{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,22]],"date-time":"2026-05-22T10:05:22Z","timestamp":1779444322708,"version":"3.53.1"},"reference-count":89,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T00:00:00Z","timestamp":1779148800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004242","name":"Princess Nourah bint Abdulrahman University Researchers Supporting Project","doi-asserted-by":"publisher","award":["PNURSP2026R460"],"award-info":[{"award-number":["PNURSP2026R460"]}],"id":[{"id":"10.13039\/501100004242","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Earthworm-derived bioactive compounds are emerging as promising pharmaceutical agents; however, the immunomodulatory effects of Eisenia fetida proteins at different developmental stages remain unclear. This study evaluated, for the first time, the stage-dependent immunomodulatory activity of E. fetida protein extracts in RAW 264.7 macrophages. Soluble proteins isolated from juvenile, mature, and senescent worms were lyophilized and tested for their effects on cell viability, phagocytic activity, nitric oxide (NO), reactive oxygen species (ROS), and inflammatory gene expression. Amino acid profiling and Western blot analysis were additionally performed to investigate biochemical composition and signaling mechanisms. Mature-stage extracts exhibited the highest protein yield, minimal cytotoxicity, enhanced macrophage phagocytosis, and significant suppression of LPS-induced NO, ROS, and proinflammatory cytokines. In contrast, juvenile-stage extracts showed moderate immunomodulatory activity, whereas senescent-stage extracts induced oxidative stress and inflammatory responses. Western blot analysis demonstrated that mature-stage proteins strongly inhibited phosphorylation of NF-\u03baB and MAPK signaling proteins, including p65, I\u03baB\u03b1, p38, ERK1\/2, and JNK, while senescent-stage extracts maintained elevated pathway activation. Amino acid analysis further revealed enriched immunologically relevant amino acids in mature-stage extracts. These findings demonstrate that developmental stage strongly influences the biological activity of E. fetida proteins and highlight mature-stage extracts as promising natural immunomodulatory agents.<\/jats:p>","DOI":"10.3390\/ijms27104568","type":"journal-article","created":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T15:17:23Z","timestamp":1779203843000},"page":"4568","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Mature-Stage Eisenia fetida Proteins Suppress Macrophage Inflammation via NF-\u03baB and MAPK Pathways"],"prefix":"10.3390","volume":"27","author":[{"given":"Hind","family":"Althagafi","sequence":"first","affiliation":[{"name":"Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hussam A.","family":"Althagafi","sequence":"additional","affiliation":[{"name":"Department of Biology, Faculty of Science, Al-Baha University, Al-Baha 65779, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fahad","family":"Alharthi","sequence":"additional","affiliation":[{"name":"Department of Biology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abdullah A. A.","family":"Alghamdi","sequence":"additional","affiliation":[{"name":"Department of Biology, Faculty of Science, Al-Baha University, Al-Baha 65779, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abdullah M.","family":"Almotayri","sequence":"additional","affiliation":[{"name":"Department of Biology, Faculty of Science, Al-Baha University, Al-Baha 65779, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ibrahim","family":"Jafri","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leena S.","family":"Alqahtani","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, College of Science, University of Jeddah, Jeddah 23445, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Atif Abdulwahab A.","family":"Oyouni","sequence":"additional","affiliation":[{"name":"Department of Biology, Faculty of Science, University of Tabuk, Tabuk 71491, Saudi Arabia"},{"name":"Biodiversity Genomics Unit, Faculty of Science, University of Tabuk, Tabuk 71491, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abdulaziz","family":"Albogami","sequence":"additional","affiliation":[{"name":"Department of Biology, Faculty of Science, Al-Baha University, Al-Baha 65779, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7164-7838","authenticated-orcid":false,"given":"Deyala M.","family":"Naguib","sequence":"additional","affiliation":[{"name":"Botany and Microbiology Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Kooch, Y., and Kuzyakov, Y. 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