委陵菜黄酮衍生物抗糖尿病活性及其作用机制研究
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摘要
目的：
建立人HepG2肝细胞及由小鼠3T3-L1前脂肪细胞株诱导而成的成熟脂肪细胞的胰岛素抵抗模型,筛选委陵菜黄酮衍生物抗糖尿病活性,并对其作用机制进行进一步探讨。
方法：
1、胰岛素抵抗模型的建立：
HepG2人肝细胞和3T3-L1脂肪细胞的胰岛素抵抗模型是研究胰岛素抵抗中最为常用的两类细胞模型,采用文献方法对胰岛素抵抗模型进行复制,以葡萄糖氧化酶—过氧化物酶法(GOD-POD法)检测细胞中葡萄糖的消耗量确立两种细胞胰岛素抵抗模型的建立。
2、抗糖尿病活性筛选：
委陵菜黄酮为中药委陵菜抗糖尿病活性的主要成分,为进一步提高其生物活性,发现先导化合物,课题组前期设计合成了一系列委陵菜黄酮衍生物,本文分别以胰岛素抵抗的HepG2细胞和3T3-L1脂肪细胞为筛选模型,通过葡萄糖氧化酶法检测各衍生物对模型细胞葡萄糖消耗量的影响,评价各衍生物的抗糖尿病活性。
3、抗糖尿病作用机制研究：
以筛选出的抗糖尿病活性最强委陵菜黄酮衍生物5f分别与HepG2细胞和3T3-L1成熟脂肪细胞共孵育,Western blot方法检测Comp.5f对5’-AMP激活蛋白激酶(AMPK),乙酰辅酶A羧化酶(Acetyl-CoA carboxylase, ACC),以及蛋白激酶B (protein kinase B, Akt)磷酸化水平的影响。检测Comp.5f对HepG2细胞中葡萄糖转运子2(GLUT2)及3T3-L1成熟脂肪细胞中葡萄糖转运子4(GLUT4)的影响。
结果：
1、本实验采用高浓度胰岛素(5×10-7mol/l)诱导HepG2细胞产生胰岛素抵抗,以1μmol/l的地塞米松作用4天诱导3T3-L1成熟脂肪细胞产生胰岛素抵抗,应用葡萄糖氧化酶—过氧化物酶法(GOD-POD法)检测各组细胞上清液中葡萄糖含量,与正常细胞相比,具有显著差异(p<0.05),确定胰岛素抵抗细胞模型的成功建立。
2、抗糖尿病活性筛选结果显示除化合物7b外,各衍生物均具有提高胰岛素抵抗模型细胞葡萄糖消耗量的作用,化合物5b、5e、5g和10b的活性较强,与市售药物二甲双胍相当,化合物5f、7d、10C抗糖尿病活性最强,优于市售药物二甲双胍。
委陵菜黄酮衍生物5a、5g、5f、5e、7d作用于胰岛素抵抗的HepG2及3T3-L1成熟脂肪细胞模型,以委陵菜黄酮衍生物5f抗糖尿病活性最强,委陵菜黄酮衍生物5f可在一定程度上提高3T3-L1脂肪细胞的2-脱氧-[3H]-葡萄糖摄取量。
3、委陵菜黄酮衍生物5f可上调HepG2肝细胞及3T3-L1成熟脂肪细胞内AMPK, ACC的磷酸化水平(p<0.05),而不影响Akt的磷酸化水平。以AMPK的特异性抑制剂Compound C与Comp.5f共同作用于HepG2细胞,结果显示Compound C能够显著降低5f刺激的AMPK、ACC磷酸化水平的上调,在3T3-L1脂肪细胞中,Compound C仅能部分抑制黄酮衍生物5f刺激的AMPK、ACC磷酸化水平的上调作用。Comp.5f对HepG2人肝细胞内的GLUT2水平有一定的下调作用。Comp.5f对3T3-L1脂肪细胞内的GLUT4蛋白表达量没有发生明显影响。
结论：
本文建立了HepG2人肝细胞及3T3-L1脂肪细胞的胰岛素抵抗模型,观察委陵菜黄酮衍生物对胰岛素抵抗的影响,结果表明黄酮类化合物均具有较强的抗糖尿病活性,活性最强的Comp.5f可显著上调HepG2细胞及3T3-L1成熟脂肪细胞内AMPK,ACC的磷酸化水平,以上结果表明,黄酮衍生物5f抗糖尿病作用的机制之一是激活AMPK信号传导通路,而不是作用于胰岛素通路。
Objective:
To establish the insulin resistant HepG2and3T3-L1adipocyte cells model. Screening of tiliroside derivantives with anti-diabetes activities. Objective to investigate the mechanism.
Methods:
1. To set up insulin resistant cell model:
Insulin resistant HepG2and3T3-L1adipocyte cells model are commonly used in the research on insulin resistance. Copy the insulin resistant cell model, whose formation was assessed by detecting the ability of uptaking glucose with GOD-POD assay
2. Evaluation of the anti-diabetes activity of the derivatives:
Tiliroside was the main anti-diabetic constituent isolated from Potentilla Chinese Ser. For the improvement of its bioactivity, we have synthesized a series of tiliroside-derivatives. All the derivatives were investgated for the glucose consumption of insulin resistance HepG2and3T3-L1adipocyte cells model to evaluate the anti-diabetes activities.
3. Study on mechanism of anti-diabetes activity:
To investigate the mechanism of Comp.5f, we detected the phosphorylation of5'-AMP-activated protein kinase (AMPK), Acetyl-CoA carboxylase (ACC), protein kinase B (Akt) by western blotting. We also detect the effect of glucose transporters2and glucose transporters4by western blotting.
Results:
1. The insulin resistant HepG2cell model was established successfully by high concentration of insulin (5×10-7mol/l) in vitro culture. The IR-3T3-L1adipocyte cell model was established successfully by dexamethasone (1μmol/1,4days). Detecting the ability of uptaking glucose with GOD-POD assay. There were significant differences (p<0.05) compared with the control group. The insulin resistant cell models were established successfully.
2. All of the derivatives could improve the glucose consumption of the insulin resistance cell except7b. Compounds5b、5e、5g and10b revealed significant activities compared with that of market drug, Metformin. Compounds5f、7d、10c showed stronger activities than Metformin.
Among tiliroside derivantives compounds5a、5g、5f、5e、7d, Compounds5f is the most active derivatives. Compounds5f strongly2-deoxidation-[3H]-glucose uptaking in3T3-L1adipocyte cell.
Compounds5f strongly phosphorylated AMPK and its substrate, ACC in both HepG2and3T3-L1adipocyte cells (p<0.05). However, Akt phosphorylation was not affected in both cells. GLUT2can be down regulated by Compounds5f in HepG2cells. Compounds5f did not affect the expression of GLUT4and AMPKa in3T3-L1adipocyte cells.
Conclusion:
The insulin resistant HepG2and3T3-L1adipocyte cell models were established successfully. Tiliroside derivatives have relatively stronger anti-diabetes activities. Compounds5f strongly phosphorylated AMPK and its substrate, ACC in both HepG2and3T3-L1adipocyte cells (p<0.05). Its mechanism involving AMPK correlation signal pathways.
建立人HepG2肝细胞及由小鼠3T3-L1前脂肪细胞株诱导而成的成熟脂肪细胞的胰岛素抵抗模型,筛选委陵菜黄酮衍生物抗糖尿病活性,并对其作用机制进行进一步探讨。
方法：
1、胰岛素抵抗模型的建立：
HepG2人肝细胞和3T3-L1脂肪细胞的胰岛素抵抗模型是研究胰岛素抵抗中最为常用的两类细胞模型,采用文献方法对胰岛素抵抗模型进行复制,以葡萄糖氧化酶—过氧化物酶法(GOD-POD法)检测细胞中葡萄糖的消耗量确立两种细胞胰岛素抵抗模型的建立。
2、抗糖尿病活性筛选：
委陵菜黄酮为中药委陵菜抗糖尿病活性的主要成分,为进一步提高其生物活性,发现先导化合物,课题组前期设计合成了一系列委陵菜黄酮衍生物,本文分别以胰岛素抵抗的HepG2细胞和3T3-L1脂肪细胞为筛选模型,通过葡萄糖氧化酶法检测各衍生物对模型细胞葡萄糖消耗量的影响,评价各衍生物的抗糖尿病活性。
3、抗糖尿病作用机制研究：
以筛选出的抗糖尿病活性最强委陵菜黄酮衍生物5f分别与HepG2细胞和3T3-L1成熟脂肪细胞共孵育,Western blot方法检测Comp.5f对5’-AMP激活蛋白激酶(AMPK),乙酰辅酶A羧化酶(Acetyl-CoA carboxylase, ACC),以及蛋白激酶B (protein kinase B, Akt)磷酸化水平的影响。检测Comp.5f对HepG2细胞中葡萄糖转运子2(GLUT2)及3T3-L1成熟脂肪细胞中葡萄糖转运子4(GLUT4)的影响。
结果：
1、本实验采用高浓度胰岛素(5×10-7mol/l)诱导HepG2细胞产生胰岛素抵抗,以1μmol/l的地塞米松作用4天诱导3T3-L1成熟脂肪细胞产生胰岛素抵抗,应用葡萄糖氧化酶—过氧化物酶法(GOD-POD法)检测各组细胞上清液中葡萄糖含量,与正常细胞相比,具有显著差异(p<0.05),确定胰岛素抵抗细胞模型的成功建立。
2、抗糖尿病活性筛选结果显示除化合物7b外,各衍生物均具有提高胰岛素抵抗模型细胞葡萄糖消耗量的作用,化合物5b、5e、5g和10b的活性较强,与市售药物二甲双胍相当,化合物5f、7d、10C抗糖尿病活性最强,优于市售药物二甲双胍。
委陵菜黄酮衍生物5a、5g、5f、5e、7d作用于胰岛素抵抗的HepG2及3T3-L1成熟脂肪细胞模型,以委陵菜黄酮衍生物5f抗糖尿病活性最强,委陵菜黄酮衍生物5f可在一定程度上提高3T3-L1脂肪细胞的2-脱氧-[3H]-葡萄糖摄取量。
3、委陵菜黄酮衍生物5f可上调HepG2肝细胞及3T3-L1成熟脂肪细胞内AMPK, ACC的磷酸化水平(p<0.05),而不影响Akt的磷酸化水平。以AMPK的特异性抑制剂Compound C与Comp.5f共同作用于HepG2细胞,结果显示Compound C能够显著降低5f刺激的AMPK、ACC磷酸化水平的上调,在3T3-L1脂肪细胞中,Compound C仅能部分抑制黄酮衍生物5f刺激的AMPK、ACC磷酸化水平的上调作用。Comp.5f对HepG2人肝细胞内的GLUT2水平有一定的下调作用。Comp.5f对3T3-L1脂肪细胞内的GLUT4蛋白表达量没有发生明显影响。
结论：
本文建立了HepG2人肝细胞及3T3-L1脂肪细胞的胰岛素抵抗模型,观察委陵菜黄酮衍生物对胰岛素抵抗的影响,结果表明黄酮类化合物均具有较强的抗糖尿病活性,活性最强的Comp.5f可显著上调HepG2细胞及3T3-L1成熟脂肪细胞内AMPK,ACC的磷酸化水平,以上结果表明,黄酮衍生物5f抗糖尿病作用的机制之一是激活AMPK信号传导通路,而不是作用于胰岛素通路。
Objective:
To establish the insulin resistant HepG2and3T3-L1adipocyte cells model. Screening of tiliroside derivantives with anti-diabetes activities. Objective to investigate the mechanism.
Methods:
1. To set up insulin resistant cell model:
Insulin resistant HepG2and3T3-L1adipocyte cells model are commonly used in the research on insulin resistance. Copy the insulin resistant cell model, whose formation was assessed by detecting the ability of uptaking glucose with GOD-POD assay
2. Evaluation of the anti-diabetes activity of the derivatives:
Tiliroside was the main anti-diabetic constituent isolated from Potentilla Chinese Ser. For the improvement of its bioactivity, we have synthesized a series of tiliroside-derivatives. All the derivatives were investgated for the glucose consumption of insulin resistance HepG2and3T3-L1adipocyte cells model to evaluate the anti-diabetes activities.
3. Study on mechanism of anti-diabetes activity:
To investigate the mechanism of Comp.5f, we detected the phosphorylation of5'-AMP-activated protein kinase (AMPK), Acetyl-CoA carboxylase (ACC), protein kinase B (Akt) by western blotting. We also detect the effect of glucose transporters2and glucose transporters4by western blotting.
Results:
1. The insulin resistant HepG2cell model was established successfully by high concentration of insulin (5×10-7mol/l) in vitro culture. The IR-3T3-L1adipocyte cell model was established successfully by dexamethasone (1μmol/1,4days). Detecting the ability of uptaking glucose with GOD-POD assay. There were significant differences (p<0.05) compared with the control group. The insulin resistant cell models were established successfully.
2. All of the derivatives could improve the glucose consumption of the insulin resistance cell except7b. Compounds5b、5e、5g and10b revealed significant activities compared with that of market drug, Metformin. Compounds5f、7d、10c showed stronger activities than Metformin.
Among tiliroside derivantives compounds5a、5g、5f、5e、7d, Compounds5f is the most active derivatives. Compounds5f strongly2-deoxidation-[3H]-glucose uptaking in3T3-L1adipocyte cell.
Compounds5f strongly phosphorylated AMPK and its substrate, ACC in both HepG2and3T3-L1adipocyte cells (p<0.05). However, Akt phosphorylation was not affected in both cells. GLUT2can be down regulated by Compounds5f in HepG2cells. Compounds5f did not affect the expression of GLUT4and AMPKa in3T3-L1adipocyte cells.
Conclusion:
The insulin resistant HepG2and3T3-L1adipocyte cell models were established successfully. Tiliroside derivatives have relatively stronger anti-diabetes activities. Compounds5f strongly phosphorylated AMPK and its substrate, ACC in both HepG2and3T3-L1adipocyte cells (p<0.05). Its mechanism involving AMPK correlation signal pathways.
引文
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