JinJin Yin1, YanQin Luo2, HouLiang Deng1, ShuMin Qin3, WaiJiao Tang1, Lu Zeng1, BenJie Zhou4. 1. Center for Drug Research and Development, Zhujiang Hospital, Southern Medical University, Guangdong, Guangzhou 510282, PR China. 2. Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China. 3. Institute of Digestive Disease, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, PR China. 4. Center for Drug Research and Development, Zhujiang Hospital, Southern Medical University, Guangdong, Guangzhou 510282, PR China. Electronic address: zhoubj163@163.com.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Hugan Qingzhi tablet (HQT), a lipid- lowering traditional Chinese medicine formula, has been used for the prevention and treatment of nonalcoholic fatty liver (NAFLD). AIM OF THE STUDY: 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. MATERIALS AND METHODS: 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). RESULTS: 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. CONCLUSION: 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.
ETHNOPHARMACOLOGICAL RELEVANCE: Hugan Qingzhi tablet (HQT), a lipid- lowering traditional Chinese medicine formula, has been used for the prevention and treatment of nonalcoholic fatty liver (NAFLD). AIM OF THE STUDY: 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. MATERIALS AND METHODS: 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). RESULTS: 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. CONCLUSION: 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.