Niacin Mitigates Cyclophosphamide-Induced Immunosuppression by Maintaining Intestinal Homeostasis and Regulating the HCAR2/NLRP3 and PTGS2/PGE2 Signaling Pathways

TL;DR

Niacin helps reduce immune suppression caused by cyclophosphamide by supporting gut health and regulating specific signaling pathways.

Contribution

This study identifies niacin's role in mitigating immunosuppression through intestinal homeostasis and specific signaling pathways.

Findings

Niacin improved immune function in mice by restoring spleen and liver indices and enhancing cytokine levels.

Niacin reduced gut inflammation and improved intestinal barrier integrity in mice.

Niacin modulated the HCAR2/NLRP3 and PTGS2/PGE2 pathways to counteract immunosuppression.

Abstract

Objectives: This study is intended to reveal whether the boost in immune function in immunocompromised mice from niacin supplementation is connected to the upkeep of intestinal homeostasis and the modulation of the hydroxycarboxylic acid receptor 2 (HCAR2)/NOD-like receptor protein 3 (NLRP3) and prostaglandin endoperoxide synthase 2 (PTGS2)/prostaglandin E2 (PGE2) signaling pathways. Methods: Balb/c mice were employed in this study as a model for immunosuppression caused by cyclophosphamide (CTX) injection. Results: The study showed that niacin supplementation restored spleen and liver indices, enhanced cytokine secretion, and increased Th1/Th2 cytokine levels. Niacin effectively enhanced the phagocytic index, natural killer cell (NK cell) activity, splenic lymphocyte activity and delayed-type hypersensitivity (DTH) reaction in immunocompromised mice. Histopathological examination showed that niacin intervention alleviated injury in mice ilea. Intestinal barrier tight junction proteins were expressed at much higher levels, while the serum concentrations of diamine oxidase (DAO) and fatty acid-binding protein 2 (FABP2) were markedly lowered. Furthermore, the expression of the intestinal HCAR2/NLRP3 signaling pathway and subsequent inflammatory mediators was significantly elevated after niacin administration compared with the CTX group. Niacin supplementation improved the composition of the gut microbiota, increasing the Firmicutes/Bacteroidetes (F/B) ratio. Spearman correlation analysis showed significant correlations between cytokine-related indices and several gut microbiotas. Within a network pharmacology framework including target screening, network construction and molecular docking, PTGS2 emerged as a candidate target of niacin, suggesting its role in counteracting immunosuppression. Further experimental findings showed that niacin markedly decreased the protein expression of PTGS2 and the levels of its downstream mediators PGE2, E-prostanoid receptor type 2 (EP2) and (E-prostanoid receptor type 4 (EP4) in the ileal tissue of mice treated with CTX. Conclusions: In conclusion, niacin supplementation alleviated CTX-induced immunosuppression by maintaining intestinal homeostasis and regulating the intestinal HCAR2/NLRP3 and PTGS2/PGE2/EP2-EP4 pathways.

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1. Introduction

As a frequently utilized cancer treatment, CTX achieves its therapeutic effects in part by enhancing anti-tumor immune responses. However, it also leads to the destruction of immune cells in the body, subsequently inducing immunosuppression [1]. CTX impairs intestinal barrier function, leading to immune cell damage, immunosuppression, increased intestinal permeability, and gut microbiota dysbiosis [2]. CTX treatment markedly lowers the levels of intestinal tight junction proteins such as Occludin and ZO-1, leading to increased intestinal permeability and enhanced bacterial translocation, thereby raising the risk of infection [3,4]. CTX damages intestinal epithelial cells, compromising the intestinal barrier’s integrity [5]. Secondly, CTX notably impacts gut microbiota by diminishing its diversity and modifying its composition, leading to fewer beneficial bacteria such as Lactobacillus and a rise in potentially harmful bacteria. The disruption in gut flora not only impacts regular intestinal functions but might also intensify immunosuppression by altering immune system control [6,7]. Modulation of the immune system and improved immune function have been documented in CTX-induced immunosuppression models. This effect is linked to the recovery of intestinal stability, which is driven by increased expression of tight junction proteins, expansion of beneficial bacterial populations and elevated production of short-chain fatty acids [8]. The effectiveness of natural compounds like Resveratrol and Anthocyanins in improving CTX-induced intestinal damage is attributed to their dual role in modulating the intestinal barrier and regulating gut microbiota. Improved intestinal immune barrier function can be achieved by boosting beneficial bacteria and suppressing associated signaling pathways [9,10].

Niacin, the main form of vitamin B3, is metabolized into the active compounds nicotinamide adenine dinucleotide (NAD^+^) and nicotinamide adenine dinucleotide phosphate (NADP^+^) in the body [11]. NAD+ is a crucial coenzyme participating in various biochemical processes, with its level fluctuations closely associated with the progression of multiple diseases [12]. NAD+ metabolism influences immune responses and inflammatory processes by modulating immune cell activity, alongside its role in energy metabolism [13,14]. By supplementing with niacin or its derivatives, endogenous NAD+ levels can be raised, which can enhance physiological functions and ameliorate disease states [15]. In HIV-infected patients, niacin could modulate immune activation and improve the recovery capacity of CD4^+^ T cells [16]. In the elderly population, niacin is considered to improve immune function, metabolic homeostasis, and antioxidant defense [17]. Recent research indicates that niacin significantly enhances intestinal homeostasis. Niacin improves intestinal barrier function by influencing intestinal immunity and the microbial community. Niacin was found to effectively alleviate body weight loss and diarrhea in weaned piglets and improve intestinal morphology and the microbial community [16]. Modulation of the NF-κB signaling pathway by niacin in grass carp leads to a downregulation of inflammatory factors and an upregulation of tight junction proteins, which in turn strengthens intestinal barrier integrity and mucosal immunity [16,18,19]. The HCAR2 receptor is a key target of niacin found on intestinal epithelial and immune cell surfaces [20]. Niacin activates the HCAR2 receptor, mitigating intestinal oxidative stress, increasing tight junction protein expression and strengthening the intestinal barrier, which collectively reduces intestinal permeability and supports intestinal health [21,22]. Niacin can alter gut microbiota composition, enhance short-chain fatty acid production (e.g., butyrate), and activate the HCAR2 receptor [23]. It remains unclear whether niacin can mitigate immunosuppression caused by CTX in mice via preserving intestinal homeostasis.

In this study, an immunosuppressive mouse model was created using intraperitoneal CTX injections, and mice were given niacin at doses of 40 and 80 mg/kg. body weight. By investigating specific and non-specific immune functions, intestinal homeostasis, and immune-related pathways in mice, we evaluated the beneficial impact of niacin on immunosuppression caused by CTX and investigated the underlying mechanism.

2. Materials and Methods

2.1. Materials and Chemical Reagents

Niacin was provided by Solarbio Co., Ltd. (Shanghai, China). Cyclophosphamide and mepenzolate bromide (MPN) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Interleukin (IL)-2, Interleukin (IL)-4, Interleukin (IL)-6, Interleukin (IL)-18, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, immunoglobulin G (IgG), DAO, FABP2 and the PGE2 ELISA kit were obtained from Jianglai Bio-Technology Co., Ltd., Shanghai, China. The CD4-FITC/CD8-PE/CD3-PC5 Kit was from Tongshengshidai Bio-Technology Co., Ltd., Beijing, China.

2.2. Animals and Experimental Design

Male BALB/c mice (4–6 weeks old, 16–20 g), sourced from Huafukang Biotechnology Co., Ltd. in Beijing under license SCXK (Beijing) 2019-0008, were specific pathogen-free. The mice were housed under specific pathogen-free (SPF) conditions with a maintained temperature of 22–24 °C, relative humidity of 55 ± 5% and a 12 h light/dark cycle. Mice had free access to animal feed and drinking water. All personnel involved in the animal experiments of this project had passed the animal experimental operation assessment and strictly adhered to the Qingdao University’s animal welfare and ethics requirements throughout the experimental procedures (No.20231116BALBC7520231227052). The optimal sample size to use was determined to ensure adequate power while minimizing animal usage. Every effort was made to minimize suffering.

Fifty specific pathogen-free male BALB/c mice were randomly divided into five groups of ten: the CON group received daily oral gavage of normal saline followed by intraperitoneal injections of saline on days 29, 30 and 31 as a blank control; the CTX group received oral saline followed by consecutive intraperitoneal injections of CTX (60 mg/kg) on these three days to establish the immunosuppression model; the LNA group and HNA group were administered 40 mg/kg and 80 mg/kg of niacin daily, respectively, followed by intraperitoneal injection of CTX (60 mg/kg); and the MPN group received high-dose niacin co-administered with the HCAR2 receptor antagonist MPN (40 mg/kg), followed by intraperitoneal injection of CTX (60 mg/kg). During the experiment, body weight was measured and feed intake was recorded daily for each group. Following the administration period, all mice were fasted for 12 h with water provided ad libitum. Subsequently, the mice were weighed and then euthanized. Mice were anesthetized via intraperitoneal sodium pentobarbital injection. Blood was subsequently obtained from the mice by performing a terminal cardiac puncture. Blood samples were centrifuged at 3000 rpm for 15 min at 4 °C and stored at −80 °C until analysis. After cervical dislocation, tissues and colon contents were extracted from each group of mice. Samples were collected in a sterile, dry tube and stored at −80 °C for microbiota analysis. Figure 1 displays the schedule for the experimental and niacin intervention protocol.

2.3. Liver, Spleen and Thymus Indexes

Following euthanasia, the liver, spleen, and thymus of the mice were immediately excised and weighed. A specific formula was used to calculate the indexes for these organs:

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2.4. ELISA

Blood samples were centrifuged at 3000 rpm for 15 min to collect the supernatant. Ileal tissues were harvested and rinsed with ice-cold PBS to remove residual blood. The tissues were then minced, and a corresponding volume of PBS containing protease inhibitors was added, followed by thorough homogenization. The homogenate underwent centrifugation at 5000 rpm for 10 min, after which the supernatant was collected for further analysis.

Serum and ileum homogenate levels of IL-2, IL-4, IL-6, IL-18, TNF-α, IFN-γ, and IgG were quantified using commercial ELISA kits from Jianglai Bio-Technology Co., Ltd. (Shanghai, China), following the manufacturer’s instructions. In the standard wells, 100 µL of the diluted standard solution was introduced. In the sample wells, 100 µL of the test serum or tissue homogenate supernatant was introduced. The plate was incubated at 37 °C for one hour. Following liquid removal, 100 µL of biotin–antibody working solution was added to each well and incubated at 37 °C for 60 min. The liquid was removed again, and the plate was washed three times. Each well was incubated with 100 µL of streptavidin-HRP working solution at 37 °C for 30 min. After the final wash, each well underwent a 15 min incubation upon the addition of 50 µL of tetramethylbenzidine substrate solution. Following this, 50 µL of stop solution was introduced into each well to halt the reaction, allowing the absorbance to be read at 450 nm for calculation and analysis.

The Th1/Th2 balance was assessed based on the respective cytokine levels: IFN-γ and IL-2 are classified as Th1-type cytokines, whereas IL-4 and IL-6 are categorized as Th2-type cytokines [24]. The balance of Th1/Th2 was analyzed by the following formula:

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2.5. Histomorphological Observation

The ileum tissue was dissected and rinsed with ice-cold physiological saline to eliminate blood residues. Subsequently, it was fixed overnight at room temperature using 4% neutral-buffered formalin. To continue fixation, the formalin solution was replaced with a fresh one. The tissues were first rinsed with distilled water; dehydration was then carried out in a graded ethanol series (75%, 85%, 90%, and 100% absolute ethanol) prior to staining. Paraffin was used to embed the ileal tissues, which were then sectioned into 5 µm slices, dried, and stained with hematoxylin and eosin (H&E). In the end, the ileal tissue’s pathological structure was observed and photographed using an optical microscope following dehydration, drying, and mounting.

2.6. Determination of Splenic Lymphocyte Proliferation

T and B lymphocyte proliferation was assessed using Concanavalin A (ConA) and lipopolysaccharide (LPS), respectively. After being processed into single-cell suspensions and subjected to erythrocyte lysis, splenocytes were resuspended in RPMI-1640 medium with 10% fetal bovine serum (FBS) at a density of 5 × 10^6^ cells/mL. Cells (100 μL) were incubated in 96-well plates with ConA (10 μg/mL) or LPS (20 μg/mL) for 72 h at 37 °C in a 5% CO_2_ environment. Methyl thiazolyl tetrazolium (MTT) (20 μL of 5 mg/mL) was incubated for 4 h, then dimethylsulfoxide (DMSO) (150 μL) was added to dissolve the crystals. The absorbance was recorded at a wavelength of 570 nm.

2.7. Delayed-Type Hypersensitivity in Mice (DTH)

Mice were sensitized through intraperitoneal injection with 0.2 mL of a 2% solution containing approximately 1 × 10^8^ sheep red blood cells (SRBC). A total of 4 days post-initiation of the immune response, the thickness of the left hind paw was measured using a vernier caliper with a precision of 0.01 mm. Subsequently, 20 μL of 20% SRBC solution (v/v) was subcutaneously administered at the same measurement site, and the paw thickness was re-evaluated 24 h after the injection. For each mouse, the average value was derived from three measurements. The change in paw thickness before and after the administration of the 20% SRBC solution (v/v) served as a quantitative measure of the delayed-type hypersensitivity reaction.

2.8. Flow Cytometry

A total of 100 μL of peripheral blood was diluted with PBS and layered onto Ficoll-Hypaque solution, followed by centrifugation for 25 min. Following collection, peripheral blood mononuclear cells (PBMCs) were isolated and cultured in medium containing 10% FBS under standard conditions (37 °C, 5% CO_2_). The antibody staining schemes were: for NK cells—FITC anti-mouse CD161a and PE anti-mouse TCRαβ; for T lymphocyte subsets—APC anti-mouse CD3, FITC anti-mouse CD4, and PE anti-mouse CD8. Following staining with diluted specific antibodies (30 min, 4 °C, dark) for NK, CD4^+^ T, and CD8^+^ T cell markers, the samples were subjected to flow cytometric analysis utilizing a Beckman Coulter instrument (Brea, CA, USA) to determine subset frequencies. Data were analyzed using FlowJo software (V.10.8.1).

2.9. Determination of Monocyte–Macrophage Function

A total of 5 mL of India ink was taken and diluted with 15 mL of normal saline. Subsequently, 0.1 mL of the diluted India ink was administered via the tail vein of mice using a micro syringe, and the time of injection was recorded. A 20 μL blood sample was obtained from the venous plexus at the medial canthus of the mice using a capillary glass tube at 2 min (t1) and 10 min (t2) after injection. These samples were immediately added with 2 mL of Na_2_CO_3_ buffer. The optical density (OD) at 600 nm was measured with a microplate reader, using Na_2_CO_3_ buffer as the blank. Upon completion of these procedures, the mice were euthanized and their liver and spleen were harvested. The organs were cleaned of blood stains on their surfaces, and their weights were measured. The phagocytic index was determined using the specified formula.

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2.10. Determination of NK Cell Activity

In the NK cell cytotoxicity assay, YAC-1 cells (ATCC, Manassas, VA, USA) served as target cells, while the effector cells were splenocytes from control or niacin-treated groups. A co-culture system was established using splenocytes and YAC-1 cells in 96-well plates, with the effector and target cells set at a 50:1 ratio. Spontaneous release wells contained 100 µL of target cells mixed with 100 µL of culture medium, while maximum release wells consisted of 100 µL of target cells combined with 100 µL of 2.5% Triton. All the above conditions were set up in triplicate. After a 4 h incubation at 37 °C in a 5% CO_2_ atmosphere, the plate was centrifuged at 1500× g for 5 min. The supernatant (100 µL/well) was then transferred to a new flat-bottomed plate, followed by the addition of an equal volume of LDH substrate solution. After a 3 min reaction, 30 µL of 1 mol/L HCl was introduced to each well. Using a microplate reader, the optical density at 490 nm was determined for the target cell control (OD1), test samples (OD2), and effector cell control (OD3). NK cell activity was determined using the formula:

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2.11. Analysis of DAO and FABP2 in Serum

Using the manufacturers’ instructions, ELISA kits were used to determine the serum concentrations of DAO and FABP2, with experimental operations conducted in line with the procedures described in Section 2.4.

2.12. Western Blot Analysis of Key Protein Expression in Intestinal Tissues

Protein extraction from mouse gut tissue was performed with a high-efficiency RIPA buffer system containing the necessary inhibitors. Protein concentrations were assessed using the BCA method, followed by mixing with loading buffer and boiling for denaturation. SDS-PAGE gels with concentrations from 8% to 15% were prepared. After loading equal protein amounts and electrophoresis at a constant voltage (terminated when the dye front reached the gel bottom), protein transfer was carried out onto a PVDF membrane with a semi-dry blotter. Following transfer, the membrane was blocked (5% skimmed milk, 1 h, 24 °C) and washed with TBST; prior to detection, it was incubated with specific primary antibodies (4 °C overnight or 24 °C for 2 h) followed by HRP-conjugated secondary antibodies (24 °C for 1 h). Protein bands were detected with an ECL reagent (Bio-Rad, Hercules, CA, USA) and visualized using a chemiluminescence imaging system. Relative expression levels were determined by analyzing the grayscale values of the target and reference bands using ImageJ software (v1.46r). The following primary antibodies were used: NLRP3, apoptosis-associated speck-like protein (ASC), IL-18, cysteinyl aspartate specific proteinase-1 (Caspase-1), HCAR2, Claudin-1, Occluden, zonula occluden-1 (ZO-1), PTGS2, EP2, EP4, GAPDH, and β-actin (all at 1:1000 dilution; Abcam, Cambridge, MA, USA). Subsequently, membranes were incubated with a goat anti-rabbit IgG–HRP secondary antibody (1:7500; Cell Signaling Technology, Danvers, MA, USA).

2.13. 16S rRNA Gene Microbiome Sequencing Analysis

Five randomly selected samples from each group underwent 16S rRNA gene sequencing. Sequencing approaches were aligned with those from prior studies [25]. For microbial community analysis, total DNA was first isolated using a commercial kit. The extracted DNA was then used as a template for PCR amplification of the 16S rRNA gene variable regions with specific primers. The V3-V4 region was amplified using the primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Sequencing of the constructed library was performed using the Illumina NovaSeq6000 platform (San Diego, CA, USA). The Biocloud Platform (https://dp.biocloud.net/microbial.html, accessed on 20 March 2025) was used for subsequent statistical data analysis. The study assessed changes in Alpha and Beta diversity, microbial abundance, and additional factors within the mouse gut microbiota.

2.14. Network Pharmacology Process

Target Screening: In the GeneCards database (http://www.genecards.org/), searches were conducted with the terms ‘immunosuppression’ and ‘niacin’. Disease-associated targets were filtered using a ‘Relevance score’ exceeding 10 as the criterion. Information on the targets and structural diagrams associated with ‘niacin’ was obtained from the SwissTargetPrediction (https://swisstargetprediction.ch/) databases. Potential targets for niacin in treating immunosuppression were identified by screening common targets using Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html, accessed on 15 November 2025) and intersecting them.

Using the STRING database (https://cn.string-db.org/), a protein–protein interaction (PPI) network was constructed, with a minimum interaction confidence score threshold set at >0.7 to ensure high reliability. Core targets were identified using Cytoscape (v3.6.1) with Degree Centrality and Maximal Clique Centrality (MCC) algorithms.

The DAVID database (https://davidbioinformatics.nih.gov/home.jsp. accessed on 15 November 2025) was employed to conduct Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses on the target gene set. The GO database was utilized for the functional annotation of genes targeted by immunosuppression and niacin. Target gene pathway enrichment was analyzed using the KEGG database. The screening criteria for the GO and KEGG analyses were established with a significance threshold of p < 0.05.

Molecular Docking: The three-dimensional structure of PTGS2 was sourced from the RCSB PDB database (https://www.rcsb.org/). Docking was conducted via the CB-Dock2 server (http://183.56.231.194:8001/cb-dock2/index.php). Niacin and PTGS2 were preprocessed by adding hydrogen and assigning charges, with the docking search area covering the enzyme’s active site. Conformations exhibiting binding affinities lower than −5.0 kcal/mol were considered to possess potential binding activity.

2.15. Statistical Analyses

Data are presented as mean ± standard error of the mean (SEM). All statistical analyses were performed using SPSS (version 20.0) and GraphPad Prism (version 7). The normality of data distribution was assessed using the Shapiro–Wilk test, and homogeneity of variances was evaluated by Levene’s test. For comparisons among multiple groups, if data met the assumptions of normality and homogeneity of variances, one-way analysis of variance (ANOVA) was used, followed by Tukey’s honestly significant difference (HSD) post hoc test for pairwise comparisons. If the assumptions were violated, the Kruskal–Wallis test was applied, followed by Dunn’s multiple comparisons test. Spearman’s rank correlation test was employed to analyze the correlations between gut microbiota and biochemical parameters or inflammatory factors, with multiple testing correction applied. The correlation heatmap was generated using the OmicStudio Cloud Platform (https://www.omicstudio.cn). Differences with p < 0.05 were considered statistically significant.

3. Results

3.1. Effects of Niacin on Body Weight and Food Intake

The weekly variations in body weight and food intake among mice in each group, as well as the changes observed before and after CTX administration, are presented in Figure 2A. Throughout the first 28 days of the experiment, all groups of mice exhibited a consistent increase in body weight, with no significant statistical differences observed between them. A significant body weight loss was observed following intraperitoneal CTX administration (p < 0.05). Food intake remained comparable across groups during the initial 28-day period but declined significantly in the CTX, LNA, HNA, and MPN groups post-CTX challenge relative to the CON group (Figure 2B; p < 0.05).

3.2. Effect of Niacin on Non-Specific Immune Function

The non-specific immune status of mice can be reflected by organ indices, phagocytic function, and NK cell activity. Figure 2C–E illustrate the impact of niacin on organ indices in CTX-treated mice. One-way ANOVA revealed significant differences among groups in liver index (F(4,35) = 11.37, p < 0.01), spleen index (F(4,35) = 7.591, p < 0.05) and thymus index (F(4,35) = 7.983, p < 0.05). The organ indices (liver, spleen, and thymus) were significantly suppressed in the CTX group relative to the CON group, indicating the inhibitory effect of CTX. This suppression was partially reversed in the HNA group, which showed increased liver and spleen indices relative to the CTX group. The MPN group, however, displayed organ indices that were intermediate between the elevated levels seen in the LNA/HNA groups and the suppressed levels of the CTX group and were statistically indistinguishable from the latter.

The influence of niacin on the clearance index, macrophage phagocytosis index (α value), and NK cell activity in mice with CTX-induced immunosuppression is shown in Figure 2F–H. One-way ANOVA revealed significant differences among groups for the clearance index (F(4,35) = 5.769, p < 0.01), phagocytic index (F(4,35) = 14.01, p < 0.05) and NK cell activity (F(4,35) = 21.99, p < 0.05). The clearance index, phagocytosis index, and NK cell activity of mice in the CTX group were significantly lower by mean differences of 63.20%, 47.35%, and 45.42%, respectively, than those in the CON group (p < 0.01). In the HNA group, the clearance index, phagocytic index and NK cell activity increased significantly by 92.23%, 44.79% and 46.10%, respectively, relative to the CTX group (p < 0.05 or p < 0.01). The MPN group exhibited significantly reduced clearance index, phagocytosis index and NK cell activity by 69.92%, 35.27% and 20.96%, respectively, compared to the HNA group (p < 0.05 or p < 0.01)

3.3. Effect of Niacin on Specific Immune Function

Flow cytometric analysis of peripheral blood (Figure 3A) revealed that CTX administration significantly altered lymphocyte subsets. One-way ANOVA showed significant differences among groups for the percentages of NK cells (F(4,35) = 116.6, p < 0.01), CD4^+^ T cells (F(4,35) = 92.22, p < 0.01), CD8^+^ T cells (F(4,35) = 22.20, p < 0.01) and the CD4^+^/CD8^+^ ratio (F(4,35) = 54.81, p < 0.01). Significant reductions relative to the CON group were observed in the CTX group: the proportions of NK cells and CD4^+^ T cells and the CD4^+^/CD8^+^ ratio were lower by 52.43%, 51.26%, and 71.94%, respectively (Figure 3B–E; p < 0.01). Niacin intervention significantly reversed these CTX-induced alterations. Relative to the CTX group, the HNA group showed significantly increased percentages of NK cells (mean increase 48.19%, p < 0.01) and CD4^+^ T cells (mean increase 55.55%, p < 0.01) and a significantly elevated CD4^+^/CD8^+^ ratio (mean increase 84.02%, p < 0.01). Conversely, the percentage of CD8^+^ T cells was significantly reduced in the HNA group compared with the CTX group (p < 0.05).

The levels of IL-2 and IFN-γ, along with those of IL-4 and IL-6, are characteristic markers of Th1 and Th2 cells, respectively [26]. The effects of niacin on multiple serum cytokines in CTX-treated mice are summarized in Figure 4A–H. One-way ANOVA revealed significant differences among groups for IL-2 (F(4,35) = 32.56, p < 0.01), IFN-γ (F(4,35) = 100.1, p < 0.01), IL-4 (F(4,35) = 93.72, p < 0.01), IL-6 (F(4,35) = 66.26, p < 0.01), IL-18 (F(4,35) = 42.05, p < 0.01), TNF-α (F(4,35) = 44.55, p < 0.01), IgG (F(4,35) = 40.88, p < 0.01) and the Th1/Th2 ratio (F(4,35) = 6.082, p < 0.01). Compared with the CON group, the CTX group exhibited significantly lower serum concentrations of IL-2, IFN-γ, IL-4, and IL-6 (p < 0.01; Figure 4A–D). Following niacin treatment, both the LNA and HNA groups showed significantly increased serum levels of these cytokines compared with the CTX group (p < 0.05 or p< 0.01; Figure 4A–D). In contrast, the HCAR2 antagonist MPN significantly attenuated these effects, as cytokine levels in the MPN group were significantly lower than those in the LNA and HNA groups (p < 0.05 or p < 0.01). Furthermore, CTX administration significantly reduced the serum Th1/Th2 ratio. Quantitative analysis revealed that the CTX group exhibited a 39.81% decrease in this ratio compared with the CON group (p < 0.01; Figure 4E). Relative to the CTX group, mean increases of 40.75% and 38.14% were observed in the LNA and HNA groups, respectively (p < 0.05 or p < 0.01; Figure 4E). These results indicate that niacin plays an immunomodulatory role by promoting both Th1 and Th2 cytokine secretion, thereby conferring a protective effect on immune function in immunosuppressed mice. Consistent with the changes in IL-2, serum concentrations of IL-18, TNF-α and IgG were also significantly altered (Figure 4F–H). Compared with the CON group, both TNF-α and IgG were significantly reduced in the CTX group (p < 0.01), and these reductions were significantly reversed by niacin intervention in the LNA and HNA groups (p < 0.05 or p < 0.01). Conversely, the MPN group showed significantly lower levels of TNF-α and IgG compared with the HNA group (p < 0.05 or p < 0.01).

The effects of niacin on the proliferative capacity of splenic lymphocytes and delayed-type allergic (DTH) activity in CTX-induced immunocompromised mice are shown in Figure 4I,J. One-way ANOVA revealed significant differences among groups for the proliferation of splenic lymphocytes (F(4,35) = 125.5, p < 0.01) and DTH activity (F(4,35) = 82.38, p < 0.01). The proliferation of splenic lymphocytes and DTH activity in the CON group were significantly higher (56.79% and 45.73%) than those in the CTX group (p < 0.01), while the proliferation of splenic lymphocytes and DTH activity in the HNA groups were significantly higher by mean differences of 90.96% and 25.33%, respectively, than those in the CTX group (p < 0.01). However, compared with the LNA and HNA groups, the DTH activity and proliferative ability of spleen lymphocytes in the MPN group were significantly decreased by mean differences of 32.87% and 32.84%, respectively (p < 0.05 or p < 0.01).

3.4. Effect of Niacin on Intestinal Barrier Function and the HCAR2/NLRP3 Signaling Pathway

3.4.1. Effect of Niacin on Histopathology of the Ileum

Histological assessment of the ileum (Figure 5A) revealed normal tissue organization with tightly packed villi and unremarkable cellular morphology in the CON group. The CTX group, however, demonstrated marked pathological alterations, such as villous blunting and loss, accompanied by detachment of the mucosal epithelium. Compared with the CTX group, after intervention with different doses of niacin, the ileum villus injury in mice was recovered. The ileum injury in the HNA group was mild, with better recovery, and most intestinal villi were arranged neatly and closely. Compared with the LNA and HNA groups, the ileum structure of the MPN group was severely damaged, with irregularly arranged intestinal glands and short, fractured intestinal villi.

Quantitative measurements of intestinal morphology are shown in Figure 5B–D. One-way ANOVA revealed significant differences among groups for villus length (F(4,35) = 50.43, p < 0.01), crypt depth (F(4,35) = 15.15, p < 0.01) and the villus length to crypt depth (V/C) ratio (F(4,35) = 19.41, p < 0.01). Compared with the CON group, CTX treatment led to significantly shorter intestinal villi (p < 0.01), while crypt depth showed no significant change; this resulted in a marked decrease in the V/C ratio (p < 0.01). Following niacin intervention, both LNA and HNA groups showed restoration of villus length versus the CTX group. While crypt depth was significantly increased only in the HNA group, the V/C ratio was significantly elevated in the LNA group (Figure 5B–D; p < 0.05). In contrast, the MPN group exhibited significantly lower villus length, crypt depth, and V/C ratio than the HNA group. Similarly, in the ileum, the MPN group had significantly decreased villus length and V/C ratio relative to the HNA group (Figure 5B–D; p < 0.05).

3.4.2. Effects of Niacin on Cytokine Production in the Ileum

The effects of niacin on the intestinal cytokine levels in mice are shown in Figure 6A–F. One-way ANOVA revealed significant differences among groups for all cytokines measured: IL-2 (F(4,35) = 61.89, p < 0.01), IL-6 (F(4,35) = 60.66, p < 0.01), IL-18 (F(4,35) = 52.84, p < 0.01), TNF-α (F(4,35) = 87.80, p < 0.01), IFN-γ (F(4,35) = 64.19, p < 0.01), and IgG (F(4,35) = 89.23, p < 0.01). Relative to CON mice, the CTX group exhibited significantly depressed intestinal levels of IL-2, IL-6, IL-18, TNF-α, IFN-γ and IgG (p < 0.01). This decrease was markedly elevated following niacin treatment in both the LNA and HNA groups (p < 0.05 or p < 0.01). However, the cytokine levels in MPN mice were significantly attenuated relative to those in the LNA and HNA groups (p < 0.05 or p < 0.01).

3.4.3. Effects of Niacin on Intestinal Permeability

Serum concentrations of the intestinal barrier markers DAO and FABP2 were quantified by ELISA (Figure 7A,B). One-way ANOVA revealed significant differences among groups for both DAO (F(4,35) = 28.21, p < 0.01) and FABP2 (F(4,35) = 12.99, p < 0.01). CTX administration led to a significant increase in both markers relative to the CON group (p < 0.01). In contrast, niacin (LNA/HNA) showed significant decreases of 18.36% and 21.87% in DAO, and 15.95% and 15.58% in FABP2. Notably, the MPN group showed a rebound, with DAO and FABP2 levels being 24.46% and 16.89% higher, respectively, than those in the HNA group (p < 0.05).

3.4.4. Effect of Niacin on Levels of Tight Junction Proteins

The expression levels of intestinal tight junction proteins were analyzed by Western blot (Figure 7C). One-way ANOVA revealed significant differences among groups for Claudin-1 (F(4,30) = 12.45, p < 0.01), Occludin (F(4,30) = 15.16, p < 0.01) and ZO-1 (F(4,30) = 27.47, p < 0.01). Compared with the CON group, CTX-treated mice exhibited significantly decreased expression of Claudin-1, Occludin, and ZO-1 (p < 0.01; Figure 7C). In contrast, niacin intervention significantly reversed these reductions: both the LNA and HNA groups showed significantly increased expression of all three tight junction proteins compared with the CTX group (p < 0.05). However, the MPN significantly attenuated these protective effects, as the MPN group exhibited significantly lower expression of Claudin-1, Occludin, and ZO-1 compared with both the LNA and HNA groups (p < 0.05).

3.4.5. Effects of Niacin on the HCAR2/NLRP3 Signaling Pathway in the Ileum

Western blot analysis was performed to investigate key proteins in the niacin-targeted HCAR2 and NLRP3 signaling pathways, including HCAR2, NLRP3, ASC, Caspase-1, and IL-18 (Figure 7D). One-way ANOVA revealed significant differences among groups for all five proteins: HCAR2 (F(4,25) = 49.21, p < 0.01), NLRP3 (F(4,25) = 27.24, p < 0.01), ASC (F(4,25) = 59.61, p < 0.01), Caspase-1 (F(4,25) = 16.74, p < 0.01) and IL-18 (F(4,25) = 11.84, p < 0.01). CTX treatment induced a significant suppression in the expression of all these proteins relative to the CON group (p < 0.01). This suppression was partially reversed by niacin intervention, as evidenced by significantly elevated protein levels in the intestinal tissues of both the LNA and HNA groups compared to CTX (p < 0.05 or p < 0.01). However, the expression levels in the MPN group were notably lower than those in the LNA and HNA groups (p < 0.05 or p < 0.01), pointing to an incomplete restoration.

3.5. Effect of Niacin on Microbial Community Composition

3.5.1. Effect of Niacin on Rarefaction Curves and the Numbers of OTUs

Each group’s Good’s coverage index, derived from OTU abundance, was greater than 99.99%. Concurrently, the flattening of the sparsity curve with increased sequence counts confirmed adequate sampling depth for subsequent analyses (Figure 8A). Community composition was analyzed using a Venn diagram based on ASV/OTU abundance, depicting both shared and unique features among groups. Figure 8B reveals that 99 OTUs were common to all four experimental groups, while unique OTUs were identified in each: CON (2061), CTX (2056), LNA (1973) and HNA (2107). These findings indicate that administering niacin significantly elevated the overall OTU numbers in mice.

3.5.2. Effect of Niacin on Alpha Diversity Analysis

Relative to the CTX group, the HNA group showed a trend toward higher alpha diversity, as measured by the Chao1, Ace, Shannon, and Simpson indices (Figure 9A–D; p < 0.05). These indices collectively reflect community richness and diversity.

3.5.3. Effect of Niacin on Beta Diversity Analysis

Principal coordinate analysis (PCoA) revealed a distinct separation of the gut microbiota communities among the four experimental groups. As depicted in Figure 10A, the gut microbiota in the CTX group showed a clear divergence from the CON group, while the microbiota in the two dose groups were more comparable to the CON group. The β-diversity heat map (Figure 10B) and UPGMA cluster map (Figure 10C) indicate that the intestinal flora composition in the LNA and HNA groups is more similar to the CON group than to the CTX group.

3.5.4. Effect of Niacin on Changes in Gut Microbiota Across Phylum and Genus Ranks

The gut microbial structure was further profiled at both the phylum and genus levels. Figure 11A showed that the intestinal microbiota was dominated by Firmicutes, Bacteroidota, Desulfobacterota, and Actinobacteriota. Within this framework, the relative abundance of the predominant Firmicutes was significantly lower in the CTX group than in CON controls but markedly higher in both niacin-treated (LNA and HNA) groups (Figure 11C). As shown in Figure 11D, the CTX-treated mice exhibited a greater proportion of Bacteroidetes compared to the CON mice, a change that was reversed by niacin supplementation. Correspondingly, the Firmicutes-to-Bacteroidota (F/B) ratio underwent marked alterations: it decreased by 58.74% in the CTX group versus the CON group, but increased by 64.18% following niacin intervention (Figure 11E; p < 0.05).

The ten most abundant bacterial genera are shown in Figure 11B, with unclassified_Muribaculaceae, Lachnospiraceae_NK4A136_group, unclassified_Lachnospiraceae, Alistipes and Alloprevotella being the primary constituents of the fecal microbiota. The CTX group exhibited opposing effects on these key genera relative to CON. Two Lachnospiraceae-associated genera (unclassified_Lachnospiraceae and Lachnospiraceae_NK4A136_group) declined by 57.85% and 47.64%, respectively, but were increased by 46.41% and 144.54% with niacin treatment (Figure 11F,G; p < 0.05 or p < 0.01). Conversely, unclassified_Muribaculaceae increased by 79.66% in the CTX group but was reduced by 39.75% and 18.02% in the niacin groups (Figure 11H; p < 0.05 or p < 0.01).

3.5.5. Effect of Niacin on LEfSe Analysis

According to the LDA score and LEfSe results in Figure 12A,B, specific bacteria varied among the four groups, with an LDA score exceeding 4. The Desulfobacterota phylum was predominant in the CON group, whereas the unclassified Muribaculaceae genus was prevalent in the HNA group.

3.5.6. The Effect of Niacin on the Correlation Between Gut Microbiota and Intestinal Inflammation Indices

Correlation analysis (Figure 13; p < 0.05, p < 0.01) revealed consistent patterns between specific gut microbes and cytokine levels. Desulfobacterota exhibited a broad positive correlation with multiple cytokines, including IL-18, IL-6, IL-4, IL-2, TNF-α, and IFN-γ. Conversely, Bacteroidota was consistently negatively correlated with all cytokines measured (IL-18, IL-2, IL-4, IL-6, TNF-α, IFN-γ). More specific associations were also observed: unclassified_Lachnospiraceae positively correlated with IL-6, IL-4, TNF-α, and IFN-γ, and Lachnospiraceae_NK4A136_group with IL-18 and IL-2, while Cyanobacteria and unclassified_Muribaculaceae showed negative correlations with IFN-γ and IL-2, respectively.

3.6. Network Pharmacology Analysis

Figure 14A presents a Venn Diagram displaying potential targets of niacin and immunosuppression. A total of 53 common targets were found in the databases and employed for network pharmacology analysis. Using Cytoscape 3.9.1, the 53 core targets were analyzed through a PPI network, where nodes represent proteins and edges depict their interactions. Darker colors signify higher degree values within the network. The key targets identified were PTGS2, PARP1, TLR4, NLRP3, IKBKB, JAK2, and CYP19A, with PTGS2 showing the highest confidence score (Figure 14B).

GO enrichment analysis revealed that the target genes were associated with 31 terms, comprising 11 Biological Processes, 10 Molecular Functions, and 10 Cellular Components. Figure 15A illustrates the top 10 GO terms per category, primarily associated with oxidoreductase activity and immune response. Figure 15B highlights significant pathway enrichment in the KEGG database, notably including cancer pathways, the NF-κB signaling pathway, and the regulation of lipolysis in adipocytes, among other signaling pathways.

Network pharmacology prediction identified PTGS2 as a key target. Molecular docking analysis further indicated that niacin could bind with low binding energy and stably occupy the active site of PTGS2 (Figure 16A), giving structural-level evidence to back this prediction. PTGS2 generates prostaglandin E2 (PGE2), which activates G protein-coupled receptors EP1-EP4, leading to immune cell inhibition and contributing significantly to immune suppression. Subsequent in vivo experiments verified that niacin intervention significantly decreased PTGS2 protein expression and its downstream signaling. The experimental findings are presented in Figure 16B,C. One-way ANOVA revealed significant differences among groups for PTGS2 (F(4,25) = 7.902, p < 0.01), PGE2 (F(4,25) = 18.17, p < 0.01), EP2 (F(4,25) = 5.134, p < 0.01) and EP4 (F(4,25) = 6.023, p < 0.01). The CTX group exhibited enhanced activity within the PGE2 pathway, evidenced by elevated PGE2 concentration and increased protein expression of PTGS2, EP2 and EP4 relative to the CON group (p < 0.01). In line with this, both the LNA and HNA groups showed significant reductions in these indices relative to the CTX group. However, the MPN group diverged from this pattern, showing markedly higher levels of both PGE2 and related proteins compared to the HNA group (p < 0.05 or p < 0.01).

4. Discussion

Niacin supplementation has the potential to mitigate CTX-induced immune dysregulation. The enhancements observed in both specific and non-specific immune functions in immunocompromised mice are linked to the prior administration of niacin. The advantageous effects are attributed to the comprehensive regulation of intestinal homeostasis: niacin strengthens the intestinal barrier, reduces permeability and modulates microbial composition. At the molecular level, it concurrently activates the HCAR2/NLRP3 pathway and inhibits the PTGS2/PGE2/EP2-EP4 pathway, improvements that ultimately mediate the alleviation of injury in immunocompromised mice (Figure 17).

Cyclophosphamide, a chemotherapeutic agent, is extensively utilized in the treatment of cancer [27]. However, its use is linked to various adverse effects, such as an increased risk of immunodeficiency disorders, gastrointestinal mucosal barrier damage, intestinal dysbiosis, and vulnerability to secondary infections [28,29,30]. CTX exerts significant effects on the development and function of immune cells within both the innate and adaptive immune systems. This is evidenced by thymic involution and a reduced number of Th1 cells, alongside compromised immune functions such as lymphocyte proliferation, cytokine and immunoglobulin production, delayed-type hypersensitivity (DTH) response, antibody response, NK cell activity, macrophage phagocytic activity, and certain neutrophil functions. Our study found that three days of CTX injection in mice led to decreased body weight and organ indices and impaired immune function, indicating the immunosuppression model was successfully established; niacin intervention improved these measures. A dynamic equilibrium between Th1 and Th2 cell activities is essential for sustaining normal cellular and humoral immune functions. CTX decreases Th1-type cytokine secretion and boosts Th2-type cytokine secretion [24,31]. Niacin can suppress proinflammatory cytokine release and maintain homeostatic control of inflammation and the innate immune system [32,33,34]. Our findings indicate that niacin can modulate the Th1/Th2 balance. Niacin treatment enhanced T splenocyte proliferation, the DTH response, peritoneal macrophage phagocytosis and NK cell activities, thereby increasing immune activity in immunosuppressed mice.

Niacin exerts its biological functions primarily through its receptor, HCAR2, with expression localized to the surface of adipocytes, macrophages, neutrophils, and intestinal epithelial cells [35,36,37,38]. Through HCAR2 signaling, niacin suppresses inflammation and promotes immune tolerance in the colon. This effect is mediated by inducing an anti-inflammatory state in macrophages and dendritic cells, as evidenced by the subsequent differentiation of Treg cells and IL-10-secreting T cells [20]. Furthermore, HCAR2 is crucial for the butyrate-induced expression of IL-18 in the colonic epithelium [39]. The use of MPN, an antagonist of the HCAR2 receptor, negated the beneficial effects of niacin [40]. Experimental findings demonstrated that both specific and non-specific immunity parameters were significantly elevated by niacin intervention. In contrast, inhibiting HCAR2 activity with MPN resulted in no significant changes. These results suggest that niacin supplementation improved immunosuppressive status in mice through the HCAR2 receptor.

The intestinal barrier plays a vital role in preserving gut health and blocking harmful microbes from entering the body [41]. It comprises intestinal epithelial and endothelial cells, featuring junctions like tight junctions that contain proteins such as occludin, claudins, and ZO-1. These proteins form a selective barrier that regulates permeability [42]. When the barrier is compromised, bacteria and their metabolites can enter the bloodstream, causing inflammation [43]. Monitoring serum levels of DAO and FABP2, along with changes in tight junction protein expression, can allow assessment of intestinal permeability and barrier function [44,45]. Niacin can effectively support the intestinal structure and mucosal immunity in weaned piglets [46]. Our data illustrate that the immunocompromised group showed severe intestinal damage and increased serum DAO and FABP2 levels, with reduced claudin-1, occludin and ZO-1 expression, indicating compromised intestinal barriers. Niacin intervention enhanced tight junction protein expression, reduced permeability and partially reversed mucosal damage, demonstrating its protective effect on the intestinal barrier. Within the specific microenvironment of the intestinal mucosa, the NLRP3/IL-18 axis is chiefly involved in epithelial restoration and barrier protection [47]. A key source of IL-18 is the intestinal epithelial cells (IECs). Rather than inducing systemic inflammation, mature IL-18 primarily targets the epithelium and immune cells in an autocrine manner to uphold barrier integrity, stimulate epithelial cell proliferation and repair, and guide Th1-type responses and NK cell functions [47,48,49,50]. The HCAR2-mediated activation of the NLRP3/IL-18 axis is an essential pathway for maintaining gut homeostasis, playing a role in reducing inflammation and supporting tolerance. This pathway promotes regulatory T cell function and facilitates the polarization of reparative macrophages [20,37]. Research showed that niacin significantly boosted the expression of HCAR2, NLRP3, ASC, Caspase-1, and IL-18 proteins, suggesting its protective role through pathway activation.

Research indicates a strong connection between gut microbiota and host immune function [51]. Intestinal microorganisms and their metabolites can affect local and systemic immune function and immune homeostasis [52]. Additionally, HCAR2 is implicated in the regulation of gut microbiota composition and microbial populations [16,53]. The results indicate a closer clustering pattern (i.e., greater similarity) between the HNA and CON groups’ microbiota, relative to the distance observed between the CTX and CON groups. Niacin administration effectively corrected the CTX-induced alterations in Firmicutes, Bacteroidetes and the Firmicutes/Bacteroidetes (F/B) ratio. Bacteroidetes generate toxins that can cause colonic cell growth and inflammation, in contrast to Firmicutes, which have beneficial bacteria that mitigate intestinal inflammation and support immune system equilibrium [53]. In this study, we observed a significant decrease in the Firmicutes/Bacteroidetes (F/B) ratio in the intestinal microbiota of mice following CTX modeling, which is consistent with findings from multiple previous studies on CTX-induced immunosuppression models [4,6]. Niacin intervention significantly restored the F/B ratio. This trend aligns with the effects reported for other substances with immunomodulatory and gut-protective properties, such as polysaccharides and probiotics, suggesting that niacin may exert its effects, at least in part, by reversing the CTX-induced structural imbalance of the microbiota at the phylum level [2,54]. Lactobacillus and Lachnospiraceae_NK4A136_group are vital for gut protection, with Lachnospiraceae being abundant in the intestine and beneficial to host health [54,55]. Niacin can enhance the gut microbiota by boosting Lactobacillus at the genus levels and Firmicutes and Bacteroidota at the phylum level [16,23]. The administration of CTX during the experiment caused a rise in the levels of unclassified_Muribaculaceae and Ligilactobacillus. Conversely, niacin enriched for beneficial genera, particularly Lachnospiraceae_NK4A136_group, Lachnospiraceae and Lactobacillus.

Our study revealed that certain experimental parameters in the MPN group remained superior to those in the CTX group, even with the administration of an HCAR2 receptor antagonist. We explored whether niacin could boost immune function in mice through different pathways by performing target screening, network construction, and molecular docking analyses using network pharmacology. The network pharmacology analysis suggested that PTGS2 could be one of the potential targets through which niacin alleviates immunosuppression. Subsequent molecular docking provided preliminary structural support for this hypothesis. Bioactive prostaglandins (PGs) are synthesized from arachidonic acid via the enzymatic actions of cyclooxygenases (PTGS) and PGE synthases. Among them, PGE2 exerts its biological effects by binding to and activating four distinct G protein-coupled receptors, designated EP1 through EP4 [56]. The PGE2-EP2/EP4 signaling pathway induces immunosuppression, which is associated with a variety of tumor traits and types, such as the inflammatory tumor microenvironment and pancreatic cancer [57]. PGE2 also modulates adaptive and innate immunity by targeting the production of IFN-γ, a critical cytokine for antiviral and antitumor responses, in NK and T cells [58]. PGE2-EP2/4 signaling amplifies inflammation by upregulating NF-kB components and their targets, and simultaneously promotes immunosuppression through the activation of mregDCs [59]. Niacin stimulated dose-dependent production of PGD2 and PGE2, while niacin-deficient mice showed increased expression of PTGS2 and PGE synthase mRNA [60,61]. Further experiments showed that the serum levels of PGE2 and PTGS2/EP2/EP4 expression were markedly increased in immunosuppressed mice. In contrast, niacin notably reduced the protein expression of PTGS2 and the concentrations of its downstream mediators PGE2, EP2, and EP4 in the ileal tissues of CTX model mice. These findings suggest that niacin can mitigate PTGS2/PGE2-EP2/EP4 signaling in CTX-treated mice.

Research has shown that niacin is a key precursor for NAD+ and is increasingly used in treating cardiovascular diseases, metabolic disorders and skin barrier conditions. This study represents the first attempt to apply niacin to an immunosuppression model and investigate its underlying mechanisms. While its generalizability requires validation across different cancer models, the present findings lay a preliminary theoretical foundation for exploring niacin as a safe and cost-effective nutritional intervention strategy to mitigate the immunotoxicity of various CTX-dependent chemotherapy regimens and improve patient prognosis. Future research will focus on validating its efficacy in more complex disease models and elucidating its mechanisms of action within different tumor immune microenvironments.

5. Conclusions

In summary, niacin restored systemic immune function in immunosuppressed mice via the combined action of two synergistic pathways: first, the restoration of intestinal homeostasis through alleviated intestinal tissue damage, repaired barrier function and a modulated microbiota composition; second, the dual regulation of the intestinal HCAR2/NLRP3 and PTGS2/PGE2/EP2-EP4 signaling pathways. Collectively, these effects ultimately alleviated pathological organ damage and promoted both specific and non-specific immune responses.