小黑药亲电成分干预混合游离脂肪酸诱导的人肝癌细胞株HepG2脂肪变性研究

采用GSH功能化磁珠靶向敲出小黑药亲电成分并用LC-MS表征,基于游离脂肪酸(FFA)诱导的HepG2细胞脂肪变性模型,评价小黑药亲电成分降低肝细胞脂质累积和氧化应激的作用和机制。结果表明:小黑药亲电成分主要集中在乙酸乙酯部位;在0.5~2.0mg/mL浓度下,小黑药石油醚部位、乙酸乙酯部位、水部位均具有降低脂肪变性细胞脂质累积和ROS生成的活性,其中乙酸乙酯部位在各浓度下效果最好;乙酸乙酯部位在各浓度下具有降低细胞内TG、TC水平和提高细胞内抗氧化酶活性的作用;经过GSH功能化磁珠靶向敲出亲电化合物后,乙酸乙酯部位在2.0μg/mL浓度下对细胞内TG水平的降低和细胞内抗氧化酶水平的提高效果显著减弱。乙酸乙酯部位萃取物上调Nrf2及下游NQO1、HO-1、GCLC基因,下调脂质合成基因SREBP1c、ACC1、FAS的表达,促进脂质分解基因PPARα、CPT1A的表达;而靶向敲出亲电成分后,乙酸乙酯部位萃取物调控脂代谢和抗氧化相关基因的作用明显下降。LC-MS表征亲电成分敲出前后乙酸乙酯部位样品,发现了7个变化的主要质谱峰,推测其为主要的亲电化合物。小黑药中亲电化合物可以改善脂肪酸诱导的脂肪变性,其机制主要与抗氧化和脂代谢相关基因的调节有关。

Study on intervention effects of electrophilic components from Xiaoheiyao against free fatty acid-induced steatosis in HepG2 cells

The electrophilic components in Xiaoheiyao were knocked out by GSH functionalized magnetic nanoparticles(GSH-MNPs)and characterized by LC-MS analysis,and the effect and mechanism of the electrophilic components in Xiaoheiyao for reducing lipid accumulation and oxidative stress in hepatocytes were evaluated,based on free fatty acid(FFA)-induced HepG2 cells.The ethyl acetate fraction was determined as the most electrophilic components-enriched part of Xiaoheiyao.At the concentration of 0.5~2.0μg/mL,the petroleum ether,ethyl acetate and water fractions of Xiaoheiyao exhibited the activities of decreasing hepatic lipid accumulation and ROS production,in which the ethyl acetate fraction showed the best activities.The ethyl acetate fraction of Xiaoheiyao decreased the intracellular levels of TG and TC while increased the activities of the antioxidant enzymes.After electrophilic components in ethyl acetate fraction were knocked out by GSH-MNPs,the activities for the decreasing TG and TC levels and increasing the activities of antioxidant enzymes were significantly compromised.After treated with ethyl acetate fraction,the expression of Nrf 2 and its downstream genes NQO 1,HO-1 and GCLC were upregulated.Moreover,the expression of genes for lipid synthesis,including SREBP 1 c,ACC 1,and FAS,were found downregulated,while the expression genes PPARαand CPT 1 A,involved in lipidolysis,were upregulated.After the electrophilic components were knocked out,the effects of the ethyl acetate fraction on regulating lipid metabolism and redox-related genes were significantly reduced.Seven mass features were found by LC-MS and concluded as electrophilic components when comparing the ethyl acetate fractions before and after treated with GSH-MNPs.The mechanism of electrophilic components in Xiaoheiyao improving the hepatic steatosis induced by FFA was associated with the regulation of the genes related to lipid metabolism and redox.