cells, and 5 Mol/LBBR the most significant effect (P0.05); 2 BBR) compared with the normal group, BBR significantly increased glucose consumption (P0.05), but the Sirt1 was inhibited by Ex527 or 1v-Sirt1 knockdown of BBR increased glucose the consumption of this effect was weakened; 3) BBR significantly increased LKB1ser428, AMPK- alpha Thr172 and ACCser79 phosphorylation and expression, and reduce the acetylation level of PGC-1 but in Sirt1 inhibition or knockdown of BBR, this effect was weakened; 4) compared with the normal group, TNF- alpha stimulated glucose absorption decreased (P0.05), after BBR treatment, the glucose uptake increased 50% (P0.05), but Sirt1 was inhibited, BBR increased glucose uptake decreased (P0.05); 5) BBR effectively restore the inflammatory factor TNF- alpha inhibition of AKTser473 activity and Sirt1 damage, inhibition or knockdown of AKT activity recovery of BBR reduction effect However, the effect of BBR on IRS-1Y612 was not significantly changed. Conclusion: Berberine is dependent on Sirt1 activation of AMPK/AKT pathway to improve the glucose metabolism of 3T3-L1 adipocytes.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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