Gastrodin extends the lifespan of Caenorhabditis elegans via the DAF-16/FOXO signaling pathway and autophagy

Gastrodin extends the lifespan of Caenorhabditis elegans via the DAF-16/FOXO signaling pathway and autophagy | 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 Research Article Gastrodin extends the lifespan of Caenorhabditis elegans via the DAF-16/FOXO signaling pathway and autophagy Bo Li, Shan Li, Haoling Chen, Dan Wu, Xingwang Cao, Mingyue Yao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7395169/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2025 Read the published version in Functional & Integrative Genomics → Version 1 posted 13 You are reading this latest preprint version Abstract Objective The progression of age-related pathologies is strongly linked to biological aging. Identifying natural anti-aging agents to mitigate disease onset and development holds substantial therapeutic value.The natural compound Gastrodin (Gas) demonstrates promising effects in retarding aging.This study aims to explore the effects of Gas on the lifespan and antioxidant capacity of Caenorhabditis elegans (C. elegans) . Additionally, it seeks to elucidate the possible mechanisms. Methods Initially, Gas was assessed for its influence on C. elegans lifespan, mobility, lipofuscin accumulation, and oxidative stress responses. Subsequent analyses focused on Gas’s modulation of the insulin/IGF-1 pathway, mitochondrial activity, autophagic processes, and gene expression to uncover its lifespan-extending mechanisms. Results Gas induced a dose-dependent lifespan extension in C. elegans , peaking at 400 µM with a 17.3% increase in longevity. Gas enhanced C. elegans mobility while suppressing age-related lipofuscin deposition.Additionally, Gas lowered ROS levels and elevated antioxidant enzyme activity in C. elegans .Mechanistic studies revealed that Gas’s anti-aging effects rely on transcription factors (DAF-16, SKN-1, HSF-1) and bolster stress resistance via HSPs activation and autophagy induction. Conclusion This study reveals the potential of Gas in extending the lifespan of C. elegans , emphasizes its mechanism of action by regulating antioxidant capacity, heat stress response, and autophagy pathway, and provides experimental evidence that supports the development of Gas as a candidate compound for lifespan extension. Gastrodin Caenorhabditis elegans Anti-aging DAF-16/FOXO signaling pathway Autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Aging is a multi-dimensional progressive decline of physiological functions that occurs over time, characterized by accumulation, universality, and gradualness. During the aging process, the body’s functions and metabolism decline, which is closely related to human diseases. Aging is a major risk factor for most chronic diseases, including cardiovascular and cerebrovascular diseases, osteoporosis, cancer, and neurodegenerative diseases [ 1 – 4 ] . As a complex biological process, aging involves the functional decline of multiple organs and systems, shows significant individual differences and organ specificity [ 5 ] , and has become a major challenge in the global public health field. With the further intensification of population aging, the incidence of age-related diseases has sharply increased, placing a heavy burden on society and economic development. Therefore, slowing down aging or promoting “healthy aging” has become an urgent problem to be solved in today’s society. As a model organism for studying the aging process and diseases, C. elegans has the advantages of a fast life cycle, easy cultivation, and well-established genetic pathways [ 6 ] . Since the 1970s, it has become an important tool in molecular biology research [ 7 ] . Not only is it easy to cultivate in the laboratory, but it also has a high genetic similarity to humans and detailed gene sequencing data, and is widely used in many fields such as genetics, gene function, aging, and drug screening [ 8 ] . In recent years, great progress has been made in aging and anti-aging research using C. elegans as an animal model, which helps people further understand the aging mechanism and provides effective strategies and methods for anti-aging. With the improvement of people’s living quality, the public’s choice of anti-aging substances gradually tends to natural active ingredients. Therefore, exploring and studying the anti-aging activity of natural active ingredients is of great significance and value for alleviating health problems caused by aging. Gas, also known as 4-hydroxymethylphenyl-beta-D-glucopyranoside hemihydrate, is the main component of Gastrodia elata Blume tubers [ 9 ] . Contemporary pharmacological studies indicate that Gas exhibits significant antioxidant, anti-inflammatory, anti-apoptotic, and antiviral activities, demonstrating therapeutic efficacy in neurological and cardiovascular disorders [ 10 – 12 ] . Recent studies have shown that Gas promotes autophagy and phagocytosis by activating the PPARα-TFEB/CD36 signaling pathway, significantly reducing oxidative stress-induced retinal pigment epithelial damage [ 13 ] . In addition, Gas enhances mitophagy by regulating the PINK1/Parkin pathway, effectively alleviating myocardial ischemia-reperfusion injury and playing a cardioprotective role [ 14 ] . However, the role of Gas in lifespan extension and its underlying molecular mechanisms remain incompletely understood. Therefore, this study aims to investigate the regulatory effects of Gas on the lifespan of C. elegans and elucidate its potential mechanisms, thereby providing new strategies for the development of lifespan-extending drugs. 2 Materials and Methods 2.1 Chemicals Gastrodin (Gas, ≥ 98% purity) was obtained from Shanghai Yuanye Bio-Technology (China). Additional reagents including 5-fluorodeoxyuridine (FUDR) and CM-H2DCFDA were acquired from Sigma-Aldrich (St. Louis, MO, USA). 2.2 Strains and Cultivation Methods of worms The Caenorhabditis Genetics Center (CGC; University of Minnesota, Minneapolis, MN) supplied all nematode strains used in this study. Standard cultivation conditions involved maintenance at 20°C with 60% relative humidity on nematode growth medium (NGM) seeded with E. coli OP50, unless otherwise indicated. The following C. elegans strains were utilized in this study:Wild-type N2;Mutant strains:CF1038 [daf-16(mu86) I],EU1 [skn-1(zu67) IV],PS3551 [hsf-1(sy441) I],RB754 [aak-2(ok524) X],RB759 [akt-1(ok525) V],VC204 [akt-2(ok393) X],CB4876 [clk-1(e2519) III];Transgenic reporter strains:CF1553 [sod-3::GFP + rol-6(su1006)],CL2166 [gst-4::GFP::NLS],SJ4100 [hsp-6::GFP],SJ4005 [hsp-4::GFP],SJ4058 [hsp-60::GFP + lin-15(+)],DA2123 [lgg-1::GFP::lgg-1 + rol-6(su1006)],TJ356 [daf-16::daf-16a/b::GFP + rol-6]. 2.3 Lifespan Assay All nematode strains underwent sequential cultivation across 2–3 generations using fresh NGM medium to prevent nutritional deprivation. Developmental-stage synchronized worms (late L4 larvae/young adults) were plated onto NGM supplemented with thermally inactivated OP50 (65°C for 30 min) and 40 µM FUDR (Sigma) to suppress offspring production.The initial transfer of synchronized worms to treatment plates marked experimental day 0.Non-motile specimens following platinum wire stimulation were classified as deceased. Vitality assessments were conducted daily until complete cohort mortality was achieved.Specimens were systematically transferred to fresh Gas-containing or control NGM plates at 48-hour intervals.Any organisms exhibiting plate migration or post-transfer hatching were excluded from longevity analysis. Each longevity trial was performed with ≥ 3 biological replicates.Experimental cohorts consisted of ≥ 60 nematodes per treatment condition. 2.4 Analysis of Aging-Related Phenotypes 2.4.1 Body Bending Behavior Test L1-stage worms were synchronized and maintained on NGM agar at 20°C under controlled incubation. Upon reaching L4 development, specimens were moved to treatment plates containing either Gas or vehicle control. At designated intervals (days 5/10 post-maturity), individuals were placed in aqueous suspension for 60-second acclimatization. Then, the frequency of body bends within 20 seconds was recorded under a microscope. One forward-backward movement of the body was counted as one bend. At least 30 worms were included in each experimental group. 2.4.2 Determination of Lipofuscin Initial specimen preparation followed identical protocols to the longevity study. Adult nematodes were sampled at days 5 and 10 post-maturation for fluorescence imaging (excitation: 360–370 nm; emission: 420–460 nm).Lipofuscin accumulation was assessed through mean fluorescence intensity measurements per specimen.Experimental cohorts contained ≥ 30 individuals per condition. 2.5 Oil Red O Staining Developmentally synchronized L1-stage nematodes were maintained on NGM agar until reaching young adult phase. Approximately 1000 specimens per condition were harvested from both treatment and control cohorts, subjected to sequential PBS rinses for complete OP50 elimination, and treated with Nile red staining reagent. Following 25-minute fixation in 4% paraformaldehyde, specimens underwent dual washes with PBS containing 1% Triton X-100. Nile red (5 mg/mL) was then applied under light-restricted conditions. Following 120-second incubation, triplicate washes with PBS-Triton solution were performed and and imaged using a Leica DFC7000T microscopy system. Image analysis was performed using ImageJ software. Each experiment was repeated three times, and at least 30 worms were used in each repetition. 2.6 Detection of Antioxidant Capacity and Reactive Oxygen Species (ROS) Level 2.6.1 Antioxidant Stress Experiment N2 strain nematodes were synchronized at the L1 larval stage and cultured on NGM agar at 20°C. Developmental-stage synchronized populations (late L4/young adult) were then transferred to Gas-treated or control NGM plates for oxidative stress assessment. At day 10 post-maturation, worms were exposed to 20 mM paraquat-containing NGM. Mortality was recorded at 24-hour intervals until complete cohort death occurred. Each experimental group contained ≥ 30 biological replicates, with three independent trials performed. 2.6.2 Determination of ROS Content Fourth-stage larval nematodes were cultured on treatment NGM agar supplemented with either Gas (400 µM), paraquat (20 mM), or N-acetyl-L-cysteine (1 mM), maintaining at 20°C for six days.Subsequently, specimens were harvested, subjected to triple M9 buffer washes, labeled with 50 µM H2DCF-DA fluorescent probe, and agitated at 35°C for one hour following manufacturer's protocol [ 15 ] . ROS levels were quantified via fluorescence microscopy. Three independent experimental replicates were performed, each comprising ≥ 30 nematodes. Statistical significance was assessed through two-tailed Student t-tests. 2.7 Heat Resistance Experiment Developmentally synchronized N2 strain nematodes at L1 stage were maintained on NGM agar under controlled 20°C incubation. Upon reaching late L4 or young adult developmental phases, specimens were allocated to Gas-treated or untreated NGM media. On the 10th day of adulthood, they were transferred to an incubator at 35°C. The death of worms was observed and counted every 2 hours until all worms died. Experimental groups contained ≥ 30 biological replicates. 2.8 Nuclear Localization In this experiment, TJ356 mutant worms, which specifically labeled DAF-16::GFP (zls356), were used. Fourth-stage larval TJ356 specimens were allocated to either Gas treatment (400 µM) or dual control conditions. Positive controls (TJ356 + OP50 on NGM) underwent 15-minute 35°C thermal stress, whereas negative controls were maintained at standard 20°C incubation. The localization of DAF-16:GFP was observed every hour under a fluorescence microscope. The green fluorescent nuclear-aggregated particles in TJ356 worms were regarded as the expression form of the DAF-16 gene in the nucleus. 2.9 Fluorescence Quantification The specimen preparation protocol for fluorescence analysis mirrored the procedures employed in the longevity assessment study. Worms strains CL2166, dvIs19[pAF15(gst-4:: GFP:: NLS)] and strain CF1553 [(pAD76) sod-3::GFP + rol6 (su1006)] were cultured until the late L4 stage or early adult stage, then transferred to a medium with or without 400 µM Gas and continued to be cultured at 20°C until the end of the observation time point. They were anesthetized with tetramisole hydrochloride (2 mM), and the expression of fluorescent proteins in different strains was observed under a fluorescence microscope. Three independent biological replicates were performed, with each treatment condition containing ≥ 30 nematodes. 2.10 Quantitative RT-PCR Detection Nematode total RNA was isolated with RNAiso Plus reagent (Takara, Japan), followed by cDNA synthesis employing the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). Quantitative real-time PCR amplifications were carried out using Power SYBR Green PCR Master Mix (Applied Biosystems) on a QuantStudio 6 Flex platform.Gene expression quantification was determined through the 2-ΔΔCT method, with normalization against the housekeeping gene cd c-42. The primers sequences are in the supplementary Materials. 2.11 Western Blot Experiment Nematode specimens were harvested following 6-day Gas treatment. Cellular proteins were isolated through ultrasonic homogenization, with protein quantification conducted via BCA assay (Beyotime Biotechnology).The experiment was performed according to a previous protocol [ 16 ] . Proteins were examined using antibodies specific for SQST − 1 (Abcam, Cambridge, UK, Cat# ab207305), LGG − 1 (Thermo Fisher Scientific, Waltham, MA, USA, Cat# PA5116410), and α - Tubulin (Abcam, Cambridge, UK, Cat# ab176560).Image analysis was performed using ImageJ software. 2.12 RNA Interference Gene silencing was achieved through RNA interference (RNAi) following established protocols [ 17 ] . HT115(DE3) Escherichia coli (Fire Laboratory) transformed with either control vector L4440 or dsRNA-producing constructs targeting atg-18 and bec-1 were used for feeding-based RNAi. Longevity analysis in RNAi-treated nematodes was conducted as described previously [ 18 ] . 2.13 Determination of Autophagy Content Autophagic activity in nematodes was assessed by quantifying GFP-labeled puncta associated with LGG-1-marked autophagosomes. The DA2123 strain exhibited cytoplasmic GFP-LGG1 fluorescence distribution across multiple tissues. The formation of autophagosome structures could be observed and quantified through the appearance of fluorescent dots, thereby evaluating the autophagy content in worms. 2.14 Statistical Methods Data were statistically analyzed with SPSS (v26.0) and GraphPad Prism (v7.0) analytical packages. Quantitative analyses of fluorescence signals, lipid deposits (Oil Red O), and protein levels were conducted via ImageJ (v1.8.0). The Kaplan-Meier method was employed for survival curve estimation and comparison. All quantitative measurements are expressed as mean ± SD, except where alternative representations are specified. Statistical analyses were performed using two-tailed Student's t-tests, with a predefined significance level of α = 0.05. 3 Results 3.1 Gas enhances longevity and ameliorates age-associated phenotypes in C. elegans To determine whether Gas can extend the lifespan of C. elegans , wild-type N2 worms were treated with different concentrations of Gas(Fig. 1 A). The experimental results showed that Gas at multiple concentrations could extend the lifespan of worms to varying degrees (Fig. 1 B). Among them, the 400 µM concentration was the most effective, with a lifespan extension rate of 17.3% (Fig. 1 C-D).The decline in locomotor ability is an important indicator of worms aging. Therefore, we investigated the effect of Gas on the locomotor ability of adult worms and found that Gas could significantly improve the locomotor ability of C. elegans on the 5th and 10th days (Fig. 1 E).Aging is accompanied by the production of free radicals, which oxidize fats and cause lysosomal degradation, leading to an increase in lysosomal digestion products and fat residues, and subsequently the accumulation of lipofuscin in worms. Thus, lipofuscin is a characteristic of the onset of cell aging, indicating a dysfunction of the intracellular clearance system and is a widely recognized aging marker [ 19 ] . Experimental data demonstrated Gas-treated nematodes exhibited reduced lipofuscin autofluorescence at both day 5 and 10 post-treatment,indicating substantial suppression of age-pigment deposition. (Fig. 1 F-G). 3.2 Gas Augments Oxidative Stress Resistance in C. elegans Age-related deterioration of antioxidant defenses coupled with reactive oxygen ROS accumulation contributes to progressive tissue dysfunction and organ pathology [ 20 ] . The C. elegans Nrf2 ortholog SKN-1, constitutively expressed during development, serves as a master regulator of oxidative stress responses [ 21 ] . Gas administration increased paraquat (20 mM) resistance in wild-type N2 strains by 24.8% relative to controls (Fig. 2A). This protective effect was abolished in skn-1 loss-of-function mutants (Fig. 2B).Environmental stressors induce persistent ROS generation. Elevated ROS levels initiate oxidative cascades, increasing cytotoxic byproducts (e.g., MDA) that compromise cellular integrity [ 22 ] . Quantitative analysis revealed Gas-mediated ROS reduction in day-6 adult N2 populations (Fig. 2C-D). The oxidative stress response in C. elegans is primarily mediated through the SKN-1/Nrf2-regulated antioxidant enzymes GST-4 and SOD-3 [ 23 ] , which function as critical cellular defense mechanisms against ROS. GST-4, a glutathione S-transferase, exhibits dual protective functions by both neutralizing lipid hydroperoxides through its glutathione peroxidase activity and detoxifying cytotoxic lipid peroxidation end-products such as 4-hydroxy-2-trans-nonenal (4-HNE) [ 24 ] . Meanwhile, the metalloenzyme SOD-3 localizes to mitochondrial membranes where it catalyzes the dismutation of superoxide radicals into hydrogen peroxide and molecular oxygen, serving as the primary scavenger of mitochondrial ROS [ 25 ] . Experimental observations revealed that Gas treatment significantly enhanced GFP reporter expression in both CL2166 [gst-4::GFP] and CF1553 [sod-3::GFP] transgenic strains (Fig. 2E-H), demonstrating a potent activation of these antioxidant pathways. These collective findings provide compelling evidence that Gas extends C. elegans lifespan through SKN-1-dependent transcriptional activation of antioxidant defense systems, resulting in improved oxidative stress resistance and reduced accumulation of oxidative damage markers. The mechanistic link between Gas exposure, SKN-1 activation, and enhanced expression/activity of downstream antioxidant enzymes establishes a clear pathway through which this compound exerts its longevity-promoting effects.Figure. 2 Gas enhances the antioxidant capacity of C. elegans . (A) Survival rate of wild-type N2 worms cultured in 20 mM paraquat after 400 µM Gas treatment.After being raised with Gas, the lifespan extension rate of C. elegans was 24.8%. (p < 0.001, log-rank test); (B) Survival curves of EU1/ skn-1 (zu67) IV worms treated with or without 400 µM Gas; (C-D) ROS levels in wild-type N2: with 400 µM Gas as the experimental group, 20 mM paraquat as the model group, and 1 mM NAC as the positive control; (E-F) Quantitative analysis of fluorescence intensity in CL2166 mutants after feeding at 20°C for 5 days with or without 400 µM Gas; (G-H) 400 µM Gas treatment significantly increases fluorescence intensity in CF1553 mutants. 3.3 Gas Enhances the Resistance of C. elegans to High-Temperature Stress High temperature can damage cell tissues, such as neuronal degeneration and heat-induced cell death, thereby increasing the content of ROS. Excessive ROS can lead to cell necrosis or apoptosis, thus shortening the lifespan [ 26 ] . Therefore, strong resistance to high-temperature stress plays an important role in the process of delaying aging. In the experiment, it was found that at an environmental temperature of 35°C, Gas could significantly extend the survival time of C. elegans (Fig. 3 A). The heat stress response is a physiological defense response commonly found in the biological world. Under the stimulation of environmental factors, the body can induce the synthesis of a group of highly conserved stress proteins, namely heat shock proteins (HSPs). HSPs facilitate proper polypeptide folding into native three-dimensional structures and mediate the renaturation of misfolded proteins, thus contributing to ROS homeostasis in plant embryos [ 27 ] . The transcription factor HSF-1 serves as a master regulator of proteostasis by controlling HSPs expression, thereby coordinating protein quality control mechanisms including synthesis, folding, and degradation [ 28 ] . Genetic evidence demonstrated the essential role of HSF-1 in Gas-mediated longevity, as hsf-1 null mutants failed to exhibit lifespan extension following treatment (Fig. 3 B). Fluorescence microscopy analysis revealed Overall upregulation of specific HSP family members, with SJ4005 [hsp-4::GFP] ,SJ4100 [hsp-6::GFP] and SJ4058 [hsp-60::GFP] transgenic strains showing markedly increased reporter expression (Fig. 3 C-H). In addition, Gas also increased the mRNA levels of the downstream genes hsp-4 , hsp-60 , and hsp-12.6 of hsf-1 (Fig. 3 I). Therefore, Gas can improve the resistance of C. elegans to high-temperature stress by promoting the high expression of hsf-1-related genes in C. elegans and activating more HSPs. 3.4 Gas Requires FOXO/DAF-16 to Extend Lifespan DAF-16 is the only direct ortholog of the forkhead box O (FOXO) transcription factor in C. elegans , which is crucial for maintaining cell homeostasis and is required for the reported lifespan extension in the daf-2 mutant strain [ 29 ] . Its pleiotropic effects include basic physiological processes such as metabolic regulation, development, aging, immune response, and cell stress resistance [ 30 ] . Within the insulin/IGF-1 signaling (IIS) cascade, AKT-1/2 kinases function as upstream regulators of the FOXO transcription factor DAF-16 [ 31 ] . To establish DAF-16 dependence, we examined Gas-mediated longevity effects in loss-of-function mutants. Genetic ablation of daf-16 (mu86) , akt-1 , or akt-2 completely abolished the lifespan-extending effects of Gas (Fig. 4 A-C), demonstrating the essential role of DAF-16 in Gas-induced longevity. Stress stimuli trigger nuclear translocation of DAF-16/FOXO, thereby activating cytoprotective gene networks [ 32 ] . We next assessed Gas-induced subcellular redistribution of DAF-16. Gas treatment markedly enhanced nuclear DAF-16::GFP accumulation in TJ356 transgenics (Fig. 4 D), concomitant with upregulated expression of canonical DAF-16 targets ( sod-3, dod-3, ctl-1, ctl-3 )(Fig. 4 E༉. These conclusions indicate that Gas requires the participation of the DAF-16/FOXO signaling pathway to extend the lifespan of C. elegans . 3.5 Gas Extends Lifespan through Mitochondrial and Fat Metabolism Pathways Previous studies have established that modulation of mitochondrial electron transport chain components can profoundly influence longevity in C. elegans [ 33 ] . To investigate potential mitochondrial involvement in Gas-mediated lifespan extension, we examined its effects in respiratory-deficient mutants. Notably, Gas failed to prolong survival in either isp-1 mutants (deficient in complex III function) or clk-1 mutants (impaired in ubiquinone biosynthesis) (Fig. 5A-B), suggesting these mitochondrial components are essential for Gas's longevity-promoting effects.Meanwhile, in recent years, intervening in the lifespan of C. elegans through lipid signaling molecules has become a hot topic in the study of anti-aging. The aak-2 , as one of the energy regulators in C. elegans , is involved in worms fat hydrolysis, fatty acid oxidation, and polyunsaturated fatty acid synthesis [ 34 – 36 ] . The results showed that Gas could not extend the lifespan of C. elegans with the aak-2 gene mutation (Fig. 5C), and it increased the mRNA content of the mitochondrial-related transcription factors xbp-1 and isp-1 (Fig. 5F), indicating that Gas extends the lifespan of C. elegans in relation to mitochondria.We fed N2 worms with or without 400 µM Gas at 20°C. After 48 h, the Oil Red O staining results showed that the fat content of C. elegans treated with Gas was lower (Fig. 5D-E), which was consistent with the mRNA content of the lipid metabolism-related target genes fat-4 , fat-5 , and fat-6 (Fig. 5F), indicating that Gas extends the lifespan of C. elegans is closely related to the fat metabolism pathway.Figure. 5 Gas extends lifespan of C. elegans through mitochondrial and lipid metabolism pathways. (A-C) Survival curves of isp-1(qm150) , clk-1(e2519) III. , and aak-2(ok524) X. worms treated with or without 400 µM Gas; (D-E) Effects of 400 µM Gas treatment on lipid content in N2 worms after 48 hours at 20°C; (F) Relative expression levels of mitochondrial and lipid genes in N2 worms after 24 hours of 400 µM Gas treatment. 3.6 Gas Extends Lifespan through Autophagy Autophagy is a lysosome-mediated process for clearing intracellular aggregated proteins and damaged organelles. It is crucial for maintaining cell homeostasis under various stress conditions such as endoplasmic reticulum (ER) stress and oxidative stress [ 37 , 38 ] and is involved in aging and various aging-related pathological processes.The experimental results showed that Gas could not extend the lifespan of C. elegans after RNA interference of the autophagy-related genes atg-18 and bec-1 (Fig. 6 A-B), suggesting that Gas may be related to autophagy in delaying worms aging. To verify this conjecture, we conducted further experiments. After treating C. elegans with Gas, we detected the fluorescence intensity of the SQST-1 protein in the BC12921 mutant strain on the 5th and 7th days and the LGG-1 protein in the DA2123 mutant strain after 48 h. We found that compared with the control group, the fluorescence intensity of SQST-1 decreased and the fluorescence intensity of LGG-1 increased in C. elegans treated with Gas (Fig. 6 C-F), indicating that Gas can enhance the autophagic activity of C. elegans . Consistent detection results were obtained for the expression intensity of the SQST-1 and LGG-1 proteins (Fig. 6 G-I). In addition, after treating the wild-type N2 strain with Gas for 24 h, the mRNA expression of vps-34 , atg-18 , and lgg-1 increased (Fig. 6 J). Meanwhile, under oxidative stress conditions, the mRNA expression of the autophagy genes lgg-1 , atg-18 , bec-1 , and vps-34 in N2 worms treated with Gas also showed an upward trend (Fig. 6 K), which is sufficient to show that Gas is closely related to autophagy in delaying the aging of C. elegans . 4 Discussion The global population is aging at an accelerating rate. The decline in global fertility rates and the significant increase in life expectancy have made age-related degenerative diseases a forefront and hot-topic issue of social concern [ 39 , 40 ] . Therefore, strategies to delay aging have become a major focus of current research.Gas has various pharmacological effects and shows good therapeutic outcomes in diseases related to the nervous, cardiovascular, and immune systems. It has been reported to have anti-cancer, anti-hypertensive, and neuroprotective effects [ 41 ] . However, the underlying mechanisms of its anti-aging effects remain unclear. Thus, we selected C. elegans as an animal model to further investigate the lifespan-extending and anti-aging effects of Gas and its potential mechanisms. Our study demonstrated that Gas extended the lifespan of C. elegans in a concentration-dependent manner, with 400 µM showing the most significant effect. Meanwhile, it improved aging-related phenotypes in C. elegans , such as body bending frequency and lipofuscin deposition.A decrease in stress resistance is also a prominent feature of aging. The lifespan of C. elegans is significantly shortened under high-temperature (35°C) and oxidative stress conditions. Treatment with 400 µM Gas can significantly enhance the stress resistance of C. elegans and extend their lifespan, providing feasibility for subsequent mechanism exploration.The insulin/IGF-1 signaling pathway (IIS) is the first signaling pathway discovered to be related to aging [ 42 ] . Multiple studies have shown that this pathway is involved in regulating aging in various species, including C. elegans , flies, mice, and humans, and is highly conserved [ 43 – 45 ] . The nematode insulin/IGF-1 signaling pathway initiates when insulin-like peptides bind to the DAF-2 receptor, triggering AGE-1/PI3K activation, subsequently modulating AKT-1/2 kinase activities via PDK-1-mediated phosphorylation [ 46 ] . These effector kinases coordinate diverse downstream processes, notably the FOXO-family transcription factor DAF-16 [ 47 ] . DAF-16, a FOXO family transcription factor encoded by the daf-16 gene, serves as a central regulator of multiple signaling pathways. It can integrate these signals, up-regulate a series of target genes, and play an important protective role in antioxidant biological processes [ 48 , 49 ] . Our results showed that Gas could not extend the lifespan of daf-16 , akt-1 , and akt-2 mutant C. elegans and could stimulate the translocation of DAF-16 into the nucleus to exert biological effects, indicating that daf-16 is essential for Gas to extend the lifespan of C. elegans . As cells and organisms age, the function of the mitochondrial respiratory chain weakens, leading to an increase in electron release and a decrease in ATP synthesis [ 50 ] , which in turn causes changes in mitochondrial function and structure [ 51 ] . Inhibiting the weakening of mitochondrial respiratory chain function helps delay host aging [ 52 , 53 ] . Our experimental results confirmed that Gas could not extend the lifespan of mitochondrial gene mutant strains and could enhance the mRNA expression of mitochondrial-related genes, suggesting that Gas extends the lifespan of C. elegans by regulating mitochondrial function. Fat accumulation in organs and impaired fatty acid utilization are both associated with the pathophysiological phenotypes of aging [ 54 ] . Unsaturated fatty acids can prevent disease progression by increasing the expression of anti-inflammatory cytokines [ 55 ] . In C. elegans , fatty acid desaturases increase with the activation of DAF-16/FOXO, promoting lipid degradation and the synthesis of unsaturated fatty acids, thereby ensuring lifespan extension [ 56 ] . In the fat metabolism experiment, Gas significantly reduced lipid accumulation in C. elegans , which was consistent with the mRNA expression levels of lipid synthesis-related genes, indicating that Gas delays worms aging by inhibiting lipid synthesis and accumulation. Autophagy is an evolutionarily conserved mechanism for cells to adapt to metabolic and environmental stresses. It aims to recycle intracellular components to maintain nutritional homeostasis, promote metabolic adaptation, prevent damage to dysfunctional organelles, and maintain genomic stability [ 57 ] .Core autophagy genes were first identified in Saccharomyces cerevisiae, with subsequent characterization in mammalian systems and evolutionarily conserved in C. elegans , including unc-51/ULK1, bec-1/ATG-6, vps-34/VPS-34, atg-18/ATG-18, lgg-1/LC3B, and sqst-1/P62 [ 58 ] . BEC-1 acts as a marker protein for the initial autophagy synthesis complex, and ATG-14 promotes the formation and nucleation of phagophores [ 59 ] . In addition, LGG-1 and ATG-18 contribute to the expansion of the autophagic membrane and the formation of autophagosomes, which then fuse with lysosomes to form autolysosomes. P62/SQST-1 is an autophagy-specific substrate-binding protein that can be degraded by lysosomal enzymes and their substrates [ 60 ] .Our experimental results showed that Gas could not extend the lifespan of C. elegans when the autophagy genes atg-18 and bec-1 were knocked out by RNAi. Moreover, Gas could promote the formation of autophagosomes involving LGG-1, indicating that the lifespan-extending effect of Gas on C. elegans requires autophagy. It should be noted that elevated autophagosome formation may not necessarily reflect enhanced autophagic flux. In specific disease states, the observed accumulation of autophagic vesicles and concomitant rise in LC3-II could alternatively indicate defective autophagosome-lysosome fusion, representing impaired autophagy completion rather than genuine activation of the autophagic process [ 61 , 62 ] .To comprehensively evaluate the autophagy-inducing effect, we detected the autophagy substrate P62/SQST-1 and found that it was effectively degraded, indicating normal autophagy activation, which is related to lifespan extension. The intricate interplay between oxidative stress and autophagy has emerged as a pivotal determinant in disease progression, with accumulating evidence indicating that autophagic activation typically functions to alleviate oxidative damage [ 63 ] . In alignment with this established relationship, Gas treatment exhibited a dual regulatory capacity by simultaneously enhancing the transcriptional expression of fundamental autophagy components (including unc-51 , vps-34 , atg-18 , and lgg-1 ) under physiological conditions while further potentiating the induction of additional autophagic factors (notably bec-1 along with recurrent activation of lgg-1 , atg-18 , and vps-34 ) under oxidative stress conditions. This comprehensive upregulation of autophagic pathways substantiates a mechanistic framework wherein Gas ameliorates age-associated oxidative stress through autophagy-dependent ROS clearance mechanisms, thereby contributing to its observed anti-aging effects. The coordinated induction of multiple autophagy-related genes across different stress conditions particularly underscores the compound's ability to reinforce cellular quality control systems through transcriptional reprogramming of autophagic flux regulators. 5 Conclusion Our findings demonstrate that Gas alleviates aging through multiple mechanisms including DAF-16/FOXO signaling pathway activation, antioxidant defense enhancement, and autophagy induction, thereby providing both strategic options and experimental evidence for developing novel anti-aging therapeutics(Fig. 7 ). But the ultimate value lies in linking invertebrate models to interventions for human aging.In this study, we exclusively employed the C. elegans model. When extrapolating these findings to humans, the multifactorial complexity of aging development and progression necessitates more meticulous and nuanced approaches. Although C. elegans serves as an established model organism due to its conserved genetic pathways and neuronal functional homology with humans [ 64 ] , we must acknowledge its inherent limitations. The worms system cannot fully recapitulate the complete spectrum of pathological alterations observed in human aging, thereby presenting potential challenges in reflecting comprehensive pathophysiological responses. To address these constraints, future investigations will extend the study of Gas's anti-aging mechanisms to more complex model organisms (suchu ac mice and zebrafish), enabling more refined and holistic characterization of its potential therapeutic effects. The parallel between DAF-16/FOXO in worms and human FOXO3 (a gene strongly associated with centenarian populations)suggests Gas could mimic genetic longevity advantages pharmacologically. Clinically, combining Gas with lifestyle interventions (such as calorie restriction) may synergistically activate FOXO and autophagy, offering a feasible strategy to delay age-related functional decline. However, rigorous phase II trials are needed to validate these speculations, particularly given the complex interplay between autophagy induction and cancer risk in mammals. Declarations Funding: This research was funded by the National Natural Science Foundation of China（82074378），the Scientific research project of Sichuan Traditional Chinese Medicine Administration（2023zd016），the Southwest Medical University integrated traditional Chinese and Western medicine special key project（2024ZXYZX01）. Author Contributions: Bo Li: Data analysis, Writing - original draft. Shan Li: Data analysis. Haoling Chen: Methodology, Supervision, Dan Wu: Images analysis. Xingwang Cao: Images analysis. Mingyue Yao and Shiying Xiong: Providing technical support. Wei Meng: Writing – review & editing. Li Dong: Conceptualization, Methodology, Supervision, Funding acquisition. All authors participated in this article. Data Availability Statement All data are in the manuscript and/or supporting information files. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Conflict of Interest The authors declare that they have no conflicts of interest. Clinical trial number Not applicable References Sfeir, J.G., et al., Skeletal Aging. Mayo Clin Proc, 2022. 97 (6): p. 1194-1208. Liberale, L., et al., Inflammation, Aging, and Cardiovascular Disease: JACC Review Topic of the Week. J Am Coll Cardiol, 2022. 79 (8): p. 837-847. Berben, L., et al., Cancer and Aging: Two Tightly Interconnected Biological Processes. Cancers (Basel), 2021. 13 (6). Hou, Y., et al., Ageing as a risk factor for neurodegenerative disease. Nat Rev Neurol, 2019. 15 (10): p. 565-581. Cai, Y., et al., The landscape of aging. Sci China Life Sci, 2022. 65 (12): p. 2354-2454. 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(A) The chemical structure of Gas; (B) Survival curves of wild-type N2 worms cultured at 20°C on NGM plates without Gas or with 50, 100, 200, 400, and 800 µM Gas; (C) Survival curves of wild-type N2 worms after hatching, cultured at 20°C on NGM plates without or with 400 µM Gas. After being raised with 400 µM Gas, the lifespan extension rate of \u003cem\u003eC. elegans\u003c/em\u003e was 17.3%.(p \u0026lt; 0.001, log-rank test); (D) The mean lifespan of N2 worms was measured after treatment with various concentrations of Gas. (E) Number of body bends in N2 worms treated with 400 µM Gas for 5 and 10 days; (F-G) Analysis of lipofuscin content in N2 worms treated with 400 µM Gas for 5 and 10 days. Relative fluorescence intensity was calculated using Image J.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7395169/v1/2743fb4e827f3fb9c9449dfb.png"},{"id":90347867,"identity":"9a5b9f26-6c5d-473a-8f7f-9d4fde176a9e","added_by":"auto","created_at":"2025-09-01 16:49:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":383510,"visible":true,"origin":"","legend":"\u003cp\u003eGas enhances the antioxidant capacity of \u003cem\u003eC. elegans\u003c/em\u003e. \u0026nbsp;(A) Survival rate of wild-type N2 worms cultured in 20 mM paraquat after 400 µM Gas treatment.After being raised with Gas, the lifespan extension rate of \u003cem\u003eC. elegans\u003c/em\u003e was 24.8%. (p \u0026lt; 0.001, log-rank test); (B) Survival curves of EU1/\u003cem\u003eskn-1 (zu67) IV\u003c/em\u003e worms treated with or without 400 µM Gas; (C-D) ROS levels in wild-type N2: with 400 µM Gas as the experimental group, 20 mM paraquat as the model group, and 1 mM NAC as the positive control; (E-F) Quantitative analysis of fluorescence intensity in CL2166 mutants after feeding at 20°C for 5 days with or without 400 µM Gas; (G-H) 400 µM Gas treatment significantly increases fluorescence intensity in CF1553 mutants.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7395169/v1/7187b5de7ec80769507882cc.png"},{"id":90348564,"identity":"de294e09-0c68-491c-9fee-0c4e99b85fd1","added_by":"auto","created_at":"2025-09-01 16:57:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":413941,"visible":true,"origin":"","legend":"\u003cp\u003eGas enhances the thermotolerance of \u003cem\u003eC. elegans\u003c/em\u003e. \u0026nbsp;(A) Survival rate of \u003cem\u003eC. elegans\u003c/em\u003e at 35°C after treatment with or without 400 µM Gas (p \u0026lt; 0.001, log-rank test); (B) Survival curves of PS3553 \u003cem\u003e(hsf-1) \u003c/em\u003eafter treatment with or without 400 µM Gas; (C-H) Quantification of fluorescence intensity of HSP-4::GFP in SJ4005, HSP-6::GFP in SJ4100, and HSP-60::GFP in SJ4058, showing that 400 µM Gas significantly enhances the expression of HSPs. Fluorescence intensity was calculated using Image J, and the data represent the mean of three independent experiments with error bars indicating SEM. *** indicates p \u0026lt; 0.001, calculated by two-tailed t-test. (I) Relative expression levels of \u003cem\u003ehsf-1\u003c/em\u003e and its downstream genes in wild-type N2 and mutant PS3553\u003cem\u003e (hsf-1)\u003c/em\u003e after 24 hours of 400 µM Gas treatment.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7395169/v1/79ceafb63dc57b563c03baab.png"},{"id":90347871,"identity":"d35e7b40-01e8-453c-a51b-23994424cb27","added_by":"auto","created_at":"2025-09-01 16:49:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":278455,"visible":true,"origin":"","legend":"\u003cp\u003eThe lifespan extension of \u003cem\u003eC. elegans\u003c/em\u003e by Gas requires FOXO/DAF-16. \u0026nbsp;(A-C) Survival curves of \u003cem\u003edaf-16(mu86) I.\u003c/em\u003e, \u003cem\u003eakt-1(ok525) V.\u003c/em\u003e, and \u003cem\u003eakt-2(ok393) X.\u003c/em\u003e worms treated with or without 400 µM Gas; (D) Nuclear localization of DAF-16 in the \u003cem\u003edaf-16(zls356) IV \u003c/em\u003estrain induced by 400 µM Gas; (E) Relative expression levels of \u003cem\u003edaf-16\u003c/em\u003eand its downstream genes in wild-type N2 and mutant CF1038 \u003cem\u003e(daf-16) \u003c/em\u003eafter 24 hours of 400 µM Gas treatment.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7395169/v1/77eb30abe70d1c22234409cf.png"},{"id":90347787,"identity":"dd3fa290-533f-4786-a8d7-0e23c727a0a7","added_by":"auto","created_at":"2025-09-01 16:41:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":556696,"visible":true,"origin":"","legend":"\u003cp\u003eGas extends lifespan of \u003cem\u003eC. elegans\u003c/em\u003e through mitochondrial and lipid metabolism pathways. \u0026nbsp;(A-C) Survival curves of \u003cem\u003eisp-1(qm150)\u003c/em\u003e, \u003cem\u003eclk-1(e2519) III.\u003c/em\u003e, and \u003cem\u003eaak-2(ok524) X. \u003c/em\u003eworms treated with or without 400 µM Gas; (D-E) Effects of 400 µM Gas treatment on lipid content in N2 worms after 48 hours at 20°C; (F) Relative expression levels of mitochondrial and lipid genes in N2 worms after 24 hours of 400 µM Gas treatment.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7395169/v1/38da7457742c70fd8a0d9a24.png"},{"id":90347793,"identity":"dfa71886-3e3f-4e4f-9df2-005bec3e3760","added_by":"auto","created_at":"2025-09-01 16:41:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":531290,"visible":true,"origin":"","legend":"\u003cp\u003eGas extends lifespan of \u003cem\u003eC. elegans\u003c/em\u003e through autophagy. \u0026nbsp;(A-B) Survival curves of atg-18 RNAi and bec-1 RNAi worms treated with or without 400 µM Gas; (C-F) Fluorescence intensity of BC12921 (SQST-1) on days 5 and 7, and DA2123 (LGG-1) after 48 hours of treatment with or without 400 µM Gas; (G-I) Expression intensity of SQST-1::GFP and LGG-1::GFP proteins after treatment with or without 400 µM Gas; (J) Relative expression levels of autophagy genes in N2 worms after 24 hours of 400 µM Gas treatment; (K) Effects of 400 µM Gas on autophagy levels in N2 worms under oxidative stress conditions.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7395169/v1/31d47bb0054badbe46089dc3.png"},{"id":90347791,"identity":"e6b01492-dd50-4e85-b7c8-adb7efa08011","added_by":"auto","created_at":"2025-09-01 16:41:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":396408,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular mechanisms of insulin-like signaling pathway, oxidative stress and autophagy in C. elegans. (A) In C. elegans, binding of insulin-like ligands to the DAF-2 receptor triggers receptor autophosphorylation and activation. The activated DAF-2 recruits and activates PI3K through insulin receptor substrate (IRS) proteins, leading to phosphorylation and activation of PDK-1, which in turn activates AKT-1/2 kinases. AKT-1/2 subsequently phosphorylates the transcription factor DAF-16, resulting in its cytoplasmic retention and loss of transcriptional activity, thereby suppressing the regulation of downstream target genes;(B)Mitochondrial dysfunction leads to excessive accumulation of ROS. Elevated ROS levels activate the transcription factor SKN-1 , which subsequently upregulates the expression of antioxidant enzyme genes, including superoxide dismutase SOD-3 and glutathione S-transferase GST-4, thereby enhancing the organism's defense against oxidative stress;(C)Upon autophagy activation, core autophagy proteins (including LC3 homolog LGG-1, ATG-18, and BEC-1/Beclin-1) coordinately promote autophagosome formation. The autophagy adapter protein SQST-1/p62 recognizes and binds to substrates destined for degradation, and these complexes are subsequently encapsulated by autophagosomes. Ultimately, autophagosomes fuse with lysosomes to form autolysosomes, accomplishing the selective clearance of damaged organelles and aberrant proteins.This diagram was drawn by BioRender.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7395169/v1/07881753d85504ad21a91628.png"},{"id":98813960,"identity":"0a375b24-b6d8-4fb1-9010-946e50d8f88f","added_by":"auto","created_at":"2025-12-22 16:08:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3790484,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7395169/v1/4009abba-7ae1-4005-a8ef-bb4f24d3eb18.pdf"},{"id":90347794,"identity":"45358a92-a467-4351-909c-7eaa7b4234d0","added_by":"auto","created_at":"2025-09-01 16:41:32","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4993755,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.zip","url":"https://assets-eu.researchsquare.com/files/rs-7395169/v1/5093b89661ea48f72149f626.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gastrodin extends the lifespan of Caenorhabditis elegans via the DAF-16/FOXO signaling pathway and autophagy","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAging is a multi-dimensional progressive decline of physiological functions that occurs over time, characterized by accumulation, universality, and gradualness. During the aging process, the body\u0026rsquo;s functions and metabolism decline, which is closely related to human diseases. Aging is a major risk factor for most chronic diseases, including cardiovascular and cerebrovascular diseases, osteoporosis, cancer, and neurodegenerative diseases\u003csup\u003e[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. As a complex biological process, aging involves the functional decline of multiple organs and systems, shows significant individual differences and organ specificity\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, and has become a major challenge in the global public health field. With the further intensification of population aging, the incidence of age-related diseases has sharply increased, placing a heavy burden on society and economic development. Therefore, slowing down aging or promoting \u0026ldquo;healthy aging\u0026rdquo; has become an urgent problem to be solved in today\u0026rsquo;s society.\u003c/p\u003e\u003cp\u003eAs a model organism for studying the aging process and diseases, \u003cem\u003eC. elegans\u003c/em\u003e has the advantages of a fast life cycle, easy cultivation, and well-established genetic pathways\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Since the 1970s, it has become an important tool in molecular biology research\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Not only is it easy to cultivate in the laboratory, but it also has a high genetic similarity to humans and detailed gene sequencing data, and is widely used in many fields such as genetics, gene function, aging, and drug screening\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. In recent years, great progress has been made in aging and anti-aging research using \u003cem\u003eC. elegans\u003c/em\u003e as an animal model, which helps people further understand the aging mechanism and provides effective strategies and methods for anti-aging.\u003c/p\u003e\u003cp\u003eWith the improvement of people\u0026rsquo;s living quality, the public\u0026rsquo;s choice of anti-aging substances gradually tends to natural active ingredients. Therefore, exploring and studying the anti-aging activity of natural active ingredients is of great significance and value for alleviating health problems caused by aging. Gas, also known as 4-hydroxymethylphenyl-beta-D-glucopyranoside hemihydrate, is the main component of Gastrodia elata Blume tubers\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Contemporary pharmacological studies indicate that Gas exhibits significant antioxidant, anti-inflammatory, anti-apoptotic, and antiviral activities, demonstrating therapeutic efficacy in neurological and cardiovascular disorders\u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Recent studies have shown that Gas promotes autophagy and phagocytosis by activating the PPARα-TFEB/CD36 signaling pathway, significantly reducing oxidative stress-induced retinal pigment epithelial damage\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In addition, Gas enhances mitophagy by regulating the PINK1/Parkin pathway, effectively alleviating myocardial ischemia-reperfusion injury and playing a cardioprotective role\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. However, the role of Gas in lifespan extension and its underlying molecular mechanisms remain incompletely understood. Therefore, this study aims to investigate the regulatory effects of Gas on the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e and elucidate its potential mechanisms, thereby providing new strategies for the development of lifespan-extending drugs.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Chemicals\u003c/h2\u003e\u003cp\u003eGastrodin (Gas, \u0026ge;\u0026thinsp;98% purity) was obtained from Shanghai Yuanye Bio-Technology (China). Additional reagents including 5-fluorodeoxyuridine (FUDR) and CM-H2DCFDA were acquired from Sigma-Aldrich (St. Louis, MO, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Strains and Cultivation Methods of worms\u003c/h2\u003e\u003cp\u003eThe Caenorhabditis Genetics Center (CGC; University of Minnesota, Minneapolis, MN) supplied all nematode strains used in this study. Standard cultivation conditions involved maintenance at 20\u0026deg;C with 60% relative humidity on nematode growth medium (NGM) seeded with E. coli OP50, unless otherwise indicated. The following \u003cem\u003eC. elegans\u003c/em\u003e strains were utilized in this study:Wild-type N2;Mutant strains:CF1038 [daf-16(mu86) I],EU1 [skn-1(zu67) IV],PS3551 [hsf-1(sy441) I],RB754 [aak-2(ok524) X],RB759 [akt-1(ok525) V],VC204 [akt-2(ok393) X],CB4876 [clk-1(e2519) III];Transgenic reporter strains:CF1553 [sod-3::GFP\u0026thinsp;+\u0026thinsp;rol-6(su1006)],CL2166 [gst-4::GFP::NLS],SJ4100 [hsp-6::GFP],SJ4005 [hsp-4::GFP],SJ4058 [hsp-60::GFP\u0026thinsp;+\u0026thinsp;lin-15(+)],DA2123 [lgg-1::GFP::lgg-1\u0026thinsp;+\u0026thinsp;rol-6(su1006)],TJ356 [daf-16::daf-16a/b::GFP\u0026thinsp;+\u0026thinsp;rol-6].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Lifespan Assay\u003c/h2\u003e\u003cp\u003eAll nematode strains underwent sequential cultivation across 2\u0026ndash;3 generations using fresh NGM medium to prevent nutritional deprivation. Developmental-stage synchronized worms (late L4 larvae/young adults) were plated onto NGM supplemented with thermally inactivated OP50 (65\u0026deg;C for 30 min) and 40 \u0026micro;M FUDR (Sigma) to suppress offspring production.The initial transfer of synchronized worms to treatment plates marked experimental day 0.Non-motile specimens following platinum wire stimulation were classified as deceased. Vitality assessments were conducted daily until complete cohort mortality was achieved.Specimens were systematically transferred to fresh Gas-containing or control NGM plates at 48-hour intervals.Any organisms exhibiting plate migration or post-transfer hatching were excluded from longevity analysis. Each longevity trial was performed with \u0026ge;\u0026thinsp;3 biological replicates.Experimental cohorts consisted of \u0026ge;\u0026thinsp;60 nematodes per treatment condition.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Analysis of Aging-Related Phenotypes\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1 Body Bending Behavior Test\u003c/h2\u003e\u003cp\u003eL1-stage worms were synchronized and maintained on NGM agar at 20\u0026deg;C under controlled incubation. Upon reaching L4 development, specimens were moved to treatment plates containing either Gas or vehicle control. At designated intervals (days 5/10 post-maturity), individuals were placed in aqueous suspension for 60-second acclimatization. Then, the frequency of body bends within 20 seconds was recorded under a microscope. One forward-backward movement of the body was counted as one bend. At least 30 worms were included in each experimental group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2 Determination of Lipofuscin\u003c/h2\u003e\u003cp\u003eInitial specimen preparation followed identical protocols to the longevity study. Adult nematodes were sampled at days 5 and 10 post-maturation for fluorescence imaging (excitation: 360\u0026ndash;370 nm; emission: 420\u0026ndash;460 nm).Lipofuscin accumulation was assessed through mean fluorescence intensity measurements per specimen.Experimental cohorts contained\u0026thinsp;\u0026ge;\u0026thinsp;30 individuals per condition.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Oil Red O Staining\u003c/h2\u003e\u003cp\u003eDevelopmentally synchronized L1-stage nematodes were maintained on NGM agar until reaching young adult phase. Approximately 1000 specimens per condition were harvested from both treatment and control cohorts, subjected to sequential PBS rinses for complete OP50 elimination, and treated with Nile red staining reagent. Following 25-minute fixation in 4% paraformaldehyde, specimens underwent dual washes with PBS containing 1% Triton X-100. Nile red (5 mg/mL) was then applied under light-restricted conditions. Following 120-second incubation, triplicate washes with PBS-Triton solution were performed and and imaged using a Leica DFC7000T microscopy system. Image analysis was performed using ImageJ software. Each experiment was repeated three times, and at least 30 worms were used in each repetition.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Detection of Antioxidant Capacity and Reactive Oxygen Species (ROS) Level\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.6.1 Antioxidant Stress Experiment\u003c/h2\u003e\u003cp\u003eN2 strain nematodes were synchronized at the L1 larval stage and cultured on NGM agar at 20\u0026deg;C. Developmental-stage synchronized populations (late L4/young adult) were then transferred to Gas-treated or control NGM plates for oxidative stress assessment. At day 10 post-maturation, worms were exposed to 20 mM paraquat-containing NGM. Mortality was recorded at 24-hour intervals until complete cohort death occurred. Each experimental group contained\u0026thinsp;\u0026ge;\u0026thinsp;30 biological replicates, with three independent trials performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.6.2 Determination of ROS Content\u003c/h2\u003e\u003cp\u003eFourth-stage larval nematodes were cultured on treatment NGM agar supplemented with either Gas (400 \u0026micro;M), paraquat (20 mM), or N-acetyl-L-cysteine (1 mM), maintaining at 20\u0026deg;C for six days.Subsequently, specimens were harvested, subjected to triple M9 buffer washes, labeled with 50 \u0026micro;M H2DCF-DA fluorescent probe, and agitated at 35\u0026deg;C for one hour following manufacturer's protocol\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. ROS levels were quantified via fluorescence microscopy. Three independent experimental replicates were performed, each comprising\u0026thinsp;\u0026ge;\u0026thinsp;30 nematodes. Statistical significance was assessed through two-tailed Student t-tests.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Heat Resistance Experiment\u003c/h2\u003e\u003cp\u003eDevelopmentally synchronized N2 strain nematodes at L1 stage were maintained on NGM agar under controlled 20\u0026deg;C incubation. Upon reaching late L4 or young adult developmental phases, specimens were allocated to Gas-treated or untreated NGM media. On the 10th day of adulthood, they were transferred to an incubator at 35\u0026deg;C. The death of worms was observed and counted every 2 hours until all worms died. Experimental groups contained\u0026thinsp;\u0026ge;\u0026thinsp;30 biological replicates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Nuclear Localization\u003c/h2\u003e\u003cp\u003eIn this experiment, TJ356 mutant worms, which specifically labeled DAF-16::GFP (zls356), were used. Fourth-stage larval TJ356 specimens were allocated to either Gas treatment (400 \u0026micro;M) or dual control conditions. Positive controls (TJ356\u0026thinsp;+\u0026thinsp;OP50 on NGM) underwent 15-minute 35\u0026deg;C thermal stress, whereas negative controls were maintained at standard 20\u0026deg;C incubation. The localization of DAF-16:GFP was observed every hour under a fluorescence microscope. The green fluorescent nuclear-aggregated particles in TJ356 worms were regarded as the expression form of the DAF-16 gene in the nucleus.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Fluorescence Quantification\u003c/h2\u003e\u003cp\u003eThe specimen preparation protocol for fluorescence analysis mirrored the procedures employed in the longevity assessment study. Worms strains CL2166, dvIs19[pAF15(gst-4:: GFP:: NLS)] and strain CF1553 [(pAD76) sod-3::GFP\u0026thinsp;+\u0026thinsp;rol6 (su1006)] were cultured until the late L4 stage or early adult stage, then transferred to a medium with or without 400 \u0026micro;M Gas and continued to be cultured at 20\u0026deg;C until the end of the observation time point. They were anesthetized with tetramisole hydrochloride (2 mM), and the expression of fluorescent proteins in different strains was observed under a fluorescence microscope. Three independent biological replicates were performed, with each treatment condition containing\u0026thinsp;\u0026ge;\u0026thinsp;30 nematodes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Quantitative RT-PCR Detection\u003c/h2\u003e\u003cp\u003eNematode total RNA was isolated with RNAiso Plus reagent (Takara, Japan), followed by cDNA synthesis employing the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). Quantitative real-time PCR amplifications were carried out using Power SYBR Green PCR Master Mix (Applied Biosystems) on a QuantStudio 6 Flex platform.Gene expression quantification was determined through the 2-ΔΔCT method, with normalization against the housekeeping gene \u003cem\u003ecd\u003c/em\u003ec-42. The primers sequences are in the supplementary Materials.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Western Blot Experiment\u003c/h2\u003e\u003cp\u003eNematode specimens were harvested following 6-day Gas treatment. Cellular proteins were isolated through ultrasonic homogenization, with protein quantification conducted via BCA assay (Beyotime Biotechnology).The experiment was performed according to a previous protocol\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Proteins were examined using antibodies specific for SQST \u0026minus;\u0026thinsp;1 (Abcam, Cambridge, UK, Cat# ab207305), LGG \u0026minus;\u0026thinsp;1 (Thermo Fisher Scientific, Waltham, MA, USA, Cat# PA5116410), and α - Tubulin (Abcam, Cambridge, UK, Cat# ab176560).Image analysis was performed using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.12 RNA Interference\u003c/h2\u003e\u003cp\u003eGene silencing was achieved through RNA interference (RNAi) following established protocols\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. HT115(DE3) Escherichia coli (Fire Laboratory) transformed with either control vector L4440 or dsRNA-producing constructs targeting \u003cem\u003eatg-18\u003c/em\u003e and \u003cem\u003ebec-1\u003c/em\u003e were used for feeding-based RNAi. Longevity analysis in RNAi-treated nematodes was conducted as described previously\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Determination of Autophagy Content\u003c/h2\u003e\u003cp\u003eAutophagic activity in nematodes was assessed by quantifying GFP-labeled puncta associated with LGG-1-marked autophagosomes. The DA2123 strain exhibited cytoplasmic GFP-LGG1 fluorescence distribution across multiple tissues. The formation of autophagosome structures could be observed and quantified through the appearance of fluorescent dots, thereby evaluating the autophagy content in worms.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Statistical Methods\u003c/h2\u003e\u003cp\u003eData were statistically analyzed with SPSS (v26.0) and GraphPad Prism (v7.0) analytical packages. Quantitative analyses of fluorescence signals, lipid deposits (Oil Red O), and protein levels were conducted via ImageJ (v1.8.0). The Kaplan-Meier method was employed for survival curve estimation and comparison. All quantitative measurements are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, except where alternative representations are specified. Statistical analyses were performed using two-tailed Student's t-tests, with a predefined significance level of α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Gas enhances longevity and ameliorates age-associated phenotypes in \u003cem\u003eC. elegans\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo determine whether Gas can extend the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e, wild-type N2 worms were treated with different concentrations of Gas(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The experimental results showed that Gas at multiple concentrations could extend the lifespan of worms to varying degrees (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Among them, the 400 \u0026micro;M concentration was the most effective, with a lifespan extension rate of 17.3% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D).The decline in locomotor ability is an important indicator of worms aging. Therefore, we investigated the effect of Gas on the locomotor ability of adult worms and found that Gas could significantly improve the locomotor ability of \u003cem\u003eC. elegans\u003c/em\u003e on the 5th and 10th days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).Aging is accompanied by the production of free radicals, which oxidize fats and cause lysosomal degradation, leading to an increase in lysosomal digestion products and fat residues, and subsequently the accumulation of lipofuscin in worms. Thus, lipofuscin is a characteristic of the onset of cell aging, indicating a dysfunction of the intracellular clearance system and is a widely recognized aging marker\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Experimental data demonstrated Gas-treated nematodes exhibited reduced lipofuscin autofluorescence at both day 5 and 10 post-treatment,indicating substantial suppression of age-pigment deposition. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Gas Augments Oxidative Stress Resistance in \u003cem\u003eC. elegans\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eAge-related deterioration of antioxidant defenses coupled with reactive oxygen ROS accumulation contributes to progressive tissue dysfunction and organ pathology\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. The \u003cem\u003eC. elegans\u003c/em\u003e Nrf2 ortholog SKN-1, constitutively expressed during development, serves as a master regulator of oxidative stress responses\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Gas administration increased paraquat (20 mM) resistance in wild-type N2 strains by 24.8% relative to controls (Fig.\u0026nbsp;2A). This protective effect was abolished in skn-1 loss-of-function mutants (Fig.\u0026nbsp;2B).Environmental stressors induce persistent ROS generation. Elevated ROS levels initiate oxidative cascades, increasing cytotoxic byproducts (e.g., MDA) that compromise cellular integrity\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Quantitative analysis revealed Gas-mediated ROS reduction in day-6 adult N2 populations (Fig.\u0026nbsp;2C-D).\u003c/p\u003e\u003cp\u003eThe oxidative stress response in \u003cem\u003eC. elegans\u003c/em\u003e is primarily mediated through the SKN-1/Nrf2-regulated antioxidant enzymes GST-4 and SOD-3\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, which function as critical cellular defense mechanisms against ROS. GST-4, a glutathione S-transferase, exhibits dual protective functions by both neutralizing lipid hydroperoxides through its glutathione peroxidase activity and detoxifying cytotoxic lipid peroxidation end-products such as 4-hydroxy-2-trans-nonenal (4-HNE)\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, the metalloenzyme SOD-3 localizes to mitochondrial membranes where it catalyzes the dismutation of superoxide radicals into hydrogen peroxide and molecular oxygen, serving as the primary scavenger of mitochondrial ROS\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Experimental observations revealed that Gas treatment significantly enhanced GFP reporter expression in both CL2166 [gst-4::GFP] and CF1553 [sod-3::GFP] transgenic strains (Fig.\u0026nbsp;2E-H), demonstrating a potent activation of these antioxidant pathways. These collective findings provide compelling evidence that Gas extends \u003cem\u003eC. elegans\u003c/em\u003e lifespan through SKN-1-dependent transcriptional activation of antioxidant defense systems, resulting in improved oxidative stress resistance and reduced accumulation of oxidative damage markers. The mechanistic link between Gas exposure, SKN-1 activation, and enhanced expression/activity of downstream antioxidant enzymes establishes a clear pathway through which this compound exerts its longevity-promoting effects.Figure. 2 Gas enhances the antioxidant capacity of \u003cem\u003eC. elegans\u003c/em\u003e. (A) Survival rate of wild-type N2 worms cultured in 20 mM paraquat after 400 \u0026micro;M Gas treatment.After being raised with Gas, the lifespan extension rate of \u003cem\u003eC. elegans\u003c/em\u003e was 24.8%. (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, log-rank test); (B) Survival curves of EU1/\u003cem\u003eskn-1 (zu67) IV\u003c/em\u003e worms treated with or without 400 \u0026micro;M Gas; (C-D) ROS levels in wild-type N2: with 400 \u0026micro;M Gas as the experimental group, 20 mM paraquat as the model group, and 1 mM NAC as the positive control; (E-F) Quantitative analysis of fluorescence intensity in CL2166 mutants after feeding at 20\u0026deg;C for 5 days with or without 400 \u0026micro;M Gas; (G-H) 400 \u0026micro;M Gas treatment significantly increases fluorescence intensity in CF1553 mutants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Gas Enhances the Resistance of \u003cem\u003eC. elegans\u003c/em\u003e to High-Temperature Stress\u003c/h2\u003e\u003cp\u003eHigh temperature can damage cell tissues, such as neuronal degeneration and heat-induced cell death, thereby increasing the content of ROS. Excessive ROS can lead to cell necrosis or apoptosis, thus shortening the lifespan\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Therefore, strong resistance to high-temperature stress plays an important role in the process of delaying aging. In the experiment, it was found that at an environmental temperature of 35\u0026deg;C, Gas could significantly extend the survival time of \u003cem\u003eC. elegans\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eThe heat stress response is a physiological defense response commonly found in the biological world. Under the stimulation of environmental factors, the body can induce the synthesis of a group of highly conserved stress proteins, namely heat shock proteins (HSPs). HSPs facilitate proper polypeptide folding into native three-dimensional structures and mediate the renaturation of misfolded proteins, thus contributing to ROS homeostasis in plant embryos\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. The transcription factor HSF-1 serves as a master regulator of proteostasis by controlling HSPs expression, thereby coordinating protein quality control mechanisms including synthesis, folding, and degradation\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Genetic evidence demonstrated the essential role of HSF-1 in Gas-mediated longevity, as \u003cem\u003ehsf-1\u003c/em\u003e null mutants failed to exhibit lifespan extension following treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Fluorescence microscopy analysis revealed Overall upregulation of specific HSP family members, with SJ4005 [hsp-4::GFP] ,SJ4100 [hsp-6::GFP] and SJ4058 [hsp-60::GFP] transgenic strains showing markedly increased reporter expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-H). In addition, Gas also increased the mRNA levels of the downstream genes \u003cem\u003ehsp-4\u003c/em\u003e, \u003cem\u003ehsp-60\u003c/em\u003e, and \u003cem\u003ehsp-12.6\u003c/em\u003e of \u003cem\u003ehsf-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Therefore, Gas can improve the resistance of \u003cem\u003eC. elegans\u003c/em\u003e to high-temperature stress by promoting the high expression of hsf-1-related genes in \u003cem\u003eC. elegans\u003c/em\u003e and activating more HSPs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Gas Requires FOXO/DAF-16 to Extend Lifespan\u003c/h2\u003e\u003cp\u003eDAF-16 is the only direct ortholog of the forkhead box O (FOXO) transcription factor in \u003cem\u003eC. elegans\u003c/em\u003e, which is crucial for maintaining cell homeostasis and is required for the reported lifespan extension in the \u003cem\u003edaf-2\u003c/em\u003e mutant strain\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Its pleiotropic effects include basic physiological processes such as metabolic regulation, development, aging, immune response, and cell stress resistance\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Within the insulin/IGF-1 signaling (IIS) cascade, AKT-1/2 kinases function as upstream regulators of the FOXO transcription factor DAF-16\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. To establish DAF-16 dependence, we examined Gas-mediated longevity effects in loss-of-function mutants. Genetic ablation of \u003cem\u003edaf-16 (mu86)\u003c/em\u003e, \u003cem\u003eakt-1\u003c/em\u003e, or \u003cem\u003eakt-2\u003c/em\u003e completely abolished the lifespan-extending effects of Gas (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C), demonstrating the essential role of DAF-16 in Gas-induced longevity.\u003c/p\u003e\u003cp\u003eStress stimuli trigger nuclear translocation of DAF-16/FOXO, thereby activating cytoprotective gene networks\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. We next assessed Gas-induced subcellular redistribution of DAF-16. Gas treatment markedly enhanced nuclear DAF-16::GFP accumulation in TJ356 transgenics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), concomitant with upregulated expression of canonical DAF-16 targets (\u003cem\u003esod-3, dod-3, ctl-1, ctl-3\u003c/em\u003e)(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eE༉. These conclusions indicate that Gas requires the participation of the DAF-16/FOXO signaling pathway to extend the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Gas Extends Lifespan through Mitochondrial and Fat Metabolism Pathways\u003c/h2\u003e\u003cp\u003ePrevious studies have established that modulation of mitochondrial electron transport chain components can profoundly influence longevity in \u003cem\u003eC. elegans\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. To investigate potential mitochondrial involvement in Gas-mediated lifespan extension, we examined its effects in respiratory-deficient mutants. Notably, Gas failed to prolong survival in either \u003cem\u003eisp-1\u003c/em\u003e mutants (deficient in complex III function) or \u003cem\u003eclk-1\u003c/em\u003e mutants (impaired in ubiquinone biosynthesis) (Fig.\u0026nbsp;5A-B), suggesting these mitochondrial components are essential for Gas's longevity-promoting effects.Meanwhile, in recent years, intervening in the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e through lipid signaling molecules has become a hot topic in the study of anti-aging. The \u003cem\u003eaak-2\u003c/em\u003e, as one of the energy regulators in \u003cem\u003eC. elegans\u003c/em\u003e, is involved in worms fat hydrolysis, fatty acid oxidation, and polyunsaturated fatty acid synthesis\u003csup\u003e[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The results showed that Gas could not extend the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e with the \u003cem\u003eaak-2\u003c/em\u003e gene mutation (Fig.\u0026nbsp;5C), and it increased the mRNA content of the mitochondrial-related transcription factors \u003cem\u003exbp-1\u003c/em\u003e and \u003cem\u003eisp-1\u003c/em\u003e (Fig.\u0026nbsp;5F), indicating that Gas extends the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e in relation to mitochondria.We fed N2 worms with or without 400 \u0026micro;M Gas at 20\u0026deg;C. After 48 h, the Oil Red O staining results showed that the fat content of \u003cem\u003eC. elegans\u003c/em\u003e treated with Gas was lower (Fig.\u0026nbsp;5D-E), which was consistent with the mRNA content of the lipid metabolism-related target genes \u003cem\u003efat-4\u003c/em\u003e, \u003cem\u003efat-5\u003c/em\u003e, and \u003cem\u003efat-6\u003c/em\u003e (Fig.\u0026nbsp;5F), indicating that Gas extends the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e is closely related to the fat metabolism pathway.Figure. 5 Gas extends lifespan of \u003cem\u003eC. elegans\u003c/em\u003e through mitochondrial and lipid metabolism pathways. (A-C) Survival curves of \u003cem\u003eisp-1(qm150)\u003c/em\u003e, \u003cem\u003eclk-1(e2519) III.\u003c/em\u003e, and \u003cem\u003eaak-2(ok524) X.\u003c/em\u003e worms treated with or without 400 \u0026micro;M Gas; (D-E) Effects of 400 \u0026micro;M Gas treatment on lipid content in N2 worms after 48 hours at 20\u0026deg;C; (F) Relative expression levels of mitochondrial and lipid genes in N2 worms after 24 hours of 400 \u0026micro;M Gas treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Gas Extends Lifespan through Autophagy\u003c/h2\u003e\u003cp\u003eAutophagy is a lysosome-mediated process for clearing intracellular aggregated proteins and damaged organelles. It is crucial for maintaining cell homeostasis under various stress conditions such as endoplasmic reticulum (ER) stress and oxidative stress\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e and is involved in aging and various aging-related pathological processes.The experimental results showed that Gas could not extend the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e after RNA interference of the autophagy-related genes atg-18 and bec-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B), suggesting that Gas may be related to autophagy in delaying worms aging. To verify this conjecture, we conducted further experiments. After treating \u003cem\u003eC. elegans\u003c/em\u003e with Gas, we detected the fluorescence intensity of the SQST-1 protein in the BC12921 mutant strain on the 5th and 7th days and the LGG-1 protein in the DA2123 mutant strain after 48 h. We found that compared with the control group, the fluorescence intensity of SQST-1 decreased and the fluorescence intensity of LGG-1 increased in \u003cem\u003eC. elegans\u003c/em\u003e treated with Gas (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-F), indicating that Gas can enhance the autophagic activity of \u003cem\u003eC. elegans\u003c/em\u003e. Consistent detection results were obtained for the expression intensity of the SQST-1 and LGG-1 proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-I). In addition, after treating the wild-type N2 strain with Gas for 24 h, the mRNA expression of \u003cem\u003evps-34\u003c/em\u003e, \u003cem\u003eatg-18\u003c/em\u003e, and \u003cem\u003elgg-1\u003c/em\u003e increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Meanwhile, under oxidative stress conditions, the mRNA expression of the autophagy genes \u003cem\u003elgg-1\u003c/em\u003e, \u003cem\u003eatg-18\u003c/em\u003e, \u003cem\u003ebec-1\u003c/em\u003e, and \u003cem\u003evps-34\u003c/em\u003e in N2 worms treated with Gas also showed an upward trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eK), which is sufficient to show that Gas is closely related to autophagy in delaying the aging of \u003cem\u003eC. elegans\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe global population is aging at an accelerating rate. The decline in global fertility rates and the significant increase in life expectancy have made age-related degenerative diseases a forefront and hot-topic issue of social concern\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Therefore, strategies to delay aging have become a major focus of current research.Gas has various pharmacological effects and shows good therapeutic outcomes in diseases related to the nervous, cardiovascular, and immune systems. It has been reported to have anti-cancer, anti-hypertensive, and neuroprotective effects\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. However, the underlying mechanisms of its anti-aging effects remain unclear. Thus, we selected \u003cem\u003eC. elegans\u003c/em\u003e as an animal model to further investigate the lifespan-extending and anti-aging effects of Gas and its potential mechanisms.\u003c/p\u003e\u003cp\u003eOur study demonstrated that Gas extended the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e in a concentration-dependent manner, with 400 \u0026micro;M showing the most significant effect. Meanwhile, it improved aging-related phenotypes in \u003cem\u003eC. elegans\u003c/em\u003e, such as body bending frequency and lipofuscin deposition.A decrease in stress resistance is also a prominent feature of aging. The lifespan of \u003cem\u003eC. elegans\u003c/em\u003e is significantly shortened under high-temperature (35\u0026deg;C) and oxidative stress conditions. Treatment with 400 \u0026micro;M Gas can significantly enhance the stress resistance of \u003cem\u003eC. elegans\u003c/em\u003e and extend their lifespan, providing feasibility for subsequent mechanism exploration.The insulin/IGF-1 signaling pathway (IIS) is the first signaling pathway discovered to be related to aging\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Multiple studies have shown that this pathway is involved in regulating aging in various species, including \u003cem\u003eC. elegans\u003c/em\u003e, flies, mice, and humans, and is highly conserved\u003csup\u003e[\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. The nematode insulin/IGF-1 signaling pathway initiates when insulin-like peptides bind to the DAF-2 receptor, triggering AGE-1/PI3K activation, subsequently modulating AKT-1/2 kinase activities via PDK-1-mediated phosphorylation\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. These effector kinases coordinate diverse downstream processes, notably the FOXO-family transcription factor DAF-16\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. DAF-16, a FOXO family transcription factor encoded by the \u003cem\u003edaf-16\u003c/em\u003e gene, serves as a central regulator of multiple signaling pathways. It can integrate these signals, up-regulate a series of target genes, and play an important protective role in antioxidant biological processes\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. Our results showed that Gas could not extend the lifespan of \u003cem\u003edaf-16\u003c/em\u003e, \u003cem\u003eakt-1\u003c/em\u003e, and \u003cem\u003eakt-2\u003c/em\u003e mutant \u003cem\u003eC. elegans\u003c/em\u003e and could stimulate the translocation of DAF-16 into the nucleus to exert biological effects, indicating that \u003cem\u003edaf-16\u003c/em\u003e is essential for Gas to extend the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eAs cells and organisms age, the function of the mitochondrial respiratory chain weakens, leading to an increase in electron release and a decrease in ATP synthesis\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e, which in turn causes changes in mitochondrial function and structure\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. Inhibiting the weakening of mitochondrial respiratory chain function helps delay host aging\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. Our experimental results confirmed that Gas could not extend the lifespan of mitochondrial gene mutant strains and could enhance the mRNA expression of mitochondrial-related genes, suggesting that Gas extends the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e by regulating mitochondrial function.\u003c/p\u003e\u003cp\u003eFat accumulation in organs and impaired fatty acid utilization are both associated with the pathophysiological phenotypes of aging\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. Unsaturated fatty acids can prevent disease progression by increasing the expression of anti-inflammatory cytokines\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003eC. elegans\u003c/em\u003e, fatty acid desaturases increase with the activation of DAF-16/FOXO, promoting lipid degradation and the synthesis of unsaturated fatty acids, thereby ensuring lifespan extension\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e. In the fat metabolism experiment, Gas significantly reduced lipid accumulation in \u003cem\u003eC. elegans\u003c/em\u003e, which was consistent with the mRNA expression levels of lipid synthesis-related genes, indicating that Gas delays worms aging by inhibiting lipid synthesis and accumulation.\u003c/p\u003e\u003cp\u003eAutophagy is an evolutionarily conserved mechanism for cells to adapt to metabolic and environmental stresses. It aims to recycle intracellular components to maintain nutritional homeostasis, promote metabolic adaptation, prevent damage to dysfunctional organelles, and maintain genomic stability\u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e.Core autophagy genes were first identified in Saccharomyces cerevisiae, with subsequent characterization in mammalian systems and evolutionarily conserved in \u003cem\u003eC. elegans\u003c/em\u003e, including unc-51/ULK1, bec-1/ATG-6, vps-34/VPS-34, atg-18/ATG-18, lgg-1/LC3B, and sqst-1/P62\u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBEC-1 acts as a marker protein for the initial autophagy synthesis complex, and ATG-14 promotes the formation and nucleation of phagophores\u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e. In addition, LGG-1 and ATG-18 contribute to the expansion of the autophagic membrane and the formation of autophagosomes, which then fuse with lysosomes to form autolysosomes. P62/SQST-1 is an autophagy-specific substrate-binding protein that can be degraded by lysosomal enzymes and their substrates\u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e.Our experimental results showed that Gas could not extend the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e when the autophagy genes \u003cem\u003eatg-18\u003c/em\u003e and \u003cem\u003ebec-1\u003c/em\u003e were knocked out by RNAi. Moreover, Gas could promote the formation of autophagosomes involving LGG-1, indicating that the lifespan-extending effect of Gas on \u003cem\u003eC. elegans\u003c/em\u003e requires autophagy. It should be noted that elevated autophagosome formation may not necessarily reflect enhanced autophagic flux. In specific disease states, the observed accumulation of autophagic vesicles and concomitant rise in LC3-II could alternatively indicate defective autophagosome-lysosome fusion, representing impaired autophagy completion rather than genuine activation of the autophagic process\u003csup\u003e[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e.To comprehensively evaluate the autophagy-inducing effect, we detected the autophagy substrate P62/SQST-1 and found that it was effectively degraded, indicating normal autophagy activation, which is related to lifespan extension.\u003c/p\u003e\u003cp\u003eThe intricate interplay between oxidative stress and autophagy has emerged as a pivotal determinant in disease progression, with accumulating evidence indicating that autophagic activation typically functions to alleviate oxidative damage\u003csup\u003e[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e. In alignment with this established relationship, Gas treatment exhibited a dual regulatory capacity by simultaneously enhancing the transcriptional expression of fundamental autophagy components (including \u003cem\u003eunc-51\u003c/em\u003e, \u003cem\u003evps-34\u003c/em\u003e, \u003cem\u003eatg-18\u003c/em\u003e, and \u003cem\u003elgg-1\u003c/em\u003e) under physiological conditions while further potentiating the induction of additional autophagic factors (notably \u003cem\u003ebec-1\u003c/em\u003e along with recurrent activation of \u003cem\u003elgg-1\u003c/em\u003e, \u003cem\u003eatg-18\u003c/em\u003e, and \u003cem\u003evps-34\u003c/em\u003e) under oxidative stress conditions. This comprehensive upregulation of autophagic pathways substantiates a mechanistic framework wherein Gas ameliorates age-associated oxidative stress through autophagy-dependent ROS clearance mechanisms, thereby contributing to its observed anti-aging effects. The coordinated induction of multiple autophagy-related genes across different stress conditions particularly underscores the compound's ability to reinforce cellular quality control systems through transcriptional reprogramming of autophagic flux regulators.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eOur findings demonstrate that Gas alleviates aging through multiple mechanisms including DAF-16/FOXO signaling pathway activation, antioxidant defense enhancement, and autophagy induction, thereby providing both strategic options and experimental evidence for developing novel anti-aging therapeutics(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBut the ultimate value lies in linking invertebrate models to interventions for human aging.In this study, we exclusively employed the \u003cem\u003eC. elegans\u003c/em\u003e model. When extrapolating these findings to humans, the multifactorial complexity of aging development and progression necessitates more meticulous and nuanced approaches. Although \u003cem\u003eC. elegans\u003c/em\u003e serves as an established model organism due to its conserved genetic pathways and neuronal functional homology with humans\u003csup\u003e[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/sup\u003e, we must acknowledge its inherent limitations. The worms system cannot fully recapitulate the complete spectrum of pathological alterations observed in human aging, thereby presenting potential challenges in reflecting comprehensive pathophysiological responses. To address these constraints, future investigations will extend the study of Gas's anti-aging mechanisms to more complex model organisms (suchu ac mice and zebrafish), enabling more refined and holistic characterization of its potential therapeutic effects.\u003c/p\u003e\u003cp\u003eThe parallel between DAF-16/FOXO in worms and human FOXO3 (a gene strongly associated with centenarian populations)suggests Gas could mimic genetic longevity advantages pharmacologically. Clinically, combining Gas with lifestyle interventions (such as calorie restriction) may synergistically activate FOXO and autophagy, offering a feasible strategy to delay age-related functional decline. However, rigorous phase II trials are needed to validate these speculations, particularly given the complex interplay between autophagy induction and cancer risk in mammals.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was funded by the National Natural Science Foundation of China（82074378），the Scientific research project of Sichuan Traditional Chinese Medicine Administration（2023zd016），the Southwest Medical University integrated traditional Chinese and Western medicine special key project（2024ZXYZX01）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003eBo Li: Data analysis, Writing - original draft. Shan Li: Data analysis. Haoling Chen: Methodology, Supervision, Dan Wu: Images analysis. Xingwang Cao: Images analysis. Mingyue Yao and Shiying Xiong: Providing technical support. Wei Meng: Writing \u0026ndash; review \u0026amp; editing. Li Dong: Conceptualization, Methodology, Supervision, Funding acquisition. All authors participated in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are in the manuscript and/or supporting information files.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSfeir, J.G., et al., \u003cem\u003eSkeletal Aging.\u003c/em\u003e Mayo Clin Proc, 2022. \u003cstrong\u003e97\u003c/strong\u003e(6): p. 1194-1208.\u003c/li\u003e\n\u003cli\u003eLiberale, L., et al., \u003cem\u003eInflammation, Aging, and Cardiovascular Disease: JACC Review Topic of the Week.\u003c/em\u003e J Am Coll Cardiol, 2022. \u003cstrong\u003e79\u003c/strong\u003e(8): p. 837-847.\u003c/li\u003e\n\u003cli\u003eBerben, L., et al., \u003cem\u003eCancer and Aging: Two Tightly Interconnected Biological Processes.\u003c/em\u003e Cancers (Basel), 2021. \u003cstrong\u003e13\u003c/strong\u003e(6).\u003c/li\u003e\n\u003cli\u003eHou, Y., et al., \u003cem\u003eAgeing as a risk factor for neurodegenerative disease.\u003c/em\u003e Nat Rev Neurol, 2019. \u003cstrong\u003e15\u003c/strong\u003e(10): p. 565-581.\u003c/li\u003e\n\u003cli\u003eCai, Y., et al., \u003cem\u003eThe landscape of aging.\u003c/em\u003e Sci China Life Sci, 2022. \u003cstrong\u003e65\u003c/strong\u003e(12): p. 2354-2454.\u003c/li\u003e\n\u003cli\u003eZhang, S., et al., \u003cem\u003eCaenorhabditis elegans as a Useful Model for Studying Aging Mutations.\u003c/em\u003e Front Endocrinol (Lausanne), 2020. \u003cstrong\u003e11\u003c/strong\u003e: p. 554994.\u003c/li\u003e\n\u003cli\u003eBrenner, S., \u003cem\u003eThe genetics of Caenorhabditis elegans.\u003c/em\u003e Genetics, 1974. \u003cstrong\u003e77\u003c/strong\u003e(1): p. 71-94.\u003c/li\u003e\n\u003cli\u003eCorsi, A.K., B. 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Shaikh, \u003cem\u003ePolyunsaturated fatty acids, specialized pro-resolving mediators, and targeting inflammation resolution in the age of precision nutrition.\u003c/em\u003e Biochim Biophys Acta Mol Cell Biol Lipids, 2021. \u003cstrong\u003e1866\u003c/strong\u003e(7): p. 158936.\u003c/li\u003e\n\u003cli\u003eGhosh, B., et al., \u003cem\u003eA Fat-Promoting Botanical Extract From Artemisia scoparia Exerts Geroprotective Effects on Caenorhabditis elegans Life Span and Stress Resistance.\u003c/em\u003e J Gerontol A Biol Sci Med Sci, 2022. \u003cstrong\u003e77\u003c/strong\u003e(6): p. 1112-1120.\u003c/li\u003e\n\u003cli\u003eSantovito, D., et al., \u003cem\u003eAutophagy, innate immunity, and cardiac disease.\u003c/em\u003e Front Cell Dev Biol, 2023. \u003cstrong\u003e11\u003c/strong\u003e: p. 1149409.\u003c/li\u003e\n\u003cli\u003eChen, Y., V. Scarcelli, and R. Legouis, \u003cem\u003eApproaches for Studying Autophagy in Caenorhabditis elegans.\u003c/em\u003e Cells, 2017. \u003cstrong\u003e6\u003c/strong\u003e(3).\u003c/li\u003e\n\u003cli\u003eFazeli, G., et al., \u003cem\u003eC. elegans midbodies are released, phagocytosed and undergo LC3-dependent degradation independent of macroautophagy.\u003c/em\u003e J Cell Sci, 2016. \u003cstrong\u003e129\u003c/strong\u003e(20): p. 3721-3731.\u003c/li\u003e\n\u003cli\u003eYu, L., Y. Chen, and S.A. Tooze, \u003cem\u003eAutophagy pathway: Cellular and molecular mechanisms.\u003c/em\u003e Autophagy, 2018. \u003cstrong\u003e14\u003c/strong\u003e(2): p. 207-215.\u003c/li\u003e\n\u003cli\u003eYoshii, S.R. and N. Mizushima, \u003cem\u003eMonitoring and Measuring Autophagy.\u003c/em\u003e Int J Mol Sci, 2017. \u003cstrong\u003e18\u003c/strong\u003e(9).\u003c/li\u003e\n\u003cli\u003eWen, Y.P., et al., \u003cem\u003eExploring the therapeutic potential of Nelumbo nucifera leaf extract against amyloid-beta-induced toxicity in the Caenorhabditis elegans model of Alzheimer\u0026apos;s disease.\u003c/em\u003e Front Pharmacol, 2024. \u003cstrong\u003e15\u003c/strong\u003e: p. 1408031.\u003c/li\u003e\n\u003cli\u003eYun, H.R., et al., \u003cem\u003eRoles of Autophagy in Oxidative Stress.\u003c/em\u003e Int J Mol Sci, 2020. \u003cstrong\u003e21\u003c/strong\u003e(9).\u003c/li\u003e\n\u003cli\u003eCuletto, E. and D.B. Sattelle, \u003cem\u003eA role for Caenorhabditis elegans in understanding the function and interactions of human disease genes.\u003c/em\u003e Hum Mol Genet, 2000. \u003cstrong\u003e9\u003c/strong\u003e(6): p. 869-77.\u003c/li\u003e\n\u003c/ol\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":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Gastrodin, Caenorhabditis elegans, Anti-aging, DAF-16/FOXO signaling pathway, Autophagy","lastPublishedDoi":"10.21203/rs.3.rs-7395169/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7395169/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eThe progression of age-related pathologies is strongly linked to biological aging. Identifying natural anti-aging agents to mitigate disease onset and development holds substantial therapeutic value.The natural compound Gastrodin (Gas) demonstrates promising effects in retarding aging.This study aims to explore the effects of Gas on the lifespan and antioxidant capacity of \u003cem\u003eCaenorhabditis elegans (C. elegans)\u003c/em\u003e. Additionally, it seeks to elucidate the possible mechanisms.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eInitially, Gas was assessed for its influence on \u003cem\u003eC. elegans\u003c/em\u003e lifespan, mobility, lipofuscin accumulation, and oxidative stress responses. Subsequent analyses focused on Gas\u0026rsquo;s modulation of the insulin/IGF-1 pathway, mitochondrial activity, autophagic processes, and gene expression to uncover its lifespan-extending mechanisms.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eGas induced a dose-dependent lifespan extension in \u003cem\u003eC. elegans\u003c/em\u003e, peaking at 400 \u0026micro;M with a 17.3% increase in longevity. Gas enhanced \u003cem\u003eC. elegans\u003c/em\u003e mobility while suppressing age-related lipofuscin deposition.Additionally, Gas lowered ROS levels and elevated antioxidant enzyme activity in \u003cem\u003eC. elegans\u003c/em\u003e.Mechanistic studies revealed that Gas\u0026rsquo;s anti-aging effects rely on transcription factors (DAF-16, SKN-1, HSF-1) and bolster stress resistance via HSPs activation and autophagy induction.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study reveals the potential of Gas in extending the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e, emphasizes its mechanism of action by regulating antioxidant capacity, heat stress response, and autophagy pathway, and provides experimental evidence that supports the development of Gas as a candidate compound for lifespan extension.\u003c/p\u003e","manuscriptTitle":"Gastrodin extends the lifespan of Caenorhabditis elegans via the DAF-16/FOXO signaling pathway and autophagy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 16:41:27","doi":"10.21203/rs.3.rs-7395169/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-30T22:04:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-18T18:09:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-17T18:11:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-05T17:31:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5482657495120924307052466445961568211","date":"2025-09-01T06:23:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"281578036289304184260479086728301472266","date":"2025-08-30T07:12:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25709040164066965420747299365685454059","date":"2025-08-26T22:18:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273306322495525425744333287577094332223","date":"2025-08-26T22:17:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104046115277267866384402959868672283394","date":"2025-08-25T06:22:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-24T22:02:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-24T20:20:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-20T23:49:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Functional \u0026 Integrative Genomics","date":"2025-08-18T02:45:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e41c5f01-19f2-406d-bd0b-55f880c4ebdf","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:01:49+00:00","versionOfRecord":{"articleIdentity":"rs-7395169","link":"https://doi.org/10.1007/s10142-025-01771-2","journal":{"identity":"functional-and-integrative-genomics","isVorOnly":false,"title":"Functional \u0026 Integrative Genomics"},"publishedOn":"2025-12-18 15:57:55","publishedOnDateReadable":"December 18th, 2025"},"versionCreatedAt":"2025-09-01 16:41:27","video":"","vorDoi":"10.1007/s10142-025-01771-2","vorDoiUrl":"https://doi.org/10.1007/s10142-025-01771-2","workflowStages":[]},"version":"v1","identity":"rs-7395169","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7395169","identity":"rs-7395169","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}