杜仲叶绿原酸提取物联合西红花苷对肝癌细胞胆固醇代谢的影响

摘要

Objective To observe the influence of chlorogenic acid extracted from Folium eucommiae (CAEF) and crocin on cholesterol metabolism in human hepatoma carcinoma HepG2 cells and to discuss the possible mechanism.Methods The HepG2 cells were randomly divided into five groups:the control group,the oleic acid group,the simvastatin group,the CAEF group and the combined administration group.In the oleic acid group,simvastatin group,CAEF group and combined administration group were added with oleic acid to induce steatosis models.At the same time,the simvastatin group was treated with 10 μmol/L simvastatin 10 μL,CAEF group was added with 25 mg/L CAEF 10 μL,the combined administration group was treated with 10 μL mixed solution including 1 μmol/L crocin,30 μmol/L chlorogenic acid,10 μmol/L geniposide and 10 μmol/L quercetin,and the control group was added with an equal volume of medium.The content of intracellular lipid droplets of HepG2 cells was observed by the oil red O staining after 48 h.The levels of triglyceride (TG) and total cholesterol (TC) in HepG2 cells were detected after 24 and 48 h.The expression of cholesterol metabolism-related genes,including ABCA1,SREBP2,CYP7A1,LXRα,AMPK2α,and HMGCR was measured by RT-PCR.The protein expression of HMGCR was detected by immunocytochemistry and Western blotting.Results The content of intracellular lipid droplets in the simvastatin group,the CAEF group and the combined administration group was significantly decreased after 48 h (the combined administration group ＞ the CAEF group ＞ the simvastatin group).Compared with the control group,the content of TC and TG was significantly increased in the other three groups after 24 h and 48 h (the combined administration group ＞ the CAEF group ＞ the simvastatin group,all P ＜ 0.05).The mRNAs expression of ABCA1,CYP7A1 and AMPK2oα was significantly increased,while the mRNAs expression of SREBP2 and HMGCR was significantly decreased in the combined administration group and in the CAEF group after 48 h (all P ＜ 0.05).The mRNAs expression of LXRα was significantly decreased in the combined administration group (P ＜ 0.05).The protein expression of HMGCR in the three groups was significantly decreased and the combined administration group showed the strongest inhibitory effect (all P ＜ 0.05).Conclusions CAEF and crocin have synergistic inhibition effect on the lipid accumulation in HepG2 cells.The regulation of cholesterol metabolism-related genes and protein expression may be one of the mechanisms.%目的 观察杜仲叶绿原酸提取物(CAEF)联合西红花苷对人肝癌细胞HepG2胆固醇代谢的影响,并探讨其可能的作用机制.方法 将人肝癌细胞HepG2随机分为空白对照组、油酸诱导组、辛伐他汀组、CAEF组、联合用药组.油酸诱导组、辛伐他汀组、CAEF组、联合用药组分别加入油酸诱导脂肪变性模型;同时,辛伐他汀组加入10 μmol/L的辛伐他汀10μL,CAEF组加入25 mg/L的CAEF 10 μL,联合用药组加入含1μmol/L西红花苷、30μmol/L绿原酸、10 μmol/L京尼平苷、10 μmol/L槲皮素的混合液10 μL;空白对照组仅加入等体积培养基.取各组干预48 h细胞,行油红O染色,检测细胞内中性脂滴含量.取干预24、48 h细胞上清液,检测TC、TG含量.采用RT-PCR法检测胆固醇代谢相关基因(ABCA1、SREBP2、CYP7A1、LXRα、AMPK2α、HMGCR)表达,免疫细胞化学法和Western blotting法检测HMGCR蛋白表达.结果 辛伐他汀组、CAEF组、联合用药组干预48 h,细胞内中性脂滴含量明显降低,其作用强度依次为联合用药组＞CAEF组＞辛伐他汀组(P均＜0.05);与对照组比较,CAEF组、联合用药组干预24、48 h时细胞外液TC、TG含量明显增加,细胞外液TC、TG含量依次为联合用药组＞CAEF组＞辛伐他汀组(P均＜0.05).CAEF组、联合用药组ABCA1、CYP7A1、AMPK2α mRNA表达明显升高,SREBP2、HMGCRmRNA表达明显降低(P均＜0.05);此外,联合用药组LXRα mRNA表达明显降低(P＜0.05).辛伐他汀组、CAEF组、联合用药组HMGCR蛋白表达明显降低,以联合用药组降低最明显(P均＜0.05).结论 CAEF联合西红花苷可协同抑制肝癌细胞脂质积聚,其作用机制可能与调控肝癌细胞胆固醇代谢相关基因和蛋白的表达有关.