Abstract：
BACKGROUND: Pueraria lobata is essential medicinal and edible homologous plants widely cultivated in Asian countries. Therefore, P. lobata is widely used in the food, health products and pharmaceutical industries and have significant domestic and international market potential and research value. P. lobata has remarkable biological activities in protecting liver, relieving alcoholism, antioxidation, anti-tumor and anti-inflammation in clinic. However, the potential mechanism of ethyl acetate extract of Pueraria lobata after 70% alcohol extraction (APL) ameliorating nonalcoholic fatty liver disease (NAFLD) has not been clarified.
OBJECTIVE: This study aimed to investigate the ameliorative effect of P. lobata extract on human hepatoma cells and injury in rats, and to evaluate its therapeutic potential for ameliorating NAFLD.
METHODS: Firstly, the effective part of P. lobata extract was determined as APL by measuring its total substances and antioxidant activity. And then the in vitro and in vivo models of NAFLD were adopted., HepG2 cells were incubated with palmitic acid (PA) and hydrogen peroxide (H2O2). In order to evaluate the effect of APL, Simvastatin and Vitamin C (VC) were used as positive control. Various parameters related to lipogenesis and fatty acid β-oxidation were studied, such as intracellular lipid accumulation, reactive oxygen species (ROS), Western Blot, mitochondrial membrane potential, apoptosis, and the mechanism of APL improving NAFLD. The chemical components of APL were further determined by HPLC and UPLC-MS, and molecular docking was carried out with Keap1/Nrf2/HO-1 pathway related proteins.
RESULTS: APL significantly reduced lipid accumulation and levels of oxidative stress-related factors in vitro and in vivo. Immunohistochemical、Western Blot and PCR analysis showed that the expressions of Nrf2 and HO-1 were up-regulated in APL treatment. The Nrf2 inhibitor ML385 can block the rescue by APL of cellular oxidative stress and lipid accumulation induced by H2O2 and PA, demonstrating its dependence on Nrf2. UPLC/MS analysis showed that there were 3\'-hydroxyl puerarin, puerarin, 3\'-methoxy puerarin, daidzein, genistin, ononin, daidzin and genistein.
CONCLUSIONS: This study further clarified the mechanism of P. lobata extract in improving NAFLD, which provided a scientific basis for developing new drugs to protect liver injury and laid a solid foundation for developing P. lobata Chinese herbal medicine resources.
OBJECTIVE: This study aimed to investigate the ameliorative effect of P. lobata extract on human hepatoma cells and injury in rats, and to evaluate its therapeutic potential for ameliorating NAFLD.
METHODS: Firstly, the effective part of P. lobata extract was determined as APL by measuring its total substances and antioxidant activity. And then the in vitro and in vivo models of NAFLD were adopted., HepG2 cells were incubated with palmitic acid (PA) and hydrogen peroxide (H2O2). In order to evaluate the effect of APL, Simvastatin and Vitamin C (VC) were used as positive control. Various parameters related to lipogenesis and fatty acid β-oxidation were studied, such as intracellular lipid accumulation, reactive oxygen species (ROS), Western Blot, mitochondrial membrane potential, apoptosis, and the mechanism of APL improving NAFLD. The chemical components of APL were further determined by HPLC and UPLC-MS, and molecular docking was carried out with Keap1/Nrf2/HO-1 pathway related proteins.
RESULTS: APL significantly reduced lipid accumulation and levels of oxidative stress-related factors in vitro and in vivo. Immunohistochemical、Western Blot and PCR analysis showed that the expressions of Nrf2 and HO-1 were up-regulated in APL treatment. The Nrf2 inhibitor ML385 can block the rescue by APL of cellular oxidative stress and lipid accumulation induced by H2O2 and PA, demonstrating its dependence on Nrf2. UPLC/MS analysis showed that there were 3\'-hydroxyl puerarin, puerarin, 3\'-methoxy puerarin, daidzein, genistin, ononin, daidzin and genistein.
CONCLUSIONS: This study further clarified the mechanism of P. lobata extract in improving NAFLD, which provided a scientific basis for developing new drugs to protect liver injury and laid a solid foundation for developing P. lobata Chinese herbal medicine resources.
摘要：
背景：葛根是亚洲国家广泛种植的重要药用和食用同源植物。因此,P.lobata被广泛用于食品中，保健品和医药行业具有重大的国内外市场潜力和研究价值。假单胞菌在保护肝脏方面具有显著的生物学活性，缓解酒精中毒,抗氧化，在临床上具有抗肿瘤和抗炎作用。然而,葛根乙酸乙酯提取物经70%乙醇提取(APL)后改善非酒精性脂肪性肝病(NAFLD)的潜在机制尚未阐明。
目的：本研究旨在探讨叶枯草提取物对大鼠肝癌细胞及损伤的改善作用。并评估其改善NAFLD的治疗潜力。
方法：首先，通过测定其总物质和抗氧化活性，确定了百叶草提取物的有效部分为APL。然后采用NAFLD的体外和体内模型。,用棕榈酸(PA)和过氧化氢(H2O2)孵育HepG2细胞。为了评估APL的效果，辛伐他汀和维生素C(VC)用作阳性对照。研究了与脂肪生成和脂肪酸β-氧化有关的各种参数，例如细胞内脂质积累，活性氧（ROS），西方印迹，线粒体膜电位，凋亡，以及APL改善NAFLD的机制。通过HPLC和UPLC-MS进一步测定APL的化学成分,并与Keap1/Nrf2/HO-1通路相关蛋白进行分子对接。
结果：APL在体外和体内均显着降低了脂质积累和氧化应激相关因子的水平。免疫组织化学、WesternBlot和PCR分析显示，Nrf2和HO-1的表达在APL治疗中上调。Nrf2抑制剂ML385可以阻断APL对H2O2和PA诱导的细胞氧化应激和脂质积累的拯救,证明了它对Nrf2的依赖性。UPLC/MS分析表明，有3'-羟基葛根素，葛根素,3\'-甲氧基葛根素，Daidzein,Genistin,ononin,Daidzin和Genistein.
结论：本研究进一步阐明了叶枯草提取物改善NAFLD的机制，为开发保护肝损伤的新药提供了科学依据，为开发中药资源奠定了坚实的基础。
目的：本研究旨在探讨叶枯草提取物对大鼠肝癌细胞及损伤的改善作用。并评估其改善NAFLD的治疗潜力。
方法：首先，通过测定其总物质和抗氧化活性，确定了百叶草提取物的有效部分为APL。然后采用NAFLD的体外和体内模型。,用棕榈酸(PA)和过氧化氢(H2O2)孵育HepG2细胞。为了评估APL的效果，辛伐他汀和维生素C(VC)用作阳性对照。研究了与脂肪生成和脂肪酸β-氧化有关的各种参数，例如细胞内脂质积累，活性氧（ROS），西方印迹，线粒体膜电位，凋亡，以及APL改善NAFLD的机制。通过HPLC和UPLC-MS进一步测定APL的化学成分,并与Keap1/Nrf2/HO-1通路相关蛋白进行分子对接。
结果：APL在体外和体内均显着降低了脂质积累和氧化应激相关因子的水平。免疫组织化学、WesternBlot和PCR分析显示，Nrf2和HO-1的表达在APL治疗中上调。Nrf2抑制剂ML385可以阻断APL对H2O2和PA诱导的细胞氧化应激和脂质积累的拯救,证明了它对Nrf2的依赖性。UPLC/MS分析表明，有3'-羟基葛根素，葛根素,3\'-甲氧基葛根素，Daidzein,Genistin,ononin,Daidzin和Genistein.
结论：本研究进一步阐明了叶枯草提取物改善NAFLD的机制，为开发保护肝损伤的新药提供了科学依据，为开发中药资源奠定了坚实的基础。
