Ethyl caffeate attefnuates Aß-induced toxicity in Caenorhabditis elegans AD models via the insulin/insulin-like growth factor-1 signaling pathway.

The pathogenesis of Alzheimer's disease (AD), a multifactorial progressive neurodegenerative disease associated with aging, is unclear. Ethyl caffeate is a plant polyphenol that has been reported to have neuroprotective effects, but the mechanisms by which it acts are unclear. In this study, for the first time, we investigated the molecular mechanism of its anti-AD properties using the Caernorhabditis elegans model. The results of our experiments showed that ethyl caffeate delayed the paralysis symptoms of CL4176 to a different extent and reduced the exogenous 5-hydroxytryptophan-induced paralysis phenotype. Further studies revealed that ethyl caffeate lowered Aß plaques and depressed the expression of Aß monomers and oligomers, but did not influence the mRNA levels of Aß. Moreover, it was able to bring paraquat-induced ROS levels down to near-standard conditions. Real-time quantitative PCR experiment showed a significant upregulation of the transcript abundance of daf-16, skn-1 and hsf-1, key factors associated with the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway (IIS), and their downstream genes sod-3, gst-4 and hsp-16.2. It was further shown that ethyl caffeate activated the translocation of DAF-16 and SKN-1 from the cytoplasm to the nucleus and enhanced the expression of sod-3::GFP, gst-4::GFP and hsp-16.2::GFP in transgenic nematodes. This meant that the protection against Aß toxicity by ethyl caffeate may be partly through the IIS signaling pathway. In addition, ethyl caffeate suppressed the aggregation of polyglutamine proteins in AM141, which indicated a potential protective effect against neurodegenerative diseases based on abnormal folding and aggregation of amyloid proteins. Taken together, ethyl caffeate is expected to develop as a potential drug for the management of AD.

The Attenuation of Insulin/IGF-1 Signaling Pathway Plays a Crucial Role in the Myo-Inositol-Alleviated Aging in Caenorhabditis elegans.

Myo-Inositol (MI) has been shown to alleviate aging in Caenorhabditis (C). elegans. However, the mechanism by which MI alleviates aging remains unclear. In this study, we investigate whether MI can modulate the PI3K so as to attenuate the insulin/IGF-1 signaling (IIS) pathway and exert the longevity effect. The wild-type C. elegans and two mutants of AKT-1 and DAF-16 were used to explore the mechanism of MI so as to extend the lifespan, as well as to improve the health indexes of pharyngeal pumping and body bend, and an aging marker of autofluorescence in the C. elegans. We confirmed that MI could significantly extend the lifespan of C. elegans. MI also ameliorated the pharyngeal pumping and body bend and decreased autofluorescence. We further adopted the approach to reveal the loss-of-function mutants to find the signaling mechanism of MI. The functions of the lifespan-extending, health-improving, and autofluorescence-decreasing effects of MI disappeared in the AKT-1 and DAF-16 mutants. MI could also induce the nuclear localization of the DAF-16. Importantly, we found that MI could dramatically inhibit the phosphoinositide 3-kinase (PI3K) activity in a dose-dependent manner with an IC50 of 90.2 µM for the p110α isoform of the PI3K and 21.7 µM for the p110ß. In addition, the downregulation of the PI3K expression and the inhibition of the AKT phosphorylation by MI was also obtained. All these results demonstrate that MI can inhibit the PI3K activity and downregulate the PI3K expression, and the attenuation of the IIS pathway plays a crucial role for MI in alleviating aging in C. elegans.

N-butanol extract of Hedyotis diffusa protects transgenic Caenorhabditis elegans from Aß-induced toxicity.

Hedyotis diffusa Willd (Rubiaceae) is a widely used and resourceful traditional Chinese medicine that exerts protection against aging and age-related diseases. However, the underlying mechanisms of the protective effects remain largely unclear. Alzheimer's disease (AD) is an age-related neurodegenerative disease, of which ß-amyloid (Aß)-induced toxicity has been suggested as a main cause. Herein, we use the transgenic Caenorhabditis elegans CL4176, CL2006, and CL2355 strains, which express human Aß1-42 peptide, to investigate the effects and the possible mechanisms of n-butanol extract of H.diffusa (HDB)-mediated protection against Aß toxicity in vivo. During the experiments, a method of quality control for HDB was established by HPLC. Additionally, we examined the effects of HBD on gene expression changes with qRT-PCR, aggregation of Aß plagues with thioflavin-S staining, and protein detection with GFP labeling. HDB improved lifespan, locomotion, and stress resistance. Further study showed that HDB decreased paralysis, the accumulation of ROS, and AChE activity. Moreover, HDB suppressed neuronal Aß-expression-induced defects in chemotaxis behavior and increased SOD activity. HDB also downregulated the Aß mRNA level and decreased the number of Aß deposits. Furthermore, HDB increased the expression levels of sod-3, daf-16, hsf-1, and hsp-16.2 gene and upregulated hsp-16.2::GFP and gst-4::GFP expression. Taken together, these results suggest that HDB may protect against Aß-induced toxicity in C. elegans via the insulin/insulin-like growth factor-1 (IGF-1) signaling pathway.

Black rice anthocyanin extract enhances the antioxidant capacity in PC12 cells and improves the lifespan by activating IIS pathway in Caenorhabditis elegans.

Black rice is rich in anthocyanins, and the antioxidant effect of anthocyanins is recognized by consumers. The aim of this study was to identify the molecular mechanisms underlying the antioxidant activity of black rice anthocyanin extract (BRAE) in PC12 cells and C. elegans. Results showed that BRAE increased antioxidant enzyme activities and decreased the accumulation of reactive oxygen species (ROS) and malondialdehyde in PC12 cells induced by H2O2. Meanwhile, BRAE extended the lifespan, enhanced resistance to stress, increased antioxidant enzyme activities, and reduced lipofuscin, ROS, and MDA accumulation in wild-type C. elegans. The polyQ40 aggregation in AM141, paralysis in CL4176, and chemotaxis deficit in CL2355 were alleviated by BRAE administration. BRAE downregulated the mRNA expression of age-1 and daf-2, while upregulated the daf-16 mRNA level and SOD-3, CTL-1, and GST-4 protein expression. Mutational lifespan tests and molecular docking showed that insulin pathway might be involved in the mechanism of lifespan extension.

Antioxidant capacity of flavonoids from Folium Artemisiae Argyi and the molecular mechanism in Caenorhabditis elegans.

ETHNOPHARMACOLOGICAL RELEVANCE: Folium Artemisiae Argyi (FAA) is one kind of Chinese herbal medicine with a long history. It has widespread pharmacological activities such as antibacterial, anti-inflammatory, antioxidative, and hemostatic, among others. FAA is traditionally used for the treatment of eczema, respiratory diseases and gynecological diseases for hundreds of years. Flavonoids are reported as the main components of them. Recent studies focused on the antioxidant effect of its flavonoids in vitro, while few studies focused on the antioxidant effect in vivo, and the underlying mechanisms have not yet been elucidated. AIM OF THE STUDY: The aim of this study was to evaluate the antioxidant activity of Folium Artemisia Argyi flavonoids (FAAF) and explore its possible molecular mechanism in Caenorhabditis elegans. The research and development of its medicinal value will beneficial to the resource utilization of FAA. MATERIALS AND METHODS: Firstly, FAAF was prepared, purified and then qualitatively and quantitatively analyzed using LC-DAD-MS. Then, 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), hydroxyl radical and ferric reducing antioxidant power (FRAP) assays were applied to investigate the antioxidant effect of FAAF in vitro. Meanwhile, a stress resistance assay was carried out to evaluate the antioxidant effect of FAAF in vivo. Moreover, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and reactive oxygen species (ROS) accumulation were determined to ascertain whether FAAF can increase the oxidant defense system of nematodes and reduce the accumulation of ROS. Lipofuscin and protein carbonylation assays were employed to test whether FAAF can increase the antioxidant capacity of nematodes to resist the growth health indicators related to antioxidation. At last, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate the expression of genes related to antioxidation. The expression of green fluorescent protein (GFP) was further investigated using a fluorescence microscope in transgenic strains (SOD-3::GFP, GST-4::GFP, and HSP-16.2::GFP). RESULTS: FAAF exhibited a strong antioxidant capacity and enhanced stress resistance in C. elegans. FAAF reduced ROS accumulation and improved the antioxidant defense system under acute stress. Moreover, FAAF prevented the accumulation of lipofuscin and protein carbonylation in C. elegans. FAAF also upregulated the gene expression levels of hsp-16.2, gst-4, sod-3, skn-1, daf-16, ctl-2, hsf-1 and increased SOD-3::GFP and GST-4::GFP expression. CONCLUSION: These results demonstrated that FAAF exerted antioxidant activity in C. elegans. It was perhaps regulated by the insulin/insulin-like growth factor-1(IGF-1) signaling pathway.