HepG2细胞胰岛素抵抗模型的建立及在筛选两色金鸡菊活性化合物中的应用

Establishment of Insulin Resistance HepG2 Cell Model and Its Application in Screening Bioactive Components of Coreopsis tinctoria

目的:体外建立人肝癌细胞(HepG2)胰岛素抵抗模型,并初步筛选两色金鸡菊中可有效改善胰岛素抵抗的萃取部位及活性化合物。方法:用不同浓度的胰岛素、棕榈酸或葡萄糖分别对HepG2细胞进行不同时间的诱导,通过MTT法对细胞活性进行评价及葡萄糖氧化酶法对HepG2细胞葡萄糖消耗量测定,明确建立稳定的HepG2胰岛素抵抗模型的试剂诱导浓度及诱导时间。模型建立后,应用不同浓度的两色金鸡菊粗提物、各萃取部位及七个单体化合物奥卡宁(Okanin,2)、3',4',8-三羟基黄酮-7-O-β-D-吡喃葡萄糖苷(3',4',8-trihydroxyflavone-7-O-β-D-glucopyranoside,3)、槲皮素(Quercetin,4)、黄诺马苷(Flavanomarein,5)、马里苷(Marein,6)、豆甾醇(Stigmasterol,13)、紫铆花素(Butein,16)预处理细胞24 h,再用55 mmol·L-1葡萄糖刺激24h,用葡萄糖氧化酶法或2-NBDG荧光标记葡萄糖法分别观察不同浓度的上述成分对胰岛素抵抗模型HepG2细胞糖消耗或糖吸收的影响。MTT法对各组细胞活性进行评价。结果:HepG2细胞在10-7mol·L-1浓度的胰岛素中作用24 h,或在55 mmol·L-1的葡萄糖中刺激24 h,细胞葡萄糖消耗量明显减少(P<0.01)且稳定性良好,并且对细胞存活率未造成影响,可作为胰岛素抵抗模型;而棕榈酸组虽然糖消耗显著下降但细胞存活率也显著降低。两色金鸡菊总提物、乙酸乙酯部位、正丁醇部位、水部位及2号、3号、5号和16号化合物均有改善细胞胰岛素抵抗作用,其中5号黄诺马苷和6号马里苷效果较好。结论:黄诺马苷和马里苷可能是两色金鸡菊中改善胰岛素抵抗的主要活性成分。

Objective：To establish human hepatoma carcinoma cell（HepG2 cell）model of insulin resistance in vitro and to initially select the effective ingredients of Coreopsis tinctoria.Methods：HepG2 cells were induced by insulin,palmic acidor glucose of different concentrations for different time.Through the estimation of the cell activity with MTT method and glucose consumption with glucose oxidase method,the effective concentrations and time of inducer in the insulin resistance cell model were ascertained.After the cell model establishment,HepG2 cells were pretreated with crude extracts of C.tinctoria,different fractions or compounds of different concentrations for 24 h,and the influences on the model cells regarding glucose consumption were observed through the glucose oxidase method or fluorescent glucose 2-NBDG and the cell viability was evaluated by the MTT method.Results：Compared with the control group,1×10^-7 mol·L^-1 insulin-stimulated cells 24 h or 55 mmol·L^-1 glucose-stimulated cells 24 h has a significant insulin resistance effect,and high concentrations of insulin or glucose did not affect the cell viability,while palmitic acid group emerges insulin resistant but the cell viability was also significantly decreased.Screening results showed that the 5th compound falavanomarein and the 6th compounds marein had better preventive activity of high glucose-induced insulin resistance.Conclusion：Insulin resistance cell models are successfully made.Falavanomarein and marein could be the main active compounds of C.tinctoria.