化瘀祛痰方及其拆方对HepG2细胞胆固醇代谢的影响及机制研究

目的:观察化瘀祛痰方药及其拆方对人肝癌细胞株HepG2胆固醇代谢的影响,探讨该方药的作用机制,并比较方药全方及各拆方的疗效及调控作用.方法:SD大鼠随机分为空白对照组、化瘀祛痰方全方组、补气组、化瘀组、祛痰组,生理盐水和相应中药予以ig,连续8d.给药量为全方组17.9g·kg-1,补气组7.5 g·kg-1,化瘀组5.4 g·kg-1,祛痰组4.2 g·kg-1,空白对照组ig等量生理盐水.第9天腹主动脉采血,分离血清.体外培养HepG2细胞,随机分为6组,即①空白对照组、②ox-LDL刺激组、③化瘀祛痰方全方组、④补气组、⑤化瘀组、⑥祛痰组.其中②组加入终质量浓度为50 mg·L-1 ox-LDL,③～⑥组用相应含药血清(10％)预处理24 h后加入终质量浓度为50 mg·L-1 ox-LDL,各组细胞培养24 h后进行各项指标测定.采用MTT法检测细胞活性；胆固醇氧化酶终点法检测HepG2细胞总胆固醇含量；反转录-聚合酶链反应(RT-PCR)定量分析3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGCR)、B类Ⅰ型清道夫受体(SR-BⅠ)、胆固醇7α-羟化酶(CYP7A1) mRNA表达.结果:50 mg·L-1 ox-LDL作用于HepG2细胞使其细胞活力显著降低至对照组的(59.37±18.21)％,细胞总胆固醇含量显著升高(幅度为71.52％).而化瘀祛痰方全方、补气方、化瘀方、祛痰方显著升高细胞活力分别至(76.52±24.38)％,(68.13±20.35)％,(72.63±21.56)％,(73.92±23.47)％(P＜0.05或P＜0.01),全方、化瘀方、祛痰方能显著降低HepG2细胞胆固醇含量,幅度分别为:28.77％,17.18％,25.09％.RT-PCR结果表明经ox-LDL诱导HepG2细胞HMGCR mRNA相对表达显著减少(P＜0.01),而SR-BⅠ和CYP7A1 mRNA相对表达显著增加(P＜0.01,P＜0.01).化瘀祛痰方及祛痰方含药血清均能显著降低HepG2细胞HMGCR mRNA水平(P＜0.01,P＜0.05),显著升高SR-BⅠ和CYP7A1mRNA水平(均P＜0.01).结论:化瘀祛痰方药全方及祛痰方可通过降低HMGCR水平和升高SR-BⅠ,CYP7A1水平从而影响胆固醇合成、摄取和转化,这可能是其调控脂质代谢发挥抗AS作用的机制之一.

Effect of Huayu Qutan Recipe and its Separated Recipes on Cholesterol Metabolism in HepG2 Cells

Objective:To observe the influence of Huayu Qutan recipe and its separated recipes on cholesterol metabolism-related genes in HepG2 cells.Method: SD rats were equally divided into five groups in random: the control,Huayu Qutan recipes,Buqi recipes,Huayu recipes and Qutan recipes groups.They were gastric perfused daily with normal saline and recipes for 8 d,respectively.Blood sample was collected from abdominal aorta at 2 h after ending administration on the 9th day,and the serum was separated for testing.The cultured HepG2 cells were divided into six groups randomly: the normal serum control,oxidized low-density lipoprotein(ox-LDL) stimulating,Huayu Qutan recipes,Buqi recipes,Huayu recipes and Qutan recipe groups.Then cell viability of HepG2 was detected by MTT method.The levels of total cholesterol in HepG2 cells were measured by a method of enzymatic reagents.RT-PCR was used to test the changes of expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGCR),scavenger receptor class B type I(SR-BⅠ)and cholesterol 7α-hydroxylase(CYP7A1) genes.Result: After stimulating by 50 mg · L-1 ox-LDL,HMGCR mRNA level was obviously decreased while SR-BⅠand CYP7A1 mRNA levels were obviously increased in HepG2 cell(all P<0.01).Compared with the ox-LDL stimulating group,the expression of HMGCR mRNA was significantly decreased while SR-BⅠand CYP7A1 mRNA significantly increased in the Huayu Qutan recipe group and Qutan recipe group.Conclusion: Huayu Qutan recipe and Qutan recipe can reduce the levels of total cholesterol in HepG2 cell.The change of cholesterol metabolism-related genes expression may be one of the mechanisms against AS.