Highly Active Probiotic Yogurt Protects Against Cyclophosphamide-Induced Immunosuppression in Mice by Regulating Immune Response and gut microbiota

Highly Active Probiotic Yogurt Protects Against Cyclophosphamide-Induced Immunosuppression in Mice by Regulating Immune Response and gut microbiota | 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 Highly Active Probiotic Yogurt Protects Against Cyclophosphamide-Induced Immunosuppression in Mice by Regulating Immune Response and gut microbiota Liyuan Yun, Jinpeng Zhang, Huping Yang, Qian Li, Shuguang Fang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6252467/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Jun, 2025 Read the published version in Probiotics and Antimicrobial Proteins → Version 1 posted 13 You are reading this latest preprint version Abstract Probiotics play a crucial role in modulating the immune system and maintaining the integrity of intestinal epithelial barrier. The study investigates the effects of high-activity probiotic yogurt on immunosuppressed mice induced by cyclophosphamide. On days 7, 8, and 9 of the experiment, ICR male mice (eight-week-old) were injected intraperitoneally with cyclophosphamide (80 mg/kg body weight/day) to establish an immunosuppressive model (n = 10). Mice fed with normal diet or high-activity probiotic yogurt for consecutive 14 days. The effect of high-activity probiotic yogurt on immunosuppressed mice was investigated by HE staining, enzyme-linked immunosorbent assay (ELISA), Western blotting, and 16s rRNA assay. Results indicated that after the treatment of high-activity probiotic yogurt, the immune organ indices, interleukin-6(IL-6), interleukin-12(IL-12), tumor necrosis factor-α (TNF-α), and intestinal structure are significantly increased in immunosuppressed mice (P < 0.05). Western blotting analysis find that high-activity probiotic yogurt improves the expression of toll-like receptor 4 (TLR4), nuclear factor kappa-B –p65(p65), TNF receptor associated factor 6(TRAF6). Furthermore, microbiota analysis showed that high-activity probiotic yogurt significantly increased the diversity and richness of the gut microbiota(P < 0.05). These findings indicated that high-activity probiotic yogurt may improve the immune function of mice by improve intestinal homeostasis and activation of TLR pathway. high-activity probiotic yogurt immune response cyclophosphamide gut microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The immune system acts as a critical defense mechanism against external pathogens, responsible for identifying and neutralizing antigenic substances. It collaborates with other bodily systems to maintain homeostasis and physiological balance[ 1 ]. Cyclophosphamide (CTX), a widely used antineoplastic agent, acts as both an alkylating compound and an immunosuppressive drug. It is known for its significant adverse effects, including damage to the intestinal mucosa, reduction in immune organ indices, and suppression of immune cell and cytokine production[ 2 ]. Studies have shown that CTX exposure increases intestinal permeability and bacterial translocation, primarily due to the proliferation of harmful bacteria, such as Proteobacteria, Sutterella , and Pseudomonas [ 3 – 5 ]. As a result, CTX is frequently employed to create animal models of immunosuppression and intestinal injury[ 6 ]. Probiotics are beneficial, active microorganisms that colonize the human gut and alter the composition of the host's microbiota at specific sites[ 7 ], thereby playing a crucial role in maintaining intestinal ecological balance and enhancing systemic immunity[ 8 ]. In recent years, the application of probiotics for modulating intestinal flora and improving host immunity has garnered significant attention[ 9 , 10 ]. Studies have demonstrated that gut microbes exert immunomodulatory effects through the adjustment of the body's internal environment or by enhancing the composition and metabolic products of gut microbiota[ 11 ]. Currently, studies on probiotic yogurt predominantly concentrate on the effects of individual or a limited number of probiotics, with little attention paid to the synergistic mechanisms among diverse strains of complex probiotics, thereby restricting our comprehension and utilization of these complex probiotics[ 12 ]. Yogurt, serving as an effective delivery system for probiotics, is deemed one of the optimal methods for administering high doses of probiotics. However, most commercially available probiotic yogurts typically contain only the fundamental fermentation strains, namely Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus [ 13 ]. Bifidobacterium is a widely employed functional strain known for its excellent tolerance in the gastrointestinal tract, and it plays a crucial role in maintaining gut health[ 14 ], as well as recognized as an immunomodulator or a biomarker for human diseases, capable of enhancing the body's immune response through the improvement of phagocytic cell function and bolstering resistance to illness[ 15 , 16 ]. Bifidobacterium strains demonstrated strain-specific biological activity, such as Bifidobacterium animalis subsp. lactis BLa80 , as an adjunct treatment for diarrhea in children[ 17 ], Bifidobacterium longum subsp. longum BL21 strengthens gut barrier integrity and possesses hypoglycemic activity[ 18 ]. However, the synergistic mechanisms of multi-strain consortia remain underexplored. In the initial phase of our study, we employed a complex probiotic blend yogourt (comprises Bifidobacterium animalis subsp. lactis BLa80 , Bifidobacterium longum subsp. longum BL21 , Bifidobacterium breve BBr16, Bifidobacterium adolescentis BAC30 , and Bifidobacterium longum subsp. infantis BI45 ) which provided by WecPro DigestBi. Our preliminary findings indicated that this complex probiotic blend demonstrated the most effective synergistic action in promoting the proliferation of viable bacteria post-fermentation, achieving a target of 100 billion highly active bacteria in the yogurt. Additionally, the fermented yogurt exhibited superior flavor characteristics. This formulation surpasses conventional yogurts limited to S. thermophilus and L. bulgaricus , offering a novel platform to investigate how multi-strain interactions enhance body health, such as improve immunity, regulate blood sugar level, and inhibit diarrhea, and so on. However, the immune -regulatory effects of this complex probiotic blend yogourt was rarely reported. Therefore, the research is necessary to clarify the immune-regulatory effects and interactions with gut microbiota associated with this high-activity probiotic yogurt. This study investigates the combinatorial effects of BLa80, BL21, BBr16, BAC30, and BI45 using in vivo models to elucidate strain-specific versus collective immunomodulatory mechanisms. The investigation encompassed the growth index, organ indices, intestinal tissue morphology, intestinal cytokine levels (including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-12, protein expression (including toll-like receptor 4 (TLR4), TNF receptor associated factor 6(TRAF6), nuclear factor kappa-B-p65(p65), and intestinal flora diversity in cyclophosphamide (CTX)-treated mice. This study not only effectively demonstrated the influence of probiotics on the enhancement of human immunity but also established a theoretical foundation for subsequent research, development, and market application of this product. Materials and methods Probiotic yogurt making The high-activity probiotic yogurt contained a mixture of the following strains: Bifidobacterium animalis subsp. lactis (BLa80), Bifidobacterium longum subsp. longum (BL21), Bifidobacterium breve (BBr16), Bifidobacterium adolescentis (BAC30), and Bifidobacterium longum subsp. infantis (BI45). These strains were provided by Wecare Probiotics Co., Ltd., located in Suzhou, Chin The basic fermentation strain WecCul®-D1, was inoculated with 30 g/T, and the complex bifidus, WecPro®-DigestBi, was inoculated at 250 g/T into the sterilized fermentation base material, which included 93.5% raw milk, 6% white granulated sugar, 0.5% complex prebiotic WecPre-S (containing xylooligosaccharide, fructooligosaccharides, isomaltooligosaccharides).The fermentation temperature was 37℃, the time was 8 h, the acidity reached 70°T to stop the fermentation, and the finished product was refrigerated at 4℃ overnight. Mice Treatment and Experimental Design Fifty male ICR mice (8-week old, 20–25 g) were purchased from Spiff (Beijing) Biotechnology Co., LTD., license No. SCXK (Beijing) 2019-0010. All animals were kept in a specific pathogen-free facility with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (8th edition). The Ethics Committee of Tianjin Agricultural University approved all animal experiments (License No. 2023LLSC08). During the study, the animals were housed in standard cages with unrestricted access to food and water for a duration of one week under controlled laboratory conditions (22–25°C and relative humidity of 40–60%). The light-dark cycle was maintained at 12 h per day. After one week adaptation period, mice were categorized into 5 groups ((n = 10 each group), which were normal control group (NC), model control group (MC), low dose compound probiotic yogurt control group (LPYC, probiotic concentration: 10 9 CFU/250 g), medium dose compound probiotic yogurt control group (MPYC, probiotic concentration: 10 10 CFU/250 g), high compound probiotic yogurt control group (HPYC, probiotic concentration: 10 11 CFU/250 g). The compound probiotic yogurt was storaged at 4℃ during the experiment. (1) NC: treat with 0.2 mL physiological saline for once a day, for 14 days; (2) MC: subjected to CTX intraperitoneal injection treatment (80 mg/kg) once a day at 7,8,9 days to induce immunosuppression, and gavaged with 0.2 mL physiological saline for once a day, for 14 days; (3)LPYC: subjected to CTX intraperitoneal injection treatment (80 mg/kg) once a day at 7,8,9 days to induce immunosuppression, and gavaged with 10 mL per kg BW per day compound probiotic yogurt which probiotic concentration was 10 9 CFU/250 g; (4)MPYC: subjected to CTX intraperitoneal injection treatment (80 mg/kg) once a day at 7,8,9 days to induce immunosuppression, and gavaged with 10 mL per kg BW per day compound probiotic yogurt which probiotic concentration was 10 10 CFU/250 g; (5)HPYC: subjected to CTX intraperitoneal injection treatment (80 mg/kg) once a day at 7,8,9 days to induce immunosuppression, and gavaged with 10 mL per kg BW per day compound probiotic yogurt which probiotic concentration was 10 11 CFU/250 g. On days 7, 8, and 9 following the initiation of the experiment, all groups except for the NC group were administered intraperitoneal injections of cyclophosphamide at a dosage of 80 mg/kg body weight for three consecutive days. Following this treatment regimen, a significant decrease in mouse body weight was observed, indicating that the model had been successfully established. The weights of the mice were recorded periodically throughout the duration of the experiment. At the conclusion of the experiment, mice were euthanized via cervical dislocation after blood collection from their eyeballs and subsequent serum extraction. The spleen and thymus were harvested and weighed; additionally, small intestinal tissue along with colonic contents was excised for further analysis. Histopathology evaluation A segment of the colon was excised, rinsed with sterile saline, and subsequently fixed in 4% paraformaldehyde (Solarbio, Beijing, China) at room temperature for 24 h. The paraformaldehyde-fixed colon tissues were then embedded in paraffin and sectioned to a thickness of 4 µm. Paraffin sections were stained with hematoxylin and eosin (Zhuhai Beso Biotechnology Co., LTD, GuangDong, China) following standard protocols. Images were captured using an upright optical microscope (Nikon Eclipse E100, Nikon, Tokyo, Japan) equipped with an imaging system (Nikon DS-U3, Nikon, Tokyo, Japan). Enzyme-linked immunosorbent assays The levels of inflammatory cytokines, including IL-6, IL-12, and TNF-α, in colon were analyzed by enzyme-linked immunosorbent assays kits (Elabscience, Wuhan, China). The measurements were conducted in accordance with the manufacturer's instructions. Western blot analysis The total cellular proteins were extracted from colon tissue samples utilizing a protein extraction kit (Beyotime, Shanghai, China), following the manufacturer's instructions meticulously. The protein concentration was quantified using the BCA assay kit (Beyotime, Shanghai, China). Equal amounts of denatured proteins were separated by SDS-PAGE and subsequently transferred onto a PVDF membrane (Millipore Co., MA, USA), which was then blocked with 5% BSA for one hour. Following this, the membrane was incubated overnight at 4°C with specific primary antibodies diluted to a ratio of 1:1000. Then, the membranes were washed three times for 10 min each in 1×TBST and subsequently incubated with HRP-conjugated secondary antibodies diluted to a ratio of 1:3000 at room temperature for one hour. After an additional three washes of 10 minutes each, the blots were analyzed using an ECL chemiluminescence detection kit (Beyotime, Shanghai, China). Densitometric analysis was performed utilizing Quantity One software version 4.6.2 (Bio-Rad Laboratories, CA, USA). Gut microbiota analysis Total bacterial DNA was extracted from fecal samples using the TIANamp Stool DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China) in accordance with the manufacturer's instructions. The PCR products were purified with AMPure XT beads (Beckman, USA), and their concentration and quality were assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) as well as 1.2% agarose gel electrophoresis. Amplification of the V3-V4 region of the bacterial 16S rRNA gene was conducted utilizing Primer F: Illumina adapter sequence ACTCCTACGGGAGGCAGCA and Primer R: Illumina adapter sequence GGACTACHVGGGTWTCTAAT. Sequencing and bioinformatics analysis of the fecal samples were performed by Tianjin Jinke Biotechnology Co., LTD (Tianjin, China) on the Illumina MiSeq platform. To analyze the structure of gut microbiota, we employed various methods including alpha-diversity (α-diversity), beta-diversity (β-diversity), Venn diagram analysis, species composition assessment, and linear discriminant analysis effect size (LEfSe). Statistical analysis The experiments were conducted in triplicate, and all data are presented as mean ± standard deviation (SD). Differences between two experimental groups were analyzed using Tukey’s test, while differences among multiple groups were evaluated through one-way analysis of variance (ANOVA) utilizing SPSS26.0 software. Graphical representations were generated using Origin 9.0 software (Origin Lab Corporation, Northampton, MA, USA). Pairwise comparisons of diversity between the experimental groups were determined using Species Richness, Simpson diversity, Chao 1 diversity measures. Microbial communities in the groups were then ordinated using weighted and unweighted UniFrac distance metrics with the Vegan package in R and visualized as a PCoA using ggplot2. The unweighted UniFrac is a qualitative measure showing the differences in the presence of bacteria. The weighted UniFrac shows the relative taxa abundance. A permutational multivariate analysis of variance (PERMANOVA) (𝛼=0.05) was run on the unweighted and weighted matrices to determine differences between groups using 999 random permutations. LEfSe was then used to validate the statistical significance and effect size of the differential abundances of taxa in the groups using the strict method (all classes differential). The experimental groups were treated as classes and a multiclass comparison was performed on closed reference operational taxonomic units using sequencing data were analyzed using the Kruskal-Wallis test and Wilcoxon rank-sum test, with a p-value cut-off of 0.05 and a linear discriminant analysis (LDA) score for discriminative features set to 2.0. The differentially abundant features were then visualized in a cladogram.[ 19 ] A p-value of less than 0.05 was considered statistically significant; lowercase letters indicate statistical significance. Results and discussion High-activity probiotic yogurt mitigated the CTX-induced structural damage to the intestines and atrophy of immune organs Cyclophosphamide is extensively used in the treatment of various malignancies and autoimmune diseases; however, it can also cause significant toxic effects on the body [ 20 ]. Intraperitoneal administration of cyclophosphamide (CTX) can be used to establish models of immunosuppression and intestinal mucosal injury. This study examined the effects of high-activity probiotic yogurt on CTX-induced immunosuppression and intestinal mucosal damage. Following three consecutive days of intraperitoneal injections of CTX, a significant decreased in body weight was observed in mice, as illustrated in Fig. 1 a. Compared to the NC group, the final body weight of mice in the MC group was significantly lower (P < 0.05) (Table 1 ). Table 1 Effects of high-activity probiotic yogurt on the body weight of immunosuppressive mice induced by Cyclophosphamide (n = 10) Group Initial body weight (g) Final body weight (g) NC 30.47 ± 1.39a 36.47 ± 1.66a MC 29.37 ± 0.71a 32.47 ± 0.94b LPYC 29.52 ± 1.71a 34.09 ± 0.75ab MPYC 31.13 ± 1.65a 35.86 ± 1.29a HPYC 30.96 ± 1.58a 35.70 ± 1.04a Note: Data are presented as mean ± SD (n = 10). Statistical analysis via unpaired t-test. Different letter identifiers indicated significant difference between groups (P < 0.05) The spleen and thymus are two vital immune organs that play a critical role in immune regulation. The spleen serves as the site for T and B cell colonization and immune responses, while also synthesizing biologically active substances. In contrast, the thymus regulates peripheral immune organs and cells, providing an environment for T cell differentiation and maturation. Consequently, indices of the spleen and thymus are recognized as important indicators reflecting immune function [ 21 ]. Compared to the NC group, both the spleen index (Fig. 1 b) and thymus index (Fig. 1 c) of mice in the MC group were significantly reduced (P < 0.05), indicating that high-activity probiotic yogurt may enhance host immunity by promoting immune organ function. Linlin Wang et al.[ 22 ] and Ji Bingjie Ge et al. [ 23 ] also reported that Bifidobacterium longum could significantly enhance host immunity by improving the spleen index (P < 0.05).In et al. research, no significant differences in the weights of the spleen were found between all groups after the treatment of Bifidobacterium longum BB536[ 24 ].Therefore, these results indicated that the high-activity probiotic yogurt effectively increased the body weight and organ index of immunocompromised mice. The intestinal tissues exhibited showed infiltration by inflammatory factors, leading to damage to the villus structure and impairment of the intestinal epithelial barrier function (Fig. 1 d). In the study conducted by Hualing Xie et al.[ 20 ], it was reported that CTX treatment resulted in significant damage to colonic epithelial tissue, accompanied by severe inflammation and atrophy of immune organs. Based on these findings, we infer that a successful immunocompromised mouse model has been established. After the administration of high-activity probiotic yoghurt, there were no significant differences in final body weight, spleen index, or intestinal tissue parameters among the LPYC, MPYC, and HPYC groups compared to the NC group (P > 0.05). These findings suggest that high-activity probiotic yogurt may effectively mitigate the side effects of CTX treatment, including weight loss, reductions in organ indices, and damage to intestinal structure. High-activity probiotic yogurt increased cytokines secretion and up-regulated the p65/TLR4/TRAF6 pathway The intestine is the largest immune organ in humans, playing a critical role in maintaining the biological barrier that regulates the internal environment. TNF-α, TLR4, and p65 are involved in the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, either directly or indirectly, contributing to immune responses and enhancing overall immunity.[ 25 ]. Therefore, enzyme-linked immunosorbent assay (ELISA) and Western blot analyses were employed to further investigate the effects of high-activity probiotic yogurt on cytokine secretion and signaling pathways within intestinal tissue. This approach aimed to assess both the expression levels of immune cytokines and related immune pathways. Various cytokines are involved in regulating intestinal mucosal inflammation and intestinal epithelial integrity [ 26 ]. Intraperitoneal injection of cyclophosphamide (CTX) significantly reduced levels of immune cytokines IL-6, IL-12, and TNF-α by factors of 0.40-, 0.45-, and 0.51-fold respectively (P < 0.05; Fig. 2 a, Fig. 2 b, Fig. 2 c). Xihong Wang et al. reported that CTX could inhibit cytokine secretion in the intestinal mucosa of mice, resulting in intestinal injury due to immune dysfunction[ 27 ]. Treatment with high-activity probiotic yogurt notably increased IL-6, IL-12, and TNF-α-fold levels by factors ranging from 0.13- to 0.21-fold for IL-6 and from 0.14-fold to 0.19-fold for IL-12. Additionally, Qiuwen He et al. found that following treatment with probiotics such as Lactiplantibacillus fermentum F6, there was a significant increase in cytokines including IL-6, interleukin1-beta (IL-1β) and TNF-α within a dextran sulfate sodium-induced colitis model group[ 9 ]. Compared to Peng Xu et al. previous studies using single Bifidobacterium animalis subsp. lactis BLa80 decreasing TNF-α by 0.3-fold[ 28 ], our multi-strain yogurt induced a more pronounced down-regulation of TNF-α(0.51-fold) suggesting that strain synergy amplifies immune activation. Toll-like receptors (TLRs) are a class of transmembrane proteins that play a critical role in regulating the immune response. They are involved in host defense against pathogens, modulate the abundance of commensal microbes, and maintain tissue integrity[ 29 ]. Among these, TLR4 is the most well-characterized member; it recognizes pathogen-associated molecular patterns (PAMPs) and activates transcription factors to produce various pro-inflammatory cytokines that aid in clearing invading pathogens[ 30 ]. Upon activation of TLR4, several intracellular signaling pathways are triggered. In this study, intraperitoneal injection of CTX resulted in down-regulation of p65, TLR4, and TRAF6 expression by approximately 0.63-, 0.80-, and 0.68-fold respectively in the MC group (Fig. 2 d). The research of Gavzy et al. showed that Bifidobacterium breve strains UCC2003 and JCM7017 regulated the NF-κB and TNFα expression [ 31 ]. Therefore, we speculated that the high-activity probiotic yogurt effect the immune may be attributable to the regulation of TLR4/NF-κB p65 signaling pathway. High-activity probiotic yogurt increased the diversity of the gut microbiota Intestinal microbiota play a critical role in the development and maintenance of the host immune system, which is essential for balancing inflammatory responses and immune tolerance[ 29 ]. In this study, we measured the richness and diversity of microbial communities through alpha-diversity analysis (Fig. 3 a- 3 b). Intraperitoneal injection of CTX resulted in a reduction in MC group in terms of microbial richness (Chao1 index) and microbial diversity (Simpson index). These findings indicated that cyclophosphamide induces significant disruption to both the diversity and richness of gut microbiota [ 32 ]. Furthermore, our results demonstrated that mice treated with high-activity probiotic yogurt exhibited higher Chao1 and Simpson indices compared to those in the MC group. Wang et al. (2022) observed modest diversity improvements with Bifidobacterium longum monotherapy, whereas our multi-strain intervention reversed CTX-induced dysbiosis to near-normal levels.Additionally, as reported by Wangdi Song et al. [ 4 ], intraperitoneal administration of CTX similarly led to decreased α-diversity within gut microbiota as evidenced by declines in Chao1 index as well as Simpson and Shannon indices. The changes in gut microbiota among the different treatment groups were further analyzed at a more granular level, specifically at the phylum and genus levels. At the phylum level (Fig. 3 c), Firmicutes and Bacteroidetes emerged as the two most predominant phyla, collectively accounting for nearly 80% of the total bacterial population. The high-activity probiotic yogurt treatment resulted in an increased relative abundance of Bacteroides , accompanied by a reduction in Firmicutes compared to the negative control group. This finding suggests that high-activity probiotic yogurt exerts regulatory effects on gut microbiota composition. Additionally, it has been reported that CTX significantly influences gut microbiota by stimulating the proliferation of Proteobacteria and Firmicutes while simultaneously decreasing the proportion of Bacteroidete s[ 33 ]. Similarly, Song et al. found that low-molecular-weight licorices enhanced immunity by modulating the microbiota, but it was mainly enriched in Firmicutes rather than Bacteroidetes[ 4 ]. Bacteroidetes , which primarily consist of lipopolysaccharides (LPS), can be recognized by TLR4. As previously mentioned, the level of TLR4 was significantly elevated following treatment with high-activity probiotic yogurt, potentially leading to the activation of NF-κB (P < 0.05). Furthermore, it has been reported that Bacteroides acidifaciens may enhance the production of IL-6 and IL-10[ 34 ]. In the present study, we observed an increased relative abundance of Bacteroidetes in the high-activity probiotic yogurt groups compared to the NC group. At the genus level (Fig. 3 d), there was a notable increase in Lactobacillus in the LPYC, MPYC, and HPYC groups when compared to the MC group. Previous reports have indicated that Lactobacillus can enhance immune responses by interacting with immune cells associated with gut health[ 35 ]. After the administration of high-activity probiotic yoghurt, a significant reduction in the bacterial genera Duncaniella and Akkermansia was observed in the LPYC, MPYC, and LPYC groups compared to the MC group (Fig. 3 e). While He et al.demonstrated that Lactiplantibacillus fermentum F6 selectively suppressed Akkermansia in colitis models[ 9 ]. This dual action—promoting beneficial taxa and suppressing pathobionts—reflects the functional diversity of the five-strain blend, as individual strains likely target distinct microbial niches For example, Bifidobacterium longum subsp. infantis BI45 may enhance mucosal integrity via glycoprotein metabolism, while Bifidobacterium breve BBr16 could competitively exclude opportunistic pathogens through bacteriocin production. Such strain-specific contributions align with Yahfoufi et al. who emphasized that multi-strain consortia achieve superior ecological and immunological outcomes by leveraging complementary metabolic and adhesion properties[ 12 ]. Therefore, the current data suggest that the modulation of gut microbiota by high-activity probiotic yogurt was closely associated with immune-regulation in CTX-induced mice, which may represent a crucial mechanism through which high-activity probiotic yogurt exerts its beneficial effects. Consequently, targeting gut microbiota modulation could emerge as a potential strategy for immunoregulation. The beta diversity analysis was conducted using principal coordinates analysis (PCoA), nonmetric multidimensional scaling analysis (NMDS), and hierarchical cluster analysis based on Bray-Curtis dissimilarity distance. As illustrated in Fig. 4 a and Fig. 4 b, the results from PCoA and NMDS demonstrated a clear separation between the MC group and the other groups. On PCo1, which accounted for 18% of the total variance, the distances among the NC, LPYC, MPYC, and HPYC groups were closely clustered. This suggests that high-activity probiotic yogurt intervention could shift the gut microbiota composition of the MC group towards that of the NC group in a dose-dependent manner. Furthermore, cluster analytical results derived from Bray-Curtis distance (Fig. 4 b) corroborated those obtained from PCoA and NMDS analyses, indicating that high-activity probiotic yogurt influenced gut microbiota composition induced by CTX. The hierarchical clustering analysis at genus level based on Bray-Curtis distance is presented in Fig. 4 c. Additionally, an Analysis of Similarities (ANOSIM) was employed to assess whether significant differences exist between intra-group variances across different groups[ 9 ]. The analysis revealed a significant difference in the gut microbial community structure between the NC and MC groups (Adonis test, R²=0.515; P = 0.0014), as well as between the MC and MPYC groups (Adonis test, R²=0.289; P = 0.06) and between the MC and HPYC groups (Adonis test, R²=0.369; P = 0.04). These findings indicate that CTX had a profound impact on the gut microbiota composition of the mice, which was subsequently restored through the consumption of high-activity probiotic yogurt. High-activity probiotic yogurt effected on the gut microbiota composition The alterations in intestinal bacterial composition across the various treatment groups are illustrated in Fig. 5 . A Venn diagram depicting the operational taxonomic units (OTUs) among different groups is presented in Fig. 5 a, revealing an overlap of 577 OTUs shared among the five sample groups. Additionally, unique OTUs were identified as follows: 3000 in the NC group, 1812 in the MC group, 2429 in the LPYC group, 1634 in the MPYC group, and 1950 in the HPYC group. Furthermore, similarities within the overall microbial community structure were assessed using principal component analysis (PCA) (Fig. 5 b), where two primary principal components accounted for 32.8% and 16.4% of total variance respectively. The results indicated that the microbial community structure of the MC group was significantly distinct from both NC and high-activity probiotic yogurt groups(P < 0.05). To further investigate relationships between gut microbiota across each experimental group, linear discriminant analysis effect size (LEfSe) was conducted (Fig. 5 c). The findings demonstrated that Nanosyncoccus, Marinifilaceae, Schaedlerella , and Saccharimonadia were predominant within the gut microbiota of the NC group; conversely, Castellaniella, Parasutterella , and Ructibacterium played key roles within the MC group. Following treatment with high-activity probiotic yogurt, specific bacteria identified included Dubosiella, Faecalibaculum, Nanosyncoccaceae, Atopobiaceae and Parabacteroides-B within the LPYC group; F0540 was found to be specific to MPYC; while Gemmatimonadota and Aerococcaceae were exclusive to HPYC. Similar observations emerged from linear discriminant analysis (LDA = 2) as shown in Fig. 5 d: there were eight dominant taxa present in both NC and MC groups; eleven taxa identified within LPYC; one taxon observed exclusively within MPYC; and nine taxa noted for HPYC. These findings collectively underscore high-activity probiotic yogurt's potential as a prebiotic agent capable of restoring gut microbial dysbiosis while regulating intestinal microbial ecology through enhanced diversity alongside modulation of microbial community structure and composition specifically observed in CTX-treated mice[ 32 ]. The correlation between the high-activity probiotic yoghurt-derived bacteria and immune detection indices was illustrated as a heat map through Spearman correlation analysis (Fig. 6 ). The results indicated that Bacteroidota and Desulfobacterota-I exhibited positive correlations with hepatic parameters, cytokines, and certain microbiota including Actinobacteriota, Campylobacterota, Proteobacteria, and Patescibacteria showed negative correlations with the mRNA levels of the relevant pathways in colon tissue. Al-Sheraji et al. reported no significant improvement in spleen weight after Bifidobacterium longum BB536 supplementation in hypercholesterolemic rats, whereas our high-dose yogurt group (HPYC) restored spleen indices to near-normal levels[ 24 ]. This disparity underscores the importance of strain synergy in amplifying host responses, as proposed by Yahfoufi et al[ 12 ]. The enriched Bacteroidetes in our study correlated with elevated TLR4 expression, a finding consistent with Masato et al. (2007), who linked Bacteroides-derived LPS to TLR4-mediated cytokine production[ 8 ]. The simultaneous activation of TLR4/NF-κB and microbiota structure may explain the compounded immune benefits. This study investigated the interactions among high-activity probiotic yoghurt-induced bacteria, chemokines, and the TLR4/NF-κB p65 signaling pathways. The findings suggest that high-activity probiotic yogurt modulates immune responses through multiple mechanisms. Conclusion In conclusion, this study demonstrated that high-activity probiotic yogurt significantly increased the levels of TLR-4 and key proteins in the NF-κB signaling pathway (specifically NF-κB p65)(P < 0.05), while also promoting the secretion of cytokines, including IL-6, IL-12, and TNF-α. Conversely, high-activity probiotic yogurt enhanced the diversity of the microbial community and regulated the overall structure of intestinal microbiota in mice induced with CTX. Additionally, it was observed that high activity probiotic yogurt elevated the abundance of Lactobacillus and COE1 , while reducing the abundance of Akkermansia and Duncaniella . In summary, the findings presented above indicate that high activity probiotic yogurt has the potential to repair damage to intestinal mucosa caused by CTX through modulation of the TLR4/NF-κB p65 signaling pathway. Therefore, high activity probiotic yogurt holds significant promise as an immune enhancer and a natural therapeutic agent. Declarations Funding We are profoundly thankful to the National Key Research and Development Program of China (2022YFF1100201); the National Natural Science Foundation of China(32172169); the Tianjin “131” Innovative Talent Team Project (201926); the Open Fund of Tianjin Key Laboratory of Intelligent Breeding of Major Crops (KLIBMC2312). Conflict of interest The authors declare no competing financial interest. References Kayisoglu O, Weiss F, Niklas C, Pierotti I Bartfeld S (2021) Location-specific cell identity rather than exposure to GI microbiota defines many innate immune signalling cascades in the gut epithelium. Gut 70, 687-697. Li XH, Lirong (2021) Polysaccharides in natural products that repair the damage to intestinal mucosa caused by cyclophosphamide and their mechanisms: A review. Carbohydrate Polymers: 261, 117876. Xincheng W, Xiao‐Jun H, Wanning M, Mingzhi L, Jinkun W, Chunhua C, Liandi L Shaoping N (2023) Bioactive polysaccharides promote gut immunity via different ways. Food & Function 14, 1387–1400. Song W, Wang Y, Li G, Xue S, Zhang G, Dang Y Wang H (2023) Modulating the gut microbiota is involved in the effect of low-molecular-weight Glycyrrhiza polysaccharide on immune function. Gut Microbes 15, 2276814. Ding Y, Yan Y, Chen D, Ran L, Mi J, Lu L, Jing B, Li X, Zeng X Cao Y (2019) Modulating effects of polysaccharides from the fruits ofLycium barbarumon the immune response and gut microbiota in cyclophosphamide-treated mice. Food & Function 10, 3671-3683. Fu YP, Feng B, Zhu ZK, Feng X, Chen SF, Li LX, Yin ZQ, Huang C, Chen XF, Zhang BZ, Jia RY, Song X, Lv C, Yue GZ, Ye G, Liang XX, He CL, Yin LZ Zou YF (2018) The Polysaccharides from Codonopsis pilosula Modulates the Immunity and Intestinal Microbiota of Cyclophosphamide-Treated Immunosuppressed Mice. Molecules 23, 1801. Vos WMD, Tilg H, Hul MV Cani PD (2022) Gut microbiome and health: mechanistic insights. Gut 71, 1020-1032. Mager LFB, Regula，Pett, Nicola，Cooke, Noah C. A.，Brown, Kirsty ，Ramay, Hena，Paik, Seungil，Stagg, John，Groves, Ryan A.，Gallo, Marco，Lewis, Ian A.，Geuking, Markus B. ，McCoy, Kathy D (2020) Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy. Science 369, 1481-1489. He Q, Zhang T, Zhang W, Feng C, Kwok LY, Zhang H Sun Z (2024) Administering Lactiplantibacillus fermentum F6 decreases intestinal Akkermansia muciniphila in a dextran sulfate sodium-induced rat colitis model. Food&Function 15, 5882-5894. Waide ML & Schmidt NW (2020) The gut microbiome, immunity, and Plasmodium severity. Current Opinion in Microbiology 58, 56-61. G LF, P W MH M (2018) Probiotics and the Gut Immune System: Indirect Regulation. Probiotics and antimicrobial proteins 10, 11-21. Yahfoufi N, Mallet J, Graham E Matar C (2018) Role of probiotics and prebiotics in immunomodulation. Current Opinion in Food Science 20, 82-91. Dennis AS & Robert WH (2020) Yogurt, cultured fermented milk, and health: a systematic review. Nutrition Reviews 79, 599-614. Pedro F-J, Gary WB, William C, Douwe van S José Luis MM (2023) Fungal β-glucan-facilitated cross-feeding activities between Bacteroides and Bifidobacterium species. Communications biology 6, 576 ME S, DJ M, G R, GR G RA R (2019) Probiotics and prebiotics in intestinal health and disease: from biology to the clinic. Nat Rev Gastroenterol Hepatol 16, 605-616. Christopher JS (2021) Breastfeeding promotes bifidobacterial immunomodulatory metabolites. Nature Microbiology 6, 1335–1336. Chen K, Jin S Ma Y, LimeiXu, PingNie, YangLuo, LiYu, QinghuaShen, YangZhou, ZengyuanLiu, Changqi (2024) Adjudicative efficacy of Bifidobacterium animalis subsp. lactis BLa80 in treating acute diarrhea in children: a randomized, double-blinded, placebo-controlled study. European Journal of Clinical Nutrition 78, 501–508. Junyu H, Yongli Z, Tao W, Rui L, Wenjie S, Jianguo Z, Shuguang F, Jie‐Ting G Min Z (2022) The antidiabetic effects of Bifidobacterium longum subsp. longum BL21 through regulating gut microbiota structure in type 2 diabetic mice. Food & Function 13, 9947-9958. Quin C, Ghosh S, Dai C, Barnett JA, Garner AM, Yoo RKH, Zandberg WF, Botta A, Gorzelak MA Gibson DL (2021) Maternal Intake of Dietary Fat Pre-Programs Offspring's Gut Ecosystem Altering Colonization Resistance and Immunity to Infectious Colitis in Mice. Mol Nutr Food Res 65, e2000635. Xie H, Fang J, Farag MA, Li Z, Sun P Shao P (2022) Dendrobium officinale leaf polysaccharides regulation of immune response and gut microbiota composition in cyclophosphamide-treated mice. Food Chemistry: X 13. Ruiqiang Z, Haoran Z, Jinsong L, Xinfu Z, Yanping W Caimei Y (2021) Rhamnolipids enhance growth performance by improving the immunity, intestinal barrier function, and metabolome composition in broilers. Journal of the Science of Food and Agriculture 102, 908-919. Linlin W, Mao C, Jialiang W, Qiangqing Y, Gang W, Hao Z, Jianxin Z Wei C (2022) Bifidobacterium longum relieves constipation by regulating the intestinal barrier of mice. Food & Function 13, 5037-5049. Bingjie G, Huajun Y, Jun M, Xiaoshuai C Zhi‐yue W (2020) Effects of Mannan Oligosaccharides and/or Bifidobacterium on Growth and Immunity in Domestic Pigeon (Columba livia domestica). Journal of Poultry Science 57, 277-283. Al-Sheraji SH, Amin I, Azlan A, Manap MY Hassan FA (2015) Effects of Bifidobacterium longum BB536 on lipid profile and histopathological changes in hypercholesterolaemic rats. Benef Microbes 6, 661-668. Hope OD, Oanh P, Lin-Xi L, Shaikh MA, Seung Joo L, Mariëtta MR, Jessica LS, Sean Paul N, Petr B, Denise MM, Andreas JB Stephen JM (2014) Toll-like Receptor and Inflammasome Signals Converge to Amplify the Innate Bactericidal Capacity of T Helper 1 Cells. Immunity 40, 213-224. Dong Y, Liao W, Tang J, Fei T, Gai Z Han M (2022) Bifidobacterium BLa80 mitigates colitis by altering gut microbiota and alleviating inflammation. AMB Express 12, 67. Wang X, Yuan Z, Zhu L, Yi X, Ou Z, Li R, Tan Z, Pozniak B, Obminska-Mrukowicz B, Wu J Yi J (2019) Protective effects of betulinic acid on intestinal mucosal injury induced by cyclophosphamide in mice. Pharmacological Reports 71, 929-939. Xu P, Cui K, Chen L, Chen S Wang Z (2023) Effect of dietary Bifidobacterium animalis subsp. lactis BLa80 on growth, immune response, antioxidant capacity, and intestinal microbiota of juvenile Japanese seabass (Lateolabrax japonicus). Aquaculture International 32, 1749-1769. Zheng D, Liwinski T Elinav E (2020) Interaction between microbiota and immunity in health and disease. Cell Research 30, 492-506. Heine H & Lien E (2003) Toll-Like Receptors and Their Function in Innate and Adaptive Immunity. International Archives of Allergy and Immunology 130, 180-192. Gavzy SJ, Kensiski A, Lee ZL, Mongodin EF, Ma B Bromberg JS (2023) Bifidobacterium mechanisms of immune modulation and tolerance. Gut Microbes 15, 2291164. Ying M, Yu Q, Zheng B, Wang H Xie M (2020) Cultured Cordyceps sinensis polysaccharides modulate intestinal mucosal immunity and gut microbiota in cyclophosphamide-treated mice. Carbohydrate Polymers 235, 115957. Shi H, Chang Y, Gao Y, Wang X Tang QJ (2017) Dietary fucoidan of Acaudina molpadioides alters gut microbiota and mitigates intestinal mucosal injury induced by cyclophosphamide. Food & Function 8, 3383-3393. Masato T, Akira H, Tsutomu Y, Satoshi H, Kazuhiro H, Kazuyoshi I, Kyôko T Shuichi K (2007) Prior stimulation of antigen-presenting cells with Lactobacillus regulates excessive antigen-specific cytokine responses in vitro when compared with Bacteroides. Cytotechnology 5, 89-101. Galdeano CM & Perdigón G (2006) The Probiotic Bacterium Lactobacillus casei Induces Activation of the Gut Mucosal Immune System through Innate Immunity. Clinical and Vaccine Immunology 13, 219-226. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 25 Jun, 2025 Read the published version in Probiotics and Antimicrobial Proteins → Version 1 posted Editorial decision: Revision requested 17 May, 2025 Reviews received at journal 17 May, 2025 Reviews received at journal 13 May, 2025 Reviews received at journal 06 May, 2025 Reviews received at journal 02 May, 2025 Reviewers agreed at journal 28 Apr, 2025 Reviewers agreed at journal 27 Apr, 2025 Reviewers agreed at journal 27 Apr, 2025 Reviewers agreed at journal 25 Apr, 2025 Reviewers invited by journal 25 Apr, 2025 Editor assigned by journal 12 Apr, 2025 Submission checks completed at journal 12 Apr, 2025 First submitted to journal 18 Mar, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6252467","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":448972791,"identity":"45dc36da-9709-4f7c-8a6e-e8e12334019a","order_by":0,"name":"Liyuan Yun","email":"","orcid":"","institution":"Tianjin Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Liyuan","middleName":"","lastName":"Yun","suffix":""},{"id":448972792,"identity":"4a2716e3-c18d-488c-859d-b6bfd2f26df1","order_by":1,"name":"Jinpeng Zhang","email":"","orcid":"","institution":"Tianjin Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jinpeng","middleName":"","lastName":"Zhang","suffix":""},{"id":448972793,"identity":"391631d5-1840-4ecd-962f-de758e4f549b","order_by":2,"name":"Huping Yang","email":"","orcid":"","institution":"Tianjin Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Huping","middleName":"","lastName":"Yang","suffix":""},{"id":448972794,"identity":"fb24ecf5-f8b9-4cb7-9dbf-36048d2f029c","order_by":3,"name":"Qian Li","email":"","orcid":"","institution":"Tianjin Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Li","suffix":""},{"id":448972795,"identity":"f984dd52-761c-4f2f-81ba-312731e0fce0","order_by":4,"name":"Shuguang Fang","email":"","orcid":"","institution":"Wecare Probiotics Co., Ltd，Suzhou","correspondingAuthor":false,"prefix":"","firstName":"Shuguang","middleName":"","lastName":"Fang","suffix":""},{"id":448972796,"identity":"0ab8fb2f-3a0d-4bf8-9297-07bf466f0cc0","order_by":5,"name":"Xiaojuan Guo","email":"","orcid":"","institution":"Wecare Probiotics Co., Ltd，Suzhou","correspondingAuthor":false,"prefix":"","firstName":"Xiaojuan","middleName":"","lastName":"Guo","suffix":""},{"id":448972797,"identity":"236eb634-826f-4e67-b5b7-ffa7eac283e5","order_by":6,"name":"Yanfeng Wu","email":"","orcid":"","institution":"Wecare Probiotics Co., Ltd，Suzhou","correspondingAuthor":false,"prefix":"","firstName":"Yanfeng","middleName":"","lastName":"Wu","suffix":""},{"id":448972798,"identity":"30e5fdf5-6897-45f1-88cd-b34aab236477","order_by":7,"name":"YunJiao Zhao","email":"","orcid":"","institution":"Wecare Probiotics Co., Ltd，Suzhou","correspondingAuthor":false,"prefix":"","firstName":"YunJiao","middleName":"","lastName":"Zhao","suffix":""},{"id":448972800,"identity":"ba751ddb-87f3-47e5-b994-e016578f5ce2","order_by":8,"name":"Min Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIie3PMQuCQBTA8ScHTpbrCyG/wu1Fn0U5sKVZmkIInJpD8cu841bxVqGG+gaOLUUaLQ2lbg33n+7B/eA9AJPpL7MSCrbL92APIkxSU0bYPgYTW8g8VSOIfyy5cmy941oTNLECt0h+E6vYtMQ5Ia8FWFmlAM/Uc4nXEewIAzZJFXAMei55EV4h1wrYfQhxvCiQWUDISQCzhhD0BFFDYpbXgstDtXaw7iF+Ee6b8LFyp1peL7d4MXezHvIRdZuO+G8ymUymbz0Bim5EZah1dmMAAAAASUVORK5CYII=","orcid":"","institution":"Tianjin Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Min","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-03-18 11:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6252467/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6252467/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12602-025-10623-1","type":"published","date":"2025-06-25T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81647066,"identity":"3c529549-4e97-4ce0-861d-7badb29214e6","added_by":"auto","created_at":"2025-04-29 14:54:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":695840,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of high activity probiotic yogurt on CTX-induced immunosuppressed in mice.(a) the change of body weight;(b)the spleen index of immunosuppressed mice;(c) the thymus index of immunosuppressed mice;(d) Representative histological images of distal colon sections stained with hematoxylin and eosin (scale bars, 200 μm). In the Whiskers boxplots, the horizontal line represents the median of the data, the lower and upper bounds of the box represent the 25th and 75th percentile of data, and the lower and upper whiskers represent the minimum and maximum of the data. Multiple comparisons were performed by using one-way ANOVA using SPSS 26.0 software. Means with different letters are significantly different (p \u0026lt;0 .05) (n=6)；NC, normal control group; MC, model control group; LPYC, low dose compound probiotic yogurt control group; MPYC, low dose compound probiotic yogurt control group; HPYC, high compound probiotic yogurt control group.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6252467/v1/6fae06c8320f27d7b5a51d91.png"},{"id":81646542,"identity":"958dd522-2b0b-43ec-8f6f-bedc3de3c11e","added_by":"auto","created_at":"2025-04-29 14:46:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":424246,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of high activity probiotic yogurt on cytokines and signaling pathways in intestinal tissues. (a) the expression of interleukin (IL)-6;(b) the expression of IL-12; (c) the expression of tumor necrosis factor-α (TNF-α);(d) the representative gene expression of p65,TLR4, TRAF6 proteins and its gray value. Multiple comparisons were performed by using one-way ANOVA using SPSS 26.0 software. Means with different letters are significantly different (p \u0026lt;0 .05)(n=6).\u003c/p\u003e\n\u003cp\u003eNC, normal control group; MC, model control group; LPYC, low dose compound probiotic yogurt control group; MPYC, low dose compound probiotic yogurt control group; HPYC, high compound probiotic yogurt control group.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6252467/v1/9408206545100bf065e995da.png"},{"id":81646549,"identity":"94f4a6d4-ad04-42c7-b400-810a60bcdf00","added_by":"auto","created_at":"2025-04-29 14:46:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":655032,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of high activity probiotic yogurt on gut microbiota structure in fecal. (a) α-diversity-Chao 1 (P=0.07); (b) α-diversity-Simpson (P=0.31); (c) microbiota composition-phylum level;(d) microbiota composition-genus level; (e) dominant microbiota in genus level(\u003cem\u003eLactobacillus;COE1; Akkermansia; Duncaniella\u003c/em\u003e). The values among groups were compared by Kruskal-Wallis rank sum test, and p \u0026lt;0.05 was considered significant (n=6). In the boxplots, the horizontal line represents the median of the data, the lower and upper bounds of the box represent the 25th and 75th percentile of data, and the lower and upper whiskers represent the minimum and maximum of the data. The scattered points in the bars and boxes represent the actual data points. NC, normal control group; MC, model control group; LPYC, low dose compound probiotic yogurt control group; MPYC, low dose compound probiotic yogurt control group; HPYC, high compound probiotic yogurt control group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6252467/v1/8f99c5a29c0a5bd26c253f09.png"},{"id":81646545,"identity":"422ca89b-c813-4e81-8f70-0fcc3551fd83","added_by":"auto","created_at":"2025-04-29 14:46:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":247459,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of high activity probiotic yogurt on β-diversity. (a) Principal coordinates analysis (PCoA) plot based on Bray-Curtis dissimilarity measures show clustering amongst diet groups; (b) Nonmetric multidimensional scaling analysis (NMDS); Each dot represents a colonic community, and the percentage of variation explained by each principal coordinate is shown; (c)Hierarchical cluster analysis, using Bray-Curtis dissimilarity distance. The values among groups were compared by Kruskal-Wallis rank sum test, and p\u0026lt;0.05 was considered significant (n=6); NC, normal control group; MC, model control group; LPYC, low dose compound probiotic yogurt control group; MPYC, low dose compound probiotic yogurt control group; HPYC, high compound probiotic yogurt control group.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6252467/v1/d22e11845eb5a55ea9d9d40f.png"},{"id":81646547,"identity":"450852b8-af38-428b-b1ef-307d42f6847d","added_by":"auto","created_at":"2025-04-29 14:46:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":334559,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of species differences species. (a) Venn diagram of OTUs in the different groups, (b) Principal coordinate analysis (PCA) plot. Circles represent 95% confidence intervals. Each dot represents a colonic community, and the percentage of variation explained by each principal coordinate is shown. (c) LEfSe taxonomic cladogram. The five groups were treated as five classes and a multiclass comparison was performed using the Kruskal-Wallis and Wilcoxon rank-sum test, with a p-value cut-off of 0.05 and an LDA score for discriminative features set to 2.0. The Cladogram shows the bacterial distribution (corresponding taxa labelled a-y) in the sample groups and differences in abundances between them were displayed as colors and circle’s diameters. (d) Histogram of the linear discriminant analysis (LDA) scores for differentially abundant genera. The values among groups were compared by Kruskal-Wallis rank sum test, and p\u0026lt;0.05 was considered significant (n=6); NC, normal control group; MC, model control group; LPYC, low dose compound probiotic yogurt control group; MPYC, low dose compound probiotic yogurt control group; HPYC, high compound probiotic yogurt control group.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-6252467/v1/dd9d8d5b9f63e37843d15b83.png"},{"id":81647067,"identity":"1b381417-adbe-4bd7-aac6-b1a07e647582","added_by":"auto","created_at":"2025-04-29 14:54:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":180132,"visible":true,"origin":"","legend":"\u003cp\u003eThe correlation between gut microbiota and relative index (analysis by spearman method). Multiple comparisons were performed by using one-way ANOVA using SPSS 26.0 software. Significant correlations are marked with *P \u0026lt; 0.05; **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-6252467/v1/dcfd24ce0add86422a22a8b8.png"},{"id":85686163,"identity":"d4cf3f83-a291-4c26-ade7-9d83c4914c53","added_by":"auto","created_at":"2025-06-30 16:04:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3300865,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6252467/v1/7808d123-f6e7-400e-b25e-4e15d08d85dc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Highly Active Probiotic Yogurt Protects Against Cyclophosphamide-Induced Immunosuppression in Mice by Regulating Immune Response and gut microbiota","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe immune system acts as a critical defense mechanism against external pathogens, responsible for identifying and neutralizing antigenic substances. It collaborates with other bodily systems to maintain homeostasis and physiological balance[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Cyclophosphamide (CTX), a widely used antineoplastic agent, acts as both an alkylating compound and an immunosuppressive drug. It is known for its significant adverse effects, including damage to the intestinal mucosa, reduction in immune organ indices, and suppression of immune cell and cytokine production[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Studies have shown that CTX exposure increases intestinal permeability and bacterial translocation, primarily due to the proliferation of harmful bacteria, such as \u003cem\u003eProteobacteria, Sutterella\u003c/em\u003e, and \u003cem\u003ePseudomonas\u003c/em\u003e[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. As a result, CTX is frequently employed to create animal models of immunosuppression and intestinal injury[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Probiotics are beneficial, active microorganisms that colonize the human gut and alter the composition of the host's microbiota at specific sites[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], thereby playing a crucial role in maintaining intestinal ecological balance and enhancing systemic immunity[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In recent years, the application of probiotics for modulating intestinal flora and improving host immunity has garnered significant attention[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Studies have demonstrated that gut microbes exert immunomodulatory effects through the adjustment of the body's internal environment or by enhancing the composition and metabolic products of gut microbiota[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, studies on probiotic yogurt predominantly concentrate on the effects of individual or a limited number of probiotics, with little attention paid to the synergistic mechanisms among diverse strains of complex probiotics, thereby restricting our comprehension and utilization of these complex probiotics[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Yogurt, serving as an effective delivery system for probiotics, is deemed one of the optimal methods for administering high doses of probiotics. However, most commercially available probiotic yogurts typically contain only the fundamental fermentation strains, namely \u003cem\u003eStreptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus\u003c/em\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eBifidobacterium\u003c/em\u003e is a widely employed functional strain known for its excellent tolerance in the gastrointestinal tract, and it plays a crucial role in maintaining gut health[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], as well as recognized as an immunomodulator or a biomarker for human diseases, capable of enhancing the body's immune response through the improvement of phagocytic cell function and bolstering resistance to illness[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. \u003cem\u003eBifidobacterium\u003c/em\u003e strains demonstrated strain-specific biological activity, such as \u003cem\u003eBifidobacterium animalis subsp. lactis BLa80\u003c/em\u003e, as an adjunct treatment for diarrhea in children[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], \u003cem\u003eBifidobacterium longum subsp. longum BL21\u003c/em\u003e strengthens gut barrier integrity and possesses hypoglycemic activity[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the synergistic mechanisms of multi-strain consortia remain underexplored.\u003c/p\u003e \u003cp\u003eIn the initial phase of our study, we employed a complex probiotic blend yogourt (comprises \u003cem\u003eBifidobacterium animalis subsp. lactis BLa80\u003c/em\u003e, \u003cem\u003eBifidobacterium longum subsp. longum BL21\u003c/em\u003e, \u003cem\u003eBifidobacterium breve BBr16, Bifidobacterium adolescentis BAC30\u003c/em\u003e, and \u003cem\u003eBifidobacterium longum subsp. infantis BI45\u003c/em\u003e) which provided by WecPro DigestBi. Our preliminary findings indicated that this complex probiotic blend demonstrated the most effective synergistic action in promoting the proliferation of viable bacteria post-fermentation, achieving a target of 100\u0026nbsp;billion highly active bacteria in the yogurt. Additionally, the fermented yogurt exhibited superior flavor characteristics. This formulation surpasses conventional yogurts limited to \u003cem\u003eS. thermophilus\u003c/em\u003e and \u003cem\u003eL. bulgaricus\u003c/em\u003e, offering a novel platform to investigate how multi-strain interactions enhance body health, such as improve immunity, regulate blood sugar level, and inhibit diarrhea, and so on. However, the immune -regulatory effects of this complex probiotic blend yogourt was rarely reported. Therefore, the research is necessary to clarify the immune-regulatory effects and interactions with gut microbiota associated with this high-activity probiotic yogurt.\u003c/p\u003e \u003cp\u003eThis study investigates the combinatorial effects of BLa80, BL21, BBr16, BAC30, and BI45 using in vivo models to elucidate strain-specific versus collective immunomodulatory mechanisms. The investigation encompassed the growth index, organ indices, intestinal tissue morphology, intestinal cytokine levels (including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-12, protein expression (including toll-like receptor 4 (TLR4), TNF receptor associated factor 6(TRAF6), nuclear factor kappa-B-p65(p65), and intestinal flora diversity in cyclophosphamide (CTX)-treated mice. This study not only effectively demonstrated the influence of probiotics on the enhancement of human immunity but also established a theoretical foundation for subsequent research, development, and market application of this product.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProbiotic yogurt making\u003c/h2\u003e \u003cp\u003eThe high-activity probiotic yogurt contained a mixture of the following strains: \u003cem\u003eBifidobacterium animalis subsp. lactis\u003c/em\u003e (BLa80), \u003cem\u003eBifidobacterium longum\u003c/em\u003e subsp. \u003cem\u003elongum\u003c/em\u003e (BL21), \u003cem\u003eBifidobacterium breve\u003c/em\u003e (BBr16), \u003cem\u003eBifidobacterium adolescentis\u003c/em\u003e (BAC30), and \u003cem\u003eBifidobacterium longum\u003c/em\u003e subsp. \u003cem\u003einfantis\u003c/em\u003e (BI45). These strains were provided by Wecare Probiotics Co., Ltd., located in Suzhou, Chin\u003c/p\u003e \u003cp\u003eThe basic fermentation strain WecCul\u0026reg;-D1, was inoculated with 30 g/T, and the complex bifidus, WecPro\u0026reg;-DigestBi, was inoculated at 250 g/T into the sterilized fermentation base material, which included 93.5% raw milk, 6% white granulated sugar, 0.5% complex prebiotic WecPre-S (containing xylooligosaccharide, fructooligosaccharides, isomaltooligosaccharides).The fermentation temperature was 37℃, the time was 8 h, the acidity reached 70\u0026deg;T to stop the fermentation, and the finished product was refrigerated at 4℃ overnight.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMice Treatment and Experimental Design\u003c/h3\u003e\n\u003cp\u003eFifty male ICR mice (8-week old, 20\u0026ndash;25 g) were purchased from Spiff (Beijing) Biotechnology Co., LTD., license No. SCXK (Beijing) 2019-0010. All animals were kept in a specific pathogen-free facility with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (8th edition). The Ethics Committee of Tianjin Agricultural University approved all animal experiments (License No. 2023LLSC08). During the study, the animals were housed in standard cages with unrestricted access to food and water for a duration of one week under controlled laboratory conditions (22\u0026ndash;25\u0026deg;C and relative humidity of 40\u0026ndash;60%). The light-dark cycle was maintained at 12 h per day.\u003c/p\u003e \u003cp\u003eAfter one week adaptation period, mice were categorized into 5 groups ((n\u0026thinsp;=\u0026thinsp;10 each group), which were normal control group (NC), model control group (MC), low dose compound probiotic yogurt control group (LPYC, probiotic concentration: 10\u003csup\u003e9\u003c/sup\u003e CFU/250 g), medium dose compound probiotic yogurt control group (MPYC, probiotic concentration: 10\u003csup\u003e10\u003c/sup\u003e CFU/250 g), high compound probiotic yogurt control group (HPYC, probiotic concentration: 10\u003csup\u003e11\u003c/sup\u003e CFU/250 g). The compound probiotic yogurt was storaged at 4℃ during the experiment.\u003c/p\u003e \u003cp\u003e(1) NC: treat with 0.2 mL physiological saline for once a day, for 14 days;\u003c/p\u003e \u003cp\u003e(2) MC: subjected to CTX intraperitoneal injection treatment (80 mg/kg) once a day at 7,8,9 days to induce immunosuppression, and gavaged with 0.2 mL physiological saline for once a day, for 14 days;\u003c/p\u003e \u003cp\u003e(3)LPYC: subjected to CTX intraperitoneal injection treatment (80 mg/kg) once a day at 7,8,9 days to induce immunosuppression, and gavaged with 10 mL per kg BW per day compound probiotic yogurt which probiotic concentration was 10\u003csup\u003e9\u003c/sup\u003e CFU/250 g;\u003c/p\u003e \u003cp\u003e(4)MPYC: subjected to CTX intraperitoneal injection treatment (80 mg/kg) once a day at 7,8,9 days to induce immunosuppression, and gavaged with 10 mL per kg BW per day compound probiotic yogurt which probiotic concentration was 10\u003csup\u003e10\u003c/sup\u003e CFU/250 g;\u003c/p\u003e \u003cp\u003e(5)HPYC: subjected to CTX intraperitoneal injection treatment (80 mg/kg) once a day at 7,8,9 days to induce immunosuppression, and gavaged with 10 mL per kg BW per day compound probiotic yogurt which probiotic concentration was 10\u003csup\u003e11\u003c/sup\u003e CFU/250 g.\u003c/p\u003e \u003cp\u003eOn days 7, 8, and 9 following the initiation of the experiment, all groups except for the NC group were administered intraperitoneal injections of cyclophosphamide at a dosage of 80 mg/kg body weight for three consecutive days. Following this treatment regimen, a significant decrease in mouse body weight was observed, indicating that the model had been successfully established. The weights of the mice were recorded periodically throughout the duration of the experiment. At the conclusion of the experiment, mice were euthanized via cervical dislocation after blood collection from their eyeballs and subsequent serum extraction. The spleen and thymus were harvested and weighed; additionally, small intestinal tissue along with colonic contents was excised for further analysis.\u003c/p\u003e\n\u003ch3\u003eHistopathology evaluation\u003c/h3\u003e\n\u003cp\u003eA segment of the colon was excised, rinsed with sterile saline, and subsequently fixed in 4% paraformaldehyde (Solarbio, Beijing, China) at room temperature for 24 h. The paraformaldehyde-fixed colon tissues were then embedded in paraffin and sectioned to a thickness of 4 \u0026micro;m. Paraffin sections were stained with hematoxylin and eosin (Zhuhai Beso Biotechnology Co., LTD, GuangDong, China) following standard protocols. Images were captured using an upright optical microscope (Nikon Eclipse E100, Nikon, Tokyo, Japan) equipped with an imaging system (Nikon DS-U3, Nikon, Tokyo, Japan).\u003c/p\u003e\n\u003ch3\u003eEnzyme-linked immunosorbent assays\u003c/h3\u003e\n\u003cp\u003eThe levels of inflammatory cytokines, including IL-6, IL-12, and TNF-α, in colon were analyzed by enzyme-linked immunosorbent assays kits (Elabscience, Wuhan, China). The measurements were conducted in accordance with the manufacturer's instructions.\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eThe total cellular proteins were extracted from colon tissue samples utilizing a protein extraction kit (Beyotime, Shanghai, China), following the manufacturer's instructions meticulously. The protein concentration was quantified using the BCA assay kit (Beyotime, Shanghai, China). Equal amounts of denatured proteins were separated by SDS-PAGE and subsequently transferred onto a PVDF membrane (Millipore Co., MA, USA), which was then blocked with 5% BSA for one hour. Following this, the membrane was incubated overnight at 4\u0026deg;C with specific primary antibodies diluted to a ratio of 1:1000. Then, the membranes were washed three times for 10 min each in 1\u0026times;TBST and subsequently incubated with HRP-conjugated secondary antibodies diluted to a ratio of 1:3000 at room temperature for one hour. After an additional three washes of 10 minutes each, the blots were analyzed using an ECL chemiluminescence detection kit (Beyotime, Shanghai, China). Densitometric analysis was performed utilizing Quantity One software version 4.6.2 (Bio-Rad Laboratories, CA, USA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGut microbiota analysis\u003c/h2\u003e \u003cp\u003eTotal bacterial DNA was extracted from fecal samples using the TIANamp Stool DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China) in accordance with the manufacturer's instructions. The PCR products were purified with AMPure XT beads (Beckman, USA), and their concentration and quality were assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) as well as 1.2% agarose gel electrophoresis. Amplification of the V3-V4 region of the bacterial 16S rRNA gene was conducted utilizing Primer F: Illumina adapter sequence ACTCCTACGGGAGGCAGCA and Primer R: Illumina adapter sequence GGACTACHVGGGTWTCTAAT. Sequencing and bioinformatics analysis of the fecal samples were performed by Tianjin Jinke Biotechnology Co., LTD (Tianjin, China) on the Illumina MiSeq platform. To analyze the structure of gut microbiota, we employed various methods including alpha-diversity (α-diversity), beta-diversity (β-diversity), Venn diagram analysis, species composition assessment, and linear discriminant analysis effect size (LEfSe).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experiments were conducted in triplicate, and all data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences between two experimental groups were analyzed using Tukey\u0026rsquo;s test, while differences among multiple groups were evaluated through one-way analysis of variance (ANOVA) utilizing SPSS26.0 software. Graphical representations were generated using Origin 9.0 software (Origin Lab Corporation, Northampton, MA, USA). Pairwise comparisons of diversity between the experimental groups were determined using Species Richness, Simpson diversity, Chao 1 diversity measures. Microbial communities in the groups were then ordinated using weighted and unweighted UniFrac distance metrics with the Vegan package in R and visualized as a PCoA using ggplot2. The unweighted UniFrac is a qualitative measure showing the differences in the presence of bacteria. The weighted UniFrac shows the relative taxa abundance. A permutational multivariate analysis of variance (PERMANOVA) (\u0026#120572;=0.05) was run on the unweighted and weighted matrices to determine differences between groups using 999 random permutations. LEfSe was then used to validate the statistical significance and effect size of the differential abundances of taxa in the groups using the strict method (all classes differential). The experimental groups were treated as classes and a multiclass comparison was performed on closed reference operational taxonomic units using sequencing data were analyzed using the Kruskal-Wallis test and Wilcoxon rank-sum test, with a p-value cut-off of 0.05 and a linear discriminant analysis (LDA) score for discriminative features set to 2.0. The differentially abundant features were then visualized in a cladogram.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] A p-value of less than 0.05 was considered statistically significant; lowercase letters indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eHigh-activity probiotic yogurt mitigated the CTX-induced structural damage to the intestines and atrophy of immune organs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCyclophosphamide is extensively used in the treatment of various malignancies and autoimmune diseases; however, it can also cause significant toxic effects on the body [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Intraperitoneal administration of cyclophosphamide (CTX) can be used to establish models of immunosuppression and intestinal mucosal injury. This study examined the effects of high-activity probiotic yogurt on CTX-induced immunosuppression and intestinal mucosal damage. Following three consecutive days of intraperitoneal injections of CTX, a significant decreased in body weight was observed in mice, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. Compared to the NC group, the final body weight of mice in the MC group was significantly lower (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of high-activity probiotic yogurt on the body weight of immunosuppressive mice induced by Cyclophosphamide (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInitial body weight (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFinal body weight (g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLPYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMPYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;10). Statistical analysis via unpaired t-test. Different letter identifiers indicated significant difference between groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe spleen and thymus are two vital immune organs that play a critical role in immune regulation. The spleen serves as the site for T and B cell colonization and immune responses, while also synthesizing biologically active substances. In contrast, the thymus regulates peripheral immune organs and cells, providing an environment for T cell differentiation and maturation. Consequently, indices of the spleen and thymus are recognized as important indicators reflecting immune function [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Compared to the NC group, both the spleen index (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and thymus index (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) of mice in the MC group were significantly reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that high-activity probiotic yogurt may enhance host immunity by promoting immune organ function. Linlin Wang et al.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and Ji Bingjie Ge \u003cem\u003eet al.\u003c/em\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] also reported that \u003cem\u003eBifidobacterium longum\u003c/em\u003e could significantly enhance host immunity by improving the spleen index (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).In et al. research, no significant differences in the weights of the spleen were found between all groups after the treatment of \u003cem\u003eBifidobacterium longum\u003c/em\u003e BB536[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].Therefore, these results indicated that the high-activity probiotic yogurt effectively increased the body weight and organ index of immunocompromised mice.\u003c/p\u003e \u003cp\u003eThe intestinal tissues exhibited showed infiltration by inflammatory factors, leading to damage to the villus structure and impairment of the intestinal epithelial barrier function (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). In the study conducted by Hualing Xie et al.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], it was reported that CTX treatment resulted in significant damage to colonic epithelial tissue, accompanied by severe inflammation and atrophy of immune organs. Based on these findings, we infer that a successful immunocompromised mouse model has been established.\u003c/p\u003e \u003cp\u003eAfter the administration of high-activity probiotic yoghurt, there were no significant differences in final body weight, spleen index, or intestinal tissue parameters among the LPYC, MPYC, and HPYC groups compared to the NC group (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These findings suggest that high-activity probiotic yogurt may effectively mitigate the side effects of CTX treatment, including weight loss, reductions in organ indices, and damage to intestinal structure.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHigh-activity probiotic yogurt increased cytokines secretion and up-regulated the p65/TLR4/TRAF6 pathway\u003c/h2\u003e \u003cp\u003eThe intestine is the largest immune organ in humans, playing a critical role in maintaining the biological barrier that regulates the internal environment. TNF-α, TLR4, and p65 are involved in the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, either directly or indirectly, contributing to immune responses and enhancing overall immunity.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, enzyme-linked immunosorbent assay (ELISA) and Western blot analyses were employed to further investigate the effects of high-activity probiotic yogurt on cytokine secretion and signaling pathways within intestinal tissue. This approach aimed to assess both the expression levels of immune cytokines and related immune pathways.\u003c/p\u003e \u003cp\u003eVarious cytokines are involved in regulating intestinal mucosal inflammation and intestinal epithelial integrity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Intraperitoneal injection of cyclophosphamide (CTX) significantly reduced levels of immune cytokines IL-6, IL-12, and TNF-α by factors of 0.40-, 0.45-, and 0.51-fold respectively (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Xihong Wang et al. reported that CTX could inhibit cytokine secretion in the intestinal mucosa of mice, resulting in intestinal injury due to immune dysfunction[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Treatment with high-activity probiotic yogurt notably increased IL-6, IL-12, and TNF-α-fold levels by factors ranging from 0.13- to 0.21-fold for IL-6 and from 0.14-fold to 0.19-fold for IL-12. Additionally, Qiuwen He et al. found that following treatment with probiotics such as \u003cem\u003eLactiplantibacillus fermentum\u003c/em\u003e F6, there was a significant increase in cytokines including IL-6, interleukin1-beta (IL-1β) and TNF-α within a dextran sulfate sodium-induced colitis model group[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Compared to Peng Xu et al. previous studies using single \u003cem\u003eBifidobacterium\u003c/em\u003e animalis subsp. lactis BLa80 decreasing TNF-α by 0.3-fold[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], our multi-strain yogurt induced a more pronounced down-regulation of TNF-α(0.51-fold) suggesting that strain synergy amplifies immune activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eToll-like receptors (TLRs) are a class of transmembrane proteins that play a critical role in regulating the immune response. They are involved in host defense against pathogens, modulate the abundance of commensal microbes, and maintain tissue integrity[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Among these, TLR4 is the most well-characterized member; it recognizes pathogen-associated molecular patterns (PAMPs) and activates transcription factors to produce various pro-inflammatory cytokines that aid in clearing invading pathogens[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Upon activation of TLR4, several intracellular signaling pathways are triggered. In this study, intraperitoneal injection of CTX resulted in down-regulation of p65, TLR4, and TRAF6 expression by approximately 0.63-, 0.80-, and 0.68-fold respectively in the MC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The research of Gavzy et al. showed that \u003cem\u003eBifidobacterium breve\u003c/em\u003e strains UCC2003 and JCM7017 regulated the NF-κB and TNFα expression [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, we speculated that the high-activity probiotic yogurt effect the immune may be attributable to the regulation of TLR4/NF-κB p65 signaling pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHigh-activity probiotic yogurt increased the diversity of the gut microbiota\u003c/h2\u003e \u003cp\u003eIntestinal microbiota play a critical role in the development and maintenance of the host immune system, which is essential for balancing inflammatory responses and immune tolerance[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this study, we measured the richness and diversity of microbial communities through alpha-diversity analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Intraperitoneal injection of CTX resulted in a reduction in MC group in terms of microbial richness (Chao1 index) and microbial diversity (Simpson index). These findings indicated that cyclophosphamide induces significant disruption to both the diversity and richness of gut microbiota [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Furthermore, our results demonstrated that mice treated with high-activity probiotic yogurt exhibited higher Chao1 and Simpson indices compared to those in the MC group. Wang et al. (2022) observed modest diversity improvements with \u003cem\u003eBifidobacterium longum\u003c/em\u003e monotherapy, whereas our multi-strain intervention reversed CTX-induced dysbiosis to near-normal levels.Additionally, as reported by Wangdi Song et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], intraperitoneal administration of CTX similarly led to decreased α-diversity within gut microbiota as evidenced by declines in Chao1 index as well as Simpson and Shannon indices.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe changes in gut microbiota among the different treatment groups were further analyzed at a more granular level, specifically at the phylum and genus levels. At the phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e emerged as the two most predominant phyla, collectively accounting for nearly 80% of the total bacterial population. The high-activity probiotic yogurt treatment resulted in an increased relative abundance of \u003cem\u003eBacteroides\u003c/em\u003e, accompanied by a reduction in \u003cem\u003eFirmicutes\u003c/em\u003e compared to the negative control group. This finding suggests that high-activity probiotic yogurt exerts regulatory effects on gut microbiota composition. Additionally, it has been reported that CTX significantly influences gut microbiota by stimulating the proliferation of Proteobacteria and Firmicutes while simultaneously decreasing the proportion of \u003cem\u003eBacteroidete\u003c/em\u003es[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Similarly, Song et al. found that low-molecular-weight licorices enhanced immunity by modulating the microbiota, but it was mainly enriched in Firmicutes rather than Bacteroidetes[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. \u003cem\u003eBacteroidetes\u003c/em\u003e, which primarily consist of lipopolysaccharides (LPS), can be recognized by TLR4. As previously mentioned, the level of TLR4 was significantly elevated following treatment with high-activity probiotic yogurt, potentially leading to the activation of NF-κB (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, it has been reported that \u003cem\u003eBacteroides\u003c/em\u003e acidifaciens may enhance the production of IL-6 and IL-10[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In the present study, we observed an increased relative abundance of Bacteroidetes in the high-activity probiotic yogurt groups compared to the NC group. At the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), there was a notable increase in \u003cem\u003eLactobacillus\u003c/em\u003e in the LPYC, MPYC, and HPYC groups when compared to the MC group. Previous reports have indicated that \u003cem\u003eLactobacillus\u003c/em\u003e can enhance immune responses by interacting with immune cells associated with gut health[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. After the administration of high-activity probiotic yoghurt, a significant reduction in the bacterial genera \u003cem\u003eDuncaniella\u003c/em\u003e and \u003cem\u003eAkkermansia\u003c/em\u003e was observed in the LPYC, MPYC, and LPYC groups compared to the MC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). While He et al.demonstrated that \u003cem\u003eLactiplantibacillus fermentum\u003c/em\u003e F6 selectively suppressed Akkermansia in colitis models[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This dual action\u0026mdash;promoting beneficial taxa and suppressing pathobionts\u0026mdash;reflects the functional diversity of the five-strain blend, as individual strains likely target distinct microbial niches For example, \u003cem\u003eBifidobacterium longum\u003c/em\u003e subsp. infantis BI45 may enhance mucosal integrity via glycoprotein metabolism, while \u003cem\u003eBifidobacterium breve\u003c/em\u003e BBr16 could competitively exclude opportunistic pathogens through bacteriocin production. Such strain-specific contributions align with Yahfoufi et al. who emphasized that multi-strain consortia achieve superior ecological and immunological outcomes by leveraging complementary metabolic and adhesion properties[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, the current data suggest that the modulation of gut microbiota by high-activity probiotic yogurt was closely associated with immune-regulation in CTX-induced mice, which may represent a crucial mechanism through which high-activity probiotic yogurt exerts its beneficial effects. Consequently, targeting gut microbiota modulation could emerge as a potential strategy for immunoregulation.\u003c/p\u003e \u003cp\u003eThe beta diversity analysis was conducted using principal coordinates analysis (PCoA), nonmetric multidimensional scaling analysis (NMDS), and hierarchical cluster analysis based on Bray-Curtis dissimilarity distance. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, the results from PCoA and NMDS demonstrated a clear separation between the MC group and the other groups. On PCo1, which accounted for 18% of the total variance, the distances among the NC, LPYC, MPYC, and HPYC groups were closely clustered. This suggests that high-activity probiotic yogurt intervention could shift the gut microbiota composition of the MC group towards that of the NC group in a dose-dependent manner. Furthermore, cluster analytical results derived from Bray-Curtis distance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) corroborated those obtained from PCoA and NMDS analyses, indicating that high-activity probiotic yogurt influenced gut microbiota composition induced by CTX. The hierarchical clustering analysis at genus level based on Bray-Curtis distance is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec. Additionally, an Analysis of Similarities (ANOSIM) was employed to assess whether significant differences exist between intra-group variances across different groups[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The analysis revealed a significant difference in the gut microbial community structure between the NC and MC groups (Adonis test, R\u0026sup2;=0.515; P\u0026thinsp;=\u0026thinsp;0.0014), as well as between the MC and MPYC groups (Adonis test, R\u0026sup2;=0.289; P\u0026thinsp;=\u0026thinsp;0.06) and between the MC and HPYC groups (Adonis test, R\u0026sup2;=0.369; P\u0026thinsp;=\u0026thinsp;0.04). These findings indicate that CTX had a profound impact on the gut microbiota composition of the mice, which was subsequently restored through the consumption of high-activity probiotic yogurt.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHigh-activity probiotic yogurt effected on the gut microbiota composition\u003c/h2\u003e \u003cp\u003eThe alterations in intestinal bacterial composition across the various treatment groups are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. A Venn diagram depicting the operational taxonomic units (OTUs) among different groups is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, revealing an overlap of 577 OTUs shared among the five sample groups. Additionally, unique OTUs were identified as follows: 3000 in the NC group, 1812 in the MC group, 2429 in the LPYC group, 1634 in the MPYC group, and 1950 in the HPYC group. Furthermore, similarities within the overall microbial community structure were assessed using principal component analysis (PCA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), where two primary principal components accounted for 32.8% and 16.4% of total variance respectively. The results indicated that the microbial community structure of the MC group was significantly distinct from both NC and high-activity probiotic yogurt groups(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). To further investigate relationships between gut microbiota across each experimental group, linear discriminant analysis effect size (LEfSe) was conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The findings demonstrated that \u003cem\u003eNanosyncoccus, Marinifilaceae, Schaedlerella\u003c/em\u003e, and \u003cem\u003eSaccharimonadia\u003c/em\u003e were predominant within the gut microbiota of the NC group; conversely, \u003cem\u003eCastellaniella, Parasutterella\u003c/em\u003e, and \u003cem\u003eRuctibacterium\u003c/em\u003e played key roles within the MC group. Following treatment with high-activity probiotic yogurt, specific bacteria identified included \u003cem\u003eDubosiella, Faecalibaculum, Nanosyncoccaceae, Atopobiaceae\u003c/em\u003e and \u003cem\u003eParabacteroides-B\u003c/em\u003e within the LPYC group; F0540 was found to be specific to MPYC; while \u003cem\u003eGemmatimonadota\u003c/em\u003e and \u003cem\u003eAerococcaceae\u003c/em\u003e were exclusive to HPYC. Similar observations emerged from linear discriminant analysis (LDA\u0026thinsp;=\u0026thinsp;2) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed: there were eight dominant taxa present in both NC and MC groups; eleven taxa identified within LPYC; one taxon observed exclusively within MPYC; and nine taxa noted for HPYC. These findings collectively underscore high-activity probiotic yogurt's potential as a prebiotic agent capable of restoring gut microbial dysbiosis while regulating intestinal microbial ecology through enhanced diversity alongside modulation of microbial community structure and composition specifically observed in CTX-treated mice[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe correlation between the high-activity probiotic yoghurt-derived bacteria and immune detection indices was illustrated as a heat map through Spearman correlation analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results indicated that Bacteroidota and Desulfobacterota-I exhibited positive correlations with hepatic parameters, cytokines, and certain microbiota including Actinobacteriota, Campylobacterota, Proteobacteria, and Patescibacteria showed negative correlations with the mRNA levels of the relevant pathways in colon tissue. Al-Sheraji et al. reported no significant improvement in spleen weight after \u003cem\u003eBifidobacterium longum\u003c/em\u003e BB536 supplementation in hypercholesterolemic rats, whereas our high-dose yogurt group (HPYC) restored spleen indices to near-normal levels[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This disparity underscores the importance of strain synergy in amplifying host responses, as proposed by Yahfoufi et al[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The enriched Bacteroidetes in our study correlated with elevated TLR4 expression, a finding consistent with Masato et al. (2007), who linked Bacteroides-derived LPS to TLR4-mediated cytokine production[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The simultaneous activation of TLR4/NF-κB and microbiota structure may explain the compounded immune benefits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis study investigated the interactions among high-activity probiotic yoghurt-induced bacteria, chemokines, and the TLR4/NF-κB p65 signaling pathways. The findings suggest that high-activity probiotic yogurt modulates immune responses through multiple mechanisms.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study demonstrated that high-activity probiotic yogurt significantly increased the levels of TLR-4 and key proteins in the NF-κB signaling pathway (specifically NF-κB p65)(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while also promoting the secretion of cytokines, including IL-6, IL-12, and TNF-α. Conversely, high-activity probiotic yogurt enhanced the diversity of the microbial community and regulated the overall structure of intestinal microbiota in mice induced with CTX. Additionally, it was observed that high activity probiotic yogurt elevated the abundance of \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eCOE1\u003c/em\u003e, while reducing the abundance of \u003cem\u003eAkkermansia\u003c/em\u003e and \u003cem\u003eDuncaniella\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn summary, the findings presented above indicate that high activity probiotic yogurt has the potential to repair damage to intestinal mucosa caused by CTX through modulation of the TLR4/NF-κB p65 signaling pathway. Therefore, high activity probiotic yogurt holds significant promise as an immune enhancer and a natural therapeutic agent.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eWe are profoundly thankful to\u0026nbsp;the\u0026nbsp;National Key Research and Development Program of China (2022YFF1100201); the\u0026nbsp;National Natural Science Foundation of China(32172169); the Tianjin \u0026ldquo;131\u0026rdquo; Innovative Talent Team Project (201926); the Open Fund of Tianjin Key Laboratory of Intelligent Breeding of Major Crops (KLIBMC2312).\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKayisoglu O, Weiss F, Niklas C, Pierotti I Bartfeld S (2021) Location-specific cell identity rather than exposure to GI microbiota defines many innate immune signalling cascades in the gut epithelium. Gut 70, 687-697.\u003c/li\u003e\n\u003cli\u003eLi XH, Lirong (2021) Polysaccharides in natural products that repair the damage to intestinal mucosa caused by cyclophosphamide and their mechanisms: A review. Carbohydrate Polymers: 261, 117876.\u003c/li\u003e\n\u003cli\u003eXincheng W, Xiao‐Jun H, Wanning M, Mingzhi L, Jinkun W, Chunhua C, Liandi L Shaoping N (2023) Bioactive polysaccharides promote gut immunity via different ways. Food \u0026amp; Function 14, 1387\u0026ndash;1400.\u003c/li\u003e\n\u003cli\u003eSong W, Wang Y, Li G, Xue S, Zhang G, Dang Y Wang H (2023) Modulating the gut microbiota is involved in the effect of low-molecular-weight Glycyrrhiza polysaccharide on immune function. Gut Microbes 15, 2276814.\u003c/li\u003e\n\u003cli\u003eDing Y, Yan Y, Chen D, Ran L, Mi J, Lu L, Jing B, Li X, Zeng X Cao Y (2019) Modulating effects of polysaccharides from the fruits ofLycium barbarumon the immune response and gut microbiota in cyclophosphamide-treated mice. Food \u0026amp; Function 10, 3671-3683.\u003c/li\u003e\n\u003cli\u003eFu YP, Feng B, Zhu ZK, Feng X, Chen SF, Li LX, Yin ZQ, Huang C, Chen XF, Zhang BZ, Jia RY, Song X, Lv C, Yue GZ, Ye G, Liang XX, He CL, Yin LZ Zou YF (2018) The Polysaccharides from Codonopsis pilosula Modulates the Immunity and Intestinal Microbiota of Cyclophosphamide-Treated Immunosuppressed Mice. Molecules 23, 1801.\u003c/li\u003e\n\u003cli\u003eVos WMD, Tilg H, Hul MV Cani PD (2022) Gut microbiome and health: mechanistic insights. Gut 71, 1020-1032.\u003c/li\u003e\n\u003cli\u003eMager LFB, Regula，Pett, Nicola，Cooke, Noah C. A.，Brown, Kirsty ，Ramay, Hena，Paik, Seungil，Stagg, John，Groves, Ryan A.，Gallo, Marco，Lewis, Ian A.，Geuking, Markus B. ，McCoy, Kathy D (2020) Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy. Science 369, 1481-1489.\u003c/li\u003e\n\u003cli\u003eHe Q, Zhang T, Zhang W, Feng C, Kwok LY, Zhang H Sun Z (2024) Administering Lactiplantibacillus fermentum F6 decreases intestinal Akkermansia muciniphila in a dextran sulfate sodium-induced rat colitis model. Food\u0026amp;Function 15, 5882-5894.\u003c/li\u003e\n\u003cli\u003eWaide ML \u0026amp; Schmidt NW (2020) The gut microbiome, immunity, and Plasmodium severity. Current Opinion in Microbiology 58, 56-61.\u003c/li\u003e\n\u003cli\u003eG LF, P W MH M (2018) Probiotics and the Gut Immune System: Indirect Regulation. Probiotics and antimicrobial proteins 10, 11-21.\u003c/li\u003e\n\u003cli\u003eYahfoufi N, Mallet J, Graham E Matar C (2018) Role of probiotics and prebiotics in immunomodulation. Current Opinion in Food Science 20, 82-91.\u003c/li\u003e\n\u003cli\u003eDennis AS \u0026amp; Robert WH (2020) Yogurt, cultured fermented milk, and health: a systematic review. Nutrition Reviews 79, 599-614.\u003c/li\u003e\n\u003cli\u003ePedro F-J, Gary WB, William C, Douwe van S Jos\u0026eacute; Luis MM (2023) Fungal \u0026beta;-glucan-facilitated cross-feeding activities between Bacteroides and Bifidobacterium species. Communications biology 6, 576 \u003c/li\u003e\n\u003cli\u003eME S, DJ M, G R, GR G RA R (2019) Probiotics and prebiotics in intestinal health and disease: from biology to the clinic. Nat Rev Gastroenterol Hepatol 16, 605-616.\u003c/li\u003e\n\u003cli\u003eChristopher JS (2021) Breastfeeding promotes bifidobacterial immunomodulatory metabolites. Nature Microbiology 6, 1335\u0026ndash;1336.\u003c/li\u003e\n\u003cli\u003eChen K, Jin S Ma Y, LimeiXu, PingNie, YangLuo, LiYu, QinghuaShen, YangZhou, ZengyuanLiu, Changqi (2024) Adjudicative efficacy of Bifidobacterium animalis subsp. lactis BLa80 in treating acute diarrhea in children: a randomized, double-blinded, placebo-controlled study. European Journal of Clinical Nutrition 78, 501\u0026ndash;508.\u003c/li\u003e\n\u003cli\u003eJunyu H, Yongli Z, Tao W, Rui L, Wenjie S, Jianguo Z, Shuguang F, Jie‐Ting G Min Z (2022) The antidiabetic effects of Bifidobacterium longum subsp. longum BL21 through regulating gut microbiota structure in type 2 diabetic mice. Food \u0026amp; Function 13, 9947-9958.\u003c/li\u003e\n\u003cli\u003eQuin C, Ghosh S, Dai C, Barnett JA, Garner AM, Yoo RKH, Zandberg WF, Botta A, Gorzelak MA Gibson DL (2021) Maternal Intake of Dietary Fat Pre-Programs Offspring\u0026apos;s Gut Ecosystem Altering Colonization Resistance and Immunity to Infectious Colitis in Mice. Mol Nutr Food Res 65, e2000635.\u003c/li\u003e\n\u003cli\u003eXie H, Fang J, Farag MA, Li Z, Sun P Shao P (2022) Dendrobium officinale leaf polysaccharides regulation of immune response and gut microbiota composition in cyclophosphamide-treated mice. Food Chemistry: X 13.\u003c/li\u003e\n\u003cli\u003eRuiqiang Z, Haoran Z, Jinsong L, Xinfu Z, Yanping W Caimei Y (2021) Rhamnolipids enhance growth performance by improving the immunity, intestinal barrier function, and metabolome composition in broilers. Journal of the Science of Food and Agriculture 102, 908-919.\u003c/li\u003e\n\u003cli\u003eLinlin W, Mao C, Jialiang W, Qiangqing Y, Gang W, Hao Z, Jianxin Z Wei C (2022) Bifidobacterium longum relieves constipation by regulating the intestinal barrier of mice. Food \u0026amp; Function 13, 5037-5049.\u003c/li\u003e\n\u003cli\u003eBingjie G, Huajun Y, Jun M, Xiaoshuai C Zhi‐yue W (2020) Effects of Mannan Oligosaccharides and/or Bifidobacterium on Growth and Immunity in Domestic Pigeon (Columba livia domestica). Journal of Poultry Science 57, 277-283.\u003c/li\u003e\n\u003cli\u003eAl-Sheraji SH, Amin I, Azlan A, Manap MY Hassan FA (2015) Effects of Bifidobacterium longum BB536 on lipid profile and histopathological changes in hypercholesterolaemic rats. Benef Microbes 6, 661-668.\u003c/li\u003e\n\u003cli\u003eHope OD, Oanh P, Lin-Xi L, Shaikh MA, Seung Joo L, Mari\u0026euml;tta MR, Jessica LS, Sean Paul N, Petr B, Denise MM, Andreas JB Stephen JM (2014) Toll-like Receptor and Inflammasome Signals Converge to Amplify the Innate Bactericidal Capacity of T Helper 1 Cells. Immunity 40, 213-224.\u003c/li\u003e\n\u003cli\u003eDong Y, Liao W, Tang J, Fei T, Gai Z Han M (2022) Bifidobacterium BLa80 mitigates colitis by altering gut microbiota and alleviating inflammation. AMB Express 12, 67.\u003c/li\u003e\n\u003cli\u003eWang X, Yuan Z, Zhu L, Yi X, Ou Z, Li R, Tan Z, Pozniak B, Obminska-Mrukowicz B, Wu J Yi J (2019) Protective effects of betulinic acid on intestinal mucosal injury induced by cyclophosphamide in mice. Pharmacological Reports 71, 929-939.\u003c/li\u003e\n\u003cli\u003eXu P, Cui K, Chen L, Chen S Wang Z (2023) Effect of dietary Bifidobacterium animalis subsp. lactis BLa80 on growth, immune response, antioxidant capacity, and intestinal microbiota of juvenile Japanese seabass (Lateolabrax japonicus). Aquaculture International 32, 1749-1769.\u003c/li\u003e\n\u003cli\u003eZheng D, Liwinski T Elinav E (2020) Interaction between microbiota and immunity in health and disease. Cell Research 30, 492-506.\u003c/li\u003e\n\u003cli\u003eHeine H \u0026amp; Lien E (2003) Toll-Like Receptors and Their Function in Innate and Adaptive Immunity. International Archives of Allergy and Immunology 130, 180-192.\u003c/li\u003e\n\u003cli\u003eGavzy SJ, Kensiski A, Lee ZL, Mongodin EF, Ma B Bromberg JS (2023) Bifidobacterium mechanisms of immune modulation and tolerance. Gut Microbes 15, 2291164.\u003c/li\u003e\n\u003cli\u003eYing M, Yu Q, Zheng B, Wang H Xie M (2020) Cultured Cordyceps sinensis polysaccharides modulate intestinal mucosal immunity and gut microbiota in cyclophosphamide-treated mice. Carbohydrate Polymers 235, 115957.\u003c/li\u003e\n\u003cli\u003eShi H, Chang Y, Gao Y, Wang X Tang QJ (2017) Dietary fucoidan of Acaudina molpadioides alters gut microbiota and mitigates intestinal mucosal injury induced by cyclophosphamide. Food \u0026amp; Function 8, 3383-3393.\u003c/li\u003e\n\u003cli\u003eMasato T, Akira H, Tsutomu Y, Satoshi H, Kazuhiro H, Kazuyoshi I, Ky\u0026ocirc;ko T Shuichi K (2007) Prior stimulation of antigen-presenting cells with Lactobacillus regulates excessive antigen-specific cytokine responses in vitro when compared with Bacteroides. Cytotechnology 5, 89-101.\u003c/li\u003e\n\u003cli\u003eGaldeano CM \u0026amp; Perdig\u0026oacute;n G (2006) The Probiotic Bacterium Lactobacillus casei Induces Activation of the Gut Mucosal Immune System through Innate Immunity. Clinical and Vaccine Immunology 13, 219-226.\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":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"high-activity probiotic, yogurt, immune response, cyclophosphamide, gut microbiota","lastPublishedDoi":"10.21203/rs.3.rs-6252467/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6252467/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProbiotics play a crucial role in modulating the immune system and maintaining the integrity of intestinal epithelial barrier. The study investigates the effects of high-activity probiotic yogurt on immunosuppressed mice induced by cyclophosphamide. On days 7, 8, and 9 of the experiment, ICR male mice (eight-week-old) were injected intraperitoneally with cyclophosphamide (80 mg/kg body weight/day) to establish an immunosuppressive model (n\u0026thinsp;=\u0026thinsp;10). Mice fed with normal diet or high-activity probiotic yogurt for consecutive 14 days. The effect of high-activity probiotic yogurt on immunosuppressed mice was investigated by HE staining, enzyme-linked immunosorbent assay (ELISA), Western blotting, and 16s rRNA assay. Results indicated that after the treatment of high-activity probiotic yogurt, the immune organ indices, interleukin-6(IL-6), interleukin-12(IL-12), tumor necrosis factor-α (TNF-α), and intestinal structure are significantly increased in immunosuppressed mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Western blotting analysis find that high-activity probiotic yogurt improves the expression of toll-like receptor 4 (TLR4), nuclear factor kappa-B \u0026ndash;p65(p65), TNF receptor associated factor 6(TRAF6). Furthermore, microbiota analysis showed that high-activity probiotic yogurt significantly increased the diversity and richness of the gut microbiota(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings indicated that high-activity probiotic yogurt may improve the immune function of mice by improve intestinal homeostasis and activation of TLR pathway.\u003c/p\u003e","manuscriptTitle":"Highly Active Probiotic Yogurt Protects Against Cyclophosphamide-Induced Immunosuppression in Mice by Regulating Immune Response and gut microbiota","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-29 14:46:38","doi":"10.21203/rs.3.rs-6252467/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-17T11:47:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-17T08:16:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-13T09:29:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-07T01:40:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-02T04:24:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"303671701121396921586557815151597438620","date":"2025-04-28T08:40:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"214284691652666037301082035782362610207","date":"2025-04-28T00:07:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"270942255696458981295779730068080756323","date":"2025-04-27T19:00:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285799586986210183059113154373012642880","date":"2025-04-26T02:20:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-25T17:57:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-12T10:42:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-12T10:40:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Probiotics and Antimicrobial Proteins","date":"2025-03-18T10:58:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c6377e11-dc68-4ee1-b4a5-9a53b3530702","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-30T16:00:11+00:00","versionOfRecord":{"articleIdentity":"rs-6252467","link":"https://doi.org/10.1007/s12602-025-10623-1","journal":{"identity":"probiotics-and-antimicrobial-proteins","isVorOnly":false,"title":"Probiotics and Antimicrobial Proteins"},"publishedOn":"2025-06-25 15:57:23","publishedOnDateReadable":"June 25th, 2025"},"versionCreatedAt":"2025-04-29 14:46:38","video":"","vorDoi":"10.1007/s12602-025-10623-1","vorDoiUrl":"https://doi.org/10.1007/s12602-025-10623-1","workflowStages":[]},"version":"v1","identity":"rs-6252467","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6252467","identity":"rs-6252467","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}