木犀草苷靶向结合PPARα改善小鼠代谢功能障碍相关脂肪性肝病

Luteolin-7-O-glucoside alleviates metabolic-associated fatty liver disease in mice through targeting peroxisome proliferator-activated receptor α

摘要:

目的探讨木犀草苷(luteolin-7-O-glucoside, LUT-7G)改善代谢功能障碍相关脂肪性肝病(metabolic dysfunction-associated fatty liver disease, MAFLD)的作用机制。 方法通过连续喂养小鼠高脂饮食(high fat diet, HFD)12周诱导小鼠MAFLD模型，后四周灌胃给予小鼠LUT-7G(30、60mg/kg)干预，给药结束后检测小鼠血清中谷丙转氨酶(alanine aminotransferase, ALT)及谷草转氨酶(aspartate aminotransferase, AST)酶活力变化、血清及肝脏中甘油三酯(triglyceride, TG)、游离脂肪酸(non-esterified fatty acids, NEFA)的含量变化，通过肝脏组织HE染色、油红O染色、F4/80及Ly6G免疫组化实验观察小鼠肝脏病理变化，通过荧光定量反转录PCR(real-time quantitative reverse transcription PCR, RT-PCR)评价肝脏中炎症因子IL-6及IL-1b的基因水平变化，评价木犀草苷给药对HFD小鼠肝脏损伤、脂质堆积及炎性浸润的改善作用。采用Western印迹法检测木犀草苷体内及NEFA(油酸∶棕榈酸=2∶1)诱导HepaRG细胞体外脂质堆积模型中给药干预对过氧化物酶体增殖物激活受体α(peroxisome proliferator-activated receptor α, PPARα)蛋白核转位的影响，Western印迹法及RT-PCR检测PPARα下游参与脂肪酸氧化因子的基因水平及肉碱棕榈酰转移酶1A(carnitine palmitoyltransferase 1A, CPT1A)的蛋白水平的变化；分子对接、微量热泳动(microscale thermophoresis, MST)以及细胞热位移实验(cellular thermal shift assay, CETSA)研究木犀草苷与PPARα的相互作用；体外通过BODIPY荧光染色评价GW6471(PPARα抑制剂)对木犀草苷降脂作用的影响。 结果LUT-7G(30、60mg/kg)能够降低MAFLD小鼠NAFLD活动度评分(NAFLD activity score, NAS)(P<0.05)，降低血清、肝脏中TG及NEFA含量(P<0.05，P<0.01，P<0.001)，减少肝脏中巨噬细胞及中性粒细胞浸润，降低炎症因子IL-1b及IL-6的基因表达(P<0.05)。木犀草苷能够在体内外激活PPARα信号通路，促进其核转位，增强下游参与脂肪酸氧化相关因子的基因表达及CPT1A的蛋白表达(P<0.05，P<0.01，P<0.001)。分子对接、MST及CETSA结果证实LUT-7G与PPARα存在直接结合(P<0.05)。BODIPY荧光染色结果显示LUT-7G的体外降脂作用被PPARα抑制剂GW6471逆转。 结论LUT-7G通过与PPARα直接结合，激活肝脏PPARα信号通路，发挥改善HFD诱导小鼠MAFLD的作用。

Abstract:

ObjectiveTo Explore the mechanism of luteolin-7-O-glucoside(LUT-7G) in alleviating metabolic dysfunction-associated fatty liver disease(MAFLD). MethodsThe C57BL/6J Mice were fed with a high-fat diet(HFD) for 12 weeks to induce the MAFLD model, and for the last four weeks, oral gavage treatment with LUT-7G(30, 60 mg/kg) was applied. The activity of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) in the serum of mice, the content of triglyceride(TG) and non-esterified fatty acids(NEFA) in the liver of mice were detected; and the staining of hematoxylin and eosin(HE), Oil Red O, F4/80 and Ly6G of liver were examined for pathological changes in the liver of the mice; the real-time quantitative reverse transcription PCR(RT-PCR) was used to detect change of mRNA levels of interleukin(IL) 6 and IL-1b; all above methods were performed to assess the alleviating efficacy of LUT-7G on liver injury, lipid accumulation, and inflammatory infiltration in HFD-fed mice. Western blotting was used to investigate the effect of luteoloside administration on the nuclear translocation of peroxisome proliferator-activated receptor α(PPARα) protein in both an in vivo model and an in vitro model of lipid accumulation induced by NEFA(oleic acid∶palmitic acid=2∶1) in HepaRG cells. Additionally, Western blotting and RT-PCR were employed to examine changes in the expression levels of genes encoding PPARα downstream factors involved in fatty acid oxidation, as well as the protein level of carnitine palmitoyltransferase 1A(CPT1A). Molecular docking, microscale thermophoresis(MST), and cellular thermal shift assay(CETSA) were used to detect the interactions of LUT-7G and PPARɑ. BODIPY fluorescence staining was used to assess the effect of GW6471 on the lipid-lowering properties of LUT-7G in vitro. ResultsLUT-7G(30,60mg/kg) reducted NAFLD activity score(NAS)(P<0.05), the TG and NEFA content in liver and serum(P<0.05, P<0.01, P<0.001), and hepatic inflammatory cell infiltration, the mRNA levels of IL-6 and IL-1b in HFD mice(P<0.05). Mechanistically, LUT-7G promoted PPARα nuclear translocation and upregulated fatty oxidation-related genes and CPT1A protein expression to increase fatty metabolism in vivo and in vitro(P<0.05, P<0.01, P<0.001); furthermore, LUT-7G directly binds PPARα to activate it(P<0.05), and the lipid-lowering effect of LUT-7G in vitro was reversed by GW6471, an inhibitor of PPARα. ConclusionLUT-7G ameliorates HFD-induced MAFLD in mice by activating the hepatic PPARα signaling pathway through direct binding to PPARα.