基于LKB1-AMPK-TFEB的白术调节溶酶体自噬抗阿尔茨海默病作用机制

Mechanism of Atractylodes macrocephala against Alzheimer′s disease via regulating lysophagy based on LKB1-AMPK-TFEB pathway

淀粉样前体蛋白(amyloid precursor protein, APP)裂解后产生Aβ是阿尔茨海默病(Alzheimer′s disease, AD)发生发展原因之一,该研究基于LKB1-AMPK-TFEB初步探讨白术(Atractylodes macrocephala,AM)降解APP抗AD作用机制。首先检测AM对AD转基因秀丽隐杆线虫CL2241记忆能力的影响,然后将APP质粒体外瞬转至小鼠神经瘤母细胞(N2a)中,设定空白对照组,APP组(模型组),阳性对照组(100μmol·L^(-1)雷帕霉素),AM低、中、高剂量组(100、200、300μg·mL),ELISA法检测细胞培养基中Aβ的分泌量;Western blot检测其对APP的降解作用,荧光显微镜分析APP荧光强度,荧光素酶报告基因法检测TFEB(transcription factor EB)的转录活性,RLuc-LC3wt/RLuc-LC3G120A检测溶酶体自噬活性,mRFP-GFP-LC3检测自噬通量,Western blot检测TFEB、LC3Ⅱ、LC3Ⅰ、LAMP2、Beclin1、LKB1、p-AMPK、p-ACC的蛋白表达量,免疫荧光检测TFEB的表达,RT-PCR法检测TFEB及下游靶基因的表达。结果显示AM可显著降低转基因线虫CL2241的趋化指数,不同浓度的AM降低细胞上清中Aβ的分泌量,AM剂量依赖性降解APP的蛋白表达量及APP的荧光强度,AM处理后TFEB转录活性增强,LC3双标质粒荧光强度均增强,RLuc-LC3wt/RLuc-LC3G120A的值也显著降低,不同浓度的AM均可以促进TFEB、LAMP2及Beclin1的蛋白水平,剂量依赖性的升高LC3Ⅱ/LC3Ⅰ的蛋白表达量比值,免疫荧光结果显示,AM促进TFEB的荧光强度及入核表达,RT-PCR结果显示各浓度的AM可提高APP转染的N2a细胞中TFEB的mRNA水平,剂量依赖性地促进LAMP2的转录水平,且高浓度的AM还可以显著提高LC3、P62的mRNA水平,不同浓度的AM升高LKB1、p-AMPK及p-ACC的蛋白水平。综上所述,白术调节自噬降解APP的机制与激活LKB1-AMPK-TFEB通路有关。

Myloid beta(Aβ) is produced by cleavage of amyloid precursor protein(APP), which is a main reason for Alzheimer′s disease(AD) occurrence and development. This study preliminarily investigated the mechanism of Atractylodes macrocephala(AM) against AD based on LKB1-AMPK-TFEB pathway. The effect of AM on memory ability of AD transgenic Caenorhabditis elegans CL2241 was detected, and then the APP plasmid was transiently transferred to mouse neuroblastoma(N2 a) cells in vitro. The mice were divided into the blank control group, APP group(model group), positive control group(100 μmol·L^(-1)rapamycin), and AM low-, medium-and high-dose groups(100, 200 and 300 μg·mL). The content of Aβin cell medium, the protein level of APP, the fluorescence intensity of APP, the transcriptional activity of transcription factor EB(TFEB), the activity of lysosomes in autophagy, and autophagy flux were determined by enzyme-linked immunosorbent assay(ELISA), Western blot, fluorescence microscope, luciferase reporter gene assay, RLuc-LC3 wt/RLuc-LC3 G120 A, and mRFP-GFP-LC3, respectively. The protein expression of TFEB, LC3Ⅱ, LC3Ⅰ, LAMP2, Beclin1, LKB1, p-AMPK and p-ACC was detected by Western blot. Immunofluorescence and reverse transcription-polymerase chain reaction(RT-PCR) were used to detect the fluorescence intensity of TFEB and the mRNA expression of TFEB and downstream target genes, respectively. The results showed that AM reduced the chemotactic index of transgenic C. elegans CL2241, and decreased the content of Aβ in the supernatant of cell culture medium at different concentrations. In addition, AM lowered the protein level of APP and the fluorescence intensity of APP in a dose-dependent manner. Transcriptional activity of TFEB and fluorescence intensity of mRFP-GFP-LC3 plasmid were enhanced after AM treatment, and the value of RLuc-LC3 wt/RLuc-LC3 G120 A was reduced. AM promoted the protein levels of TFEB, LAMP2 and Beclin1 at different concentrations, and increased the protein expression ratio of LC3Ⅱ/LC3Ⅰ in a dose-dependent manner. Immunofluorescence results revealed that AM improved the fluorescence intensity and nuclear expression of TFEB, and RT-PCR results indicated that AM of various concentrations elevated the mRNA expression of TFEB in APP transfected N2 a cells and promoted the transcription level of LAMP2 in a dose-dependent manner, and high-concentration AM also increased the mRNA levels of LC3 and P62. The protein levels of LKB1, p-AMPK and p-ACC were elevated by AM of different concentrations. In summary, AM regulating lysophagy and degrading APP are related to the activation of LKB1-AMPK-TFEB pathway.