大黄素和小檗碱对胰岛素抵抗HepG2细胞的实验研究
摘要
目的胰岛素抵抗(IR)是非酒精性脂肪肝(NAFLD)的病理生理基础,改善肝脏IR对防治NAFLD起着至关重要的作用。大黄素和小檗碱分别是中药大黄和黄连的主要有效成分,具有改善IR的作用。本研究旨在探讨高浓度的游离脂肪酸(FFA)诱导HepG2细胞为IR的机制以及大黄素和小檗碱对IR的改善作用,为防治NAFLD提供新的理论依据。
方法用高浓度的FFA培养HepG2细胞,诱导为IR的细胞模型,测定培养液中葡萄糖浓度及细胞内糖原含量作为模型鉴定指标;四甲基偶氮噻唑蓝(MTT)实验分别检测大黄素和小檗碱的最适药物浓度;逆转录-聚合酶链反应(RT-PCR)方法检测各组细胞中脂联素受体2(AdipoR2)、过氧化物酶体增殖物激活受体γ(PPARγ)、磷酸烯醇式丙酮酸羧激酶(PEPCK)的mRNA表达。
结果(一)MTT实验显示:浓度为10μmol/L大黄素时的细胞存活率为92.5%。浓度为10μmol/L小檗碱时的细胞存活率为90.3%,故选择10μmol/L为大黄素和小檗碱的工作浓度。
(二)预防组1.IR模型组培养液中葡萄糖含量较正常组明显升高( P < 0.01),当同时加入中药和FFA后即:大黄素(10μmol/L)+FFA组、小檗碱(10μmol/L)+FFA组培养液中葡萄糖含量较模型组显著降低,与正常组比较无统计学差异。
2. IR模型组细胞内糖原含量较正常组明显降低( P < 0.01);而大黄素+FFA组、小檗碱+FFA组细胞内糖原含量显著高于模型组,与正常组比较无统计学差异。
3.正常HepG2细胞可表达AdipoR2、PPARγ、PEPCK。IR模型组较正常组AdipoR2、PPARγmRNA表达减少,PEPCKmRNA表达增高( P < 0.01),而大黄素+FFA组、小檗碱+FFA组AdipoR2、PPARγmRNA表达增高,PEPCKmRNA表达减少。
(三)治疗组1. IR的模型组培养液中葡萄糖含量以及PEPCK mRNA表达明显高于正常对照组( P < 0.01),细胞内糖原含量、AdipoR2mRNA、PPARγmRNA表达显著减少( P < 0.01)。
2.培养液中分别加入大黄素(10μmol/L)、小檗碱(10μmol/L)、吡格列酮(10μmol/L)后,可明显改善IR。表现为:培养液中葡萄糖含量及PEPCKmRNA表达较IR模型组显著降低,而细胞内糖原含量、AdipoR2mRNA、PPARγmRNA表达升高。且以上三组分别与正常组比较均无统计学差异(P>0.01)。
结论高浓度的FFA培养HepG2细胞,能够诱导为IR的细胞模型,大黄素和小檗碱能阻断该模型的形成并显著改善FFA引起的IR,且二者可能通过AdipoR2、PPARγ、PEPCK而起作用,从而为下一步防治NAFLD提供了新的方法和思路。
Objective insulin resistance(IR) is the pathophysiologic foundation of nonalcoholic fatty liver disease (NAFLD). Improving IR is very important role in prevention of NAFLD. Emodin and berberine are separately main active ingredients of Rhubarb and Coptis, with the improvement of IR. This experiment aims to study on the mechanism of IR for HepG2 cells induced by high concentrations of free fatty acid (FFA) and the prevention effect of emodin and berberine. It would provide new methods and ideas for the prevention and treatment of NAFLD.
Methods The model of IR was established with HepG2 cells cultured at high concentrations of FFA,the glucose contents and hepatic glycogen contents were measured. The optimal concentration of emodin and berberine were determined by MTT.The expression of adiponectin receptor2 (AdipoR2)mRNA,peroxisome proliferator-activated receptor gamma (PPARγ)mRNA and phosphoenolpyruvate carboxykinase (PEPCK) mRNA in HepG2 cell were detected by RT-PCR.
Results (一) MTT experiments showed that the cell survival rate with the concentration of 10μmol / L emodin is 92.5%..The cell survival rate with the concentration of 10μmol / L berberine is 90.3%. Therefore, we selected 10μmol / L for the experimental concentration of emodin and berberine.
(二)Prevention Group 1. The glucose contents in IR model group was obviously higher than those in normal group (P <0.01). The glucose contents was significantly lower compared with IR model group when emodin and FFA or berberine and FFA were simultaneously added in culture medium.It was considered no statistical difference compared with control group.
2.Hepatic glycogen contents in IR model group was lower than control group.But the hepatic glycogen contents in both drug groups higher than model group.
3. The expression of AdipoR2Mrna, PPARγmRNA and PEPCKmRNA were detected in normal HepG2 cells . The expression of AdipoR2 and PPARγmRNA strongly reduced in model group of IR.But the expression of PEPCKmRNA were significantly higher than those in controlgroup.Th- -e expression of AdipoR2mRNAd and PPARγmRNA were increased as emodin and FFA or berberine and FFA were simultaneously added in culture medium,But the expression of PEPCKmRNA was decreased.
(三) Treatment group :1.The glucose contents in culture medium and the expression of PEPCKmRNA in model group were significantly higher than those in control group ,but hepatic glycogen contents and the expression of AdipoR2 and PPARγmRNA strongly reduced in model group of IR( P < 0.01).
2.The effect of improving IR could be obvious as emodin(10μmol/L)or berberine(10μmol/L)or pioglitazone(10μmol/L)was respectively added in culture medium.It was discovered that the glucose contents in culture medium and the expression of PEPCKmRNA after adding these drugs were significantly lower than model group. But the hepatic glycogen contents and the expression of AdipoR2mRNA and PPARγmRNA increased. And there was no statistical difference that three groups respectively compared with the normal group(P>0.01) .
Conclusions These results suggest that HepG2 cells cultured at high concentrations of FFA,could successfully induce hepatic IR. Emodin berberine and pioglitazone can respectively improve IR,which may be resulted from AdipoR2、PPARγand PEPCK.So it would provide new methods and ideas for the prevention and treatment of NAFLD.
方法用高浓度的FFA培养HepG2细胞,诱导为IR的细胞模型,测定培养液中葡萄糖浓度及细胞内糖原含量作为模型鉴定指标;四甲基偶氮噻唑蓝(MTT)实验分别检测大黄素和小檗碱的最适药物浓度;逆转录-聚合酶链反应(RT-PCR)方法检测各组细胞中脂联素受体2(AdipoR2)、过氧化物酶体增殖物激活受体γ(PPARγ)、磷酸烯醇式丙酮酸羧激酶(PEPCK)的mRNA表达。
结果(一)MTT实验显示:浓度为10μmol/L大黄素时的细胞存活率为92.5%。浓度为10μmol/L小檗碱时的细胞存活率为90.3%,故选择10μmol/L为大黄素和小檗碱的工作浓度。
(二)预防组1.IR模型组培养液中葡萄糖含量较正常组明显升高( P < 0.01),当同时加入中药和FFA后即:大黄素(10μmol/L)+FFA组、小檗碱(10μmol/L)+FFA组培养液中葡萄糖含量较模型组显著降低,与正常组比较无统计学差异。
2. IR模型组细胞内糖原含量较正常组明显降低( P < 0.01);而大黄素+FFA组、小檗碱+FFA组细胞内糖原含量显著高于模型组,与正常组比较无统计学差异。
3.正常HepG2细胞可表达AdipoR2、PPARγ、PEPCK。IR模型组较正常组AdipoR2、PPARγmRNA表达减少,PEPCKmRNA表达增高( P < 0.01),而大黄素+FFA组、小檗碱+FFA组AdipoR2、PPARγmRNA表达增高,PEPCKmRNA表达减少。
(三)治疗组1. IR的模型组培养液中葡萄糖含量以及PEPCK mRNA表达明显高于正常对照组( P < 0.01),细胞内糖原含量、AdipoR2mRNA、PPARγmRNA表达显著减少( P < 0.01)。
2.培养液中分别加入大黄素(10μmol/L)、小檗碱(10μmol/L)、吡格列酮(10μmol/L)后,可明显改善IR。表现为:培养液中葡萄糖含量及PEPCKmRNA表达较IR模型组显著降低,而细胞内糖原含量、AdipoR2mRNA、PPARγmRNA表达升高。且以上三组分别与正常组比较均无统计学差异(P>0.01)。
结论高浓度的FFA培养HepG2细胞,能够诱导为IR的细胞模型,大黄素和小檗碱能阻断该模型的形成并显著改善FFA引起的IR,且二者可能通过AdipoR2、PPARγ、PEPCK而起作用,从而为下一步防治NAFLD提供了新的方法和思路。
Objective insulin resistance(IR) is the pathophysiologic foundation of nonalcoholic fatty liver disease (NAFLD). Improving IR is very important role in prevention of NAFLD. Emodin and berberine are separately main active ingredients of Rhubarb and Coptis, with the improvement of IR. This experiment aims to study on the mechanism of IR for HepG2 cells induced by high concentrations of free fatty acid (FFA) and the prevention effect of emodin and berberine. It would provide new methods and ideas for the prevention and treatment of NAFLD.
Methods The model of IR was established with HepG2 cells cultured at high concentrations of FFA,the glucose contents and hepatic glycogen contents were measured. The optimal concentration of emodin and berberine were determined by MTT.The expression of adiponectin receptor2 (AdipoR2)mRNA,peroxisome proliferator-activated receptor gamma (PPARγ)mRNA and phosphoenolpyruvate carboxykinase (PEPCK) mRNA in HepG2 cell were detected by RT-PCR.
Results (一) MTT experiments showed that the cell survival rate with the concentration of 10μmol / L emodin is 92.5%..The cell survival rate with the concentration of 10μmol / L berberine is 90.3%. Therefore, we selected 10μmol / L for the experimental concentration of emodin and berberine.
(二)Prevention Group 1. The glucose contents in IR model group was obviously higher than those in normal group (P <0.01). The glucose contents was significantly lower compared with IR model group when emodin and FFA or berberine and FFA were simultaneously added in culture medium.It was considered no statistical difference compared with control group.
2.Hepatic glycogen contents in IR model group was lower than control group.But the hepatic glycogen contents in both drug groups higher than model group.
3. The expression of AdipoR2Mrna, PPARγmRNA and PEPCKmRNA were detected in normal HepG2 cells . The expression of AdipoR2 and PPARγmRNA strongly reduced in model group of IR.But the expression of PEPCKmRNA were significantly higher than those in controlgroup.Th- -e expression of AdipoR2mRNAd and PPARγmRNA were increased as emodin and FFA or berberine and FFA were simultaneously added in culture medium,But the expression of PEPCKmRNA was decreased.
(三) Treatment group :1.The glucose contents in culture medium and the expression of PEPCKmRNA in model group were significantly higher than those in control group ,but hepatic glycogen contents and the expression of AdipoR2 and PPARγmRNA strongly reduced in model group of IR( P < 0.01).
2.The effect of improving IR could be obvious as emodin(10μmol/L)or berberine(10μmol/L)or pioglitazone(10μmol/L)was respectively added in culture medium.It was discovered that the glucose contents in culture medium and the expression of PEPCKmRNA after adding these drugs were significantly lower than model group. But the hepatic glycogen contents and the expression of AdipoR2mRNA and PPARγmRNA increased. And there was no statistical difference that three groups respectively compared with the normal group(P>0.01) .
Conclusions These results suggest that HepG2 cells cultured at high concentrations of FFA,could successfully induce hepatic IR. Emodin berberine and pioglitazone can respectively improve IR,which may be resulted from AdipoR2、PPARγand PEPCK.So it would provide new methods and ideas for the prevention and treatment of NAFLD.
引文
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