大萼金丝桃素B抑制mTORC1信号通路改善非酒精性脂肪肝炎的作用和机制研究

Hypercalin B alleviates nonalcoholic steatohepatitis progression via suppressing mTORC1 signaling pathway

非酒精性脂肪肝炎(nonalcoholic steatohepatitis,NASH)全球发病率持续攀升,其发病机制复杂,临床上尚无有效治疗手段。前期研究成果显示,RNA解旋酶DDX蛋白5(DEAD box protein 5,DDX5)可显著缓解小鼠NASH进程,本研究对课题组天然产物库进行筛选,发现中药黄花香(Hypericum beanii N.Robson)活性成分大萼金丝桃素B(hypercalin B,HB)可剂量依赖性上调DDX5蛋白表达量。本实验通过构建棕榈酸(palmitic acid,PA)刺激的NASH体外模型以及胆碱−蛋氨酸缺乏饮食(methionine-and choline-deficient diet,MCD)诱导的NASH动物模型,采用不同浓度的HB处理,考察HB缓解NASH进程的效果及作用机制。本文中所有动物实验都获得中国药科大学伦理学委员会批准(批准号:2021-02-003)。体外模型结果显示,HB可显著减少游离脂肪酸(free fatty acid,FFA)诱导的细胞内脂质沉积。动物实验结果显示,HB可显著减少NASH小鼠肝脏中脂质沉积;降低血清谷草转氨酶(aspartate transaminase,AST)和谷丙转氨酶(alanine transaminase,ALT)水平,改善肝损伤;减少肝脏促炎因子白介素6(interleukin 6,IL-6)、白介素1β(interleukin 1β,IL-1β)和肿瘤坏死因子α(tumor necrosis factorα,TNFα)等mRNA表达量,抑制肝脏炎症激活。进一步研究表明,HB可减少雷帕霉素靶蛋白(mechanistic target of rapamycin,mTOR)磷酸化水平,降低脂肪酸合成关键蛋白固醇调节元件结合蛋白1(sterol regulatory element-binding protein 1,SREBP1)和脂肪酸合成酶(fatty acid synthase,FASN)表达量,从而改善脂代谢,缓解NASH进程,且HB该作用依赖于DDX5。综上,HB通过上调DDX5蛋白抑制雷帕霉素靶蛋白复合物1(mechanistic target of rapamycin complex 1,mTORC1)信号通路激活,改善脂代谢紊乱、抑制炎症,在体内外表现出良好的抗NASH活性。

The global incidence rate of nonalcoholic steatohepatitis(NASH)continues to rise.The pathogenesis of NASH is complex,and there is no effective clinical treatment.Previous study has shown that DEAD box protein 5(DDX5)can significantly alleviate the NASH process in mice.This study screened the natural product library of the research group and found that the active compound hypercalin B(HB)in Hypericum beanii N.Robson,a traditional Chinese medicine,can upregulate the expression of DDX5 protein in a dose-dependent manner.In this study,an in vitro model of NASH stimulated by palmitic acid(PA)and an animal model of NASH induced by the methionine-and choline-deficient diet(MCD)were constructed.Different concentrations of HB were used to investigate the effect and mechanism of HB in alleviating NASH progression.All animal experiments in this paper were approved by the Ethics Committee of China Pharmaceutical University(NO:2021-02-003).In vitro model results showed that HB significantly reduced the intracellular lipid deposition induced by free fatty acid(FFA).Animal experiments showed that HB improved liver injury by significantly reducing lipid accumulation in the liver of NASH mice,and reducing serum aspartate transaminase(AST)and alanine transaminase(ALT)levels.Moreover,HB could inhibit liver inflammation by reducing the mRNA levels of liver pro-inflammatory cytokines including interleukin 6(IL-6),interleukin 1β(IL-1β),and tumor necrosis factorα(TNFα).Further research showed that HB could reduce the phosphorylation level of the mechanical target of rapamycin(mTOR)and reduce the expression of sterol regulatory element binding protein 1(SREBP1)and fatty acid synthase(FASN),thereby improving lipid metabolism and alleviating NASH progression,and the effects of HB against NASH were dependent on DDX5.In conclusion,HB can improve lipid metabolism and inhibit inflammatory activation by suppressing mTORC1 pathway via upregulating DDX5 protein,and showed promising anti-NASH activity in vitro and in vivo.