Molecular mechanism of culinary herb Artemisia argyi in promoting lifespan and stress tolerance

Molecular mechanism of culinary herb Artemisia argyi in promoting lifespan and stress tolerance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Molecular mechanism of culinary herb Artemisia argyi in promoting lifespan and stress tolerance Jinsong Wang, Hailin Cui, Yan Xu, Shuyou Shang, Yuanxin Miao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5028259/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Dec, 2024 Read the published version in npj Science of Food → Version 1 posted 10 You are reading this latest preprint version Abstract Artemisia argyi Lévl. et Vant. (A. argyi) leaf possesses various health promoting functions contributed by its main bioactive flavonoids.In this study, the anti-aging effect and mechanism of Artemisia argyi leaf extract (AALE) were identified using Caenorhabditis elegans (C. elegans) as a model. The results showed that the AALE promoted the lifespan and stress resistance of C. elegans. Meanwhile, the AALE treated C. elegans had high physical activity and low lipofuscin accumulation without negative impact on body size. It was found that the AALE boosted the expression of oxidative stress-related proteins by regulating the insulin/ IGF-1 signaling (IIS) pathway, which then activated the transcription factors DAF-16/FOXO. The results of RNA-sequence analysis indicated that the changes of genes in nematodes treated with AALE were associated with the responses against oxidative stress, cell maturation, and immune reaction, and stress. The qPCR results indicated that the AALE obviously up-regulated the expression of genes related to antioxidant activity and lipid and carbohydrate metabolisms. These findings reveal the mechanism of health prompting function of Artemisia argyi leaf at molecular genetic level. The positive results obtained from the highly conserved signaling pathways of C. elegans model suggest that Artemisia argyi leaf could have the robust benefits for improving healthy aging as well as preventing aging-related diseases in the human body. Biological sciences/Drug discovery Biological sciences/Physiology Artemisia argyi C. elegans aging stress resistance flavonoids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Aging is an inevitably biological process characterized as deterioration of intrinsic physiological functions. Meanwhile, it leads to increased susceptibility to many chronic diseases, including cardiovascular disorders, type 2 diabetes, Alzheimer and Parkinson diseases (Campisi et al., 2019). Therefore, the demand for attenuating the aging process and promoting healthy aging has become widespread attention. Compared with synthetic anti-aging medicines, natural antioxidant plant components or extracts have been very attractive alternatives due to their great potential to modulate aging-related disorders with fewer side effects (Li et al., 2024; Liu et al., 2024; Wang et al., 2024). Artemisia argyi Lévl. et Vant. ( A. argyi ) leaf is edible and used in culinary for aroma enhancement or food pigment in Asia. To date, several bioactive groups have been identified in A. argyi leaf including essential oils, flavonoids, polysaccharides, and organic acids (Song et al., 2019).5 The primary flavonoids in A. argyi leaf are a group antioxidant phytochemicals or phenolics, anti-inflammatory, anti-tumor, anticoagulant activities (Gan et al., 2022; Lv et al., 2018; Seo et al., 2003; Zhong et al., 2022). C. elegans is a classic model used as an in vivo study and has widely been applied to identify bioactive compounds with anti-aging effects and elucidate biological mechanisms of anti-aging and stress resistance due to their short lifespan, easy maintenance, and conserved signalling pathways (Ye et al., 2020). For example, the critical genes and signalling pathways that regulate the aging process are evolutionarily conserved. In C. elegans , the IIS pathway is well known to mediate longevity, metabolism, and development (Lapierre & Hansen, 2012). DAF-16, an ortholog of human Forkhead box protein O (FOXO), regulates the downstream IIS pathway responding stress (Murphy & Hu, 2013). Inhibiting of IIS pathway could allow the DAF-16/FOXO to access the nucleus and initiate its target genes that prolong lifespan and enhance resistance to various stresses. The combination of different antioxidant phytochemicals in the AALE may exert a positively synergetic effect which can enhance the benefits for health promoting and preventing age-associated diseases in the human body. In this study, the effects and mechanism of Artemisia argyi Lévl. leaf extract (AALE) on promoting lifespan and stress tolerance were evaluated using C. elegans model. The antioxidant phytochemical profile of AALE was determined by HPLC-MS/MS method. The physiological status of nematodes including motility, body size, and lipofuscin accumulation were monitored after treated with different concentration of the extract. RNA-sequence and RT-PCR techniques and a series of genetic analysis were applied to explore the molecular mechanisms of health benefit of AALE related to longevity and stress tolerance. Our findings would be very helpful to fully understand the health promoting function of Artemisia argyi leaf and plant antioxidant phytochemicals. In addition to aroma and color decoration, the extract of Artemisia argyi leaf could be used as a health promoting food ingredient or supplement. 2. Materials And Methods 2.1 Chemicals and materials 5-Fluoro-2-deoxyuridine (FUdR 98%) and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (St. Louis, MO, U.S.A). The strains used in this study including N2, EU1 skn-1 (zu67) IV CF1038 daf-16 (mu86) I, VC345 sgk-1(ok538) X, TJ1052 age-1 (hx546) II, CB1370 daf-2 (e1370) III, PS3551 hsf-1(sy441) I, CL2070 (dvIs70 [hsp-16.2p::GFP + rol-6(su1006)]), TJ356 (zIs356IV [daf-16p::daf-16a/b::GFP +rol-6 (su1006)]), and CF1553 (muIs84 [(pAD76)sod-3p::GFP+ rol-6(su1006)]) were purchased from the Caenorhabditis Genetics Center at the University of Minnesota (MN, USA). Artemisia argyi leaves were obtained from Qichun Qiaikang Material Medical Technology Co., Ltd (Hubei, China). 2.2 Preparation of AALE Artemisia argyi Lévl. leaf extract (AALE) was prepared according to a previous method (Na et al., 2008). Dried Artemisia argyi leaves were extracted twice with 50% ethanol at 50 o C for 2 hours. After evaporating the solvent, the extract was loaded onto a column packed with resin AB-8 (Yuan Ye Biological Technology Co. Ltd, Shanghai, China) for further purification. The eluted fraction with 50% ethanol was collected and then concentrated and lyophilized to obtain final dried AALE. The AALE was dissolved in DMSO to obtain an AALE stock solution at concentration of 60 mg/mL and stored at -80℃. 2.3 Identification and quantification of major flavonoids in AALE using HPLC-MS/MS AALE was analyzed by an HPLC system with a column (HSS T3 C18, pore size 1.8 μm, length 2.1 × 100 mm). Mobile phase A and B were 0.04% acetic acid and acetonitrile with 0.04% acetic acid, respectively. The gradient program of mobile phase was 5% B at 0 min, ramped 95% B in 12.0 min and maintained 5% B from 12.1 min to 15.0 min with a constant flowrate of 0.35 mL/min. The Full-Scan mode by Q Exactive Focus Orbitrap LC-MS/MS (Thermo Scientific, USA) was applied. The ESI source operation parameters were as follows: nebulizing gas flow, 3 L/min; heating gas flow, 10 L/min; interface temperature, 550 o C; DL temperature, 250 o C; heat block temperature, 400 o C; drying gas flow, 10 L/min. Parent ions and base fragment ions were used to carry out identification and quantification. 2.4 Lifespan, body size, motility, lipofuscin, and stress resistance assays The lifespan assays were conducted according to the methods previously described (Li et al., 2021). Age-synchronized L4 larvae were transferred to a new 96-well plate (liquid S-completed medium added) and treated with 0.6% DMSO (control) and different concentrations (60, 240, and 360 μg/mL) of AALE. FUdR (150 μM) was also added to inhibit the reproduction of progeny. The survival rates of worms were recorded every other day. Worms were cultured as outlined above. On the 3, 6, and 9 day of adulthood, the body size, pharyngeal pumping rates, and body bending rates of worms were determined according to our previous reports (Li et al., 2022). For lipofuscin determination, on the 10 th day of adulthood, the worms were paralyzed with sodium azide (2%) and photographed by a fluorescence microscope (Olympus, Japan) with excitation and emission wavelengths at 485 nm and 528 nm, respectively. The fluorescence intensity of each worm was quantified using ImageJ software. For oxidative stress assay, the day 7 adult worms were transferred to a fresh 96-well plate containing 1mM hydrogen peroxide and incubated at 20°C (Xu et al., 2022). For the thermal stress assay, the worms were transferred to a pre-heated 96-well plate on the 7 th day of adulthood and then subjected to the heat stress at 37°C (Xu et al., 2022). The survivals were recorded every 2 h until all the worms died. 2.5 DAF-16::GFP subcellular localization assay Synchronized L4 larvae of strain TJ356 (a GFP reporter for DAF-16) were treated with 240 μg/mL AALE or 0.4% DMSO for 1 h, and then observed and photographed using a fluorescence microscope (Olympus, Japan). “cytosolic”, “intermediate” and “nuclear” were utilized to identify the expression patterns of DAF-16::GFP (Jiang et al., 2021; Tao et al., 2022). The worms were counted and analyzed to express as percentages in each group. 2.6 Fluorescence measurements in transgenic strains Age-synchronized L1 larvae of CF1553 (SOD-3 fused GFP protein) were cultured with AALE (240 μg/mL) or 0.4% DMSO for 72 h. The worms were anesthetized with 2% sodium azide and imaged with a fluorescence microscope (Wan et al., 2020). Prior to microscopy observation, the young adult mutants of CL2070 (HSP-16.2 fused GFP protein) were exposed to heat shock at 37℃ for 2 h and allowed to recover at 20℃ for 4 h (Kim et al., 2014). The GFP fluorescence intensity per worm was analyzed by ImageJ software. 2.7 RNA sequencing analysis Worms were collected on the 3 rd day of adulthood and washed with M9 buffer to remove E. coli OP50. RNA sequencing experiment was performed through Majorbio BioTech Co. (Shanghai, China) using the Illumina Novaseq 6000 platform following the manufacturer’s recommendations (Xue et al., 2022). One μg of RNA per sample was used as input material for the RNA sample preparation. The reference genome used for analysis was as follows (https://www.ncbi.nlm.nih.gov/datasets/taxonomy/6239). Transcripts with an adjusted p value ≤ 0.05 and fold change ≥ 2 were considered significantly differential expression. 2.8 Gene expression analysis by quantitative real-time polymerase chain reaction (RT-PCR) Age-synchronized L4 larvae of wild-type worms were treated with or without 240 μg/mL AALE for 72 h at 20°C. Total RNA was extracted according to the standard protocols (Tiangen Biotech, China) and converted to cDNA using reverse transcription kit (Vazyme Biotech, China). Afterwards, the qRT-PCR reaction was carried out in a QuantStudio 3.0 PCR system (ABI, USA) along with SYBR Green PCR PreMix (Tiangen Biotech, China). Actin-1 was chosen as reference gene and gene expression was analyzed using the 2 -ΔΔCT method. 2.9 Statistical analysis Statistical analyses were conducted utilizing Graph Pad Prism version 9.0 (San Diego, CA, U.S.A) and SPSS 21.0 (SPSS Inc., Chicago, USA). Statistical significance was evaluated by ANOVA or t -tests, and significance was defined as p < 0.05. 3. Results And Discussion 3.1 Effects of AALE on lifespan, body size, motility, lipofuscin, and stress resistance of C. elegans A total of 22 flavonoid compounds were identified in AALE. Among them, the major flavonoids were kaempferol, L-epicatechin, apigenin, catechin, quercetin, luteolin, formononetin, and naringenin (Table 1). These flavonoids could provide health-promoting activity individually or synergistically. Several studies reported that Artemisia argyi demonstrates a variety of beneficial bioactivities, including antioxidant, antimicrobial, anti-inflammatory, and neuroprotection activities due to its abundant flavonoids (Hu et al., 2021; Kang et al., 2019; Lee et al., 2023). However, the effects and mechanism of Artemisia argyi on these health benefits have not been well understood. The survival curves of C. elegans (wild-type N2 worms) in control and treated with different concentrations are shown in Figure 2. The survival rate of C. elegans at each experimental time was the lowest among the four groups, while the rate of C. elegans in the treatment group with 240 μg/mL was the highest after two weeks. The mean lifespan of each treatment group was significantly higher than control (Table 1). The C. elegans with AALE at 60, 240, and 360 μg/mL treatments significantly prolonged the mean lifespan by 12.06% ( p value 0.0013), 23.19% ( p value 0.0001), and 19.07% ( p value 0.0001), respectively. Compared with a similar study of evaluating flavonoids-rich Ginkgo biloba leaf extract, AALE had higher lifespan, especially at lower dosage. However, the mean lifespans of 240 and 360μg/mL were not significantly different. It indicated that there might be an optimal range of dosage for AALE in extending the lifespan. The fitness indices including (A) body bending rates, (B) pharyngeal pumping rates, (C) body length and (D) body width of C. elegans in the control and treatment groups are shown in Figure 1. As illustrated in Figure 2A and 2B, AALE-treated worms exhibited significantly higher levels of both body bending and pharyngeal pumping in comparison to the control at each tested stage. Meanwhile there were no significant differences in the body length and body width between AALE-treated worms and control worms (Figure 2C and 2D). It suggested that AALE improved the motility of C. elegans without change of its body size . The change of body size of C. elegans is usually associated with the toxicity in their diets (Peixoto et al., 2016). AALE treatment significantly declined the lipofuscin accumulation level of worms by 11.31%, 16.18%, and 23.38% at doses of 60, 240, 360 μg/mL, respectively (Figure 2E and 2F). The reduction of lipofuscin accumulation delays the aging process of C. elegans (Yu et al., 2023). These results demonstrated AALE significantly prolonged the youthfulness and promoted healthiness in aging of C. elegans . In C. elegans , their low resistance against external stress sharply declines their lifespan (Monickaraj et al., 2013). Most of the observed lifespan extension phenotypes are associated with increased stress resistance such as thermal and oxidative stresses (Denzel et al., 2019). As shown in Figure 3A, AALE treatments prolonged the lifespan of worms under oxidative stress induced by 1 mM hydrogen peroxide ( p< 0.01) with increases of mean lifespan by 9.76%, 17.35%, and 13.80% at concentrations of 60, 240, and 360 μg/mL, respectively. In the thermo-resistance at 37℃, the mean lifespans of worms treated with 60, 240, and 360 μg/mL of AALE were increased by 8.84%, 16.65%, and 11.26%, respectively ( p < 0.01) (Figure 3B). Similar to the survival rates without induced stress (Figure 1), the survival rate of worm treated with 240 μg/mL of AALE was the highest, while the rate of worm treated with 360 μg/mL of AALE which was the highest dosage used in this study was the second among the four group. Therefore, based on the results of assays, concentration of 240 μg/mL AALE was selected and used in the subsequent experiments. 3.2 AALE prolonged lifespan by activating transcription factor DAF-16/FOXO and IIS pathway The mammalian FOXO (Forkhead box O transcription factor) orthologue DAF-16 was reported to mediate longevity, lipogenesis, heat shock survival and oxidative stress responses in C. elegans (Tia et al., 2018). The nuclear localizations of DAF-16 in TJ356 strains treated with and without AALE were examined. The nuclear proportion of DAF-16 was enhanced from 19.69% to 53.13% ( p < 0.001), while the fraction in cytoplasm declined from 48.21% to 17.71% ( p < 0.001) after the strain treated with 240 μg/mL AALE compared with the strain in control group (Figure 4A and 4B). The mRNA expression levels of daf-16 and its downstream target genes, sod-3 (superoxide dismutase) and hsp-16.2 (heat shock protein), were obviously up-regulated in AALE- treated worms (Figure 4C, p < 0.05). In C. elegans , the heat shock proteins (HSPs) are closely associated with thermo-tolerance and can serve as a biomarker of aging (Lund et al., 2002). It is in agreement with our results that AALE-treated worms exhibited higher fluorescence intensity of HSP-16.2::GFP than that in control group. The expression levels of SOD-3::GFP and HSP-16.2::GFP were significantly improved by 24.37% and 19.31% in CF1553 and CL2070 strains treated with AALE, respectively (Figure 4D and 4E, p < 0.01). Therefore, it indicated that the AALE-mediated longevity promotion was also dependent on the activation of DAF-16. It is well-known that the insulin/IGF-1 signaling (IIS) pathway is highly conserved and involved in metabolism, growth, development and longevity (Murphy & Hu, 2013). In C. elegans , the insulin-like receptor DAF-2 signals through the phosphatidylinositol 3-kinase/Akt kinase pathway to regulate of DAF-16/FOXO (Lin et al., 2001). Considering that AALE extended the lifespan by activating DAF-16 which is a central transcription factor of IIS pathway, AALE might extend the lifespan through the IIS pathway (Murphy et al., 2003). Our study showed that AALE failed to extend the lifespans of the loss of function mutants including daf-2 , age-1 , sgk-1 , daf-16 , skn-1 , and hsf-1 mutants(Figure 5A-5F, p > 0.05). It was confirmed that the IIS pathway was crucial to AALE-mediated lifespan for C. elegans . Therefore, AALE extended the lifespan of C. elegans by regulating the IIS pathway and activating DAF-16/FOXO. This is consistent with a previous finding that flavonoid-rich Gastrodia elata extract extended lifespan and reduced oxidative stress by regulating the IIS pathway as well (Shi et al., 2023). 3.3 Differential expressed genes identification and functional distribution RNA-sequencing analysis was conducted on the 3 rd day of N2 worms treated with or without AALE. As shown in Figure 6A, there were 218 upregulated and 254 downregulated genes based on the criteria of fold change ≥ 2 and p ≤ 0.05. Moreover, annotations and enrichment of GO (gene ontology) pathway were analyzed based on the differentially expressed genes (DEGs) of AALE-treated worms. The results showed that molecular functions primarily include transporter activity, binding, and catalytic activity. The cellular components mainly consist of membrane part, cell part, organelle, membrane, and extracellular regions. The biological processes primarily involved in response to stimulus, cellular process, multi-organism process, immune system process, metabolic process, biological regulation, and developmental process (Figure 6B). The GO enrichment analysis of DEGs was conducted by Goatools and the enrichment results of top 25 are shown according to the degree of significance (Figure 6C, p < 0.05). It exhibited that the DEGs in AALE-treated worms were mainly enriched in regulation of mitogen-activated protein kinase (MAPK) cascade. The MAPK cascade includes the responses to oxidative stress, cell maturation, developmental maturation, immune reaction, and stress. All of them involve in the regulation of aging, stress resistance, and development of C. elegans . In C. elegans , MAPK cascade regulates cellular adaptive response to environmental changes and SKN-1 has been considered as a key factor for the p38 MAPK pathway to exert stress-resistance function (Okuyama et al., 2010). It was found that AALE failed to extend the lifespan of skn-1 null mutants, suggesting that the lifespan extension effect of AALE also relied on skn-1 . In summary, the above results further confirmed that AALE promoted longevity mainly by regulating the MAPK pathway. It was further supported the capability of AALE in enhancing lifespan, healthy aging, and stress resistance of C. elegans by the molecular genetic results. 3.4 KEGG analysis and validation of DEGs To systematically analyze gene function and explore the relationship between genomic information and functional information, KEGG function annotation was performed on DEGs. The results showed that DEGs of AALE-treated worms were mainly related to lipid and amino acid metabolism, signal transduction, transport and catabolism, aging, nervous and immune system, neurodegenerative disease, and cancer (Figure 7A). Furthermore, the results of KEGG enrichment analysis revealed that DEGs were mainly enriched in the lysosome, peroxisome proliferator-activated receptor (PPAR) signaling pathway, fatty acid degradation, longevity regulation pathway, FOXO signaling pathway, autophagy, and insulin resistance (Figure 7B). The results were in accordance with that AALE activated the DAF-16 and regulated the IIS pathway. Furthermore, ten differentially expressed genes were selected to measure their expression levels by qPCR to support the RNA-seq results. The results showed that AALE significantly increased the expressions of gst-38 (Glutathione S-Transferase), ugt-43 (UDP-Glucuronosyl Transferase), abf-2 (Antibacterial factor-related peptide 2), clec-63 (C-type LECtin), and lys-1 (Lysozyme-like protein 1) (Figure 7C, p <0.05). AALE decreased the expressions of acs-18 (AMP-binding domain-containing protein), ins-30 (INSulin related), comt-4 (Catechol-O-Methyl Transferase family), gpx-3 (Glutathione peroxidase 3), and far-4 (Fatty Acid/Retinol binding protein)(Figure 7C, p <0.05). These findings were in agreement with the RNA-seq results. In C. elegans , gst-38 and ugt-43 are related to glutathione metabolism and phase II detoxification, respectively (Asif et al., 2024). Thus, AALE significantly upregulated the expression of gst-38 and ugt-43 to enhance the anti-oxidant activity and oxidative stress resistance of worms. The genes lys-1 , abf-2 , and clec-63 which were remarkably upregulated in AALE-treated worms contribute to the antimicrobial activity and immune response as well (Shao & Wang, 2020; Yang et al., 2023). A previous study suggested that A rtemisia argyi exerted antimicrobial effect in goldfish and Candida albicans models (Shi et al., 2017). Our results showed that AALE significantly downregulated comt-4 , which might be beneficial to the enhancement of the body bending and pharyngeal pumping rates in AALE-treated worms. Similar to the results, it was reported that inhibition of comt-4 could decrease the dopamine levels and promote locomotory behaviors of nematodes (Rodríguez-Ramos et al., 2017). Moreover, the significantly decreased expressions of ins-3 , acs-18 , and far-4 by AALE were associated with insulin secretion and fatty acid metabolism (Balasubramanian et al., 2016). Based on the results, it was suggested that AALE may mediate glycolipid metabolism in nematodes by influencing the activity of key metabolic enzymes and synthesis, catabolism, and oxidation of fatty acids. 4. Conclusion AALE was rich in a variety of flavonoids and significantly improved the lifespan and fitness of C. elegans via IIS and FOXO signaling pathway. The MAPK was enriched in AALE treated worms to response against oxidative stress, cell maturation, developmental maturation, immune reaction, and stress. KEGG analysis showed that the changes of genes in treated worms were also related to the lipid and amino acid metabolism, signal transduction, transport and catabolism, aging, nervous and immune system, neurodegenerative disease, and cancer. The results of qPCR indicated that AALE significantly upregulated the expression of genes related to the antioxidant activity and oxidative stress resistance of worms. Because of the high conservation of these pathways between mammals and C. elegans , our findings could suggest that Artemisia argyi also has a great activity in promoting healthy aging for humans. Declarations Acknowledgements This work was supported by Key Research and Development Project of Hubei Province (2022BCE057、2022BEC031), Hubei Engineering Research Center for Specialty Flowers Biological Breeding (2023ZD006), and Doctoral Research Initiation Fund of Jingchu University of Technology (YY202403). Author contribution Jinsong Wang: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. Hailin Cui: Data curation, Formal analysis. Yan Xu: Data curation, Conceptualization. Shuyou Shang: Formal analysis. Yuanxin Miao: Conceptualization, Methodology, Data curation. Rong Li: Project administration, Resources, Writing – review & editing. Zhimin Xu: Conceptualization, Writing – review & editing. Conflict of interest The authors declare no conflict of interest. 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A promising strategy for investigating the anti-aging effect of natural compounds: a case study of caffeoylquinic acids. Food Funct. 12, 8583–8593 (2021). Li, R. et al. Artemisia selengensis Turcz. leaf extract promotes longevity and stress resistance in Caenorhabditis elegans. J. Sci. Food Agric. 102, 4532–4541 (2022). Xu, T. et al. Longevity-promoting properties of ginger extract in Caenorhabditis elegans via the insulin/IGF-1 signaling pathway. Food Funct. 13, 9893–9903 (2022). Jiang, S. et al. Rhodiola extract promotes longevity and stress resistance of Caenorhabditis elegans via DAF-16 and SKN-1. Food Funct. 12, 4471–4483 (2021). Tao, M. et al. Vitexin and Isovitexin Act through Inhibition of Insulin Receptor to Promote Longevity and Fitness in Caenorhabditis elegans. Mol. Nutr. Food Res. 66, e2100845 (2022). Wan, Q. et al. Hypotaurine promotes longevity and stress tolerance via the stress response factors DAF-16/FOXO and SKN-1/NRF2 in Caenorhabditis elegans. Food Funct. 11, 347–357 (2020). Kim, D. K. et al. 4-Hydroxybenzoic acid-mediated lifespan extension in Caenorhabditis elegans. J. Funct. Foods. 7, 630–640 (2014). Xue, D. et al. Deoxynivalenol triggers porcine intestinal tight junction disorder through hijacking SLC5A1 and PGC1α-mediated mitochondrial function. Food Chem. Toxicol. 163, 112921 (2022). Tables Table 1. Major flavonoids in AALE identified and quantified by HPLC-MS/MS Compounds Mass Main fragments (m/z) Concentration (%) L-Epicatechin 291.0000 139.0803, 122.8801 39.16 ± 2.40 Catechin 291.0790 139.0606, 122.8931 21.70 ± 1.33 Naringenin 273.0690 153.0639, 147.08 7.20 ± 0.66 Apigenin 271.0530 215.0778, 243.1533 25.71 ± 2.39 Luteolin 287.1000 287.0799, 186.963 15.01 ± 1.38 Kaempferol 287.0480 286.9807, 165.141 52.84 ± 4.89 Quercetin 303.0430 303.0053, 257.0263 16.55 ± 1.49 Formononetin (4'-O-methyldaidzein) 269.0740 269.1, 105 8.60 ± 0.80 Table 2. Effect of AALE on the lifespan of wild-type (N2) worms at 20 o C Treatment Mean lifespan (days ± SEM) Percentage change Number of worms p value Control 17.25 ± 0.46 a - 167 - 60 μg/mL AALE 19.33 ± 0.50 b 12.06% 161 0.0013 240 μg/mL AALE 21.25 ± 0.54 c 23.19% 158 < 0.0001 360 μg/mL AALE 20.54 ± 0.52 bc 19.07% 158 < 0.0001 Different letters (a, b, and c) indicated a significant difference between two groups. p value was determined by log-rank test using the Kaplan–Meier survival analysis. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 24 Dec, 2024 Read the published version in npj Science of Food → Version 1 posted Editorial decision: Revision requested 02 Nov, 2024 Reviews received at journal 02 Nov, 2024 Reviews received at journal 25 Oct, 2024 Reviewers agreed at journal 15 Oct, 2024 Reviewers agreed at journal 12 Oct, 2024 Reviewers agreed at journal 10 Oct, 2024 Reviewers invited by journal 10 Oct, 2024 Editor assigned by journal 30 Sep, 2024 Submission checks completed at journal 09 Sep, 2024 First submitted to journal 03 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5028259","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":373360436,"identity":"5d44e94c-24b3-4c16-bfba-dcd92f255eb7","order_by":0,"name":"Jinsong Wang","email":"","orcid":"","institution":"Jingchu University of Technology Jingmen","correspondingAuthor":false,"prefix":"","firstName":"Jinsong","middleName":"","lastName":"Wang","suffix":""},{"id":373360438,"identity":"a6f032f8-4d87-4b8c-8925-8c21598be5e0","order_by":1,"name":"Hailin Cui","email":"","orcid":"","institution":"Jingchu University of Technology Jingmen","correspondingAuthor":false,"prefix":"","firstName":"Hailin","middleName":"","lastName":"Cui","suffix":""},{"id":373360441,"identity":"f888645b-2d09-49ff-9acf-cd8169cce35f","order_by":2,"name":"Yan Xu","email":"","orcid":"","institution":"Jingchu University of Technology Jingmen","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Xu","suffix":""},{"id":373360442,"identity":"f8a39292-870e-46a7-b3e8-5a23f06f7e7b","order_by":3,"name":"Shuyou Shang","email":"","orcid":"","institution":"Wuhan Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"Shuyou","middleName":"","lastName":"Shang","suffix":""},{"id":373360444,"identity":"15afd83d-7a23-4615-8646-baaed5fb9cf4","order_by":4,"name":"Yuanxin Miao","email":"","orcid":"","institution":"Jingchu University of Technology Jingmen","correspondingAuthor":false,"prefix":"","firstName":"Yuanxin","middleName":"","lastName":"Miao","suffix":""},{"id":373360449,"identity":"f788254e-dec4-4f18-8b55-a0cba41822d0","order_by":5,"name":"Rong Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIie3RPQuCQBjA8TsOzuWxW43Az3Bxa9BXUYImB6dmI7BF2/sWtjUaQZM1t2UITQ2NGUL50nw6Bt1/uJPjftwDIqRS/WA9Um8jYNoi5s2ZJSe0IVOzHxyskvAOpNn2gp+d6noXounXzH0R24udh5v7BWJaafOtbDBNiPWK2nPvGA1Dn6N+cOc4TGSE0oEegL3AYSRwSaoJCfZbiWH7BNKajDsReHIBFFDzitFOiNA9yzSA8mFwEmAkN3cXSghjB5xB8YbxJbvx58w02XKySXMJqfuOQaufAtVX3ALKinolaftNlUql+sc+FgFEu6fE1wkAAAAASUVORK5CYII=","orcid":"","institution":"Jingchu University of Technology Jingmen","correspondingAuthor":true,"prefix":"","firstName":"Rong","middleName":"","lastName":"Li","suffix":""},{"id":373360452,"identity":"31cacb8c-3fd5-44e5-9091-433443594427","order_by":6,"name":"Zhimin Xu","email":"","orcid":"","institution":"Louisiana State University","correspondingAuthor":false,"prefix":"","firstName":"Zhimin","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2024-09-04 03:21:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5028259/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5028259/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41538-024-00358-8","type":"published","date":"2024-12-24T15:57:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69638174,"identity":"c860892c-b564-4fd9-a497-31a6deadaea7","added_by":"auto","created_at":"2024-11-22 13:35:38","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90696,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of AALE on the lifespan of \u003cem\u003eC. elegans\u003c/em\u003eexposed to AALE at 0, 60, 240, and 360 μg/mL.*The log-rank (Kaplan–Meier) test was utilized for statistical analysis.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5028259/v1/e4cb3528da899030daf79c98.jpg"},{"id":69637764,"identity":"3b6648d2-83ca-4726-9463-554cb29b0e6f","added_by":"auto","created_at":"2024-11-22 13:27:38","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":150259,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Body bending rates, (B) Pharyngeal pumping rates, (C) Body length, and (D) Body width of N2 worms treated with 60, 240, 360μg/mL AALE or control on the days 3, 6, 9 of adulthood. (E) Images of intestinal autofluorescence from lipofuscin of worms on the day 10 of adulthood. (F) Relative fluorescence intensity of lipofuscin. *The images were analyzed with ImageJ software and numerical data were analyzed by two-way ANOVA. Data were represented as mean ± SD. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. ns., no significance.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5028259/v1/e2eb4b0d73e0b5ceb6857cc8.jpg"},{"id":69637758,"identity":"4adbb6ca-018b-4f60-b7c7-9901a020944b","added_by":"auto","created_at":"2024-11-22 13:27:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":118217,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of AALE on the stress resistances of N2 worms (A) survival curves of N2 nematodes under oxidative stress. (B) survival curves of the N2 nematodes under thermal stress.*Survival rates were analyzed by the log-rank (Kaplan–Meier) test using GraphPad Prism 9.0.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5028259/v1/97237b58a7806d831609a231.jpg"},{"id":69637759,"identity":"afdeb66f-fff7-4d91-aad8-da9c270b6af3","added_by":"auto","created_at":"2024-11-22 13:27:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":140200,"visible":true,"origin":"","legend":"\u003cp\u003eAALE extended the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e by activating DAF-16. (A) Distribution of DAF-16::GFP in TJ356: cytosolic, intermediate, and nuclear. (B) Quantification of DAF-16::GFP location. (C) The mRNA expression of \u003cem\u003edaf-16\u003c/em\u003eand its downstream genes. (D) The images and quantification of fluorescence intensity in CF1553 strain. (E) The images and quantification of fluorescence intensity in CL2070 strain. Data were represented as mean ± SD. Statistical significance at * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** p \u0026lt; 0.01, and ***\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.001 by student’s t-test. ns., no significance.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5028259/v1/2e417fb638d858b1811ba85c.jpg"},{"id":69638172,"identity":"9abf8d6b-2541-4ab1-ae0a-1320083e646f","added_by":"auto","created_at":"2024-11-22 13:35:38","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":170843,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival curves of (A) \u003cem\u003edaf-2 (e1370)\u003c/em\u003e mutants, (B) \u003cem\u003eage-1(hx546)\u003c/em\u003emutants, (C) \u003cem\u003esgk-1 (ok538)\u003c/em\u003e mutants, (D)\u003cem\u003edaf-16(mu86) \u003c/em\u003emutants, (E) \u003cem\u003eskn-1(zu67)\u003c/em\u003emutants, and (F) \u003cem\u003ehsf-1 (sy441) \u003c/em\u003emutants treated with 240 μg/mL AALE and control. *Survival rates were analyzed by the log-rank (Kaplan–Meier) test using GraphPad Prism 9.0.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5028259/v1/2d18cb136487af3c1c3efc43.jpg"},{"id":69637760,"identity":"f700a15c-04f3-4800-be9f-d45c2af6ed05","added_by":"auto","created_at":"2024-11-22 13:27:38","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":207743,"visible":true,"origin":"","legend":"\u003cp\u003eGo analysis of AALE-treated \u003cem\u003eC. elegans\u003c/em\u003e (A) differential expression volcano plot - the blue dots represent downregulated differentially expressed genes, red dots represent upregulated differentially expressed genes, and gray dots represent genes with no change, (B) GO annotations analysis of differentially expressed genes - the horizontal coordinate is the number of genes, and the vertical coordinate is the GO classification, and (C) GO enrichment analysis of differentially expressed genes - the horizontal coordinate is the ratio of the genes of interest annotated in the entry to the number of all differentially expressed genes and the vertical coordinate is each GO annotation entry.\u003cstrong\u003e \u003c/strong\u003eThe size of the dots represents the number of differentially expressed genes in the GO annotation entry, and the color of the dots represents the \u003cem\u003ep\u003c/em\u003e-adjust of the hypergeometric test.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5028259/v1/262fcdb3f0934e047b36843f.jpg"},{"id":69637763,"identity":"aefc5be5-e031-4fc7-8505-8531de3071f8","added_by":"auto","created_at":"2024-11-22 13:27:38","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":252703,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG analysis and validation of differentially expressed genes (A) KEGG functional annotations of differentially expressed genes - the horizontal coordinate is the name of the KEGG annotations classification, and the vertical coordinate is the number of genes, (B) KEGG enrichment analysis of differentially expressed genes - the horizontal coordinate is the ratio of the genes of interest annotated in the entry to the number of all differentially expressed genes, and the vertical coordinate is each KEGG pathway - the size of the dots represents the number of differentially expressed genes annotated in the pathway, and the color of the dots represents the p-adjust of the hypergeometric test, and (C) The mRNA expression of several differentially expressed genes. Data were represented as mean ± SD. Statistical significance at * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** p \u0026lt; 0.01, and ***\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.001 by student’s t-test.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5028259/v1/9d0c27c4c23539070c8fd22a.jpg"},{"id":72640640,"identity":"2ffded33-a1c3-49ca-89d2-3e0c0c115add","added_by":"auto","created_at":"2024-12-30 16:07:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1514316,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5028259/v1/a1b7b6e3-b218-4e5c-90c5-3dfce2f8dc1f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular mechanism of culinary herb Artemisia argyi in promoting lifespan and stress tolerance","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAging is an inevitably biological process characterized as deterioration of intrinsic physiological functions. Meanwhile, it leads to increased susceptibility to many chronic diseases, including cardiovascular disorders, type 2 diabetes, Alzheimer and Parkinson diseases (Campisi et al., 2019). Therefore, the demand for attenuating the aging process and promoting healthy aging has become widespread attention. Compared with synthetic anti-aging medicines, natural antioxidant plant components or extracts have been very attractive alternatives due to their great potential to modulate aging-related disorders with fewer side effects (Li et al., 2024; Liu et al., 2024; Wang et al., 2024).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eArtemisia argyi\u003c/em\u003e L\u0026eacute;vl. et Vant. (\u003cem\u003eA. argyi\u003c/em\u003e) leaf is edible and used in culinary for aroma enhancement or food pigment in Asia. To date, \u0026nbsp;several bioactive groups have been identified in \u003cem\u003eA. argyi\u003c/em\u003e leaf including essential oils, flavonoids, polysaccharides, and organic acids (Song et al., 2019).5 The primary flavonoids in \u003cem\u003eA. argyi\u003c/em\u003e leaf are a group antioxidant phytochemicals or phenolics, anti-inflammatory, anti-tumor, anticoagulant activities (Gan et al., 2022; Lv et al., 2018; Seo et al., 2003; Zhong et al., 2022).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003eis a classic model used as an \u003cem\u003ein vivo\u003c/em\u003e study and has widely been applied to identify bioactive compounds with anti-aging effects and elucidate biological mechanisms of anti-aging and stress resistance due to their short lifespan, easy maintenance, and conserved signalling pathways (Ye et al., 2020). For example, the critical genes and signalling pathways that regulate the aging process are evolutionarily conserved. In \u003cem\u003eC. elegans\u003c/em\u003e, the IIS pathway is well known to mediate longevity, metabolism, and development (Lapierre \u0026amp; Hansen, 2012). DAF-16, an ortholog of human Forkhead box protein O (FOXO), regulates the downstream IIS pathway responding stress (Murphy \u0026amp; Hu, 2013). Inhibiting of IIS pathway could allow the DAF-16/FOXO to access the nucleus and initiate its target genes that prolong lifespan and enhance resistance to various stresses. The combination of different antioxidant phytochemicals in the AALE may exert a positively synergetic effect which can enhance the benefits for health promoting and preventing age-associated diseases in the human body.\u003c/p\u003e\n\u003cp\u003eIn this study, the effects and mechanism of \u003cem\u003eArtemisia argyi\u003c/em\u003e L\u0026eacute;vl. leaf extract (AALE) on promoting lifespan and stress tolerance were evaluated using \u003cem\u003eC. elegans\u003c/em\u003e model. The antioxidant phytochemical profile of AALE was determined by HPLC-MS/MS method. The physiological status of nematodes including motility, body size, and lipofuscin accumulation were monitored after treated with different concentration of the extract. RNA-sequence and RT-PCR techniques and a series of genetic analysis were applied to explore the molecular mechanisms of health benefit of AALE related to longevity and stress tolerance. Our findings would be very helpful to fully understand the health promoting function of \u003cem\u003eArtemisia argyi\u003c/em\u003e leaf and plant antioxidant phytochemicals. In addition to aroma and color decoration, the extract of \u003cem\u003eArtemisia argyi\u003c/em\u003e leaf could be used as a health promoting food ingredient or supplement.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cp\u003e2.1\u0026nbsp;Chemicals and materials\u003c/p\u003e\n\u003cp\u003e5-Fluoro-2-deoxyuridine (FUdR 98%) and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (St. Louis, MO, U.S.A). The strains used in this study including N2, EU1 skn-1 (zu67) IV CF1038 daf-16 (mu86) I, VC345 sgk-1(ok538) X, TJ1052 age-1 (hx546) II, CB1370 daf-2 (e1370) III, PS3551 hsf-1(sy441) I, CL2070 (dvIs70 [hsp-16.2p::GFP + rol-6(su1006)]), TJ356 (zIs356IV [daf-16p::daf-16a/b::GFP +rol-6 (su1006)]), and CF1553 (muIs84 [(pAD76)sod-3p::GFP+ rol-6(su1006)]) were purchased from the Caenorhabditis Genetics Center at the University of Minnesota (MN, USA). Artemisia argyi leaves were obtained from Qichun Qiaikang Material Medical Technology Co., Ltd (Hubei, China).\u003c/p\u003e\n\u003cp\u003e2.2 Preparation of AALE\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eArtemisia argyi L\u0026eacute;vl. leaf extract (AALE) was prepared according to a previous method\u0026nbsp;(Na et al., 2008).\u0026nbsp;Dried \u003cem\u003eArtemisia argyi\u0026nbsp;\u003c/em\u003eleaves were extracted twice with 50% ethanol at 50\u003csup\u003eo\u003c/sup\u003eC for 2 hours. After evaporating the solvent, the extract was loaded onto a column packed with resin AB-8 (Yuan Ye Biological Technology Co. Ltd, Shanghai, China) for further purification. The eluted fraction with 50% ethanol was collected and then concentrated and lyophilized to obtain final dried AALE. The AALE was dissolved in DMSO to obtain an AALE stock solution at concentration of 60 mg/mL and stored at -80℃.\u003c/p\u003e\n\u003cp\u003e2.3 Identification and quantification of major flavonoids in AALE using HPLC-MS/MS\u003c/p\u003e\n\u003cp\u003eAALE was analyzed by an HPLC system with a column (HSS T3 C18, pore size 1.8 \u0026mu;m, length 2.1 \u0026times; 100 mm). Mobile phase A and B were 0.04% acetic acid and acetonitrile with 0.04% acetic acid, respectively. The gradient program of mobile phase was 5% B at 0 min, ramped 95% B in 12.0 min and maintained 5% B from 12.1 min to 15.0 min with a constant flowrate of 0.35 mL/min. The Full-Scan mode by Q Exactive Focus Orbitrap LC-MS/MS (Thermo Scientific, USA) was applied. The ESI source operation parameters were as follows: nebulizing gas flow, 3 L/min; heating gas flow, 10 L/min; interface temperature, 550\u003csup\u003eo\u003c/sup\u003eC; DL temperature, 250\u003csup\u003eo\u003c/sup\u003eC; heat block temperature, 400\u003csup\u003eo\u003c/sup\u003eC; drying gas flow, 10 L/min. Parent ions and base fragment ions were used to carry out identification and quantification.\u003c/p\u003e\n\u003cp\u003e2.4 Lifespan, body size, motility, lipofuscin, and stress resistance assays\u003c/p\u003e\n\u003cp\u003eThe lifespan assays were conducted according to the methods previously described\u0026nbsp;(Li et al., 2021). Age-synchronized L4 larvae were transferred to a new 96-well plate (liquid S-completed medium added) and treated with 0.6% DMSO (control) and different concentrations (60, 240, and 360 \u0026mu;g/mL) of AALE. FUdR (150 \u0026mu;M) was also added to inhibit the reproduction of progeny. The survival rates of worms were recorded every other day.\u003c/p\u003e\n\u003cp\u003eWorms were cultured as outlined above. On the 3, 6, and 9 day of adulthood, the body size, pharyngeal pumping rates, and body bending rates of worms were determined according to our previous reports\u0026nbsp;(Li et al., 2022). For lipofuscin determination, on the 10\u003csup\u003eth\u003c/sup\u003e day of adulthood, the worms were paralyzed with sodium azide (2%) and photographed by a fluorescence microscope (Olympus, Japan) with excitation and emission wavelengths at 485 nm and 528 nm, respectively. The fluorescence intensity of each worm was quantified using ImageJ software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor oxidative stress assay, the day 7 adult worms were transferred to a fresh 96-well plate containing 1mM hydrogen peroxide and incubated at 20\u0026deg;C\u0026nbsp;(Xu et al., 2022). For the thermal stress assay, the worms were transferred to a pre-heated 96-well plate on the 7\u003csup\u003eth\u003c/sup\u003e day of adulthood and then subjected to the heat stress at 37\u0026deg;C\u0026nbsp;(Xu et al., 2022). The survivals were recorded every 2 h until all the worms died.\u003c/p\u003e\n\u003cp\u003e2.5 DAF-16::GFP subcellular localization assay\u003c/p\u003e\n\u003cp\u003eSynchronized L4 larvae of strain TJ356\u0026nbsp;(a GFP reporter for DAF-16)\u0026nbsp;were treated with 240 \u0026mu;g/mL AALE or 0.4% DMSO for 1 h, and then observed and photographed using a fluorescence microscope (Olympus, Japan). \u0026ldquo;cytosolic\u0026rdquo;, \u0026ldquo;intermediate\u0026rdquo; and \u0026ldquo;nuclear\u0026rdquo; were utilized to identify the expression patterns of DAF-16::GFP\u0026nbsp;(Jiang et al., 2021; Tao et al., 2022). The worms were counted and analyzed to express as percentages in each group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.6 Fluorescence measurements in transgenic strains\u003c/p\u003e\n\u003cp\u003eAge-synchronized L1 larvae of CF1553 (SOD-3 fused GFP protein) were cultured with AALE (240 \u0026mu;g/mL) or 0.4% DMSO for 72 h. The worms were anesthetized with 2% sodium azide and imaged with a fluorescence microscope\u0026nbsp;(Wan et al., 2020). Prior to microscopy observation, the young adult mutants of CL2070 (HSP-16.2 fused GFP protein) were exposed to heat shock at 37℃\u0026nbsp;for 2 h and allowed to recover at 20℃\u0026nbsp;for 4 h\u0026nbsp;(Kim et al., 2014). The GFP fluorescence intensity per worm was analyzed by ImageJ software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.7 RNA sequencing analysis\u003c/p\u003e\n\u003cp\u003eWorms were collected on the 3\u003csup\u003erd\u003c/sup\u003e day of adulthood and washed with M9 buffer to remove \u003cem\u003eE. coli\u003c/em\u003e OP50. RNA sequencing experiment was performed through Majorbio BioTech Co. (Shanghai, China) using the Illumina Novaseq 6000 platform following the manufacturer\u0026rsquo;s recommendations\u0026nbsp;(Xue et al., 2022). One \u0026mu;g of RNA per sample was used as input material for the RNA sample preparation. The reference genome used for analysis was as follows (https://www.ncbi.nlm.nih.gov/datasets/taxonomy/6239). Transcripts with an adjusted \u003cem\u003ep\u0026nbsp;\u003c/em\u003evalue \u0026le; 0.05 and fold change \u0026ge; 2 were considered significantly differential expression.\u003c/p\u003e\n\u003cp\u003e2.8 Gene expression analysis by quantitative real-time polymerase chain reaction (RT-PCR)\u003c/p\u003e\n\u003cp\u003eAge-synchronized L4 larvae of wild-type worms were treated with or without 240 \u0026mu;g/mL AALE for 72 h at 20\u0026deg;C. Total RNA was extracted according to the standard protocols (Tiangen Biotech, China) and converted to cDNA using reverse transcription kit (Vazyme Biotech, China). Afterwards, the qRT-PCR reaction was carried out in a QuantStudio 3.0 PCR system (ABI, USA) along with SYBR Green PCR PreMix (Tiangen Biotech, China). \u003cem\u003eActin-1\u003c/em\u003e was chosen as reference gene and gene expression was analyzed using the 2\u003csup\u003e-\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003e2.9 Statistical analysis\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted utilizing Graph Pad Prism version 9.0 (San Diego, CA, U.S.A) and SPSS 21.0 (SPSS Inc., Chicago, USA). Statistical significance was evaluated by ANOVA or \u003cem\u003et\u003c/em\u003e-tests, and significance was defined as \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"3. Results And Discussion","content":"\u003cp\u003e3.1 Effects of AALE on lifespan, body size, motility, lipofuscin, and stress resistance of \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA total of 22 flavonoid compounds were identified in AALE. Among them, the major flavonoids were kaempferol, L-epicatechin, apigenin, catechin, quercetin, luteolin, formononetin, and naringenin (Table 1). These flavonoids could provide health-promoting activity individually or synergistically. Several studies reported that \u003cem\u003eArtemisia argyi\u003c/em\u003e demonstrates a variety of beneficial bioactivities, including antioxidant, antimicrobial, anti-inflammatory, and neuroprotection activities due to its abundant flavonoids\u0026nbsp;(Hu et al., 2021; Kang et al., 2019; Lee et al., 2023). However, the effects and mechanism of \u003cem\u003eArtemisia argyi\u003c/em\u003e on these health benefits have not been well understood.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe survival curves of \u003cem\u003eC. elegans\u003c/em\u003e (wild-type N2 worms) in control and treated with different concentrations are shown in Figure 2. The survival rate of \u003cem\u003eC. elegans\u003c/em\u003e at each experimental time was the lowest among the four groups, while the rate of \u003cem\u003eC. elegans\u003c/em\u003e in the treatment group with 240 \u0026mu;g/mL was the highest after two weeks. The mean lifespan of each treatment group was significantly higher than control (Table 1). The \u003cem\u003eC. elegans\u003c/em\u003e with AALE at 60, 240, and 360 \u0026mu;g/mL treatments significantly prolonged the mean lifespan by 12.06% (\u003cem\u003ep\u003c/em\u003e value\u0026nbsp;0.0013), 23.19% (\u003cem\u003ep\u003c/em\u003e value 0.0001), and 19.07% (\u003cem\u003ep\u003c/em\u003e value 0.0001), respectively. Compared with a similar study of evaluating flavonoids-rich Ginkgo biloba leaf extract, AALE had higher lifespan, especially at lower dosage. However, the mean lifespans of 240 and 360\u0026mu;g/mL were not significantly different. It indicated that there might be an optimal range of dosage for AALE in extending the lifespan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe fitness indices including (A) body bending rates, (B) pharyngeal pumping rates, (C) body length and (D) body width of \u003cem\u003eC. elegans\u003c/em\u003e in the control and treatment groups are shown in Figure 1. As illustrated in Figure 2A and 2B, AALE-treated worms exhibited significantly higher levels of both body bending and pharyngeal pumping in comparison to the control at each tested stage. Meanwhile there were no significant differences in the body length and body width between AALE-treated worms and control worms (Figure 2C and 2D). It suggested that AALE improved the motility of \u003cem\u003eC. elegans\u003c/em\u003e without change of its body size\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThe change of body size of \u003cem\u003eC. elegans\u003c/em\u003e is usually associated with the toxicity in their diets\u0026nbsp;(Peixoto et al., 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAALE treatment significantly declined the lipofuscin accumulation level of worms by 11.31%, 16.18%, and 23.38% at doses of 60, 240, 360 \u0026mu;g/mL, respectively (Figure 2E and 2F). The reduction of lipofuscin accumulation delays the aging process of \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003e(Yu et al., 2023). These results demonstrated AALE significantly prolonged the youthfulness and promoted healthiness in aging of \u003cem\u003eC. elegans\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eC. elegans\u003c/em\u003e, their low resistance against external stress sharply declines their lifespan\u0026nbsp;(Monickaraj et al., 2013). Most of the observed lifespan extension phenotypes are associated with increased stress resistance such as thermal and oxidative stresses (Denzel et al., 2019).\u0026nbsp;As shown in Figure 3A, AALE treatments prolonged the lifespan of worms under oxidative stress induced by 1 mM hydrogen peroxide (\u003cem\u003ep\u0026lt;\u003c/em\u003e0.01) with increases of mean lifespan by 9.76%, 17.35%, and 13.80% at concentrations of 60, 240, and 360 \u0026mu;g/mL, respectively. In the thermo-resistance at 37℃, the mean lifespans of worms treated with 60, 240, and 360 \u0026mu;g/mL of AALE were increased by 8.84%, 16.65%, and 11.26%, respectively (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01) (Figure 3B). Similar to the survival rates without induced stress (Figure 1), the survival rate of worm treated with 240 \u0026mu;g/mL of AALE was the highest, while the rate of worm treated with 360 \u0026mu;g/mL of AALE which was the highest dosage used in this study was the second among the four group. Therefore, based on the results of assays, concentration of 240 \u0026mu;g/mL AALE was selected and used in the subsequent experiments.\u003c/p\u003e\n\u003cp\u003e3.2 AALE prolonged lifespan by activating transcription factor DAF-16/FOXO and IIS pathway\u003c/p\u003e\n\u003cp\u003eThe mammalian FOXO (Forkhead box O transcription factor) orthologue DAF-16 was reported to mediate longevity, lipogenesis, heat shock survival and oxidative stress responses in \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003e(Tia et al., 2018).\u0026nbsp;The nuclear localizations of DAF-16 in TJ356 strains treated with and without AALE were examined. The nuclear proportion of DAF-16 was enhanced from 19.69% to 53.13% (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001), while the fraction in cytoplasm declined from 48.21% to 17.71% (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) after the strain treated with 240 \u0026mu;g/mL AALE compared with the strain in control group (Figure 4A and 4B). The mRNA expression levels of \u003cem\u003edaf-16\u003c/em\u003e and its downstream target genes, \u003cem\u003esod-3\u003c/em\u003e (superoxide dismutase) and \u003cem\u003ehsp-16.2\u003c/em\u003e (heat shock protein), were obviously up-regulated in AALE- treated worms (Figure 4C, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05). In \u003cem\u003eC. elegans\u003c/em\u003e, the heat shock proteins (HSPs) are closely associated with thermo-tolerance and can serve as a biomarker of aging\u0026nbsp;(Lund et al., 2002). It is in agreement with our results that AALE-treated worms exhibited higher fluorescence intensity of HSP-16.2::GFP than that in control group. The expression levels of SOD-3::GFP and HSP-16.2::GFP were significantly improved by 24.37% and 19.31% in CF1553 and CL2070 strains treated with AALE, respectively (Figure 4D and 4E, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01). Therefore, it\u0026nbsp;indicated that the AALE-mediated longevity promotion was also dependent on the activation of DAF-16.\u003c/p\u003e\n\u003cp\u003eIt is well-known that the insulin/IGF-1 signaling (IIS) pathway is highly conserved and involved in metabolism, growth, development and longevity\u0026nbsp;(Murphy \u0026amp; Hu, 2013). In \u003cem\u003eC. elegans\u003c/em\u003e, the insulin-like receptor DAF-2 signals through the phosphatidylinositol 3-kinase/Akt kinase pathway to regulate of DAF-16/FOXO\u0026nbsp;(Lin et al., 2001).\u0026nbsp;Considering that AALE extended the lifespan by activating DAF-16 which is a central transcription factor of IIS pathway, AALE might extend the lifespan through the IIS pathway\u0026nbsp;(Murphy et al., 2003). Our study showed that AALE failed to extend the lifespans of the loss of function mutants including \u003cem\u003edaf-2\u003c/em\u003e, \u003cem\u003eage-1\u003c/em\u003e, \u003cem\u003esgk-1\u003c/em\u003e, \u003cem\u003edaf-16\u003c/em\u003e, \u003cem\u003eskn-1\u003c/em\u003e, and \u003cem\u003ehsf-1\u0026nbsp;\u003c/em\u003emutants(Figure 5A-5F, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05). It was confirmed that the IIS pathway was crucial to AALE-mediated lifespan for \u003cem\u003eC. elegans\u003c/em\u003e. Therefore, AALE extended the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e by regulating the IIS pathway and activating DAF-16/FOXO. This is consistent with a previous finding that flavonoid-rich \u003cem\u003eGastrodia elata\u003c/em\u003e extract extended lifespan and reduced oxidative stress by regulating the IIS pathway as well\u0026nbsp;(Shi et al., 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.3 Differential expressed genes identification and functional distribution\u003c/p\u003e\n\u003cp\u003eRNA-sequencing analysis was conducted on the 3\u003csup\u003erd\u003c/sup\u003e day of N2 worms treated with or without AALE. As shown in Figure 6A, there were 218 upregulated and 254 downregulated genes based on the criteria of fold change \u0026ge; 2 and\u003cem\u003e\u0026nbsp;p\u003c/em\u003e \u0026le; 0.05. Moreover, annotations and enrichment of GO\u0026nbsp;(gene ontology) pathway were analyzed based on the differentially expressed genes (DEGs) of AALE-treated worms. The results showed that molecular functions primarily include transporter activity, binding, and catalytic activity. The cellular components mainly consist of membrane part, cell part, organelle, membrane, and extracellular regions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe biological processes primarily involved in response to stimulus, cellular process, multi-organism process, immune system process, metabolic process, biological regulation, and developmental process (Figure 6B). The GO enrichment analysis of DEGs was conducted by Goatools and the enrichment results of top 25 are shown according to the degree of significance (Figure 6C, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). It exhibited that the DEGs in AALE-treated worms were mainly enriched in regulation of mitogen-activated protein kinase (MAPK) cascade. The MAPK cascade includes the responses to oxidative stress, cell maturation, developmental maturation, immune reaction, and stress. All of them involve in the regulation of aging, stress resistance, and development of\u003cem\u003e\u0026nbsp;C. elegans\u003c/em\u003e. In \u003cem\u003eC. elegans\u003c/em\u003e, MAPK cascade regulates cellular adaptive response to environmental changes and SKN-1 has been considered as a key factor for the p38 MAPK pathway to exert stress-resistance function\u0026nbsp;(Okuyama et al., 2010). It was found that AALE failed to extend the lifespan of \u003cem\u003eskn-1\u003c/em\u003e null mutants, suggesting that the lifespan extension effect of AALE also relied on \u003cem\u003eskn-1\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, the above results further confirmed that AALE promoted longevity mainly by regulating the MAPK pathway. It was further supported the capability of AALE in enhancing lifespan, healthy aging, and stress resistance of \u003cem\u003eC. elegans\u003c/em\u003e by the molecular genetic results.\u003c/p\u003e\n\u003cp\u003e3.4 KEGG analysis and validation of DEGs\u003c/p\u003e\n\u003cp\u003eTo systematically analyze gene function and explore the relationship between genomic information and functional information, KEGG function annotation was performed on DEGs. The results showed that DEGs of AALE-treated worms were mainly related to lipid and amino acid metabolism, signal transduction, transport and catabolism, aging, nervous and immune system, neurodegenerative disease, and cancer (Figure 7A). Furthermore, the results of KEGG enrichment analysis revealed that DEGs were mainly enriched in the lysosome, peroxisome proliferator-activated receptor (PPAR) signaling pathway, fatty acid degradation, longevity regulation pathway, FOXO signaling pathway, autophagy, and insulin resistance (Figure 7B). The results were in accordance with that AALE activated the DAF-16 and regulated the IIS pathway.\u003c/p\u003e\n\u003cp\u003eFurthermore, ten differentially expressed genes were selected to measure their expression levels by qPCR to support the RNA-seq results. The results showed that AALE significantly increased the expressions of \u003cem\u003egst-38\u0026nbsp;\u003c/em\u003e(Glutathione S-Transferase), \u003cem\u003eugt-43\u0026nbsp;\u003c/em\u003e(UDP-Glucuronosyl Transferase), \u003cem\u003eabf-2\u0026nbsp;\u003c/em\u003e(Antibacterial factor-related peptide 2), \u003cem\u003eclec-63\u0026nbsp;\u003c/em\u003e(C-type LECtin), and \u003cem\u003elys-1\u003c/em\u003e(Lysozyme-like protein 1) (Figure 7C, \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05). AALE decreased the expressions of \u003cem\u003eacs-18\u0026nbsp;\u003c/em\u003e(AMP-binding domain-containing protein), \u003cem\u003eins-30\u0026nbsp;\u003c/em\u003e(INSulin related), \u003cem\u003ecomt-4\u003c/em\u003e (Catechol-O-Methyl Transferase family), \u003cem\u003egpx-3\u0026nbsp;\u003c/em\u003e(Glutathione peroxidase 3), and \u003cem\u003efar-4\u0026nbsp;\u003c/em\u003e(Fatty Acid/Retinol binding protein)(Figure 7C, \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05). These findings were in agreement with the RNA-seq results.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In \u003cem\u003eC. elegans\u003c/em\u003e, \u003cem\u003egst-38\u003c/em\u003e and \u003cem\u003eugt-43\u003c/em\u003e are related to glutathione metabolism and phase II detoxification, respectively\u0026nbsp;(Asif et al., 2024). Thus, AALE significantly upregulated the expression of \u003cem\u003egst-38\u003c/em\u003e and \u003cem\u003eugt-43\u003c/em\u003e to enhance the anti-oxidant activity and oxidative stress resistance of worms. The genes \u003cem\u003elys-1\u003c/em\u003e, \u003cem\u003eabf-2\u003c/em\u003e, and \u003cem\u003eclec-63\u003c/em\u003e which were remarkably upregulated in AALE-treated worms contribute to the antimicrobial activity and immune response as well\u0026nbsp;(Shao \u0026amp; Wang, 2020; Yang et al., 2023). A previous study suggested that A\u003cem\u003ertemisia argyi\u003c/em\u003e exerted antimicrobial effect in goldfish and \u003cem\u003eCandida albicans\u0026nbsp;\u003c/em\u003emodels\u0026nbsp;(Shi et al., 2017). Our results showed that AALE significantly downregulated \u003cem\u003ecomt-4\u003c/em\u003e, which might be beneficial to the enhancement of the body bending and pharyngeal pumping rates in AALE-treated worms. Similar to the results, it was reported that inhibition of \u003cem\u003ecomt-4\u003c/em\u003e could decrease the dopamine levels and promote locomotory behaviors of nematodes\u0026nbsp;(Rodr\u0026iacute;guez-Ramos et al., 2017). Moreover, the significantly decreased expressions of \u003cem\u003eins-3\u003c/em\u003e, \u003cem\u003eacs-18\u003c/em\u003e, and \u003cem\u003efar-4\u003c/em\u003e by AALE were associated with insulin secretion and fatty acid metabolism (Balasubramanian et al., 2016). Based on the results, it was suggested that AALE may mediate glycolipid metabolism in nematodes by influencing the activity of key metabolic enzymes and synthesis, catabolism, and oxidation of fatty acids.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eAALE was rich in a variety of flavonoids and significantly improved the lifespan and fitness of \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003evia IIS and FOXO signaling pathway. The MAPK was enriched in AALE treated worms to response against oxidative stress, cell maturation, developmental maturation, immune reaction, and stress. KEGG analysis showed that the changes of genes in treated worms were also related to the lipid and amino acid metabolism, signal transduction, transport and catabolism, aging, nervous and immune system, neurodegenerative disease, and cancer. The results of qPCR indicated that AALE significantly upregulated the expression of genes related to the antioxidant activity and oxidative stress resistance of worms. Because of the high conservation of these pathways between mammals and \u003cem\u003eC. elegans\u003c/em\u003e, our findings could suggest that \u003cem\u003eArtemisia argyi\u0026nbsp;\u003c/em\u003ealso\u003cem\u003e\u0026nbsp;\u003c/em\u003ehas a great activity in promoting healthy aging for humans.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by Key Research and Development Project of Hubei Province (2022BCE057、2022BEC031), Hubei Engineering Research Center for Specialty Flowers Biological Breeding (2023ZD006), and Doctoral Research Initiation Fund of Jingchu University of Technology (YY202403).\u003c/p\u003e\n\u003cp\u003eAuthor contribution\u003c/p\u003e\n\u003cp\u003eJinsong Wang: Conceptualization, Data curation, Formal analysis, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Hailin Cui: Data curation, Formal analysis. Yan Xu: Data curation, Conceptualization. Shuyou Shang: Formal analysis. Yuanxin Miao: Conceptualization, Methodology, Data curation. Rong Li: Project administration, Resources, Writing \u0026ndash; review \u0026amp; editing. Zhimin Xu: Conceptualization, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eEthical Statement\u003c/p\u003e\n\u003cp\u003eThe experiment of this study was in accordance with the State Code of Practice for the Care and Use of Animals for Scientific Purposes of China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCampisi, J. et al. From discoveries in ageing research to therapeutics for healthy ageing. Nature 571, 183\u0026ndash;192 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, R. et al. 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Artemisia argyi exhibits anti-aging effects through decreasing the senescence in aging stem cells. Aging-Us 14, 6187\u0026ndash;6201 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, Y. et al. Artemisia argyi extract exerts antioxidant properties and extends the lifespan of Drosophila melanogaster. J. Sci. Food Agric. 104, 3926\u0026ndash;3935 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe, Y. et al. Potential of Caenorhabditis elegans as an antiaging evaluation model for dietary phytochemicals: A review. Compr. Rev. Food Sci. Food Saf. 19, 3084\u0026ndash;3105 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLapierre, L. R. \u0026amp; Hansen, M. Lessons from C. elegans: signaling pathways for longevity. Trends Endocrinol. Metlab. 23, 637\u0026ndash;644 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, H. \u0026amp; Lee, S. V. 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Sci. 56, 281\u0026ndash;287 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKenyon, C. The plasticity of Aging: Insights from Long-lived Mutants. Cell, 120, 449\u0026ndash;460 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin, K. et al. Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling. Nat. Genet. 28, 139\u0026ndash;145 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsif, M. Z. et al. Role of UDP-Glycosyltransferase (ugt) Genes in Detoxification and Glycosylation of 1-Hydroxyphenazine (1-HP) in Caenorhabditis elegans. Chem. Res. Toxicol. 37, 590\u0026ndash;599 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu, L. et al. Insecticidal activity and mechanism of cinnamaldehyde in C. elegans. Fitoterapia, 146, 104687 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShao, H. \u0026amp; Wang, D. Long-term and low-dose exposure to nanopolystyrene induces a protective strategy to maintain functional state of intestine barrier in nematode Caenorhabditis elegans. Environ. Pollut. 258, 113649 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, W. H. et al. Impaired immune response and barrier function in GSPD-1-deficient C. elegans infected with Klebsiella pneumoniae. Curr. Res. Microb. Sci. 4, 100181 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi, G. X. et al. Activity of essential oil extracted from Artemisia argyi in inducing apoptosis of Candida albicans. Zhongguo Zhong Yao Za Zhi. 42, 3572\u0026ndash;3577 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-Ramos, \u0026Aacute;. et al. Impaired Dopamine-Dependent Locomotory Behavior of C. elegans Neuroligin Mutants Depends on the Catechol-O-Methyltransferase COMT-4. Behav. 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Vitexin and Isovitexin Act through Inhibition of Insulin Receptor to Promote Longevity and Fitness in Caenorhabditis elegans. Mol. Nutr. Food Res. 66, e2100845 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan, Q. et al. Hypotaurine promotes longevity and stress tolerance via the stress response factors DAF-16/FOXO and SKN-1/NRF2 in Caenorhabditis elegans. Food Funct. 11, 347\u0026ndash;357 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, D. K. et al. 4-Hydroxybenzoic acid-mediated lifespan extension in Caenorhabditis elegans. J. Funct. Foods. 7, 630\u0026ndash;640 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXue, D. et al. Deoxynivalenol triggers porcine intestinal tight junction disorder through hijacking SLC5A1 and PGC1α-mediated mitochondrial function. Food Chem. Toxicol. 163, 112921 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Major flavonoids in AALE identified and quantified by HPLC-MS/MS\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"642\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27.1028%;\"\u003e\n \u003cp\u003eCompounds\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.757%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Mass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33.6449%;\"\u003e\n \u003cp\u003eMain fragments (m/z)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4953%;\"\u003e\n \u003cp\u003eConcentration (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27.1028%;\"\u003e\n \u003cp\u003eL-Epicatechin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.757%;\"\u003e\n \u003cp\u003e291.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33.6449%;\"\u003e\n \u003cp\u003e139.0803, 122.8801\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4953%;\"\u003e\n \u003cp\u003e39.16 \u0026plusmn; 2.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27.1028%;\"\u003e\n \u003cp\u003eCatechin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.757%;\"\u003e\n \u003cp\u003e291.0790\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33.6449%;\"\u003e\n \u003cp\u003e139.0606, 122.8931\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4953%;\"\u003e\n \u003cp\u003e21.70 \u0026plusmn; 1.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27.1028%;\"\u003e\n \u003cp\u003eNaringenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.757%;\"\u003e\n \u003cp\u003e273.0690\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33.6449%;\"\u003e\n \u003cp\u003e153.0639, 147.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4953%;\"\u003e\n \u003cp\u003e7.20 \u0026plusmn; 0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27.1028%;\"\u003e\n \u003cp\u003eApigenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.757%;\"\u003e\n \u003cp\u003e271.0530\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33.6449%;\"\u003e\n \u003cp\u003e215.0778, 243.1533\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4953%;\"\u003e\n \u003cp\u003e25.71 \u0026plusmn; 2.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27.1028%;\"\u003e\n \u003cp\u003eLuteolin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.757%;\"\u003e\n \u003cp\u003e287.1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33.6449%;\"\u003e\n \u003cp\u003e287.0799, 186.963\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4953%;\"\u003e\n \u003cp\u003e15.01 \u0026plusmn; 1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27.1028%;\"\u003e\n \u003cp\u003eKaempferol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.757%;\"\u003e\n \u003cp\u003e287.0480\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33.6449%;\"\u003e\n \u003cp\u003e286.9807, 165.141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4953%;\"\u003e\n \u003cp\u003e52.84 \u0026plusmn; 4.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27.1028%;\"\u003e\n \u003cp\u003eQuercetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.757%;\"\u003e\n \u003cp\u003e303.0430\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33.6449%;\"\u003e\n \u003cp\u003e303.0053, 257.0263\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4953%;\"\u003e\n \u003cp\u003e16.55 \u0026plusmn; 1.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27.1028%;\"\u003e\n \u003cp\u003eFormononetin (4\u0026apos;-O-methyldaidzein)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.757%;\"\u003e\n \u003cp\u003e269.0740\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33.6449%;\"\u003e\n \u003cp\u003e269.1, 105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4953%;\"\u003e\n \u003cp\u003e8.60 \u0026plusmn; 0.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 2. Effect of AALE\u0026nbsp;on the lifespan of wild-type (N2) worms at 20\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"636\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.2704%;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.956%;\"\u003e\n \u003cp\u003eMean lifespan\u003c/p\u003e\n \u003cp\u003e(days \u0026plusmn; SEM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.9811%;\"\u003e\n \u003cp\u003ePercentage\u003c/p\u003e\n \u003cp\u003echange\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.5975%;\"\u003e\n \u003cp\u003eNumber of\u003c/p\u003e\n \u003cp\u003eworms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.195%;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.2704%;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.956%;\"\u003e\n \u003cp\u003e17.25 \u0026plusmn; 0.46\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.9811%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.5975%;\"\u003e\n \u003cp\u003e167\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.195%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.2704%;\"\u003e\n \u003cp\u003e60 \u0026mu;g/mL AALE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.956%;\"\u003e\n \u003cp\u003e19.33 \u0026plusmn; 0.50\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.9811%;\"\u003e\n \u003cp\u003e12.06%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.5975%;\"\u003e\n \u003cp\u003e161\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.195%;\"\u003e\n \u003cp\u003e0.0013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.2704%;\"\u003e\n \u003cp\u003e240 \u0026mu;g/mL AALE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.956%;\"\u003e\n \u003cp\u003e21.25 \u0026plusmn; 0.54\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.9811%;\"\u003e\n \u003cp\u003e23.19%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.5975%;\"\u003e\n \u003cp\u003e158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.195%;\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.2704%;\"\u003e\n \u003cp\u003e360 \u0026mu;g/mL AALE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.956%;\"\u003e\n \u003cp\u003e20.54 \u0026plusmn; 0.52\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.9811%;\"\u003e\n \u003cp\u003e19.07%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.5975%;\"\u003e\n \u003cp\u003e158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.195%;\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eDifferent letters (a, b, and c) indicated a significant difference between two groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e value was determined by log-rank test using the Kaplan\u0026ndash;Meier survival analysis.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-science-of-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjscifood","sideBox":"Learn more about [npj Science of Food](http://www.nature.com/npjscifood/)","snPcode":"41538","submissionUrl":"https://submission.springernature.com/new-submission/41538/3","title":"npj Science of Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Artemisia argyi, C. elegans, aging, stress resistance, flavonoids","lastPublishedDoi":"10.21203/rs.3.rs-5028259/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5028259/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Artemisia argyi Lévl. et Vant. (A. argyi) leaf possesses various health promoting functions contributed by its main bioactive flavonoids.In this study, the anti-aging effect and mechanism of Artemisia argyi leaf extract (AALE) were identified using Caenorhabditis elegans (C. elegans) as a model. The results showed that the AALE promoted the lifespan and stress resistance of C. elegans. Meanwhile, the AALE treated C. elegans had high physical activity and low lipofuscin accumulation without negative impact on body size. It was found that the AALE boosted the expression of oxidative stress-related proteins by regulating the insulin/ IGF-1 signaling (IIS) pathway, which then activated the transcription factors DAF-16/FOXO. The results of RNA-sequence analysis indicated that the changes of genes in nematodes treated with AALE were associated with the responses against oxidative stress, cell maturation, and immune reaction, and stress. The qPCR results indicated that the AALE obviously up-regulated the expression of genes related to antioxidant activity and lipid and carbohydrate metabolisms. These findings reveal the mechanism of health prompting function of Artemisia argyi leaf at molecular genetic level. The positive results obtained from the highly conserved signaling pathways of C. elegans model suggest that Artemisia argyi leaf could have the robust benefits for improving healthy aging as well as preventing aging-related diseases in the human body.","manuscriptTitle":"Molecular mechanism of culinary herb Artemisia argyi in promoting lifespan and stress tolerance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-22 13:27:33","doi":"10.21203/rs.3.rs-5028259/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-03T00:47:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-02T23:27:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-25T05:36:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"304635345577810111787784893588198076012","date":"2024-10-15T04:34:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104046115277267866384402959868672283394","date":"2024-10-12T16:44:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312345638176538025706004054413115473225","date":"2024-10-11T03:20:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-10T18:57:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-01T03:37:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-09T07:27:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Science of Food","date":"2024-09-04T03:17:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-science-of-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjscifood","sideBox":"Learn more about [npj Science of Food](http://www.nature.com/npjscifood/)","snPcode":"41538","submissionUrl":"https://submission.springernature.com/new-submission/41538/3","title":"npj Science of Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"426ef874-3c19-493e-9d94-1de3a31d5e8b","owner":[],"postedDate":"November 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39740130,"name":"Biological sciences/Drug discovery"},{"id":39740132,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2024-12-30T16:02:11+00:00","versionOfRecord":{"articleIdentity":"rs-5028259","link":"https://doi.org/10.1038/s41538-024-00358-8","journal":{"identity":"npj-science-of-food","isVorOnly":false,"title":"npj Science of Food"},"publishedOn":"2024-12-24 15:57:39","publishedOnDateReadable":"December 24th, 2024"},"versionCreatedAt":"2024-11-22 13:27:33","video":"","vorDoi":"10.1038/s41538-024-00358-8","vorDoiUrl":"https://doi.org/10.1038/s41538-024-00358-8","workflowStages":[]},"version":"v1","identity":"rs-5028259","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5028259","identity":"rs-5028259","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}