甲基阿魏酸通过microRNA-378b介导CaMKK2-AMPK通路改善乙醇诱导的L02细胞脂肪变性  

Methyl ferulic acid ameliorates ethanol-induced L02 cell steatosis through microRNA-378b-mediated CaMKK2-AMPK pathway

作　　者：黄萍 陈杏 蒙荣华 卢君 张言 李丽[3] 李勇文[1,2] HUANG Ping;CHEN Xing;MENG Rong-hua;LU Jun;ZHANG Yan;LI Li;LI Yong-wen(School of Pharmacy,Guilin Medical University,Guilin 541199,China;Guangxi Key Laboratory of Diabetic Systems Medicine,Guilin 541199,China;School of Basic Medicine,Guilin Medical University,Guilin 541199,China)

机构地区：[1]桂林医学院药学院,广西桂林541199 [2]广西糖尿病系统医学研究中心重点实验室,广西桂林541199 [3]桂林医学院基础医学院,广西桂林541199

出　　处：《中国中药杂志》2023年第1期193-201,共9页China Journal of Chinese Materia Medica

基　　金：广西壮族自治区重点研究课题(2018GXNSFDA281012);国家自然科学基金项目(82060673)。

摘　　要：酒精性肝病(alcoholic liver disease, ALD),随着其发病率和病死率的不断上升,已严重且广泛地影响着全世界人民的健康。甲基阿魏酸(methyl ferulic acid, MFA)在前期已被证实可通过腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)信号显著抑制乙醇诱导的L02细胞内脂质生成,但深层作用机制尚不清楚。该研究旨在现有基础上,进一步阐明MFA可通过microRNA-378b(miR-378b)介导钙/钙调蛋白依赖性蛋白激酶激酶2(calcium/calmodulin-dependent protein kinase kinase 2,CaMKK2)-AMPK信号通路改善乙醇诱导的L02细胞脂质积累的机制。通过100 mmol·L^(-1)乙醇诱导L02细胞48 h建立体外ALD模型,并给予不同浓度(100、50、25μmol·L^(-1))的MFA处理。采用电穿孔法将miR-378b质粒(含过表达质粒-miR-378b mimics、沉默质粒-miR-378b inhibitor及各自的阴性对照-miR-378b NCs)转染进L02肝细胞以上调或下调细胞中miR-378b水平。采用市售诊断试剂盒和全自动生化分析仪检测细胞中胆固醇(total cholesterol, TC)、甘油三酯(triglyceride, TG)水平。采用定量实时聚合酶链反应(quantitative reverse transcription-polymerase chain reaction, qRT-PCR)检测L02细胞中miR-378b的表达水平,采用PCR检测CaMKK2 mRNA水平,采用Western blot检测脂质代谢过程中参与脂质合成、分解和转运的相关因子的蛋白表达。结果显示乙醇能显著诱导细胞内TG、TC水平的上升,而MFA则能显著降低TG、TC水平。乙醇使miR-378b的水平急剧上调,而MFA有效地抑制了miR-378b水平。miR-378b过表达刺激了乙醇诱导的L02细胞中的脂质积累,miR-378b沉默改善了乙醇诱导的脂质沉积,而MFA通过降低miR-378b来激活CaMKK2-AMPK信号通路,调节脂质合成、分解与转运,从而改善L02细胞内脂代谢紊乱。实验结果表明,MFA通过miR-378b调控CaMKK2-AMPK通路改善L02细胞内脂质沉积。Alcoholic liver disease(ALD), with its increasing morbidity and mortality, has seriously and extensively affected the health of people worldwide. Methyl ferulic acid(MFA) has been proven to significantly inhibit alcohol-induced lipid production in L02 cells through the AMP-activated protein kinase(AMPK) pathway, but its in-depth mechanism remains unclear. This study aimed to further clarify the mechanism of MFA in improving lipid accumulation in L02 cells through the microRNA-378b(miR-378b)-mediated calcium/calmodulin-dependent protein kinase kinase 2(CaMKK2)-AMPK signaling pathway based on existing researches. L02 cells were induced by 100 mmol·L^(-1)ethanol for 48 h to establish the model of ALD in vitro, and 100, 50, and 25 μmol·L^(-1)concentration of MFA was treated. MiR-378b plasmids(containing the overexpression plasmid-miR-378b mimics, silence plasmid-miR-378b inhibitor, and their respective negative control-miR-378b NCs) were transfected into L02 cells by electroporation to up-regulate or down-regulate the levels of miR-378b in L02 cells. The levels of total cholesterol(TC) and triglyceride(TG) in cells were detected by commercial diagnostic kits and automatic biochemical analyzers. The expression levels of miR-378b in L02 cells were detected by real-time quantitative polymerase chain reaction(qRT-PCR). CaMKK2 mRNA levels were detected by PCR, and protein expressions of related factors involved in lipid synthesis, decomposition, and transport in lipid metabolism were detected by Western blot. The results displayed that ethanol significantly increased TG and TC levels in L02 cells, while MFA decreased TG and TC levels. Ethanol up-regulated the miR-378b level, while MFA effectively inhibited the miR-378b level. The overexpression of miR-378b led to lipid accumulation in ethanol-induced L02 cells, while the silence of miR-378b improved the lipid deposition induced by ethanol. MFA activated the CaMKK2-AMPK signaling pathway by lowering miR-378b, thus improving lipid synthesis, decomposition, and transport,

关 键 词：甲基阿魏酸 miR-378b CaMKK2 肝脂积累 酒精性肝病 

分 类 号：R285[医药卫生—中药学]