基于PPARα/TFEB的川续断干预阿尔茨海默病秀丽隐杆线虫模型作用机制  

Dipsacus asper Treats Alzheimer's Disease in Caenorhabditis elegans by Regulating PPARα/TFEB Pathway

作　　者：王萌萌[1] 赵建平 吴丽敏 初双 黄艳丽 崔正浩 孙意冉 王潘 王辉[1] 张振强[1] 谢治深 WANG Mengmeng;ZHAO Jianping;WU Limin;CHU Shuang;HUANG Yanli;CUI Zhenghao;SUN Yiran;WANG Pan;WANG Hui;ZHANG Zhenqiang;XIE Zhishen(Academy of Chinese Medicine Sciences,Henan University of Chinese Medicine,Zhengzhou 450046,China;School of Pharmacy,Henan University of Chinese Medicine,Zhengzhou 450046,China)

机构地区：[1]河南中医药大学中医药科学院,郑州450046 [2]河南中医药大学药学院,郑州450046

出　　处：《中国实验方剂学杂志》2025年第5期104-114,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82274612);河南省高等学校青年骨干教师资助计划项目(2021GGJS081);河南省高校科技创新人才支持计划项目(22HASTIT048);河南省中医药科学研究专项重大专项(2022ZYZD21)。

摘　　要：目的:利用转基因秀丽线虫模式生物探讨川续断抗阿尔茨海默病(AD)作用,并基于过氧化物酶体增殖物激活受体α(PPARα)/转录因子EB(TFEB)通路探讨其可能作用机制。方法:将转基因AD秀丽隐杆线虫分为空白组、模型组、阳性药WY14643(20μmol·L^(-1))组及川续断低、中、高剂量组(100、200、400 mg·L^(-1)),检测肌肉细胞内形成寡聚态β淀粉样蛋白42(Aβ42)并产生瘫痪现情况、线虫头部Aβ沉积的影响。使用BV2细胞模型,利用Rluc-LC3wt/G120A检测对溶酶体自噬的影响;蛋白免疫印迹法(Western blot)检测微管相关蛋白1轻链3(LC3)Ⅰ、LC3Ⅱ、溶酶体相关膜蛋白2(LAMP2)、TFEB蛋白表达量;实时荧光定量聚合酶链式反应(Real-time PCR)检测对PPARα/TFEB下游自噬相关基因自噬效应蛋白1(Beclin1)、自噬相关基因5(Atg5)及溶酶体相关基因LAMP2、自噬相关基因2(CLN2)的影响。报告基因评估PPARα、TFEB转录活性,免疫荧光检测PPARα的荧光强度,使用超高液相色谱-质谱(UPLC-MS)检测川续断醇提物的活性成分,利用RCSB PDB和中药系统药理数据库和分析平台(TCMSP)及Autodock软件进行分子对接,Docking分析川续断成分和PPARα配体调节结构域的结合。结果:与CL4176模型组比较,WY14643组和200、400 mg·L^(-1)川续断组可以明显延长秀丽线虫的瘫痪时间(P<0.05);各给药组均可以显著减少秀丽线虫头部Aβ沉积(P<0.01)。MTT结果显示,50~500 mg·L^(-1)浓度范围内川续断不影响BV2细胞活力;同时川续断还可以增强自噬活性(P<0.05),明显提高Beclin1、Atg5、LAMP2、CLN2 mRNA的表达(P<0.05,P<0.01),明显促进TFEB核易位(P<0.05),明显提高LAMP2蛋白表达量和自噬通量(P<0.05,P<0.01),并显著增强PPARα和TFEB转录活性(P<0.01),免疫荧光结果显示WY14643组和200、400 mg·L^(-1)川续断组可以显著增强PPARα在BV2细胞核的荧光强度(P<0.01)。UPLCMS检测出川续断的9个已知化合物,筛选出8个川续断活性成分,�Objective:To investigate the anti-Alzheimer's disease(AD)effect of Dipsacus asper(DA)in the Caenorhabditis elegans model,and decipher the underlying mechanism via the peroxisome proliferator-activated receptorα(PPARα)/transcription factor EB(TFEB)pathway.Methods:First,transgenic AD C.elegans individuals were assigned into the blank control,model,positive control(WY14643,20μmol·L^(-1)),and low-,medium-,and high-dose(100,200,and 400 mg·L^(-1),respectively)DA groups.The amyloidβ-42(Aβ42)formation in the muscle cells,the paralysis time,and the deposition of amyloidβ-protein(Aβ)in the head were detected.The lysosomal autophagy in the BV2 cell model was examined by Rluc-LC3wt/G120A.The expression levels of lysosomal autophagy-related proteins LC3Ⅱ,LC3I,LAMP2,and TFEB were detected by Western blot.Real-time quantitative polymerase chain reaction(Real-time PCR)was employed to determine the mRNA levels of autophagy-related genes beclin1 and Atg5 and lysosome-related genes LAMP2 and CLN2 downstream of PPARα/TFEB.A reporter gene assay was used to detect the transcriptional activities of PPARαand TFEB.Immunofluorescence was used to detect the fluorescence intensity of PPARα,and the active components of the ethanol extract of DA were identified by UPLC-MS.RCSB PDB,Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),and Autodock were used to analyze the binding between the active components and PPARα-ligand-binding domain(LBD).Results:Compared with the model group,the positive control group and 200 and 400 mg·L^(-1) DA groups showed prolonged paralysis time(P<0.05),and all the treatment groups showed decreased Aβdeposition in the head(P<0.01).DA within the concentration range of 50-500 mg·L^(-1) did not affect the viability of BV2 cells.In addition,DA enhanced the autophagy flux(P<0.05),up-regulated the mRNA levels of beclin1,Atg5,LAMP2,and CLN2(P<0.05,P<0.01),promoted the nuclear translocation of TFEB(P<0.05),increased LAMP2 expression and autophagy flux(P<0.05,P<0.01),and

关 键 词：川续断 阿尔茨海默病 秀丽隐杆线虫 过氧化物酶体增殖物激活受体α(PPARα) 转录因子EB(TFEB) 

分 类 号：R285[医药卫生—中药学] R289[医药卫生—中医学] R287