Hederagenin improves Alzheimer's disease through PPARα/TFEB-mediated autophagy.

BACKGROUND: Autophagic flux is coordinated by a network of master regulatory genes, which centered on transcription factor EB (TFEB). The disorders of autophagic flux are closely associated with Alzheimer's disease (AD), and thus restoring autophagic flux to degrade pathogenic proteins has become a hot therapeutic strategy. Hederagenin (HD), a triterpene compound, isolated from a variety food such as Matoa (Pometia pinnata) Fruit, Medicago sativa, Medicago polymorpha L. Previous studies have shown that HD has the neuroprotective effect. However, the effect of HD on AD and underlying mechanisms are unclear. PURPOSE: To determine the effect of HD on AD and whether it promotes autophagy to reduce AD symptoms. STUDY DESIGN: BV2 cells, C. elegans and APP/PS1 transgenic mice were used to explore the alleviative effect of HD on AD and the molecular mechanism in vivo and in vitro. METHODS: The APP/PS1 transgenic mice at 10 months were randomized into 5 groups (n = 10 in each group) and orally administrated with either vehicle (0.5% CMCNa), WY14643 (10 mg/kg/d), low-dose of HD (25 mg/kg/d), high-dose of HD (50 mg/kg/d) or MK-886 (10 mg/kg/d) + HD (50 mg/kg/d) for consecutive 2 months. The behavioral experiments including morris water maze test, object recognition test and Y maze test were performed. The effects of HD on Aß deposition and alleviates Aß pathology in transgenic C. elegans were operated using paralysis assay and fluorescence staining assay. The roles of HD in promoting PPARα/TFEB-dependent autophagy were investigated using the BV2 cells via western blot analysis, real-time quantitative PCR (RT-qPCR), molecular docking, molecular dynamic (MD) simulation, electron microscope assay and immunofluorescence. RESULTS: In this study, we found that HD upregulated mRNA and protein level of TFEB and increased the distribution of TFEB in the nucleus, and the expressions of its target genes. HD also promoted the expressions of LC3BII/LC3BI, LAMP2, etc., and promoted autophagy and the degradation of Aß. HD reduced Aß deposition in the head area of C. elegans and Aß-induced paralysis. HD improved cognitive impairment and pathological changes in APP/PS1 mice by promoting autophagy and activating TFEB. And our results also showed that HD could strongly target PPARα. More importantly, these effects were reversed by treatment of MK-886, a selective PPARα antagonist. CONCLUSION: Our present findings demonstrated that HD attenuated the pathology of AD through inducing autophagy and the underlying mechanism associated with PPARα/TFEB pathway.

[Mechanism of Atractylodes macrocephala against Alzheimer's disease via regulating lysophagy based on LKB1-AMPK-TFEB pathway].

Myloid beta(Aß) 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~(-1)). The content of Aß_(1-42) in 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 Aß 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.

Sesquiterpene Lactams and Lactones With Antioxidant Potentials From Atractylodes macrocephala Discovered by Molecular Networking Strategy.

Atractylodes macrocephala rhizome (called Bái-zhú in China) has a long history as a functional food and herbal medicine in East Asia, especially China. Sesquiterpenoids are one of the main active compounds of Atractylodes macrocephala rhizome. This study aimed to explore the unknown sesquiterpenoids of A. macrocephala rhizome using a molecular networking strategy. Two new nitrogen-containing sesquiterpenoids, atractylenolactam A (1) and atractylenolactam B (2), and 2 new sesquiterpene lactones, 8-methoxy-atractylenolide V (6) and 15-acetoxyl atractylenolide III (7), along with 12 known analogs (3-5 and 8-16) were discovered and isolated. All the structures were assigned based on detailed spectroscopic analyses. The absolute configurations of 1, 2, 6, and 7 were established by time-dependent density functional theory ECD (TDDFT-ECD) calculations. All these compounds had different degrees of concentration-dependent activating effects on nuclear-factor-E2-related factor-2 (Nrf2).

Amelioration by Idesia polycarpa Maxim. var. vestita Diels. of Oleic Acid-Induced Nonalcoholic Fatty Liver in HepG2 Cells through Antioxidant and Modulation of Lipid Metabolism.

Idesia polycarpa Maxim. var. vestita Diels (I. polycarpa) is well known as an edible oil plant which contains abundant linoleic acid and polyphenols. The objective of this study was to maximize the by-product of defatted fruit of I. polycarpa. We found that the fraction D of ethyl acetate extract (EF-D) contained more polyphenols, which contribute to its strong antioxidant activity by antioxidant assays (DPPH, ABTS, and FRAP). Meanwhile, EF-D showed a significant lipid-lowering effect on oleic acid- (OA-) induced hepatic steatosis in HepG2 cells through enhancing antioxidant activity, reducing liver damage, and regulating lipid metabolism, antioxidant, and inflammation-related gene expression. The SOD and T-AOC levels significantly increased, but the levels of MDA, AST, and ALT decreased obviously when treated with EF-D. In general, EF-D improved the antioxidant enzyme activities and decreased the hepatic injury activities. Besides, treatment with EF-D for NAFLD influenced lipid metabolism and inflammation by activating PPARα which was associated with the increased expression of CPT1 and decreased expression of SCD, NF-κB, and IL-1. Moreover, EF-D improved the oxidative stress system through activation of the Nrf2 antioxidant signal pathways and upregulated its target genes of HO-1, NQO1, and GSTA2. The results highlighted the EF-D from the defatted fruit of I. polycarpa regarding lipid-lowering, proving it to be a potential drug resource of natural products for treating the nonalcoholic fatty liver disease (NAFLD).