本文關鍵詞：小檗堿通過Sirt1改善3T3-L1脂肪細胞糖代謝的機制研究　出處：《南京中醫(yī)藥大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 小檗堿 Sirt1 3T3-L1脂肪細胞 胰島素抵抗 糖代謝

【摘要】：小檗堿(BBR)是常用清熱解毒藥黃連的主要活性成分,能顯著降低糖尿病患者和動物血糖,減輕體重,改善糖耐量和胰島素抵抗。體內(nèi)沉默信息調(diào)節(jié)因子1(Sirt1)是一種去乙�；�,在抗炎和調(diào)節(jié)敏感性脂肪組織糖脂代謝相關信號通路中與BBR作用靶點和生物學效應基本一致。因此,本文以3T3-L1脂肪細胞葡萄糖代謝紊亂為切入點,體外干擾Sirt1表達,探明BBR改善糖代謝是否依賴于Sirt1,為BBR的作用機制提供新的線索。目的:體外培養(yǎng)3T3-L1脂肪細胞,探明小檗堿對脂肪細胞Sirt1蛋白表達和糖代謝及其相關通路蛋白表達的影響是否依賴于Sirt1的介導。方法:1)Western Blot和熒光定量PCR檢測不同濃度(1、5、10μMol/L)小檗堿對3T3-L1脂肪細胞中Sirt1表達的影響;2)將分化成熟的3T3-L1脂肪細胞用Sirt1抑制劑Ex527或慢病毒1v-Sirt1干擾Sirt1蛋白表達后分為四組,對照組,BBR組,Ex527(或1v-Sirt1)組,Ex527(或1v-Sirt1)+BBR組,作用24h后更換含1nM胰島素和0.2%BSA的無酚紅低糖培養(yǎng)基作用1Oh,收取細胞上清液,采用葡萄糖氧化酶法檢測細胞的上清液中葡萄糖含量,以無酚紅高糖培養(yǎng)基中糖含量相減,即為各組細胞葡萄糖消耗量;3)按2)將分化成熟的3T3-L1脂肪細胞分為四組,提取蛋白采用Western Blot檢測脂肪細胞中LKB1ser428、AMPK-αThr172、ACCser79的磷酸化蛋白表達,免疫沉淀檢測PGC-1α的乙�；磉_;4)將誘導成熟的3T3-L1脂肪細胞分為六組,其中三組用Ex527預處理1h,同時加入TNF-α或合并加入5μMol/LBBR,分別為對照組,TNF-α組,TNF-α+BBR組,Ex527 組,Ex527+TNF-α 組,Ex527+TNF-α+BBR 組,作用 24 后,棄上清,用含有 1nmol/L Insulin 的 0.2%BSA DMEM 處理 15min 后加入 30μM 2-NBDG 和 1 μg/mlDAP2熒光探針的PBS 37℃孵育2h,用高內(nèi)涵細胞成像儀拍照并計算脂肪細胞平均熒光強度;5)按4)將誘導成熟的3T3-L1脂肪細胞分為六組,分別為對照組,TNF-α組,TNF-α+BBR組,Ex527組,Ex527+TNF-α組,Ex527+TNF-α+BBR組,作用24h,Western Blot檢測AKTser 473與IRS-1Y612磷酸化表達。結(jié)果:1)BBR可顯著上調(diào)3T3-L1脂肪細胞中Sirt1蛋白和基因的表達,且5μMol/LBBR作用最為顯著(P0.05);2)BBR與正常組相比,BBR明顯增加葡萄糖消耗(P0.05),但在Sirt1被Ex527抑制或被1v-Sirt1敲減后,BBR增加葡萄糖消耗這一作用明顯減弱;3)BBR 明顯增加 LKB1ser428、AMPK-αThr172、ACCser79 的磷酸化表達,并降低 PGC-1乙�；�,但在Sirt1被抑制或敲減后,BBR這一作用被減弱;4)與正常組比較,TNF-α刺激后葡萄糖吸收減弱(P0.05),BBR處理后,葡萄糖攝取增加50%(P0.05),然而Sirt1被抑制后,BBR增加葡萄糖攝取作用減弱(P0.05);5)BBR有效恢復炎癥因子TNF-α抑制下AKTser473活性損傷,而Sirt1被抑制或敲減后,BBR恢復AKT活性的作用減弱,然而BBR對IRS-1Y612的影響未見明顯變化。結(jié)論:小檗堿依賴于Sirt1激活AMPK/AKT通路改善3T3-L1脂肪細胞糖代謝。
[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.
【學位授予單位】：南京中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285

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