{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T16:26:49Z","timestamp":1791822409716,"version":"4.3.4"},"reference-count":37,"publisher":"MDPI AG","issue":"20","license":[{"start":{"date-parts":[[2024,10,19]],"date-time":"2024-10-19T00:00:00Z","timestamp":1729296000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key R&amp;D Program of China","doi-asserted-by":"publisher","award":["2023YFE0114200"],"award-info":[{"award-number":["2023YFE0114200"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Nutrients"],"abstract":"<jats:p>Obejectives: This study explored the immunomodulatory effects of a prebiotic formula consisting of 2\u2032-fucosyllactose (2\u2032-FL), galacto-oligosaccharides (GOSs), and fructo-oligosaccharides (FOSs) (hereinafter referred to as 2FGF) in cyclophosphamide (CTX)-induced immunosuppressed BALB\/c mice and its underlying mechanisms. Methods: Sixty healthy female BALB\/c mice were randomly divided into the following groups: normal control (NC) group; CTX treatment (CTX) group; 2FGF low-dose (2FGF-L) group; 2FGF medium-dose (2FGF-M) group; and 2FGF high-dose (2FGF-H) group. An immunosuppressed model was established in the 2FGF-H group by intraperitoneal injection of 80 mg\/kg CTX. After 30 days of 2FGF intervention, peripheral blood, spleen tissue, thymus tissue, and intestinal tissue from the mice were collected and analyzed. The changes in weight and food intake of the mice were recorded weekly. Hematoxylin-eosin (HE) staining was used to observe the histological change of the spleen tissue. Enzyme-linked immunosorbent assay (ELISA) was employed to detect cytokine levels in peripheral blood. Flow cytometry was used to analyze T lymphocyte subgroup ratio of splenic lymphocytes. Western blot analysis was conducted on intestinal tissues to assess the expression of proteins involved in the tight junction, toll-like receptor 4 (TLR4), mitogen-activated protein kinase (MAPK), and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-\u03baB) signaling pathways. Additionally, molecular techniques were used to analyze the intestinal microbiota. Results: The results showed that 2FGF restored CTX-induced splenic injury, increased the number of splenic T lymphocytes, and elevated serum cytokines such as interleukin-4 (IL-4) and IL-10. In the intestine, 2FGF upregulated the expression of intestinal epithelial tight junction proteins such as Claudin-1 and zonula occludens 1 (ZO-1), thereby enhancing intestinal barrier function and activating the MAPK and NF-\u03baB pathways via TLR4. Furthermore, 2FGF elevated the \u03b1-diversity (Shannon and Simpson indices) of the gut microbiota in CTX-induced immunosuppressed mice, enriching bacteria species positively correlated with anti-inflammatory cytokines (e.g., IL-4) such as g_Streptomyces and g_Bacillus and negatively correlated with pro-inflammatory cytokines (e.g., IL-1\u03b2) such as g_Saccharomyces. The results suggest that 2FGF may enhance immunity via the gut\u2013immune axis. Conclusions: The 2FGF prebiotic formula showed an immunomodulatory effect in CTX-induced immunosuppressed mice, and the mechanism of which might involve optimizing the gut flora, enhancing intestinal homeostasis, strengthening the intestinal barrier, and promoting the expression of immune factors by regulating the TLR-4\/MAPK\/NF-\u03baB pathway.<\/jats:p>","DOI":"10.3390\/nu16203552","type":"journal-article","created":{"date-parts":[[2024,10,21]],"date-time":"2024-10-21T08:53:11Z","timestamp":1729500791000},"page":"3552","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Immunomodulatory Effects of a Prebiotic Formula with 2\u2032-Fucosyllactose and Galacto- and Fructo-Oligosaccharides on Cyclophosphamide (CTX)-Induced Immunosuppressed BALB\/c Mice via the Gut\u2013Immune Axis"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-7901-746X","authenticated-orcid":false,"given":"Wanyun","family":"Ye","sequence":"first","affiliation":[{"name":"Department of Nutrition and Food Hygiene, School of Public Health, Peking University, No. 38 Xueyuan Road, Beijing 100083, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3335-0237","authenticated-orcid":false,"given":"Hanxu","family":"Shi","sequence":"additional","affiliation":[{"name":"Department of Nutrition and Food Hygiene, School of Public Health, Peking University, No. 38 Xueyuan Road, Beijing 100083, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wentao","family":"Qian","sequence":"additional","affiliation":[{"name":"Mengniu Hi-Tech Dairy Products (Beijing) Co., Ltd., Beijing 101100, China"},{"name":"Inner Mongolia Mengniu Dairy (Group) Co., Ltd., Hohhot 011500, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liping","family":"Meng","sequence":"additional","affiliation":[{"name":"Mengniu Hi-Tech Dairy Products (Beijing) Co., Ltd., Beijing 101100, China"},{"name":"Inner Mongolia Mengniu Dairy (Group) Co., Ltd., Hohhot 011500, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meihua","family":"Wang","sequence":"additional","affiliation":[{"name":"Inner Mongolia Mengniu Dairy (Group) Co., Ltd., Hohhot 011500, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9085-1487","authenticated-orcid":false,"given":"Yalin","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Nutrition and Food Hygiene, School of Public Health, Peking University, No. 38 Xueyuan Road, Beijing 100083, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhang","family":"Wen","sequence":"additional","affiliation":[{"name":"Department of Nutrition and Food Hygiene, School of Public Health, Peking University, No. 38 Xueyuan Road, Beijing 100083, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muke","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Nutrition and Food Hygiene, School of Public Health, Peking University, No. 38 Xueyuan Road, Beijing 100083, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yile","family":"Peng","sequence":"additional","affiliation":[{"name":"Department of Nutrition and Food Hygiene, School of Public Health, Peking University, No. 38 Xueyuan Road, Beijing 100083, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongliang","family":"Li","sequence":"additional","affiliation":[{"name":"Mengniu Hi-Tech Dairy Products (Beijing) Co., Ltd., Beijing 101100, China"},{"name":"Inner Mongolia Mengniu Dairy (Group) Co., Ltd., Hohhot 011500, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5791-4629","authenticated-orcid":false,"given":"Yajun","family":"Xu","sequence":"additional","affiliation":[{"name":"Department of Nutrition and Food Hygiene, School of Public Health, Peking University, No. 38 Xueyuan Road, Beijing 100083, China"},{"name":"Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Peking University, No. 38 Xueyuan Road, Beijing 100083, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,10,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1146\/annurev-immunol-090222-102035","article-title":"Microbial Metabolites and Gut Immunology","volume":"42","author":"Takeuchi","year":"2024","journal-title":"Annu. Rev. Immunol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1038\/nature11551","article-title":"Reciprocal interactions of the intestinal microbiota and immune system","volume":"489","author":"Maynard","year":"2012","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"681","DOI":"10.4161\/19490976.2014.972237","article-title":"Prebiotics in infant formula","volume":"5","author":"Vandenplas","year":"2014","journal-title":"Gut Microbes"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"\u0160uligoj, T., Vigsn\u00e6s, L.K., Abbeele, P.V.D., Apostolou, A., Karalis, K., Savva, G.M., McConnell, B., and Juge, N. (2020). Effects of Human Milk Oligosaccharides on the Adult Gut Microbiota and Barrier Function. Nutrients, 12.","DOI":"10.3390\/nu12092808"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Azagra-Boronat, I., Massot-Cladera, M., Knipping, K., Van\u2019t Land, B., Stahl, B., Garssen, J., Rodr\u00edguez-Lagunas, M.J., Franch, \u00c0., Castell, M., and P\u00e9rez-Cano, F.J. (2018). Supplementation with 2\u2019-FL and scGOS\/lcFOS Ameliorates Rotavirus-Induced Diarrhea in Suckling Rats. Front. Cell. Infect. Microbiol., 8.","DOI":"10.3389\/fcimb.2018.00372"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"856","DOI":"10.1093\/jn\/nxz006","article-title":"The Combination of 2\u2019-Fucosyllactose with Short-Chain Galacto-Oligosaccharides and Long-Chain Fructo-Oligosaccharides that Enhance Influenza Vaccine Responses Is Associated with Mucosal Immune Regulation in Mice","volume":"149","author":"Xiao","year":"2019","journal-title":"J. Nutr."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Tian, B., Jiang, Y., Liu, R., Hamed, Y.S., Rayan, A.M., Xu, S., Sun, P., and Yang, K. (2024). Positive effects of extracellular polysaccharides from Paecilomyces hepiali on immune-enhancing properties by regulating gut microbiota in cyclophosphamide-induced mice. Int. J. Biol. Macromol., 274.","DOI":"10.1016\/j.ijbiomac.2024.133390"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"100351","DOI":"10.1016\/j.xpro.2021.100351","article-title":"Analysis of T cells in mouse lymphoid tissue and blood with flow cytometry","volume":"2","author":"Skordos","year":"2021","journal-title":"STAR Protoc."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Ma, H., Mueed, A., Liu, D., Ali, A., Wang, T., Ibrahim, M., Su, L., and Wang, Q. (2024). Polysaccharides of Floccularia luteovirens regulate intestinal immune response, and oxidative stress activity through MAPK\/Nrf2\/Keap1 signaling pathway in immunosuppressive mice. Int. J. Biol. Macromol., 277.","DOI":"10.1016\/j.ijbiomac.2024.134140"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1038\/nature13568","article-title":"Alterations of the human gut microbiome in liver cirrhosis","volume":"513","author":"Qin","year":"2014","journal-title":"Nature"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2145843","DOI":"10.1080\/19490976.2022.2145843","article-title":"Lactobacillus johnsonii alleviates colitis by TLR1\/2-STAT3 mediated CD206+ macrophagesIL-10 activation","volume":"14","author":"Jia","year":"2022","journal-title":"Gut Microbes"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"110756","DOI":"10.1016\/j.foodres.2021.110756","article-title":"Sulfated modification enhances the immunomodulatory effect of Cyclocarya paliurus polysaccharide on cyclophosphamide-induced immunosuppressed mice through MyD88-dependent MAPK\/NF-\u03baB and PI3K-Akt signaling pathways","volume":"150","author":"Yu","year":"2021","journal-title":"Food Res. Int."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Mao, J.H., Wang, Y., Chen, W.M., Wang, X.M., Liu, J., Shao, Y.H., and Tu, Z.C. (2024). Galacto-oligosaccharides modified whey protein isolate ameliorates cyclophosphamide-induced immunosuppression. Int. J. Biol. Macromol., 278.","DOI":"10.1016\/j.ijbiomac.2024.134642"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"480","DOI":"10.1038\/31002","article-title":"T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions","volume":"393","author":"Schoenberger","year":"1998","journal-title":"Nature"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1557","DOI":"10.1182\/blood-2008-05-078154","article-title":"CD4 T cells: Fates, functions, and faults","volume":"112","author":"Zhu","year":"2008","journal-title":"Blood"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/j.carbpol.2018.10.120","article-title":"Curdlan oligosaccharides having higher immunostimulatory activity than curdlan in mice treated with cyclophosphamide","volume":"207","author":"Tang","year":"2019","journal-title":"Carbohydr. Polym."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1038\/nri.2017.118","article-title":"T(H)2 cell development and function","volume":"18","author":"Walker","year":"2018","journal-title":"Nat. Rev. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1038\/nri2711","article-title":"The regulation of IL-10 production by immune cells","volume":"10","author":"Saraiva","year":"2010","journal-title":"Nat. Rev. Immunol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"109793","DOI":"10.1016\/j.foodres.2020.109793","article-title":"Bacillus coagulans 13002 and fructo-oligosaccharides improve the immunity of mice with immunosuppression induced by cyclophosphamide through modulating intestinal-derived and fecal microbiota","volume":"140","author":"Zhao","year":"2021","journal-title":"Food Res. Int."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"356","DOI":"10.4014\/jmb.2211.11049","article-title":"The Human Milk Oligosaccharide 2\u2019-Fucosyllactose Shows an Immune-Enhancing Effect in a Cyclophosphamide-Induced Mouse Model","volume":"33","author":"Jo","year":"2023","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Yang, Y., Li, H., Fotopoulou, C., Cunnea, P., and Zhao, X. (2022). Toll-like receptor-targeted anti-tumor therapies: Advances and challenges. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.1049340"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1038\/nrc2694","article-title":"Signal integration by JNK and p38 MAPK pathways in cancer development","volume":"9","author":"Wagner","year":"2009","journal-title":"Nat. Rev. Cancer"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"17023","DOI":"10.1038\/sigtrans.2017.23","article-title":"NF-\u03baB signaling in inflammation","volume":"2","author":"Liu","year":"2017","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"23075","DOI":"10.1074\/jbc.M114.591495","article-title":"\u03b2-Arrestin 2 negatively regulates Toll-like receptor 4 (TLR4)-triggered inflammatory signaling via targeting p38 MAPK and interleukin 10","volume":"289","author":"Li","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1620","DOI":"10.1248\/bpb.b18-00963","article-title":"2\u2019-Fucosyllactose Attenuates Particulate Matter-Induced Inflammation via Inhibition of Hypoxia-Inducible Factor in Keratinocytes","volume":"42","author":"Lee","year":"2019","journal-title":"Biol. Pharm. Bull."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1007\/s11739-023-03374-w","article-title":"Gut microbiota, intestinal permeability, and systemic inflammation: A narrative review","volume":"19","author":"Petito","year":"2024","journal-title":"Intern. Emerg. Med."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"121722","DOI":"10.1016\/j.carbpol.2023.121722","article-title":"Fucosylated chondroitin sulfate from sea cucumber Stichopus chloronotus alleviate the intestinal barrier injury and oxidative stress damage in vitro and in vivo","volume":"328","author":"Yin","year":"2024","journal-title":"Carbohydr. Polym."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"112072","DOI":"10.1016\/j.foodres.2022.112072","article-title":"Differential responses on gut microbiota and microbial metabolome of 2\u2019-fucosyllactose and galactooligosaccharide against DSS-induced colitis","volume":"162","author":"Liu","year":"2022","journal-title":"Food Res. Int."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"9844","DOI":"10.1039\/D1FO01147B","article-title":"Fructooligosaccharide supplementation alleviated the pathological immune response and prevented the impairment of intestinal barrier in DSS-induced acute colitis mice","volume":"12","author":"Liao","year":"2021","journal-title":"Food Funct."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1867","DOI":"10.1039\/D3FO05158G","article-title":"Oat protein isolate-Pleurotus ostreatus \u03b2-glucan conjugate nanoparticles bound to \u03b2-carotene effectively alleviate immunosuppression by regulating gut microbiota","volume":"15","author":"Zhong","year":"2024","journal-title":"Food Funct."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"5480","DOI":"10.1002\/fsn3.4195","article-title":"Fermenting kale (Brassica oleracea L.) enhances its functional food properties by increasing accessibility of key phytochemicals and reducing antinutritional factors","volume":"12","author":"Subedi","year":"2024","journal-title":"Food Sci. Nutr."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1038\/s41420-023-01666-w","article-title":"The role of Lactobacillus in inflammatory bowel disease: From actualities to prospects","volume":"9","author":"Li","year":"2023","journal-title":"Cell Death Discov."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"9349","DOI":"10.1021\/acs.jafc.2c08814","article-title":"Galactooligosaccharide or 2\u2019-Fucosyllactose Modulates Gut Microbiota and Inhibits LPS\/TLR4\/NF-\u03baB Signaling Pathway to Prevent DSS-Induced Colitis Aggravated by a High-Fructose Diet in Mice","volume":"71","author":"Chen","year":"2023","journal-title":"J. Agric. Food Chem."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Tandon, D., Haque, M.M., Gote, M., Jain, M., Bhaduri, A., Dubey, A.K., and Mande, S.S. (2019). A prospective randomized, double-blind, placebo-controlled, dose-response relationship study to investigate efficacy of fructo-oligosaccharides (FOS) on human gut microflora. Sci. Rep., 9.","DOI":"10.1038\/s41598-019-41837-3"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Liu, Z., Sun, W., Hu, Z., Wang, W., and Zhang, H. (2024). Marine Streptomyces-Derived Novel Alkaloids Discovered in the Past Decade. Mar. Drugs, 22.","DOI":"10.3390\/md22010051"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"112863","DOI":"10.1016\/j.intimp.2024.112863","article-title":"Bacillus coagulans BACO-17 ameliorates in vitro and in vivo progression of Rheumatoid arthritis","volume":"141","author":"Kuo","year":"2024","journal-title":"Int. Immunopharmacol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"112738","DOI":"10.1016\/j.intimp.2024.112738","article-title":"Saccharomyces boulardii (CNCM I-745) alleviates collagen-induced arthritis by partially maintaining intestinal mucosal integrity through TLR2\/MYD88\/NF-\u03baB pathway inhibition","volume":"139","author":"Ahmad","year":"2024","journal-title":"Int. Immunopharmacol."}],"container-title":["Nutrients"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6643\/16\/20\/3552\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:16:51Z","timestamp":1760113011000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6643\/16\/20\/3552"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,10,19]]},"references-count":37,"journal-issue":{"issue":"20","published-online":{"date-parts":[[2024,10]]}},"alternative-id":["nu16203552"],"URL":"https:\/\/doi.org\/10.3390\/nu16203552","relation":{},"ISSN":["2072-6643"],"issn-type":[{"value":"2072-6643","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,10,19]]}}}