Yin JinJin, Luo YanQin, Deng HouLiang, Qin ShuMin, Tang WaiJiao, Zeng Lu, Zhou BenJie
Center for Drug Research and Development, Zhujiang Hospital, Southern Medical University, Guangdong, Guangzhou 510282, PR China.
Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China.
J Ethnopharmacol. 2014 May 28;154(1):229-39. doi: 10.1016/j.jep.2014.04.011. Epub 2014 Apr 13.
Hugan Qingzhi tablet (HQT), a lipid- lowering traditional Chinese medicine formula, has been used for the prevention and treatment of nonalcoholic fatty liver (NAFLD).
This study was realized to evaluate the effects of HQT-medicated serum on hepatic steatosis using in vitro experiments with cells and explore the relevant mechanisms with method of serum pharmacology.
A model of hepatic steatosis in the L02 and HepG2 cells was induced by free fatty acid (FFA). The components in the HQT-medicated serum were assayed by high-performance liquid chromatography. Intracellular lipid droplets were detected by Oil Red O staining, and their ultrastructure was examined by transmission electron microscope. The biochemical parameters, including triglyceride (TG), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), total antioxidant capacity (T-AOC), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH), were measured with commercial kits. Furthermore, the expression of adiponectin, AMP-activated protein kinase (AMPK) phosphorylation, sterol regulatory element-binding protein 1 (SREBP-1), peroxisome proliferator activated receptor-α (PPARα), carnitine palmitoyltransferase 1 (CPT-1), and acetyl-CoA oxidase 1 (ACOX1) was analyzed by Western blot and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR).
Moderate- and high-dose HQT-medicated serum reduced (P<0.05 or P<0.01) the accumulation of lipid droplets and the cellular TG content in L02 and HepG2 cells. They caused significant reductions (P<0.01) in LDH, AST, ALT and MDA and significant increase (P<0.05 or P<0.01) in T-AOC in the culture medium. They also caused increase (P<0.05 or P<0.01) in GSH level and SOD activity in FFA-induced steatotic L02 and HepG2 cells. Furthermore, moderate- and high-dose HQT-medicated serum enhanced (P<0.01) adiponectin expression in a concentration-dependent manner and increased (P<0.05 or P<0.01) the phosphorylation of AMPK and the expression of PPARα, CPT-1, and ACOX1, and reduced (P<0.05 or P<0.01) the expression of SREBP-1.
The results suggested that HQT-medicated serum exerts a preventive effect against hepatic steatosis, and the potential mechanism might be activation of AMPK and PPARα pathways.
护肝清脂片（HQT）是一种降脂中药配方，已用于非酒精性脂肪肝（NAFLD）的防治。
本研究通过细胞体外实验评估HQT含药血清对肝脂肪变性的影响，并采用血清药理学方法探讨相关机制。
用游离脂肪酸（FFA）诱导L02和HepG2细胞建立肝脂肪变性模型。采用高效液相色谱法测定HQT含药血清中的成分。用油红O染色检测细胞内脂滴，并用透射电子显微镜观察其超微结构。使用商业试剂盒测定生化参数，包括甘油三酯（TG）、乳酸脱氢酶（LDH）、天冬氨酸转氨酶（AST）和丙氨酸转氨酶（ALT）、总抗氧化能力（T-AOC）、丙二醛（MDA）、超氧化物歧化酶（SOD）和谷胱甘肽（GSH）。此外，通过蛋白质免疫印迹法和/或定量逆转录-聚合酶链反应（qRT-PCR）分析脂联素、AMP激活蛋白激酶（AMPK）磷酸化、固醇调节元件结合蛋白1（SREBP-1）、过氧化物酶体增殖物激活受体-α（PPARα）、肉碱棕榈酰转移酶1（CPT-1）和乙酰辅酶A氧化酶1（ACOX1）的表达。
中、高剂量HQT含药血清可降低（P<0.05或P<0.01）L02和HepG2细胞中脂滴的积累和细胞内TG含量。它们可使培养基中LDH、AST、ALT和MDA显著降低（P<0.01），T-AOC显著升高（P<0.05或P<0.01）。它们还可使FFA诱导的脂肪变性L02和HepG2细胞中GSH水平和SOD活性升高（P<0.05或P<0.01）。此外，中、高剂量HQT含药血清以浓度依赖性方式增强（P<0.01）脂联素表达，增加（P<0.05或P<0.01）AMPK磷酸化以及PPARα、CPT-1和ACOX1的表达，并降低（P<0.05或P<0.01）SREBP-1的表达。
结果表明，HQT含药血清对肝脂肪变性具有预防作用，其潜在机制可能是激活AMPK和PPARα途径。
