解毒化痰法干预神经酰胺和iNOS诱导非酒精性脂肪性肝病脂凋亡信号通路的研究

Based on toxicity of fat fat apoptosis caused nonalcoholic fatty liver disease (NAFLD) point of view and amide (ceramide) and nitric oxide (NO) plays an important role, this study based on Chinese medicine "balm", "phlegm turbid" and "fat" theory, combined with early and establish the basis of the work "jiedu phlegm method" intervention ceramide and iNOS NAFLD occurs halloysite toxicity induced apoptosis signaling pathways of possible working hypothesis, with NAFLD rat model and cell model as the research object, using thin layer chromatography detection of NAFLD rat liver tissue, effective thin layer chromatography (TLC) analysis (HPTCL) determination of NAFLD ceramide content in cells, rt-pcr, Western blot detection of NAFLD rat liver tissue and cytoplasm NF - KB, iNOS, BCL - 2, bax mRNA and protein expression, electron microscope observation of organelles and lipid drops, detection and lipid metabolism related triglycerides, free fatty acids and other indicators, in order to reveal the toxin and eliminating phlegm as the therapeutic principle of traditional Chinese medicine preparation "arrowhead disappear fat pill" prevention and control of the mechanism of action of NAFLD, to reduce fat liver lesion and the clinical application of promotion to provide theoretical support.

基于脂毒性脂凋亡引起非酒精性脂肪性肝病(NAFLD)的观点及神经酰胺（ceramide）和一氧化氮（NO）所起的重要作用，本研究依据中医"痰浊/脂毒"理论，结合前期工作基础建立"解毒化痰法" 干预神经酰胺和iNOS诱导NAFLD发生脂毒性脂凋亡信号通路防治NAFLD工作假说，以NAFLD大鼠模型和细胞模型为研究对象，采用薄膜层析法检测NAFLD大鼠肝组织、高效薄层色谱法（HPTCL）测定分析NAFLD细胞中神经酰胺含量，RT-PCR、Western blot检测NAFLD大鼠肝组织与胞浆NF-KB、iNOS、bcl-2、bax mRNA和蛋白表达，电镜观察细胞器和脂滴，检测与脂代谢有关的甘油三酯、游离脂肪酸等指标，以期揭示以解毒化痰法为治则的中药制剂"慈菇消脂丸"防治NAFLD的作用机制，为减少肝脏脂肪病变及临床应用推广提供理论支撑。

本项目研究基于脂毒性脂凋亡引起非酒精性脂肪性肝病(NAFLD)的观点及神经酰胺（ceramide）和一氧化氮（NO）所起的重要作用，本研究依据中医“痰浊/脂毒”理论，结合前期工作基础建立“解毒化痰法”干预神经酰胺和iNOS诱导NAFLD发生脂毒性脂凋亡信号通路防治NAFLD工作假说，以NAFLD大鼠模型和细胞模型为研究对象，采用薄膜层析法检测NAFLD大鼠肝组织、高效薄层色谱法（HPTCL）测定分析NAFLD细胞中神经酰胺含量，RT-PCR、Western blot检测NAFLD大鼠肝组织与胞浆NF-KB、iNOS、bcl-2、bax mRNA和蛋白表达，电镜观察细胞器和脂滴，检测与脂代谢有关的甘油三酯、游离脂肪酸等指标，项目研究始终按照计划开展工作，完成了全部研究任务，实现了预期研究成果。本项目研究已经取得一定影响的成果，项目研究成果在国内期刊共发表学术论文 7 篇，其中 CSCD 目录期刊发表论文 2 篇，省级期刊 5 篇。著作1部。参加国家级学术会议8次。慈菇消脂原始处方申请国家专利1项（实审阶段待答辩）。项目实施中培养硕士研究生4名。.本研究运用解毒化痰为治法的慈菇消脂丸以化痰解毒、消降浊脂，从整体—细胞—分子水平揭示中医药干预神经酰胺、iNOS在NAFLD组织中介导细胞凋亡信号通路，通过对慈菇消脂丸干预神经酰胺和iNOS诱导NAFLD发生脂毒性脂凋亡信号通路的基础研究，为进一步揭示以解毒化痰法为指导的慈菇消脂丸治疗NAFLD的作用机制，充分发挥中医药的优势和特色，为寻找确切的中医药复方治疗NAFLD的作用药物靶点和机制提供理论依据，亦为应用中医药防治NAFLD提供有效方法，阐明以解毒化痰法为治则的中药制剂“慈菇消脂丸”防治NAFLD的作用机制的科学意义，为减少肝脏脂肪病变及临床应用推广提供理论支撑。