Ethanol extract of Lentinus edodes exerted antioxidant activity in Caenorhabditis elegans by regulating insulin DAF-16 signaling pathway

Ethanol extract of Lentinus edodes exerted antioxidant activity in Caenorhabditis elegans by regulating insulin DAF-16 signaling pathway | 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 Ethanol extract of Lentinus edodes exerted antioxidant activity in Caenorhabditis elegans by regulating insulin DAF-16 signaling pathway Quan-Cen Li, Na Li, Wen-Wen Cai, Bin Liu, Feng Zeng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1937791/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Lentinus edodes ( L. edodes ) was a fungus plant, which was widely grown and eaten as food in China. As an antioxidant, the polysaccharide of L. edodes has been widely studied in terms of antioxidant activity, however, the effect of action and underlying mechanism of L. edodes ethanol extracts (LEEE) in antioxidant aspects were largely unknown. In this study, the antioxidant activity of LEEE was investigated in vitro . In addition, the antioxidant activity of LEEE was estimated in vivo through Caenorhabditis elegans model. It was found that LEEE had excellent antioxidant activity in vitro by scavenging DPPH, ABTS and hydroxyl radicals. At the same time, the anti-heat stress abilities of C. elegans were enhanced after supplementation with different concentrations of LEEE. The activities of antioxidant enzymes such as T-SOD, GSH-PX and CAT were significantly enhanced and the levels of MDA and ROS were significantly down-regulated. However, the level of apoptosis of C. elegans could not be improved after supplementation with LEEE. In the study of LEEE antioxidant-related signaling pathways through q-PCR experiments, it was found that LEEE exerted antioxidant activity in C. elegans by up-regulating MTL-1, DAF-16 and SOD-3 mRNA expression and down-regulating DAF-2 mRNA expression. This provided a solid foundation for the active substances of Lentinus edodes in improving oxidation-related diseases. Ethanol extract Lentinus edodes Antioxidant Caenorhabditis elegans Insulin DAF-16 signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Oxidation of organisms was an inevitable process. Under normal conditions, oxidation and anti-oxidation were in a dynamic equilibrium relationship [ 1 ]. When the organism was stimulated by the external environment or the increase of age, this balance was broken, the expression of reactive oxygen species (ROS) free radicals in mitochondria increased, causing an increase in malondialdehyde (MDA) content and inhibited the expression of organismal antioxidant enzymes such as total superoxide dismutase (T-SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) [ 2 , 3 ]. The malignant changes of these biochemical indicators have been confirmed to be closely related to the adverse effects of aging, inflammation, lipid metabolism disorders, and immune weakness of the organism [ 4 – 6 ]. Therefore, it had an important significance for improving the homeostasis of the organism and regulating the health of the body by exploring the antioxidant activity of active substances in natural products. Lentinus edodes ( L. edodes ), a fungal plant widely cultivated in China, was rich in a variety of active substances such as polysaccharides, organic bases, purines and ribonucleic acid, which played an important role in anti-aging, anti-oxidation, regulating lipid metabolism, improving immunity and anti-inflammatory activities [ 7 – 9 ]. In the study of lentinan by Liu et al. [ 10 ], it was found that lentinan can prolong the lifespan of C. elegans and improve the antioxidant capacity by up-regulating the expression of DAF-16 and SKN-1 signaling factors. Cao et al. [ 11 ] found that Lentinus edodes mycelia polysaccharide ameliorated cell damage by modulating the MAPK pathway to regulate lipid metabolism. However, polysaccharide was the focus of research on the nutritional activity of the active substances from L. edodes in recent years, and There were few studies on alcohol extracts with polyphenols as main components. In this study, the antioxidant activity of ethanol extract of Lentinus edodes (LEEE) was explored by determining the scavenging activity of free radicals in vitro . In addition, the antioxidant activity of LEEE on C. elegans was evaluated through estimating the survival time under oxidative stress conditions, the content of related enzymes and the level of cell apoptosis. At the same time, the signaling factors related to oxidative stress were explored. These results might provide value for exploring the antioxidant activity of fungi plants and related mechanisms of action. Materials And Methods The material and methods section were presented as supplementary information. Results And Discussion Antioxidant activity of LEEE in vitro The result and discussion of antioxidant activity in vitro of LEEE were presented as supplementary information. The stress resistance of C. elegans The survival time of C. elegans treated with LEEE under heat stress and acute oxidative stress was the most intuitive way to evaluate the antioxidant capacity of LEEE [10]. In the study of heat stress resistance of C. elegans in an environment of 37 °C, it was found that although the maximum lifespan of C. elegans treated with different concentrations of LEEE did not change, the median lifespan was significantly increased (Fig 1A, B). The median lifespan of the LEEE-L, LEEE-M and LEEE-H groups increased by 7.55% ( p ＜0.01), 9.08% ( p ＜0.01) and 5.01% ( p ＜0.05) respectively compared to the NFD group(Fig 1B). However, in experiments on LEEE-treated C. elegans against acute oxidative stress, it was found that the median lifespan of nematodes had an upward trend, but there was no significant change (Fig 1C, D). It might be caused by the short time or low concentration of LEEE supplementation before the stress study [12]. In conclusion, LEEE had antioxidant activity by increasing the survival time of C. elegans under heat stress conditions. However, it did not improve the lifespan of C. elegans under acute oxidative stress conditions. The activity of anti-oxidant enzymes, and MDA and ROS levels ROS was a strong oxidant, its expression increased sharply when the organism was stimulated by the external environment, which caused lipid and protein peroxidation [10,13]. At the same time, the level of MDA was increased in the organism and the levels of antioxidant enzymes such as T-SOD, GSH-PX and CAT were decreased, which indicated that damaged to the organism [13]. Therefore, the activity of anti-oxidant enzymes, and MDA and ROS levels of C. elegans by supplement with DIPH were determined in this study (Fig 2). The activities of antioxidant enzymes were significantly improved, while the content of MDA and the relative expression of ROS were significantly decreased in C. elegans treated with LEEE for 72 h. After 72 hours of LEEE treatment, the MDA level of C. elegans showed a downward trend. Compared with the NFD group, the MDA level of C. elegans decreased by 40.19%, 84.22% ( p ＜0.01) and 85.03% ( p ＜0.01) in the LEEE-L, LEEE-M and LEEE-H groups, respectively (Fig 2A). In addition, there was a positive dose-response relationship between T-SOD antioxidant enzyme levels of C. elegans and the concentration of LEEE [10, 13]. Compared with the NFD group, the T-SOD levels of the LEEE-L, LEEE-M, and LEEE-H groups were increased by 2.00, 3.50 and 6.74 times, respectively ( p ＜0.01) (Fig 2B). However, in the study of LEEE improving the GSH-PX and CAT antioxidant enzyme activities of C. elegans , it was found that the nematodes in the LEEE-L group had the best improvement effect, which was 0.98 times ( p ＜0.01) and 1.09 times ( p ＜0.05) higher than the NFD group, respectively (Fig 2C, D) [10,14]. But there were no significant changes in the LEEE-M and LEEE-H groups (Fig 2C, D). In the regulation of ROS level, with the increase of LEEE concentration, the relative expression of ROS of C. elegans showed a downward trend. Meanwhile, the relative expression of ROS in the LEEE-H group decreased by 20.59% compared with the NFD group ( p ＜0.05) (Fig 2E). In conclusion, these results implied that LEEE had excellent antioxidant activity by up-regulating the levels of antioxidant enzymes such as T-SOD, GSH-PX and CAT, and down-regulating the MDA content and the relative expression of ROS. Cell apoptosis The degree of DNA damage increased with age, and this process was closely related to cell apoptosis [13]. As a stain, acridine orange could enter into the cell through the cell membrane of apoptotic cells and stain green under a fluorescence microscope [15]. With the greater the degree of DNA damage and the more apoptotic cells, the stronger the green fluorescence intensity and the larger the fluorescence area observed under the fluorescence microscope [15]. It was found that there was no significant change in the staining effect of acridine orange under a fluorescence microscope to C. elegans treated with different concentrations of LEEE for 72h (Fig 3A-H). However, after using Image J software to analyze the relative fluorescence intensity of the pictures, it was found that the relative fluorescence intensity had a downward trend with the increase of LEEE concentration, but there was no significant change (Fig 3I). These results demonstrated that it did not exhibit antioxidant activity at the level of apoptosis in C. elegans treated with different concentrations of LEEE (0.1-1.0 mg/mL). Effect of LEEE on mRNA expressions As one of the lifespan regulators of C. elegans , DAF-16 played an important role in anti-oxidation, anti-aging and regulation of immunity [14, 16]. At the same time, DAF-2, AGE-1, MTL-1, SOD-3 are its upstream and downstream signaling factors, and their changes play an important role in clarifying the signaling pathway and mechanism of LEEE antioxidant effect in C. elegans [17] . In addition, SKN-1 has been shown to be related to the expression of reactive oxygen species, and it was regulated by a variety of factors, including the MAPK signaling pathway, which could be activated by phosphorylating SKN-1 [18]. The specific signaling factors were verified from the mRNA level in order to explore the signaling pathway and mechanism of LEEE regulating the antioxidant activity of C. elegans (Fig 4). In the mRNA expression of DAF-16 and MTL-1, it was found that compared with NFD group, the signal factors in the LEEE-M group were significantly increased, which increased by 1.49 and 2.82 times ( p ＜0.01) (Fig 4A, B). The regulation of DAF-2 signaling factor showed a negative dose-effect relationship of C. elegans treated with different concentrations of LEEE, and the mRNA expression of DAF-2 decreased with the increase of LEEE concentration. Compared with the NFD group, the relative expression levels of DAF-2 in the LEEE-M group and LEEE-H group were significantly down-regulated, which decreased by 59.45% and 72.31%, respectively ( p ＜0.01) (Fig 4B). At the same time, it was found that although supplementation of LEEE could up-regulate its relative expression in the study of the regulation of SOD-3 downstream factors, it did not show a positive dose-effect dependence, and the LEEE-M group had the highest relative expression increase, which increased by 1.12 times ( p ＜0.01) (Fig 4C). However, it was found that its antioxidant activity was not related to the mRNA of AGE-1 and SKN-1 in the study of LEEE regulating the expression of antioxidant-related genes in C. elegans , because it had no significant expression of these two genes for the nematodes treated with different concentrations of LEEE (Fig 4A, C). Conclusion Lentinula edodes was widely planted in China, rich in nutritional value and inexpensive fungi, and were often used as food by humans. It is rich in various active substances with excellent biological activity. In this study, the antioxidant activity of LEEE were explored in vitro. In addition, the specific regulatory mechanism of LEEE in terms of antioxidant activity was explored through using the C. elegans model. The excellent antioxidant activity in vitro was found on LEEE by scavenging DPPH, ABTS and hydroxyl radicals. At the same time, the lifespan of C. elegans in heat stress environments was extended by supplementation with LEEE. In addition, the antioxidant enzyme activities such as T-SOD, GSH-PX and CAT were increased and the levels of MDA and ROS were down-regulated. However, the level of cell apoptosis in C. elegans did not change significantly after supplementation with LEEE. The study of related mRNA expression by gene expression level found that LEEE exerted antioxidant effect by up-regulating the mRNA expression of MTL-1, DAF-16 and SOD-3 and down-regulating the mRNA expression of DAF-2 of C. elegans. These studies provided a theoretical basis for the research on antioxidation and related diseases of edible and medicinal fungi represented by Lentinula edodes. Declarations Funding This work was financially supported by Key Research and Development Project of Jiangxi Province (20192ACB60008). Conflict of Interest The authors declare that they have no conflict of interest. Data Availability Not applicable. Authors' contributions Quan-cen Li and Na Li conducted the study, drafted the first manuscript; Wen-wen Cai analyzed the data; Bin Liu and Feng Zeng conceived and designed the study, critically reviewed the manuscript. All authors approved the final manuscript for submission. Ethics approval Not applicable. Consent to participate The present paper has been approved by all named authors. Consent for publication The present paper, which is original, has not been published before and is not currently being considered for publication elsewhere. References Lennicke C, Cochemé HM (2021) Redox metabolism: ROS as specific molecular regulators of cell signaling and function. Mol Cell 81:3691–3707. https://doi.org/10.1016/j.molcel.2021.08.018 Iqbal H, Yadav P, Verma AK, Mishra D, Vamadevan B, Singh D, Luqman S, Negi AS, Chanda D (2022) Anti-inflammatory, anti-oxidant and cardio-protective properties of novel fluorophenyl benzimidazole in L-NAME-induced hypertensive rats. Eur J Pharmacol 929:175132. https://doi.org/10.1016/j.ejphar.2022.175132 Lee SA, Lim WH, Van Le V, Ko SR, Kim B, Oh HM, Ahn CY (2022) Lifespan extension and anti-oxidant effects of carotenoid pigments in Caenorhabditis elegans . Bioresource Technol Rep 17:100962. https://doi.org/10.1016/j.biteb.2022.100962 Goshtasbi H, Pakchin PS, Movafeghi A, Barar J, Castejon AM, Omidian H, Omidi Y (2022) Impacts of oxidants and antioxidants on the emergence and progression of Alzheimer's disease. Neurochem Int 153:105268. https://doi.org/10.1016/j.neuint.2021.105268 Wassie T, Cheng B, Zhou TT, Gao LM, Lu Z, Wang JL, Mulu B, Taye M, Wu X (2022) Enteromorpha polysaccharide and yeast glycoprotein mixture improves growth, antioxidant activity, serum lipid profile and regulates lipid metabolism in broiler chickens. Poult Sci 102064. https://doi.org/10.1016/j.psj.2022.102064 Hemmati-Dinarvand M, Saedi S, Valilo M, Kalantary-Charvadeh A, Alizadeh Sani M, Kargar R, Safari H, Samadi N (2019) Oxidative stress and Parkinson’s disease: Conflict of oxidant-antioxidant systems. Neurosci Lett 709:134296. https://doi.org/10.1016/j.neulet.2019.134296 Trivedi S, Patel K, Belgamwar V, Wadher K (2022) Functional polysaccharide lentinan: Role in anti-cancer therapies and management of carcinomas. Pharmacol Res - Mod Chin Med 2:100045. https://doi.org/10.1016/j.prmcm.2022.100045 Rao ZL, Dong YT, Zheng XJ, Tang KY, Liu J (2021) Extraction, purification, bioactivities and prospect of lentinan: A review. Biocatal Agric Biotechnol 37:102163. https://doi.org/10.1016/j.bcab.2021.102163 Cui LL, Liu YH, Liu M, Ren MJ, Ahmed AF, Kang WY (2022) Identification of phytochemicals from Lentinus edodes and Auricularia auricula with UPLC-Q-Exactive Orbitrap MS. J Future Foods 2:253–260. https://doi.org/10.1016/j.jfutfo.2022.06.006 Liu HM, Wang Y, Zhang WP, Sun WC, Ji XX, Zhang SA, Qiao K (2022) Lentinan extends lifespan and increases oxidative stress resistance through DAF-16 and SKN-1 pathways in Caenorhabditis elegans . Int J Biol Macromol 202:286–295. https://doi.org/10.1016/j.ijbiomac.2022.01.071 Cao XY, Xia Y, Liu D, He YL, Mu T, Huo YP, Liu JL (2020) Inhibitory effects of Lentinus edodes mycelia polysaccharide on α-glucosidase, glycation activity and high glucose-induced cell damage. Carbohyd Polym 246:116659. https://doi.org/10.1016/j.carbpol.2020.116659 Xiao X, Zhou YR, Tan C, Bai J, Zhu Y, Zhang JY, Zhou XH, Zhao YS (2021) Barley β-glucan resist oxidative stress of Caenorhabditis elegans via daf-2/daf-16 pathway. Int J Biol Macromol 193:1021–1031. https://doi.org/10.1016/j.ijbiomac.2021.11.067 Chen YH, Wan XZ, Wu DS, Ouyang YZ, Gao LY, Chen ZX, El-Seedi HR, Wang MF, Chen XH, Zhao C (2020) Characterization of the structure and analysis of the anti-oxidant effect of microalga Spirulina platensis polysaccharide on Caenorhabditis elegans mediated by modulating microRNAs and gut microbiota. Int J Biol Macromol 163:2295–2305. https://doi.org/10.1016/j.ijbiomac.2020.09.041 Shao XY, He T, Lai YL, Chen M, Tong ZH (2022) Water-soluble polysaccharides extracted from Pueraria lobata delay aging of Caenorhabditis elegans under heat stress. Plant Food Hum Nutr 77:220–225. https://doi.org/10.1007/s11130-022-00964-5 Lin GP, Wu DS, Xiao XW, Huang QY, Chen HB, Liu D, Fu HQ, Chen XH, Zhao C (2020) Structural characterization and antioxidant effect of green alga Enteromorpha prolifera polysaccharide in Caenorhabditis elegans via modulation of microRNAs. Int J Biol Macromol 150:1084–1092. https://doi.org/10.1016/j.ijbiomac.2019.10.114 Uno M, Tani Y, Nono M, Okabe E, Kishimoto S, Takahashi C, Abe R, Kurihara T, Nishida E (2021) Neuronal DAF-16-to-intestinal DAF-16 communication underlies organismal lifespan extension in C. elegans . iScience 24:102706. https://doi.org/10.1016/j.isci.2021.102706 Wang EH, Wang NB, Zou Y, Fahim M, Zhou YT, Yang H, Liu Y, Li HY (2022) Black mulberry ( Morus nigra ) fruit extract alleviated AD-Like symptoms induced by toxic Aβ protein in transgenic Caenorhabditis elegans via insulin DAF-16 signaling pathway. Food Res Int 160:111696. https://doi.org/10.1016/j.foodres.2022.111696 Wu CG, Karakuzu O, Garsin DA (2021) Tribbles pseudokinase NIPI-3 regulates intestinal immunity in Caenorhabditis elegans by controlling SKN-1/Nrf activity. Cell Rep 36:109529. https://doi.org/10.1016/j.celrep.2021.109529 Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1937791","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":127671076,"identity":"f47cc1e4-2e2b-45b7-822b-3be6b0096daf","order_by":0,"name":"Quan-Cen Li","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Quan-Cen","middleName":"","lastName":"Li","suffix":""},{"id":127671077,"identity":"21ada631-8c61-46ca-bde5-3494563ea1e1","order_by":1,"name":"Na Li","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Li","suffix":""},{"id":127671080,"identity":"510c25e6-2da6-4dfd-9684-c2a568e8902d","order_by":2,"name":"Wen-Wen Cai","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wen-Wen","middleName":"","lastName":"Cai","suffix":""},{"id":127671082,"identity":"1f11c9b4-bdde-4993-951e-fdc71f8eab77","order_by":3,"name":"Bin Liu","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Liu","suffix":""},{"id":127671083,"identity":"8b5e7bb2-a705-4a89-ab69-0fcf3784b726","order_by":4,"name":"Feng Zeng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYBAC9gY4g/nAgQ8/iNDCcwDOYEs8OLOHNC08xoc52IjRwt57+OWPijt2PfxnPhxm4GGQ5xc7QEALz7k0C4kzz5J7GM5uOFxgwWA4c3YCfi32EjlmBoZth5PtGXs3HJ7Bw5BgcJuAFh75N2YGif8OJ/Mw8zw4zMNGjBYJHuMHBxsO2/Gw8TAQqYUnx4yx4djhBB4eNgNgIEsQ9gsP+xnjjz9qDtvz8B9+/OHDDxt5fmkCWoCATQJIJDZAOBIElYMA8wcgYU+U0lEwCkbBKBiZAACuk0X7YDBIUgAAAABJRU5ErkJggg==","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Zeng","suffix":""}],"badges":[],"createdAt":"2022-08-07 10:29:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1937791/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1937791/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":25101516,"identity":"950edc6c-5332-46e6-9ed6-38b64b2aeb79","added_by":"auto","created_at":"2022-08-11 17:24:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":101478,"visible":true,"origin":"","legend":"\u003cp\u003eThe activity of stress resistance by different concentrations of LEEE (0, 0.1, 0.5 and 1 mg/mL) groups of \u003cem\u003eC. elegans\u003c/em\u003e. Percent survival (A) and median lifespan (B) of \u003cem\u003eC. elegans\u003c/em\u003e at 37℃. Percent survival (C) and median lifespan (D) of \u003cem\u003eC. elegans\u003c/em\u003e under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05and\u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05, compared to the NFD group.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1937791/v1/2384bf1ac151e45d13554eee.png"},{"id":25101118,"identity":"d457e5d9-b885-4112-8176-109a167e631b","added_by":"auto","created_at":"2022-08-11 17:19:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81710,"visible":true,"origin":"","legend":"\u003cp\u003e\tThe activity of anti-oxidant enzymes (B-D), and MDA (A) and ROS (E) levels by different concentrations of LEEE (0, 0.1, 0.5 and 1 mg/mL) groups of \u003cem\u003eC. elegans\u003c/em\u003e. Bars indicated means ± SD (n=3). \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05and\u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05, compared to the NFD group.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1937791/v1/22624fdda3aa09f1cde5e37b.png"},{"id":25101122,"identity":"be418847-dbde-423e-9938-ae6b7b924182","added_by":"auto","created_at":"2022-08-11 17:19:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":282003,"visible":true,"origin":"","legend":"\u003cp\u003e\tApoptotic cells in the NFD (A-B), LEEE-L (C-D), LEEE-M (E-F) and LEEE-H (G-H) groups observed with a fluorescence microscope. The fluorescence intensity of different concentrations of LEEE (0, 0.1, 0.5 and 1 mg/mL) groups of \u003cem\u003eC. elegans\u003c/em\u003e cells stained with acridine orange (I). Bars indicated means ± SD (n=3).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1937791/v1/c98dbff93d301c329ecf77db.png"},{"id":25101120,"identity":"da4d4421-ef03-4e2a-a396-bcd374a7953b","added_by":"auto","created_at":"2022-08-11 17:19:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":55328,"visible":true,"origin":"","legend":"\u003cp\u003e\tEffects of LEEE on the expressions of mRNAs: AGE-1 and MTL-1 (A), DAF-2 and DAF-16 (B) and SOD-3 and SKN-1 (C) in \u003cem\u003eC. elegans.\u003c/em\u003e \u003csup\u003e⁎\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 and \u003csup\u003e⁎⁎\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, compared to the NFD group.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1937791/v1/d9c98271cdc496c148586062.png"},{"id":27208336,"identity":"ec148d49-6b35-44fa-8263-cb5bacbea00d","added_by":"auto","created_at":"2022-09-30 19:44:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":674232,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1937791/v1/2b987848-664c-4fca-a87b-efb3b53295c3.pdf"},{"id":25101517,"identity":"c98223ad-417b-4e85-a1c7-0b62ef311b70","added_by":"auto","created_at":"2022-08-11 17:24:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1139578,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-1937791/v1/47d712a50ed357cd7a99574d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ethanol extract of Lentinus edodes exerted antioxidant activity in Caenorhabditis elegans by regulating insulin DAF-16 signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOxidation of organisms was an inevitable process. Under normal conditions, oxidation and anti-oxidation were in a dynamic equilibrium relationship [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. When the organism was stimulated by the external environment or the increase of age, this balance was broken, the expression of reactive oxygen species (ROS) free radicals in mitochondria increased, causing an increase in malondialdehyde (MDA) content and inhibited the expression of organismal antioxidant enzymes such as total superoxide dismutase (T-SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The malignant changes of these biochemical indicators have been confirmed to be closely related to the adverse effects of aging, inflammation, lipid metabolism disorders, and immune weakness of the organism [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, it had an important significance for improving the homeostasis of the organism and regulating the health of the body by exploring the antioxidant activity of active substances in natural products.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLentinus edodes\u003c/em\u003e (\u003cem\u003eL. edodes\u003c/em\u003e), a fungal plant widely cultivated in China, was rich in a variety of active substances such as polysaccharides, organic bases, purines and ribonucleic acid, which played an important role in anti-aging, anti-oxidation, regulating lipid metabolism, improving immunity and anti-inflammatory activities [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the study of lentinan by Liu et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], it was found that lentinan can prolong the lifespan of C. elegans and improve the antioxidant capacity by up-regulating the expression of DAF-16 and SKN-1 signaling factors. Cao et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] found that \u003cem\u003eLentinus edodes\u003c/em\u003e mycelia polysaccharide ameliorated cell damage by modulating the MAPK pathway to regulate lipid metabolism. However, polysaccharide was the focus of research on the nutritional activity of the active substances from \u003cem\u003eL. edodes\u003c/em\u003e in recent years, and There were few studies on alcohol extracts with polyphenols as main components.\u003c/p\u003e \u003cp\u003eIn this study, the antioxidant activity of ethanol extract of \u003cem\u003eLentinus edodes\u003c/em\u003e (LEEE) was explored by determining the scavenging activity of free radicals \u003cem\u003ein vitro\u003c/em\u003e. In addition, the antioxidant activity of LEEE on \u003cem\u003eC. elegans\u003c/em\u003e was evaluated through estimating the survival time under oxidative stress conditions, the content of related enzymes and the level of cell apoptosis. At the same time, the signaling factors related to oxidative stress were explored. These results might provide value for exploring the antioxidant activity of fungi plants and related mechanisms of action.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003eThe material and methods section were presented as supplementary information.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003e\u003cstrong\u003eAntioxidant activity of LEEE\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe result and discussion of antioxidant activity\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e of LEEE were presented as supplementary information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe stress resistance of \u003cem\u003eC. elegans\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe survival time of \u003cem\u003eC. elegans\u003c/em\u003e treated with LEEE under heat stress and acute oxidative stress was the most intuitive way to evaluate the antioxidant capacity of LEEE [10]. In the study of heat stress resistance of \u003cem\u003eC. elegans\u003c/em\u003e in an environment of 37 \u0026deg;C, it was found that although the maximum lifespan of \u003cem\u003eC. elegans\u003c/em\u003e treated with different concentrations of LEEE did not change, the median lifespan was significantly increased (Fig 1A, B). The median lifespan of the LEEE-L, LEEE-M and LEEE-H groups increased by 7.55% (\u003cem\u003ep\u003c/em\u003e＜0.01), 9.08% (\u003cem\u003ep\u003c/em\u003e＜0.01) and 5.01% (\u003cem\u003ep\u003c/em\u003e＜0.05) respectively compared to the NFD group(Fig 1B). However, in experiments on LEEE-treated \u003cem\u003eC. elegans\u003c/em\u003e against acute oxidative stress, it was found that the median lifespan of nematodes had an upward trend, but there was no significant change (Fig 1C, D). It might be caused by the short time or low concentration of LEEE supplementation before the stress study [12]. In conclusion, LEEE had antioxidant activity by increasing the survival time of \u003cem\u003eC. elegans\u003c/em\u003e under heat stress conditions. However, it did not improve the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e under acute oxidative stress conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe activity of anti-oxidant enzymes, and MDA and ROS levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eROS was a strong oxidant, its expression increased sharply when the organism was stimulated by the external environment, which caused lipid and protein peroxidation [10,13]. At the same time, the level of MDA was increased in the organism and the levels of antioxidant enzymes such as T-SOD, GSH-PX and CAT were decreased, which indicated that damaged to the organism [13]. Therefore, the activity of anti-oxidant enzymes, and MDA and ROS levels of\u003cem\u003e\u0026nbsp;C. elegans\u003c/em\u003e by supplement with DIPH were determined in this study (Fig 2). The activities of antioxidant enzymes were significantly improved, while the content of MDA and the relative expression of ROS were significantly decreased in \u003cem\u003eC. elegans\u003c/em\u003e treated with LEEE for 72 h. After 72 hours of LEEE treatment, the MDA level of \u003cem\u003eC. elegans\u003c/em\u003e showed a downward trend. Compared with the NFD group, the MDA level of \u003cem\u003eC. elegans\u003c/em\u003e decreased by 40.19%, 84.22% (\u003cem\u003ep\u003c/em\u003e＜0.01)\u0026nbsp;and 85.03% (\u003cem\u003ep\u003c/em\u003e＜0.01) in the LEEE-L, LEEE-M and LEEE-H groups, respectively (Fig 2A). In addition, there was a positive dose-response relationship between T-SOD antioxidant enzyme levels of \u003cem\u003eC. elegans\u003c/em\u003e and the concentration of LEEE [10, 13]. Compared with the NFD group, the T-SOD levels of the LEEE-L, LEEE-M, and LEEE-H groups were increased by 2.00, 3.50 and 6.74 times, respectively (\u003cem\u003ep\u003c/em\u003e＜0.01)\u0026nbsp;(Fig 2B). However, in the study of LEEE improving the GSH-PX and CAT antioxidant enzyme activities of \u003cem\u003eC. elegans\u003c/em\u003e, it was found that the nematodes in the LEEE-L group had the best improvement effect, which was 0.98 times (\u003cem\u003ep\u003c/em\u003e＜0.01) and 1.09 times (\u003cem\u003ep\u003c/em\u003e＜0.05) higher than the NFD group, respectively (Fig 2C, D) [10,14]. But there were no significant changes in the LEEE-M and LEEE-H groups (Fig 2C, D). In the regulation of ROS level, with the increase of LEEE concentration, the relative expression of ROS of \u003cem\u003eC. elegans\u003c/em\u003e showed a downward trend. Meanwhile, the relative expression of ROS in the LEEE-H group decreased by 20.59% compared with the NFD group (\u003cem\u003ep\u003c/em\u003e＜0.05) (Fig 2E). In conclusion, these results implied that LEEE had excellent antioxidant activity by up-regulating the levels of antioxidant enzymes such as T-SOD, GSH-PX and CAT, and down-regulating the MDA content and the relative expression of ROS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe degree of DNA damage increased with age, and this process was closely related to cell apoptosis [13]. As a stain, acridine orange could enter into the cell through the cell membrane of apoptotic cells and stain green under a fluorescence microscope [15]. With the greater the degree of DNA damage and the more apoptotic cells, the stronger the green fluorescence intensity and the larger the fluorescence area observed under the fluorescence microscope [15]. It was found that there was no significant change in the staining effect of acridine orange under a fluorescence microscope to \u003cem\u003eC. elegans\u003c/em\u003e treated with different concentrations of LEEE for 72h (Fig 3A-H). However, after using Image J software to analyze the relative fluorescence intensity of the pictures, it was found that the relative fluorescence intensity had a downward trend with the increase of LEEE concentration, but there was no significant change (Fig 3I). These results demonstrated that it did not exhibit antioxidant activity at the level of apoptosis in\u003cem\u003e\u0026nbsp;C. elegans\u003c/em\u003e treated with different concentrations of LEEE (0.1-1.0 mg/mL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of LEEE on mRNA expressions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs one of the lifespan regulators of \u003cem\u003eC. elegans\u003c/em\u003e, DAF-16 played an important role in anti-oxidation, anti-aging and regulation of\u0026nbsp;immunity\u0026nbsp;[14, 16]. At the same time, DAF-2, AGE-1, MTL-1, SOD-3 are its upstream and downstream signaling factors, and their changes play an important role in clarifying the signaling pathway and mechanism of LEEE antioxidant effect in \u003cem\u003eC. elegans\u003c/em\u003e [17]\u003cem\u003e.\u003c/em\u003e In addition, SKN-1 has been shown to be related to the expression of reactive oxygen species, and it was regulated by a variety of factors, including the MAPK signaling pathway, which could be activated by phosphorylating SKN-1 [18]. The specific signaling factors were verified from the mRNA level in order to explore the signaling pathway and mechanism of LEEE regulating the antioxidant activity of\u003cem\u003e\u0026nbsp;C. elegans\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Fig 4).\u0026nbsp;In the mRNA expression of DAF-16 and MTL-1, it was found that compared with NFD group, the signal factors in the LEEE-M group were significantly increased, which increased by 1.49 and 2.82 times (\u003cem\u003ep\u003c/em\u003e＜0.01) (Fig 4A, B).\u0026nbsp;The regulation of DAF-2 signaling factor showed a negative dose-effect relationship of\u003cem\u003e\u0026nbsp;C. elegans\u003c/em\u003e treated with different concentrations of LEEE, and the mRNA expression of DAF-2 decreased with the increase of LEEE concentration.\u0026nbsp;Compared with the NFD group, the relative expression levels of DAF-2 in the LEEE-M group and LEEE-H group were significantly down-regulated, which decreased by 59.45% and 72.31%, respectively (\u003cem\u003ep\u003c/em\u003e＜0.01) (Fig 4B). At the same time, it was found that although supplementation of LEEE could up-regulate its relative expression in the study of the regulation of SOD-3 downstream factors, it did not show a positive dose-effect dependence, and the LEEE-M group had the highest relative expression increase, which increased by 1.12 times (\u003cem\u003ep\u003c/em\u003e＜0.01) (Fig 4C). However, it was found that its antioxidant activity was not related to the mRNA of AGE-1 and SKN-1 in the study of LEEE regulating the expression of antioxidant-related genes in \u003cem\u003eC. elegans\u003c/em\u003e, because it had no significant expression of these two genes for the nematodes treated with different concentrations of LEEE (Fig 4A, C).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cem\u003eLentinula edodes\u003c/em\u003e was widely planted in China, rich in nutritional value and inexpensive fungi, and were often used as food by humans. It is rich in various active substances with excellent biological activity. In this study, the antioxidant activity of LEEE were explored \u003cem\u003ein vitro.\u003c/em\u003e In addition, the specific regulatory mechanism of LEEE in terms of antioxidant activity was explored through using the \u003cem\u003eC. elegans\u003c/em\u003e model. The excellent antioxidant activity \u003cem\u003ein vitro\u003c/em\u003e was found on LEEE by scavenging DPPH, ABTS and hydroxyl radicals. At the same time, the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e in heat stress environments was extended by supplementation with LEEE. In addition, the antioxidant enzyme activities such as T-SOD, GSH-PX and CAT were increased and the levels of MDA and ROS were down-regulated. However, the level of cell apoptosis in \u003cem\u003eC. elegans\u003c/em\u003e did not change significantly after supplementation with LEEE. The study of related mRNA expression by gene expression level found that LEEE exerted antioxidant effect by up-regulating the mRNA expression of MTL-1, DAF-16 and SOD-3 and down-regulating the mRNA expression of DAF-2 of \u003cem\u003eC. elegans.\u003c/em\u003e These studies provided a theoretical basis for the research on antioxidation and related diseases of edible and medicinal fungi represented by \u003cem\u003eLentinula edodes.\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by Key Research and Development Project of Jiangxi Province (20192ACB60008).\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 conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuan-cen Li and Na Li conducted the study, drafted the first manuscript; Wen-wen Cai analyzed the data; Bin Liu and Feng Zeng conceived and designed the study, critically reviewed the manuscript. All authors approved the final manuscript for submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present paper has been approved by all named authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present paper, which is original, has not been published before and is not currently being considered for publication elsewhere.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eLennicke C, Cochem\u0026eacute; HM (2021) Redox metabolism: ROS as specific molecular regulators of cell signaling and function. Mol Cell 81:3691\u0026ndash;3707. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.molcel.2021.08.018\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eIqbal H, Yadav P, Verma AK, Mishra D, Vamadevan B, Singh D, Luqman S, Negi AS, Chanda D (2022) Anti-inflammatory, anti-oxidant and cardio-protective properties of novel fluorophenyl benzimidazole in L-NAME-induced hypertensive rats. Eur J Pharmacol 929:175132. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ejphar.2022.175132\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLee SA, Lim WH, Van Le V, Ko SR, Kim B, Oh HM, Ahn CY (2022) Lifespan extension and anti-oxidant effects of carotenoid pigments in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e. Bioresource Technol Rep 17:100962. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biteb.2022.100962\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGoshtasbi H, Pakchin PS, Movafeghi A, Barar J, Castejon AM, Omidian H, Omidi Y (2022) Impacts of oxidants and antioxidants on the emergence and progression of Alzheimer\u0026apos;s disease. Neurochem Int 153:105268. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.neuint.2021.105268\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWassie T, Cheng B, Zhou TT, Gao LM, Lu Z, Wang JL, Mulu B, Taye M, Wu X (2022) \u003cem\u003eEnteromorpha\u003c/em\u003e polysaccharide and yeast glycoprotein mixture improves growth, antioxidant activity, serum lipid profile and regulates lipid metabolism in broiler chickens. Poult Sci 102064. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.psj.2022.102064\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHemmati-Dinarvand M, Saedi S, Valilo M, Kalantary-Charvadeh A, Alizadeh Sani M, Kargar R, Safari H, Samadi N (2019) Oxidative stress and Parkinson\u0026rsquo;s disease: Conflict of oxidant-antioxidant systems. Neurosci Lett 709:134296. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.neulet.2019.134296\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTrivedi S, Patel K, Belgamwar V, Wadher K (2022) Functional polysaccharide lentinan: Role in anti-cancer therapies and management of carcinomas. Pharmacol Res - Mod Chin Med 2:100045. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.prmcm.2022.100045\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRao ZL, Dong YT, Zheng XJ, Tang KY, Liu J (2021) Extraction, purification, bioactivities and prospect of lentinan: A review. Biocatal Agric Biotechnol 37:102163. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bcab.2021.102163\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCui LL, Liu YH, Liu M, Ren MJ, Ahmed AF, Kang WY (2022) Identification of phytochemicals from \u003cem\u003eLentinus edodes\u003c/em\u003e and \u003cem\u003eAuricularia auricula\u003c/em\u003e with UPLC-Q-Exactive Orbitrap MS. J Future Foods 2:253\u0026ndash;260. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jfutfo.2022.06.006\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLiu HM, Wang Y, Zhang WP, Sun WC, Ji XX, Zhang SA, Qiao K (2022) Lentinan extends lifespan and increases oxidative stress resistance through DAF-16 and SKN-1 pathways in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e. Int J Biol Macromol 202:286\u0026ndash;295. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2022.01.071\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCao XY, Xia Y, Liu D, He YL, Mu T, Huo YP, Liu JL (2020) Inhibitory effects of \u003cem\u003eLentinus edodes\u003c/em\u003e mycelia polysaccharide on \u0026alpha;-glucosidase, glycation activity and high glucose-induced cell damage. Carbohyd Polym 246:116659. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.carbpol.2020.116659\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eXiao X, Zhou YR, Tan C, Bai J, Zhu Y, Zhang JY, Zhou XH, Zhao YS (2021) Barley \u0026beta;-glucan resist oxidative stress of \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e via daf-2/daf-16 pathway. Int J Biol Macromol 193:1021\u0026ndash;1031. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2021.11.067\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eChen YH, Wan XZ, Wu DS, Ouyang YZ, Gao LY, Chen ZX, El-Seedi HR, Wang MF, Chen XH, Zhao C (2020) Characterization of the structure and analysis of the anti-oxidant effect of microalga \u003cem\u003eSpirulina platensis\u003c/em\u003e polysaccharide on \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e mediated by modulating microRNAs and gut microbiota. Int J Biol Macromol 163:2295\u0026ndash;2305. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2020.09.041\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eShao XY, He T, Lai YL, Chen M, Tong ZH (2022) Water-soluble polysaccharides extracted from \u003cem\u003ePueraria lobata\u003c/em\u003e delay aging of \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e under heat stress. Plant Food Hum Nutr 77:220\u0026ndash;225. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11130-022-00964-5\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLin GP, Wu DS, Xiao XW, Huang QY, Chen HB, Liu D, Fu HQ, Chen XH, Zhao C (2020) Structural characterization and antioxidant effect of green alga \u003cem\u003eEnteromorpha prolifera\u003c/em\u003e polysaccharide in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e via modulation of microRNAs. Int J Biol Macromol 150:1084\u0026ndash;1092. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2019.10.114\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eUno M, Tani Y, Nono M, Okabe E, Kishimoto S, Takahashi C, Abe R, Kurihara T, Nishida E (2021) Neuronal DAF-16-to-intestinal DAF-16 communication underlies organismal lifespan extension in \u003cem\u003eC. elegans\u003c/em\u003e. iScience 24:102706. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.isci.2021.102706\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWang EH, Wang NB, Zou Y, Fahim M, Zhou YT, Yang H, Liu Y, Li HY (2022) Black mulberry (\u003cem\u003eMorus nigra\u003c/em\u003e) fruit extract alleviated AD-Like symptoms induced by toxic A\u0026beta; protein in transgenic \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e via insulin DAF-16 signaling pathway. Food Res Int 160:111696. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodres.2022.111696\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWu CG, Karakuzu O, Garsin DA (2021) Tribbles pseudokinase NIPI-3 regulates intestinal immunity in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e by controlling SKN-1/Nrf activity. Cell Rep 36:109529. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.celrep.2021.109529\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ethanol extract, Lentinus edodes, Antioxidant, Caenorhabditis elegans, Insulin DAF-16 signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-1937791/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1937791/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eLentinus edodes\u003c/em\u003e (\u003cem\u003eL. edodes\u003c/em\u003e) was a fungus plant, which was widely grown and eaten as food in China. As an antioxidant, the polysaccharide of \u003cem\u003eL. edodes\u003c/em\u003e has been widely studied in terms of antioxidant activity, however, the effect of action and underlying mechanism of \u003cem\u003eL. edodes\u003c/em\u003e ethanol extracts (LEEE) in antioxidant aspects were largely unknown. In this study, the antioxidant activity of LEEE was investigated \u003cem\u003ein vitro\u003c/em\u003e. In addition, the antioxidant activity of LEEE was estimated \u003cem\u003ein vivo\u003c/em\u003e through \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e model. It was found that LEEE had excellent antioxidant activity \u003cem\u003ein vitro\u003c/em\u003e by scavenging DPPH, ABTS and hydroxyl radicals. At the same time, the anti-heat stress abilities of \u003cem\u003eC. elegans\u003c/em\u003e were enhanced after supplementation with different concentrations of LEEE. The activities of antioxidant enzymes such as T-SOD, GSH-PX and CAT were significantly enhanced and the levels of MDA and ROS were significantly down-regulated. However, the level of apoptosis of\u003cem\u003e C. elegans\u003c/em\u003e could not be improved after supplementation with LEEE. In the study of LEEE antioxidant-related signaling pathways through q-PCR experiments, it was found that LEEE exerted antioxidant activity in \u003cem\u003eC. elegans\u003c/em\u003e by up-regulating MTL-1, DAF-16 and SOD-3 mRNA expression and down-regulating DAF-2 mRNA expression. This provided a solid foundation for the active substances of \u003cem\u003eLentinus edodes\u003c/em\u003e in improving oxidation-related diseases.\u003c/p\u003e","manuscriptTitle":"Ethanol extract of Lentinus edodes exerted antioxidant activity in Caenorhabditis elegans by regulating insulin DAF-16 signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-08-11 17:19:33","doi":"10.21203/rs.3.rs-1937791/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fa3a15df-9db4-453d-9d94-3dba764c7519","owner":[],"postedDate":"August 11th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-09-30T19:44:20+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-11 17:19:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1937791","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1937791","identity":"rs-1937791","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}