Microcin C7 Prevents Cyclophosphamide-Induced Immunosuppression and Intestinal Injury by Modulating T-Cell Differentiation and Gut Microbiota Composition in Mice

Abstract

Microcin C7 (McC7) is a ribosomally synthesized antimicrobial peptide that has emerged as a promising candidate due to its dual antibacterial and immunomodulatory activities. This study evaluated the preventive effect of McC7 against cyclophosphamide (CTX)-induced immunosuppression and intestinal injury. An immunosuppression model was established by intraperitoneal CTX injection in mice, which were randomly allocated into five groups (n = 15): a negative control, a CTX model group, and three McC7 treatment groups receiving dietary McC7 at 100, 200, or 400 mg/kg both before and during CTX exposure. Body weight and feed intake were monitored throughout the study. Organ indices, serum biochemical parameters, immune and antioxidant markers, and intestinal morphology were assessed. Splenic T-cell subsets were analyzed by flow cytometry, and gut microbiota composition was evaluated by 16S rRNA sequencing. McC7 supplementation significantly attenuated the CTX-induced reduction in body weight, feed intake, and organ indices, ameliorated markers of hepatic and renal injury, and restored the splenic CD4⁺/CD8⁺ T-cell ratio. McC7 enhanced intestinal mucosal barrier integrity, increased the abundance of beneficial bacteria such as Candidatus Arthromitus and ASF356, and reduced the abundance of the potentially pathogenic genus Bilophila. In conclusion, our results demonstrate that McC7 alleviates CTX-induced immunosuppression by regulating T-cell differentiation, maintaining cytokine homeostasis, and modulating gut microbial composition to support intestinal health.

1. Introduction

The immune system serves as the primary defense mechanism against pathogenic invasion, and plays a critical role in maintaining homeostasis, preventing infection, and suppressing tumorigenesis [1,2]. Under various pathological conditions, the immune system may become functionally suppressed [3,4]. This state of immunosuppression increases susceptibility to opportunistic infections and compromises overall health, underscoring the importance of developing safe and effective immunomodulatory agents to restore immune competence [5].

Antimicrobial peptides (AMPs) are host-defense molecules with broad-spectrum antimicrobial activity and a lower propensity to induce microbial resistance than conventional antibiotics [6,7,8]. Beyond their direct antimicrobial role, a key advancement in the field is the recognition that many AMPs exhibit potent immunomodulatory functions, including the regulation of inflammatory responses, promotion of tissue repair, and direct interaction with immune cells [9]. This dual functionality positions AMPs as promising therapeutic candidates capable of simultaneously targeting pathogens and modulating host immunity. Microcin C7 (McC7), a ribosomally synthesized and post-translationally modified heptapeptide–nucleotide conjugate produced by Escherichia coli, exemplifies this dual potential. Its established mechanism involves the potent inhibition of bacterial protein synthesis with minimal host cytotoxicity [10,11]. Notably, emerging evidence indicates that McC7 can also modulate cytokine expression and immune cell activity, suggesting its utility as a dual-action agent against both infection and immune dysfunction [12].

Cyclophosphamide (CTX) is a widely used alkylating chemotherapeutic agent that induces potent, reproducible immunosuppression by targeting proliferating lymphocytes [13,14,15]. However, CTX administration also causes significant off-target toxicity to the rapidly renewing intestinal epithelium. The resulting compromise in mucosal barrier integrity facilitates microbial translocation, thereby triggering a cascade of systemic inflammation and contributing to a complex state of immune dysregulation, intestinal injury, and gut microbiota dysbiosis [16]. While the antibacterial activity of McC7 is established, its potential to protect against this multifaceted CTX-induced pathology—particularly through mechanisms that simultaneously address immune imbalance, intestinal damage, and microbial ecology—remains largely unexplored. To address this, the present study aimed to systematically evaluate whether and how McC7 prevents CTX-induced immunosuppression and intestinal injury. We specifically focused on its dose-dependent effects on immune homeostasis, intestinal barrier integrity, and the composition and predicted function of the gut microbiota. This integrated approach seeks to provide a holistic understanding of McC7’s protective potential and lay the groundwork for its application as a microbiota-targeting immunomodulatory agent.

2. Materials and Methods

2.1. Animals and Ethics Statement

Male ICR mice (age 3 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. All animal procedures were approved by the Institutional Animal Care and Use Committee of Southwest University of Science and Technology, Mianyang, Sichuan, China (Permit Number: L2024023).

2.2. McC7 Source and Experimental Diet Preparation

McC7 was provided by the Chongqing Academy of Animal Sciences (Chongqing, China). It is a synthetic heptapeptide-nucleotide conjugate with a purity of >95%, as determined by high-performance liquid chromatography (HPLC). For the preparation of experimental diets, the powdered McC7 was thoroughly and evenly blended into the standard basal diet at target concentrations of 100, 200, and 400 mg per kilogram of feed. The homogeneity of the diet mixtures was confirmed before use.

2.3. Experimental Design

A total of 75 healthy male ICR mice weighing 18–20 g were randomly assigned to five groups of 15 mice each, with one mouse per replicate cage: negative control (NC), positive control (CTX), low-dose McC7 (CTX + L Microcin C7), medium-dose McC7 (CTX + M Microcin C7), and high-dose McC7 (CTX + H Microcin C7). The experiment was commenced after a 7-day acclimation period. The NC and CTX groups were fed a basal diet, whereas the McC7-treated CTX + L Microcin C7, CTX + M Microcin C7, and CTX + H Microcin C7 groups received the basal diet supplemented with 100, 200, and 400 mg/kg McC7, respectively. From days 15 to 17, the CTX, CTX + L Microcin C7, CTX + M Microcin C7, and CTX + H Microcin C7 groups were intraperitoneally injected with 0.2 mL CTX solution (80 mg/kg) for three consecutive days to establish the immunosuppressive model. The NC group was injected with 0.2 mL physiological saline. Body weight was recorded at the start of the experimental period and on days 14 and 18, and daily feed intake was recorded throughout. The average daily gain (ADG) and average daily feed intake (ADFI) were calculated using the following formulas:

ADG (g/d) = Average weight gain (g)/Number of feeding days (d).

ADFI (g/d) = Average feed intake (g)/Number of feeding days (d).

2.4. Sample Collection

Twenty-four hours after final intraperitoneal injection of CTX, mice were anesthetized and blood samples were collected via orbital bleeding. The blood was allowed to stand and then centrifuged to obtain serum, which was stored at −80 °C until further analysis. The liver, spleen, and thymus were excised and weighed. A portion of the liver (approximately 100 mg per mouse) was snap-frozen and preserved at −80 °C for the determination of antioxidant indices. Intestinal segments, including the duodenum, jejunum, ileum, and colon, were collected and fixed in 4% paraformaldehyde. Under sterile conditions, cecal contents were collected into cryotubes and stored at −80 °C.

2.5. Determination of Serum Biochemical Indexes and Immune Indexes

Serum levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, total protein, albumin, total cholesterol (CHO), and triglycerides (TGs) were measured using a Biobase 400 fully automated biochemical analyzer (Jinan Biobase Biological Technology Co., Ltd., Jinan, China). Serum levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-4, IL-10, and IgG were measured by ELISA (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). All assays were performed in duplicate.

2.6. Determination of Antioxidant Indexes in Serum and Liver

Total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT), and malondialdehyde (MDA) in mouse serum and liver were determined using specific commercial assay kits (T-AOC: Catalog No. A015-1-2; T-SOD: Catalog No. A001-1-2; CAT: Catalog No. A007-1-1; MDA: Catalog No. A003-1-1) purchased from Jiancheng Bioengineering Institute (Nanjing, China).

2.7. Preparation and Observation of Intestinal Tissue Slices

Following a 48 h fixation in 4% paraformaldehyde, intestinal tissues were trimmed into flat longitudinal strips for standardized embedding. The samples were dehydrated through an ethanol gradient, cleared in xylene, and infiltrated with a xylene–paraffin mixture. Tissue embedding was performed using a biological tissue embedding machine (KD-BM; Jinhua Kedi Instrument Co., Ltd., Jinhua, China). After the paraffin solidified, 5-μm thick sections were cut with a microtome (KD-2260; Jinhua Kedi Instrument Co., Ltd., Jinhua, China) and mounted on slides. Hematoxylin and eosin staining was used to observe and measure intestinal villus height (VH) and crypt depth (CD), and VH/CD ratio was calculated. For goblet cell quantification, five intact, well-oriented villi per intestinal segment per animal were selected. Goblet cells were counted along the entire epithelial lining of each villus (from the crypt-villus junction to the tip) under 400× magnification using a light microscope. The count was expressed as the mean number of goblet cells per villus for each segment. All morphological assessments and cell counts were performed by an observer blinded to the experimental group assignments.

2.8. Flow Cytometry of Splenic Lymphocyte Subsets

On day 18, mice were euthanized, and spleens were aseptically collected into sterile PBS on ice for immediate preparation of single-cell suspensions. The spleen tissues were minced, gently homogenized, and passed through a cell strainer to obtain single-cell suspensions. After washing with PBS, the harvested splenocytes were resuspended in 100 μL PBS for staining. The following fluorescently labeled antibodies were added: anti-CD3-FITC (for total T cells), anti-CD4-PE (for helper T cells), and anti-CD8-APC (for cytotoxic T cells). The cell suspension was incubated with antibodies for 30 min at 4 °C in the dark. Following incubation, cells were washed and resuspended in 500 μL PBS prior to flow cytometry (SFL0 2; Hangzhou POWCLIN Medical Technology Co., Ltd., Hangzhou, China). Flow cytometry was performed by gating on the lymphocyte population based on forward scatter and side scatter properties. In the lymphocyte gate, CD3⁺ T cells were identified. CD4⁺ and CD8⁺ T cell subsets were distinguished using a CD4-PE versus CD8-APC dot plot. Data were analyzed using FlowJo software (10.8.1), and results are presented as bar graphs showing subset proportions and CD4⁺/CD8⁺ ratios across experimental groups.

2.9. Gut Microbiota Analysis

Samples of cecal content were stored at −80 °C prior to analysis to prevent DNA degradation. Genomic DNA was extracted from mouse cecal microbiota using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). DNA concentration and purity were measured using a NanoDrop One/Oneᶜ spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), with an A260/A280 ratio close to 1.8 considered indicative of pure DNA. The integrity of DNA was assessed by agarose gel electrophoresis, and its accurate concentration was determined using a Qubit assay system (Qubit 4.0, Thermo Fisher Scientific, Waltham, MA, USA). The V4 region of the 16S rRNA gene was amplified with primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), followed by sequencing on the Illumina NovaSeq platform (Illumina, San Diego, CA, USA). A minimum of 10,000 reads per sample were typically processed. Raw reads from each sample were assembled and filtered, followed by alignment and taxonomic classification against the SILVA database. Chimeric sequences were detected and subsequently removed. Finally, denoising was performed using DADA2 in QIIME2 to generate Amplicon Sequence Variants (ASVs). Finally, ASVs with a relative abundance below 0.1% were excluded to minimize false positives. Data analysis was performed on the NovoMagic platform (https://magic-plus.novogene.com, accessed on 18 September 2025), including assessments of species abundance, alpha diversity, beta diversity (principal coordinates analysis; PCoA), and LDA effect size (LEfSe).

2.10. Statistical Analysis

Statistical analysis was performed using SPSS 26.0. One-way ANOVA followed by Duncan’s post hoc test was applied for multiple comparisons. Data are presented as mean ± SEM, and p < 0.05 was considered statistically significant. Bar graphs were generated using GraphPad Prism 8.0 software. In microbiome analysis, alpha diversity was assessed using the Kruskal–Wallis rank-sum test. Beta diversity was visualized via PCoA based on the weighted unifrac distance, and group differences were tested by Adonis analysis. LEfSe was performed with an LDA score threshold of 2.

Based on 16S rRNA gene sequencing data, PICRUSt2 (version 2.3.0) was used to predict the functional potential of the cecal microbiota by referencing known microbial genomic information. For Spearman correlation analyses, p-values were adjusted for multiple comparisons using the Benjamini–Hochberg false discovery rate (FDR) correction, with a significance threshold of q < 0.05. Subsequently, a t-test (p < 0.01, minimum abundance threshold = 0.001) was performed to compare the KEGG level 3 functions between the CTX group and the CTX + H Microcin C7 group. Spearman correlation analysis was conducted using the psych package (version 1.9.12.31) in R (version 3.5.1). Microbial communities between groups were tested for significant differences using analysis of similarities (ANOSIM) and the multiple reaction permutation program, while rank sum tests were used for species analyses between groups.

3. Results

3.1. Effects of McC7 on Body Weight and Feed Intake in Mice

To evaluate whether the immunosuppressive model was established effectively, and the immunomodulatory effects of McC7, body weight and feed intake were recorded and analyzed across different groups (

Table 1. Effects of microcin C7 on body weight and feed intake in mice.

Items	NC	CTX	CTX + L Microcin C7	CTX + M Microcin C7	CTX + H Microcin C7	SEM	p-Value
Body weight (g)
Day 1	21.91	21.83	21.49	21.15	21.05	0.115	0.058
Day 14	33.61	33.90	33.66	34.42	35.38	0.317	0.370
Day 18	35.68 a	31.86 b	33.07 b	33.94 ab	33.99 ab	0.357	0.012
Average daily gain (g/d)
Day 1–14	0.83	0.86	0.87	0.94	1.02	0.022	0.053
Day 15–18	0.21 a	−0.92 c	−0.07 b	−0.20 b	−0.19 b	0.051	<0.001
Average daily feed intake (g/d)
Day 1–14	4.44	4.23	4.10	4.32	4.45	0.110	0.886
Day 15–18	4.25 a	3.73 b	4.38 a	4.22 a	4.28 a	0.055	<0.001

NC: mice were intraperitoneally injected with normal saline and fed a basal diet; CTX: mice were intraperitoneally injected with CTX and fed a basal diet; CTX + L Microcin C7: mice were intraperitoneally injected with CTX and fed a basal diet containing 100 mg/kg McC7; CTX + M Microcin C7: mice were intraperitoneally injected with CTX and fed a basal diet containing 200 mg/kg McC7; CTX + H Microcin C7: mice were intraperitoneally injected with CTX and fed a basal diet containing 400 mg/kg McC7. ^{a, b, c} Means in the same row with different superscripts differ (p < 0.05).

). No significant differences were observed among the groups in terms of ADG, ADFI from day 1 to 14, or body weight on day 14 (p > 0.05). During days 15–18, corresponding to the intraperitoneal CTX injection phase, ADG and ADFI in the CTX group were significantly lower than in the NC, CTX + L Microcin C7, CTX + M Microcin C7, and CTX + H Microcin C7 groups (p < 0.001). ADG in the CTX + L Microcin C7, CTX + M Microcin C7, and CTX + H groups remained significantly lower than in the NC group (p < 0.001). By day 18, only the CTX and CTX + L Microcin C7 groups showed a significant reduction in body weight compared with the NC group (p < 0.05).

3.2. Effect of McC7 on Organ Indexes in Mice

Changes in organ weight reflected the health status of the mice. The organ indices of the liver, spleen, and thymus were measured and compared among the groups (Figure 1). In the CTX group (treated with CTX), the organ indices of the liver, spleen, and thymus were significantly decreased (p < 0.05). Among the McC7-treated groups, only the CTX + M Microcin C7 group showed a significant increase in organ indices compared to the CTX group (p < 0.05), and only the liver index recovered to a level comparable to that of the NC group (p > 0.05).

3.3. Effect of McC7 on Serum Biochemical Indexes in Mice

To further evaluate the overall health status of the mice in each group, we measured their serum biochemical parameters. Serum levels of ALP, ALT, AST, CHO, and TG in the CTX group were significantly increased compared with those in the NC, CTX + L Microcin C7, CTX + M Microcin C7, and CTX + H Microcin C7 groups (p < 0.05) (Figure 2A–C,G,H). Compared with the NC group, the CTX + L Microcin C7 and CTX + H Microcin C7 groups had significantly lower ALP levels (p < 0.05) (Figure 2A); ALT levels were significantly reduced in the CTX + L Microcin C7 and CTX + M Microcin C7 groups (p < 0.05) (Figure 2B); AST levels were significantly decreased in the CTX + M Microcin C7 group (p < 0.05) (Figure 2C); and TG levels were significantly lower in the CTX + L Microcin C7 group (p < 0.05) (Figure 2H). In contrast, urea levels were significantly higher in all other groups compared with the NC group (p < 0.05) (Figure 2F). Only the CTX + L Microcin C7 and CTX + H Microcin C7 groups had significantly lower urea levels than the CTX group had (p < 0.05) (Figure 2F).

3.4. Effect of McC7 on Antioxidant Properties of Serum and Liver Tissue in Mice

We measured the antioxidant indices in serum and liver tissues of mice from each group. Compared with the NC group, serum T-AOC and T-SOD levels, as well as liver T-SOD and CAT levels, were significantly decreased in the CTX, CTX + L Microcin C7 and CTX + H Microcin C7 groups (p < 0.05) (Figure 3A,B,F,G). Serum CAT levels were significantly reduced in the CTX and CTX + L Microcin C7 groups (p < 0.05) (Figure 3C). In comparison with the CTX group, serum T-AOC and CAT levels were significantly higher in the CTX + M Microcin C7 and CTX + H Microcin C7 groups (p < 0.05) (Figure 3A,C). Serum T-SOD and liver CAT levels were significantly elevated in the CTX + H Microcin C7 group (p < 0.05) (Figure 3B,G). Liver T-SOD levels were significantly increased in the CTX + L Microcin C7 and CTX + M Microcin C7 groups compared with the CTX group (p < 0.05) (Figure 3F). Serum MDA levels were significantly higher in the CTX and CTX + L Microcin C7 groups than in the other groups (p < 0.05) (Figure 3D). No significant differences were observed in liver T-AOC or MDA levels among the groups (p > 0.05) (Figure 3E,H).

3.5. Effects of McC7 on Serum Immune Indexes in Mice

Serum levels of cytokines and immunoglobulins are important indicators of health status in mice. The CTX group had significantly higher levels of TNF-α, IFN-γ, and IgG, and significantly lower levels of IL-4 and IL-10 compared with the other groups (p < 0.05) (Figure 4A–E). With increasing dose of McC7, the concentrations of TNF-α, IFN-γ, and IgG progressively decreased; all being significantly lower than in the CTX group but significantly higher than in the NC group (p < 0.05) (Figure 4A,B,E). Conversely, the levels of IL-4 and IL-10 gradually increased with higher McC7 doses, showing significantly higher values than in the CTX group and significantly lower values than in the NC group (p < 0.05) (Figure 4C,D).

3.6. Effects of McC7 on Intestinal Morphology and Number of Goblet Cells in Mice

Compared with the NC group, the CTX group showed significant reductions in VH in the jejunum, and number of goblet cells in the duodenum, jejunum, and colon (p < 0.05) (Figure 5A,D) (representative histological images of all intestinal segments are provided in Supplementary Figure S1). In contrast, CD was significantly increased in the duodenum and jejunum of the CTX group (p < 0.05) (Figure 5B). In mice supplemented with McC7, the CTX + L Microcin C7 and CTX + H Microcin C7 groups showed significantly greater duodenal VH and VH/CD ratio compared with the other groups (p < 0.05) (Figure 5A,C). The CTX + L Microcin C7, CTX + M Microcin C7, and CTX + H Microcin C7 groups demonstrated significantly higher VH in the ileum and jejunum, VH/CD ratio in the ileum, and number of goblet cells in the duodenum, jejunum, and ileum compared with the CTX group (p < 0.05) (Figure 5A,C,D). The CTX + M Microcin C7 and CTX + H Microcin C7 groups showed significantly increased colon number of goblet cells compared with the CTX group (p < 0.05) (Figure 5D). Conversely, CD in the duodenum was significantly lower in the CTX + L Microcin C7 and CTX + H Microcin C7 groups, and jejunal CD was significantly reduced in the CTX + H Microcin C7 group, compared with in the CTX group (p < 0.05) (Figure 5B).

3.7. Effect of McC7 on Splenic T Cells in Mice

Compared with the NC group, the CTX group showed a significant increase in the proportions of CD3⁺ and CD8⁺ T cells (p < 0.05) (Figure 6A–C,E), along with a significant decrease in the CD4⁺/CD8⁺ ratio (p < 0.05) (Figure 6B,F). In comparison with the CTX group, no significant changes were observed in the proportion of CD3⁺ T cells in the CTX + L Microcin C7, CTX + M Microcin C7, and CTX + H Microcin C7 groups (p > 0.05) (Figure 6C). However, the CTX + L Microcin C7 and CTX + H Microcin C7 groups showed a significant reduction in the proportion of CD8⁺ T cells and a significant increase in CD4⁺/CD8⁺ ratio (p < 0.05) (Figure 6E,F). The proportion of CD4⁺ T cells in the CTX + H Microcin C7 group was significantly higher than in the NC and CTX groups (p < 0.05) (Figure 6D).

3.8. Effect of McC7 on Cecal Microbiome in Mice

Microbial community analysis revealed no significant differences in alpha diversity (Chao1, observed features, Shannon Index, and Simpson Index) among the groups (Figure 7A–D). PCoA demonstrated minimal separation between samples from different groups, indicating similar overall composition of microbial communities (Figure 7E). Analysis at the phylum and genus levels revealed variations in the abundance of Bacillota, Bacteroidota, and Thermodesulfobacteriota, as well as unclassified Muribaculaceae, Ligilactobacillus, and Desulfovibrio among the groups (Figure 7F,G). LEfSe analysis indicated that Ligilactobacillus and Lactobacillus were significantly enriched in the NC group, Bilophila in the CTX group, Eubacterium xylanophilum group in the CTX + L Microcin C7 group, Erysipelotrichaceae UCG 003 in the CTX + M Microcin C7 group, and Candidatus Arthromitus and ASF356 in the CTX + H Microcin C7 group (Figure 7H). Most taxa significantly enriched in the NC group belonged to the class Bacilli, order Lactobacillales, and family Lactobacillaceae, whereas those enriched in the CTX + H Microcin C7 group were primarily affiliated with the order Clostridiales and family Clostridiaceae (Figure 7I).

3.9. Functional Prediction of Gut Microbiota and Its Correlation with Host Parameters

Functional prediction via PICRUSt2, derived from 16S rRNA sequencing data, was performed by first identifying statistically significant differential pathways (p < 0.01) between the CTX and CTX + H Microcin C7 groups. Subsequent analysis demonstrated distinct microbial functional profiles based on this stringent screening. The Mean-Difference plot (Figure 8A) illustrates the differentially abundant predicted gene functions. Key functional categories enriched in the CTX + H Microcin C7group included transcriptional regulators (LacI family and PadR family transcriptional regulators), substrate-binding proteins involved in transport (peptide/nickel and multiple sugar transport systems), and core cellular machinery (elongation factor G and DNA-binding protein HU-beta). Additionally, microbial metabolic pathways such as formate C-acetyltransferase and nucleotide metabolism-related functions (tRNA pseudouridine synthase and putative GTP pyrophosphokinase) were significantly differentially abundant, indicating a broad restructuring of microbial metabolic potential in response to the combined treatment.

To explore the functional relevance of these microbial shifts, Spearman correlation analysis was performed between the predicted functional abundances (filtered at p < 0.01) and a panel of host phenotypic markers related to immune status, oxidative stress, and antioxidant capacity (Figure 8B). The analysis revealed significant associations between specific microbial functions and host physiology. Notably, functions central to microbial growth and integrity (elongation factor G and DNA-binding protein HU-beta) exhibited strong positive correlations with pro-inflammatory markers (TNF-α) and oxidative stress indicators (MDA), while showing negative correlations with anti-inflammatory cytokines such as IL-10. Furthermore, the predicted abundance of formate C-acetyltransferase, a key enzyme in fermentative metabolism, was positively associated with the CD4⁺/CD8⁺ ratio and negatively with antioxidant markers (T-SOD, T-AOC). Transport-related functions and transcriptional regulators also showed significant correlation patterns with multiple host parameters. It is important to emphasize that these functional insights are predictive and derived from 16S rRNA gene sequencing data. While PICRUSt2 provides valuable hypotheses, the inferred upregulation of pathways such as formate C-acetyltransferase and their correlation with host parameters require direct validation through metagenomic sequencing, metabolomic profiling, or in vitro functional assays.

4. Discussion

Our findings confirm and extend the understanding of McC7’s protective role against chemotherapy-induced toxicity across immunological, intestinal, and microbial dimensions. Immunologically, McC7 dose-dependently restored the CD4⁺/CD8⁺ T-cell ratio and rebalanced Th1/Th2 cytokines. Intestinal integrity was preserved along the entire tract, with increased goblet cell numbers. Most notably, McC7 remodeled the gut microbiota, enriching beneficial taxa (e.g., Candidatus Arthromitus, ASF356) and suppressing Bilophila. Exploratory PICRUSt2 analysis linked these shifts to predicted upregulation of acetate-synthesis pathways, which correlated with T-cell homeostasis—generating the testable hypothesis that McC7 may promote immune recovery partly via microbiota-mediated metabolic modulation. These integrated insights point to a microbiota-involved mechanism warranting further validation.

The protective efficacy of McC7 followed a clear dose–response relationship. The low dose (100 mg/kg) provided fundamental mitigation, primarily attenuating CTX-induced declines in body weight and feed intake, and partially restoring intestinal villus structure. The medium dose (200 mg/kg) elicited more robust improvements in systemic antioxidant capacity and significantly restored the indices of immune organs. Critically, the high dose (400 mg/kg) conferred the most comprehensive benefits, demonstrating superior efficacy in fully normalizing the CD4⁺/CD8⁺ T-cell ratio and cytokine balance, enriching key beneficial gut microbiota, and strengthening the correlation between predicted microbial metabolic shifts and immune recovery.

Building upon this dose-dependent framework, we next detail the mechanisms of immune recovery. The establishment of CTX-induced immunosuppression was confirmed by significant reductions in body weight, feed intake, and the indices of key immune organs (spleen and thymus). McC7 supplementation effectively mitigated these declines. More importantly, McC7 restored the disrupted systemic immune equilibrium. Flow cytometric analysis revealed that McC7 normalized the CTX-altered CD4⁺/CD8⁺ T-cell ratio, a critical indicator of immunotoxicity [17,18]. This cellular rebalancing was further evidenced by a dose-dependent shift in the serum cytokine profile, wherein McC7 suppressed pro-inflammatory Th1 cytokines (TNF-α, IFN-γ) while elevating anti-inflammatory Th2 cytokines (IL-4, IL-10). This shift aligns with the known immunomodulatory properties of certain host-defense peptides [9,10,11,12]. A critical interpretation of these immune markers is required. The elevated levels of TNF-α, IFN-γ, and IgG in the CTX group are indicative of a state of immune dysregulation and compensatory inflammation [4,19], likely triggered by chemotherapy-induced tissue damage and loss of gut barrier integrity. Therefore, McC7’s dose-dependent reduction in these markers signifies a restoration of immune homeostasis—by attenuating collateral inflammation and correcting lymphocyte imbalance—rather than generalized immunosuppression [11]. This clarifies that McC7 facilitates true immune recovery, although future studies incorporating T-cell functional assays and antigen-specific humoral challenges would provide deeper mechanistic insight [17]. Concurrently, McC7 alleviated CTX-induced hepatorenal injury, as reflected in the attenuation of elevated serum markers of hepatic damage (ALP, ALT, AST) and renal stress (urea). This organ-protective effect was, at least in part, mediated through a significant enhancement of systemic antioxidant defenses. By reducing the oxidative burden, which is known to exacerbate immune dysfunction [20], McC7 helps to create a more favorable microenvironment for immune recovery.

CTX administration significantly compromised the intestinal mucosal barrier, as expected. McC7 treatment markedly ameliorated this damage, improving villus architecture, crypt depth ratio, and goblet cell numbers across all intestinal segments examined. This morphological restoration is consistent with prior reports that McC7 upregulates tight junction proteins and mucosal defense components [12], underscoring its potent role in preserving intestinal barrier integrity.

Analysis of the cecal microbiota revealed that while neither CTX nor McC7 drastically altered overall microbial diversity, both induced specific, functionally relevant taxonomic shifts. CTX administration led to a dysbiotic state characterized by an increased Firmicutes/Bacteroidota ratio [19], and promoted the expansion of pro-inflammatory genera such as Desulfovibrio [21,22] and Bilophila [23], while reducing beneficial Ligilactobacillus [24,25]. Supplementation with a high dose of McC7 prevented the CTX-induced rise in the Firmicutes/Bacteroidota ratio and the increase in Desulfovibrio. Furthermore, McC7 enriched for beneficial taxa including Candidatus Arthromitus, which enhances mucosal immunity and barrier function [26], and ASF356, involved in microbiota stability via bile acid metabolism [27]. These results indicate that McC7 counteracts CTX-induced dysbiosis not by restructuring overall community diversity, but by selectively modulating key bacterial populations to favor a microbiota composition associated with improved intestinal and immune homeostasis.

To infer the functional implications of these taxonomic changes, we employed PICRUSt2-based prediction [28]. This in silico analysis revealed that McC7 treatment was associated with the predicted upregulation of microbial formate C-acetyltransferase, a key enzyme in acetate synthesis. Strikingly, the predicted abundance of this metabolic pathway showed a significant positive correlation with the normalized splenic CD4⁺/CD8⁺ T cell ratio. The observed correlation between the predicted abundance of microbial formate C-acetyltransferase and the normalized CD4⁺/CD8⁺ ratio is intriguing and generates a plausible, yet speculative, mechanistic hypothesis. It suggests that McC7-induced immune benefits might be partially mediated by shifting the gut microbiota towards a metabolic profile that favors acetate production, a metabolite known to influence T-cell regulation [29,30]. Future studies designed to measure acetate levels and T-cell receptor expression are warranted to test this hypothesis directly.

In conclusion, our integrated analysis demonstrates that McC7 prevents CTX-induced immunosuppression and intestinal injury through multi-faceted mechanisms involving the restoration of T-cell homeostasis, enhancement of intestinal barrier integrity, and beneficial modulation of gut microbiota composition and predicted function. We acknowledge several limitations of this study that also point to future directions. While the statistical approaches employed are standard for comparative animal studies, more advanced models could be applied to complex datasets like those derived from microbiome sequencing. Although overall microbial alpha and beta diversity were not drastically altered by CTX or McC7, the specific, differential abundance of key taxa (identified via LEfSe) suggests targeted microbial restructuring, which warrants deeper investigation. Furthermore, the functional predictions generated by PICRUSt2 and the exploratory correlation analyses, while hypothesis-generating, require direct validation through metagenomic sequencing, metabolomic profiling, and mechanistic studies in gnotobiotic models. Finally, the preventive design of our study establishes McC7’s prophylactic efficacy; evaluating its therapeutic potential in a rescue protocol remains an important next step. Despite these limitations, the present work provides compelling evidence for McC7 as a promising microbiota-targeting immunomodulator and lays a solid foundation for its further development.

5. Conclusions

Our findings demonstrated that dietary supplementation with McC7 effectively prevents CTX-induced immunosuppression in mice, as demonstrated by improvements in growth performance, immune status in the serum, spleen, and liver, as well as restoration of intestinal mucosal barrier integrity and microbial homeostasis. McC7 modulates systemic immune function by regulating the differentiation of T cells and rebalancing anti-inflammatory and proinflammatory cytokine profiles. Additionally, it enhances gut barrier function and immunity by altering the abundance of specific intestinal microbiota. These results highlight McC7 as a promising immunomodulatory agent with dual protective functions and provide mechanistic insights into the potential of antimicrobial peptides in restoring immune homeostasis via the gut–immune axis.