Article pubs.acs.org/JAFC
Monascin from Monascus-Fermented Products Reduces Oxidative Stress and Amyloid‑β Toxicity via DAF-16/FOXO in Caenorhabditis elegans Yeu-Ching Shi,† Tzu-Ming Pan,‡ and Vivian Hsiu-Chuan Liao*,† †
Department of Bioenvironmental Systems Engineering and ‡Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan S Supporting Information *
ABSTRACT: Amyloid-β (Aβ)-induced oxidative stress and toxicity are leading risk factors for Alzheimer’s disease (AD). Monascin (MS) is a novel compound proposed for antioxidative stress applications and is derived from an edible fungus secondary metabolite. This study assessed the effects of MS on oxidative stress, paralysis, Aβ accumulation, and lifespan in the nematode Caenorhabditis elegans and investigated its underlying mechanisms of action. The results showed that MS increased the survival of C. elegans under juglone-induced oxidative stress and attenuated endogenous levels of reactive oxygen species. Furthermore, MS induced a decline in Aβ-induced paralysis phenotype and Aβ deposits in the transgenic strains CL4176 and CL2006 of C. elegans, which expresses human muscle-specific Aβ1−42 in the cytoplasm of body wall muscle cells. In addition, mRNA levels of strain CL4176 of several antioxidant genes (sod-1, sod-2, sod-3, hsp16.2) and daf-16 were up-regulated by MS treatment when compared to the nontreated controls. Further evidence showed that MS treatment in C. elegans strains lacking DAF-16/FOXO did not affect paralysis or lifespan phenotypes. The findings indicate that MS reduces oxidative stress and Aβ toxicity via DAF-16 in C. elegans, suggesting that MS can be used for the prevention of AD-associated oxidative stress complications. KEYWORDS: Alzheimer’s disease (AD), amyloid-β, monascin, fungus secondary metabolite, Caenorhabditis elegans, oxidative stress, DAF-16
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“amyloid cascade hypothesis”, which points out that Aβ and its aggregated forms (including oligomers, protofibril, and fibrils) trigger downstream oxidative stress and neurotoxic events, leading to neuro-degeneration, neuronal dysfunction, and cell death.16 Two common Aβ species have been found in the AD brain, including Aβ1−40 and Aβ1−42.17 Several studies in the literature have found that Aβ1−42 is the predominant component of plaques, and it is also more toxic than Aβ1−40, and these monomeric peptides are generated through the cleavage of amyloid precursor protein (APP) by β-secretases and γ-secretases.17 The association between ROS and Aβ that results in neural injury has been reported in a recent study.18 In 2013, an estimated 5 million people in the United States had AD, and the number may rise to 13.8 million by 2050.19 Alzheimer’s disease was the sixth leading cause of death among American people in 2013.20 Consequently, finding the risk factors and novel therapeutic compounds for treating AD seems more and more important. Several functional foods and materials have been used or proposed for the inhibition of AD development, including ice plant,21 resveratrol,22 and Gingko biloba.23 Monascus-fermented products may suppress risk factors of AD in rats,8,24 and MS has been reported to prevent AD development in Aβ40-induced AD rats and improve
INTRODUCTION Monascin (MS), the major yellow pigment of secondary metabolites in extracts of Monascus-fermented rice, is a potent inhibitor of carcinogenesis.1 MS has been shown to have several beneficial health effects, such as inhibiting skin carcinogenetic activity in mice,2 diabetic symptom delay,3 antiatherosclerosis effect,4 acting as an Nrf2 activator with PPARγ-agonist,5 attenuating lung oxidative inflammation via Nrf-2 and PPARγ,6 and inhibiting the activation of RBL-2H3 mast cells.7 Recently, Monascus-fermented products had been reported to attenuate amyloid-β (Aβ) in Alzheimer’s disease (AD) in vivo,8 and MS has also shown antioxidative effects with the forkhead transcription factor DAF-16/FOXO activity in the Caenorhabditis elegans model.9 Oxidative stress results in the production of excessive quantities of free radicals and reactive oxygen species (ROS), which overwhelm the natural antioxidant defense ability of the cells, leading to biomolecular injury, dysfunction, or apoptosis.10 The genes that influence oxidative stress have been identified through studies of the pathophysiology of a variety of diseases and aging.11 Oxidative stress has been associated with human age-related diseases, such as AD and Parkinson’s disease (PD),12,13 and some major factors in the insulin-like pathway, such as daf-2, age-1, and daf-16, are involved in invertebrates.14 AD is a progressive neurodegenerative disease of the aged population. It has been demonstrated that Aβ peptide deposition is enhanced in the brains of Alzheimer’s patients.15 One of the most well-accepted hypotheses of AD is the © 2016 American Chemical Society
Received: Revised: Accepted: Published: 7114
June 21, 2016 August 24, 2016 August 24, 2016 August 24, 2016 DOI: 10.1021/acs.jafc.6b02779 J. Agric. Food Chem. 2016, 64, 7114−7120
Article
Journal of Agricultural and Food Chemistry
min, then de-stained with 50% ethanol, mounted on slides for microscopy, and analyzed for fluorescence images.26 The quantification of Aβ plaques was achieved by counting the thioflavin S-reactive deposits in the anterior of the pharyngeal bulb in transgenic strain CL2006. At least three independent trials were performed. RNA Extraction and Real-Time PCR Analysis. Transgenic CL4176 worms were incubated for 50 h at 16 °C from eggs to L3 larvae, then shifted to 23 °C, and harvested at 33 h after being temperature-shifted. Worms were washed by PBS and collected. RNA extraction and cDNA synthesis were performed by following the manufacturer’s instructions in the kits (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed using the Power SYBR Green PCR Master Mix (Qiagen) and Applied Biosystems step one plus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). Primer sequences used for qRT-PCR are listed in STable 1 (Supporting Information). The relative quantities of mRNA were determined using comparative cycle threshold methods and normalized against the housekeeping gene (ama-1) mRNA. C. elegans RNA Interference (RNAi) Assay. daf-16 expression was knocked down by feeding the nematodes E. coli HT115(DE3) bacteria carrying daf-16 dsRNA. Worms were placed onto NGM agar plates that had been supplemented with 50 μg/mL carbenicillin and 1 mM IPTG and stored for 12 h or more for the bacterial lawn to grow. The L4 larvae were transferred to fresh daf-16 RNAi plates and allowed to lay eggs. Subsequently, the resultant adult worms were chosen for the paralysis assays. Statistical Analyses. Statistical comparisons between treated and untreated worms were done with the unpaired Student’s t test. Data are expressed as the mean ± standard error of the mean (SEM). A p value
