Nicotinamide Mononucleotide Modulates Endothelin-1 via NR4A1 and Histone Modifications in Canine Intestinal Epithelial Cells

TL;DR

NMN improves canine gut health by reducing inflammation and aging through NR4A1 and changes in gene regulation.

Contribution

NMN's regulation of EDN-1 via NR4A1 and histone modifications in canine intestinal cells is newly identified.

Findings

NMN boosts cell growth and reduces oxidative stress in canine intestinal cells.

NMN decreases EDN-1 and MAPK13 gene activity, linked to inflammation and aging.

NMN alters histone modifications at the EDN-1 gene, reducing its expression.

Abstract

This study investigated how Nicotinamide Mononucleotide (NMN), a natural compound found in foods like avocados and broccoli, influences the health of canine intestinal cells. Using a comprehensive gene activity analysis, we found that NMN significantly promotes cell growth and vitality. It also reduces harmful oxidative stress by increasing key antioxidants (like SOD and GSH) and boosting cellular energy (ATP) production. Furthermore, NMN treatment decreased the activity of specific genes linked to inflammation and aging, such as EDN-1 and MAPK13. These beneficial effects of NMN treatment occur through a specific regulator protein called NR4A1, which in turn influences how other genes are expressed. Additionally, NMN alters epigenetic markers specifically histone modifications like H3K27ac and H3K27me3 at the EDN-1 gene, effectively turning its activity down. Together, these findings suggest that NMN supports canine gut health by mitigating inflammation and cellular aging pathways, highlighting its potential as a dietary supplement for improving intestinal well-being in dogs as they age.

This work conducted a transcriptome analysis of canine intestinal epithelial cells (cIECs) treated with nicotinamide mononucleotide (NMN), a physiologically active nucleotide with a pyridine base known for its anti-aging and anti-inflammatory effects. In our experiment, cIECs were cultured and segregated into a control group (Ctrl) and an NMN-treated group. The finding demonstrated that NMN significantly affects cell proliferation in cIECs in comparison to the Ctrl. The transcriptome analysis indicated a high enrichment of genes associated with the cell cycle, proliferation, cellular senescence, and inflammatory pathways in NMN-treated cIECs, showing that NMN has the capacity to modify these biological processes. Compared to the Ctrl group, NMN treatment significantly increased ATP, SOD, CAT and GSH levels and decreased the activities of ROS and MDA. NMN treatment also significantly increased the activity of the relative complex I, III and V enzymes compared to the Ctrl group. Furthermore, the expression of MAPK13, EDN1, TNFAIP6, TNFSF15 and SLC7A11 were decreased significantly, while ACOX2, CPT1C, CCNA1 and CCNE1 were increased significantly in NMN-5μM treatment compared to Ctrl. NMN-treated significantly decreased the expression of Hdac2, Hdac6 and Hdac8, while increasing the expression of Kdm5a, Kdm5b and Kdm5c compared to the Ctrl group. Additionally, ChIP-qPCR use discovered that NMN-treatment significantly downregulated the enrichment of EDN-1 at target loci of NR4A1, SRC1, P300, Pol II and Ser5- Pol II compared to the Ctrl group. Expression of the NR4A1 gene suggests that its exert in biological activities by inhibiting inflammatory responses and anti-aging pathways. Then, we detected the transcriptional activation linked histone markers and found that H3K23ac and H3K27ac were significantly downregulated, while H3K27me3 was significantly upregulated in the NMN-treatment compared to the Ctrl group. We conclude that NMN regulates EDN-1 expression in cIECs through mechanisms involving NR4A1 and histone modifications, highlighting its potential role in canine intestinal health.

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1. Introduction

Nicotinamide mononucleotide (NMN) is an important precursor to nicotinamide adenine dinucleotide (NAD+) [1]. Currently, NMN is primarily produced via fermentation, chemical synthesis, or enzyme-catalyzed reactions [2]. Furthermore, NMN is also found naturally in foods such as edamame, avocados and broccoli [3], which supports the possibility of these sources as dietary sources of NMN. Previous research has shown that NMN has a variety of biological activities and offers substantial potential in anti-aging [1], anti-inflammatory [4], and cardioprotective roles [5]. NMN enhances gut health by lowering the levels of pro-inflammatory cytokines, including TNF-α and IL-6, and repairing the intestinal mucosal barrier [6]. Additionally, NMN has been demonstrated to enhance the Wnt/β-catenin signaling pathway in the small intestine, thereby promoting its anti-aging biological effects [7]. NMN also modulates the gut microbiota, increasing the abundance of probiotics and other beneficial bacteria, thereby helping to maintain microbial balance and gut health [8].

As human living-conditions have improved, companion animals’ lifespans have gradually increased [9]. According to research, the appearance and function of the gastrointestinal system change as we age [10]. The small intestine plays an important role in nutrition [11], immunity, and overall physiological processes, including digestion and absorption of nutrients and health [12]. However, NMN’s effects on cIECs are not entirely understood. To investigate the impacts of NMN on cIECs and their underlying mechanisms, this study employs transcriptome analysis to determine to elucidate the regulatory effects of NMN on these cells.

Beyond its role in aging and inflammation, NMN may also influence oxidative stress pathways, which are critical for intestinal epithelial homeostasis. Oxidative stress occurs when the antioxidant system fails to keep up with the production of reactive oxygen species (ROS) caused by exposure to metals [13]. This imbalance alters the redox state of cells, harms macromolecules, and controls gene expression [14]. The initial line of defense against oxidative stress is SOD activity, which transforms superoxide anion into hydrogen peroxide (H_2_O_2_) [15]. Catalase (CAT) and glutathione peroxidase (GPx) then detoxify H_2_O_2_ to water and oxygen. CAT also regulates the production of cellular ROS, which is important for controlling cellular signaling [16]. Additionally, CAT is very important for keeping H_2_O_2_ levels low, which helps cells maintain homeostasis and adapt to stress. Genetic variations in CAT have been shown to alter oxidative stress markers associated with obesity in children. This suggests that CAT plays a crucial role in redox regulation [17]. To detoxify hydrogen peroxide, which can cause intestinal damage and apoptosis, cIECs require a strong CAT activity [18]. In general, the health and functionality of cIECs depend on preserving the balance between oxidative stress and antioxidant defenses. Inflammation and serious intestinal damage can result from a dysregulation in this balance.

Endothelin-1 (EDN-1) is a potent vasoconstrictor and mitogen composed of 21 amino acids, first isolated from the supernatant of porcine aortic endothelial cells (ECs) in 1988 [19]. Subsequently, EDN-1 has been found to be produced by a variety of cell types, including pulmonary epithelial cells [20] and keratinocytes [21]. EDN-1 serves several physiological and pathological functions. In recent years, researchers have focused on its role in disease [22]. It performs a variety of physiological activities, including as attracting immune cells to inflammatory regions and controlling ion transport in the digestive system. When the gene is transcribed, it produces a 2.8 kb mRNA that is translated into a 212-amino-acid preproendothelin-1. Several transcription factors, including HIF-1, EDN-1, and AP-1 [23], can affect the transcription of EDN-1, enabling a wide range of physiological and pathological conditions to control its production in various cells and tissues. Furthermore, mRNA stability and epigenetic regulation can be employed to control EDN-1 expression [23]. NR4A1, an orphan nuclear receptor, is a transcription factor that regulates inflammation and cellular stress responses. It has anti-proliferative actions on smooth muscle cells (SMCs). It has been proposed to reduce intestinal SMC proliferation and muscle thickening in inflammatory diseases like Crohn’s disease (CD) [24]. Additionally, NR4A1 has been implicated in other epithelial diseases. For example, by participating in the mesenchymal–epithelial transition (MET) of endometrial stromal cells, NR4A1 might alter endometrial receptivity [25]. The p38 MAPK family, which includes MAPK13, is known to be involved in stress and inflammatory responses. Studies show that MAPK13 regulates disease progression and structural remodeling following epithelial injury [26]. It has been shown that the activation of p38 MAPK, which comprises MAPK13, is involved in EDN-1-induced tubular epithelial–mesenchymal transition in rat renal cells. This suggests a potential regulatory connection between the MAPK and ET-1 pathways in epithelial cells, where EDN-1 signaling may activate MAPK13, resulting in subsequent effects on remodeling, proliferation, or inflammation [27].

Epigenetic mechanisms, such as histone modifications and DNA methylation, regulate the rate and level of gene expression [28]. DNA methylation usually happens in the promoter region, where it stops transcription factor binding and represses gene expression [29]. In addition to DNA methylation, histone modifications can impact chromosomal regions and modify the transcriptional regulation of specific genes [30]. Acetylation of histone H3 on lysine 9 and lysine 27 (H3K9ac and H3K27ac) is typically associated with active transcription [31]. Lysine methylation can be influenced by the location and degree of methylation. While tri-methylation on histone H3 on lys9 and lys27 (H3K9me3 and H3K27me3) denotes transcriptional suppression, H3K4me3 is linked to transcriptional activation [32]. Many inflammatory diseases typically exhibit dysregulation of histone modifications, highlighting their importance as possible treatment targets [33]. The function of HDACs in controlling inflammation has been emphasized in numerous studies. HDAC3, a class I HDAC, is an essential modulator of inflammatory processes, and its inhibition can lower inflammation in many cases [34]. Similarly, HDACs influence inflammatory pathways by regulating the activity of non-histone proteins, including transcription factors, nuclear hormone receptors, and cytokine receptors [35]. Nonetheless, there has been limited research on how NR4A1 and histone modifications influence EDN-1 in cIECs. This study aimed to elucidate the impact of NMN on EDN-1 expression in cIECs, focusing on the role of NR4A1 and histone modifications as potential regulatory mechanisms. Understanding these pathways may offer valuable insights into preserving gut health and formulating treatment approaches for intestinal diseases.

2. Materials and Methods

2.1. Cell Culture and Treatment

cIECs were cultured in high-glucose DMEM medium (Cytiva, Marlborough, MA, USA) supplemented with 1% penicillin-streptomycin (Solarbio, Beijing, China), 5% fetal bovine serum (Hyclone, Logan, UT, USA), 10 ng/mL EGF (Tongli Haiyuan, Suzhou, China), 5 μg/mL insulin (Vicente Biotech, Nanjing, China), and 20 mM HEPES (Beyotime, Shanghai, China). The cells were maintained in a humidified incubator at 37 °C with 5% CO_2_. The experimental design included two treatment groups: Cells were treated with NMN-5 μM (purity > 98%, sourced from Bidde, Shanghai, China) for 48 h, alongside a Ctrl group. In vitro and in vivo researches often use a concentration of 5 μM NMN to study its protective or modulatory effects. In this study, we selected NMN-5 μM based on previous research that revealed that NMN-5 μM alleviates heat stress-induced oxidative stress and apoptosis in BMECs by decreasing mitochondrial damage and endoplasmic reticulum stress [36].

2.2. Cell Number and Cell Viability Detections

The number of live cells was counted at 0, 24, 48, 72 and 96 h using a hemocytometer and a microscope. Caspase-3/7 activity was assessed using a commercial assay kit, which measures the cleavage of the substrate Ac-DEVD-pNA into p-nitroaniline (pNA), and absorbance was measured at 405 nm.

2.3. Antioxidant Indexes, Hepatic Complexes I, III and V Activities, and ATP Content Assay

We used a PU 8720UV/VIS scanning spectrophotometer to measure the levels of MDA, CAT, GSH and SOD in the cIECs using commercial assay kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China) according to the manufacturer’s instructions. We employed Intracellular ROS levels were determined using the OxySelect In Vitro ROS/RNS Assay Kit (Cell Biolabs, STA-347, San Diego, CA, USA) to do the analysis, following the manufacturer’s protocol. This assay uses the cell-permeable fluorogenic probe DCFH-DA. Upon oxidation by ROS, non-fluorescent DCFH is converted to highly fluorescent DCF, which was measured with excitation/emission at 480/530 nm. The activities of mitochondrial respiratory chain complexes I, III and V were measured using commercial assay kits (Comin Technologies, Co., Ltd., Suzhou, China). We used an ATP assay kit to measure the ATP content in the cIECs (Beotime, S0026, Shanghai, China).

2.4. RNA Extraction and Sequencing

We used 1 mL of Trizol (Invitrogen, Waltham, MA, USA) to extract total RNA from cells cultured in 6-well plates for both the treatment and Ctrl groups. The Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA) was then used to check the RNA’s quality. High-throughput sequencing was performed by Wuhan Yingzi Gene Technology Co., Ltd. (Wuhan, China) using the BGI T7 platform. We utilized Xshell8 and Xftp8 to analyze the raw data, and gene expression was quantified using the FPKM (Fragments Per Kilobase of exon per Million reads mapped) method. We followed the instructions from Vazyme in Nanjing, China, perform quantitative real-time PCR for mRNA expression analysis and used the 2^−∆∆CT^ method to calculate relative expression levels.

2.5. Western Blot Analysis

Phosphatase and protease inhibitors were added to a cell lysis solution to homogenize cell (Biosharp, BL509A Hefei, China). After equilibration of the protein content, samples were separated using 10% SDS-PAGE gels. Samples were transferred to PVDF membranes (Millipore, IPVH00010, Burlington, CA, USA) and blocked for one hour with 5% skim milk against NR4A1 (Product # MA5-55301), MAPK13 (Product # MA5-26206), EDN-1 (Product # MA3-005), and β-actin (Product # MA1-1140) as the loading control. Membranes were incubated at 4 °C for 12 h with primary antibodies before being treated with HRP-conjugated secondary antibodies (Product # A18763). Chemiluminescence was measured using a Dannon 5200 multi-imaging system and a high-sensitivity ECL kit (NCM Biotech, P2300, Suzhou, China).

2.6. Gene Enrichment Analysis

Gene set enrichment analysis (GSEA v4.1.0) was used to identify pathway enrichment. We utilized the Metascape database (http://metascape.org/ (accessed on 7 October 2025)) and DAVID (https://david.ncifcrf.gov/ (accessed on 14 October 2025)) to categorize and classify the biological processes or pathways of differentially expressed genes (DEGs). We used online tools for generating Venn diagrams, KEGG enrichment bubble charts, and GO pathway enrichment result charts (http://www.bioinformatics.com.cn, accessed on 19 October 2025). Moreover, functional interactions among proteins encoded by differentially expressed genes were examined by protein–protein interaction network analysis using the STRING online network tool (https://cn.string-db.org/, accessed on 20 October 2025).

2.7. ChIP-qPCR

Cells were cross-linked with 1% formaldehyde for 12 min at room temperature. The reaction was quenched by adding glycine to a final concentration of 0.125 M for 10 min. Cells were washed twice with cold PBS, scraped, and pelleted by centrifugation at 2000 rpm for 5 min at 4 °C. Cell pellets were resuspended in lysis buffer (50 mM HEPES pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and incubated on ice for 10 min. Nuclei were pelleted (2000 rpm, 5 min, 4 °C), washed once with wash buffer (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), and then resuspended in shearing buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.1% SDS). Chromatin was sheared by sonication to an average fragment size of 200–500 bp and cleared by centrifugation.

The sheared chromatin was incubated overnight at 4 °C with magnetic beads conjugated to antibodies against NR4A1, SRC1, SRC3, P300, Pol II, Ser5-Pol II and Ser2-Pol II. This schematic (Figure S1) aids in understanding the coordinated regulation of EDN-1 forward (5′GGCGTCTGCCTCTGAAGTTA-3′) and reverse (5′-TAACTTCAGAGGCAGCGCC-3′) transcription by these factors. In addition, H3K9ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, or H3K27me3 were also investigated. Immune complexes were washed sequentially with low-salt, high-salt, LiCl wash buffer (500 mM LiCl, 1% NP-40, 0.5% sodium deoxycholate, 100 mM Tris, pH 7.5), and TE buffer. Cross-links were reversed, and DNA was purified by treatment with Proteinase K and RNase A, followed by extraction. Enrichment of specific genomic regions was quantified by qPCR.

2.8. Statistical Analysis

Student’s t-test was used to compare groups, with a p-value of less than 0.05 being statistically significant. Non-parametric statistical techniques were employed to ascertain the significance of group differences, given the restricted sample size. Mean ± SEM was used to show the data. Graphical and statistical analyses were performed using GraphPad Prism 9.0.

3. Results

3.1. NMN Promotes Proliferation and Viability in cIECs

To examine the effects of NMN on cell growth, we evaluated the cell number and viability. From 0 to 24 h, cell counts did not differ significantly between the NMN-5μM and Ctrl groups. However, at 48, 72 and 96 h, the NMN-5μM group’s cell numbers were significantly (p < 0.05) higher in comparison to the Ctrl group, as shown in Figure 1A. Cell viability did not differ substantially between groups at 0, 24 and 72 h; however, between 72 and 96 h, the NMN-5μM group’s viability was significantly (p < 0.05) higher than the Ctrl group’s (Figure 1B). Furthermore, as shown in Figure 1C, NMN-5μM treatment had no effect on the activity of caspase 3/7, a crucial marker of apoptosis. Additionally, compared to the Ctrl group, the relative DNA contents were significantly (p < 0.05) greater in the NMN-treated group (Figure 1D).

Transcriptome sequencing identified 14,366 annotated genes across all samples. FPKM (Fragments Per Kilobase of transcript per Million mapped reads) was used to identify differentially expressed genes (DEGs) in the NMN-5μM group compared to the Ctrl group. Fold change (FC) > 1.2 was used as the criterion for upregulated DEGs, whereas FC < 0.8 was used for downregulated genes. We identified 756 downregulated and 1009 upregulated DEGs. IRX3, LOC100688918, CDC42EP5, HCN3, TSTD1, ACSBG1, CDC42BPG, SEC16B, UCP3 and KCNK2 were the top ten genes that were elevated. PRSS53, MYO1F, NOTUM, PRELP, PTPRN2, LOC100688100, RASSF2, ANKRD34B, HJV and BNIPL were the top ten genes that were downregulated. Gene Set Enrichment Analysis (GSEA) identified the cell cycle and cell proliferation as the most significantly altered pathways (Figure 1E). When compared to the Ctrl group (Figure 1F), NMN-5μM treatment caused significant alterations in genes related to the cell cycle and cell proliferation.

3.2. NMN Ameliorates Oxidative Stress and Enhances Antioxidant Capacity in cIECs

As shown in Figure 2, the cellular ATP, SOD, CAT and GSH levels increased significantly (p < 0.05) in the NMN-5μM group compared to Ctrl (Figure 2A,D–F), while levels of MDA significantly (p < 0.05) decreased in the NMN-5μM group compared to Ctrl (Figure 2B,C). Furthermore, NMN-5μM treatment significantly (p < 0.05) increases the activity of mitochondrial complexes I, III and V enzymes in comparison to Ctrl (Figure 2G–I). Additionally, as shown in (Figure 2J), the fatty acid beta oxidation and GO-BP response to oxidative stress pathways were significantly (p < 0.05) enriched in the NMN-5μM treatment cells using GSEA. Heatmap of core gene expression of fatty acid beta oxidation, and GOBP response to oxidative stress is represented in (Figure 2K).

3.3. Pathway Enrichment and Interaction of DEGs and Gene Expression Analysis

To elucidate the principal transcriptional pathways modulated by NMN, transcriptome analysis was conducted on cIECs from both the NMN-treated and Ctrl groups. Differentially expressed genes (DEGs) exhibiting substantial expression variations (fold change FC > 1.2 for upregulated genes and FC < 0.8 for downregulated genes) underwent Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The upregulated DEGs were significantly enriched in pathways such as Endocytosis, Tight junction, Phagosome, and Wnt signaling pathway (Figure 3A). In contrast, the downregulated DEGs were predominantly enriched in pathways such as the MAPK signaling pathway, cellular senescence, and autophagy—animal (Figure 3B). Notably, the cellular senescence pathway and the inflammation-related MAPK signaling pathway showed the highest enrichment factors and the lowest p values. Research has indicated that NMN contributes significantly to the inflammatory process [37] and is involved in restoring processes related to oxidative stress and inflammation [38]. Additionally, NMN has anti-aging effects since it raises the levels of NAD+ inside cells [39]. This study demonstrates that the substantial downregulation of the cellular senescence pathway in NMN-treated cIECs indicates NMN’s potential to mitigate aging in these cells.

We performed Gene Ontology (GO) analysis on DEGs (FC > 1.2 for upregulated genes and FC < 0.8 for downregulated genes) in comparison to the Ctrl group. The up-regulated DEGs were mostly found in pathways such as the regulation of the Wnt signaling pathway, the circadian regulation of gene expression, and the transition between cell cycle phases (Figure 3C). In contrast, the downregulated DEGs were mainly enriched in pathways such as cytosolic transport, mitochondrial transcription, and histone modification (Figure 3D). The NMN/Ctrl group genes were highly enriched in pathways related to cellular senescence, tumor necrosis factor alpha, and inflammatory response (Figure 3E). Additionally, given the known role of NR4A1 in intestinal inflammation [40] and senescence [24], we examined its potential network. STRING analysis of key DEGs from the inflammatory response, TNFA-via-NF-κB, and cellular senescence pathways predicted a high degree of functional interaction (57 edges), with NFKB1, MAPK8, SP1 and NR4A1 forming central nodes (Figure 3F). These findings suggest that NMN may exert its anti-inflammatory and anti-aging effects through NR4A1. Heatmaps were generated for DEGs related to tumor necrosis factor alpha (TNFA-Via-NF-KB), cellular senescence, and inflammation, showing a downregulation trend in these pathways following NMN treatment (Figure 3G).

3.4. NMN Regulates Gene Expression, and Histone Modifications to Modulate EDN1 and NR4A1

As shown in Figure 4, the mRNA expression of MAPK13, EDN1, TNFAIP6, TNFSF15 and SLC7A11 was decreased significantly (p < 0.05) in NMN-5μM treatment compared to Ctrl, while, ACOX2, CPT1C, CCNA1 and CCNE1 were increased significantly (p < 0.05), yet the NR4A1 has not been affected by NMN-5μM treatment (Figure 4A). However, to further confirm the expression of NR4A1, MAPK13 and EDN-1, we conducted Western blot analysis, and the result shows that the protein levels of MAPK13 and EDN-1 were significantly decreased in the NMN-5μM treatment compared to the Ctrl group (Figure 4B). Moreover, the mRNA expression of (HDACs) for example, (Hdac1, Hdac10, Hdac11, Hdac1-ps, Hdac2, Hdac3, Hdac4, Hdac5, Hdac6, Hdac7, Hdac8 and Hdac9) has been conducted, and the result shows that Hdac2, Hdac6 and Hdac8 were significantly decreased in the NMN-5μM treatment compared to the Ctrl group (Figure 4C). Additionally, we measured expression of the histone lysine demethylase (KDMs) family of (Kdm1a, Kdm1b, Kdm2a, Kdm2b, Kdm3a, Kdm3b, Kdm4a, Kdm4b, Kdm4c, Kdm4d, Kdm5a, Kdm5b, Kdm5c, Kdm5d, Kdm6a, Kdm6b, Kdm6bos, Kdm7a and Kdm8), the only Kdm5a, Kdm5b and Kdm5c were significantly increased in the NMN-5μM treatment compared to Ctrl group (Figure 4D).

We next examined the binding of transcriptional regulators and histone marks at the EDN-1 gene locus by ChIP-qPCR. NMN treatment significantly reduced the enrichment of EDN-1 at the target loci of NR4A1, SRC1, P300, Pol II and Ser5-Pol II compared to the Ctrl group (Figure 4E). We also analyzed ChIP-qPCR to detect the transcriptional activation-linked histone marks H3K9ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3 and H3K27me3. In the NMN-5μM treatment group, the histone marks H3K23ac and H3K27ac were dramatically downregulated (p < 0.05), while H3K27me3 was significantly elevated (p < 0.05) compared to the Ctrl group (Figure 4F). The NMN-5μM treatment did not significantly affect H3K9ac, H3K18ac, H3K4me1, H3K4me2 and H3K4me3 enrichment.

4. Discussion

NMN is a bioactive substance that has anti-aging and anti-inflammatory properties [41], and it has been identified as a health supplement for anti-aging and alleviating chronic inflammation [42]. According to earlier research, NMN can reduce inflammation and intestinal epithelial cell death [43]. Compared with the Ctrl group, cells treated with NMN exhibited significantly downregulated inflammatory responses. In this study, we conducted a transcriptomic analysis of the effects of NMN on cIECs, which revealed NMN-mediated regulation of genes and pathways related to inflammation and cellular senescence. These findings provide a theoretical basis for the role of NMN in promoting canine intestinal health.

In this study, NMN treatment significantly increased cell counts at 48, 72 and 96 h, as well as cell survival at 72 and 96 h. However, there was no increase in caspase 3/7 activity implicated in apoptosis, and relative DNA was higher than in the Ctrl group. Our transcriptomic analysis revealed that NMN regulates genes and pathways involved in inflammation and cellular senescence in cIECs. KEGG enrichment analysis of the DEGs was performed to investigate the functions of genes and pathways. This analysis revealed that pathways and genes are related to cell cycle, cell proliferation, fatty acid beta oxidation, response to oxidative stress, TNFA-Via-NF-KB, and cellular senescence, and inflammatory responses were significantly regulated. KEGG analysis was performed on differentially expressed genes (DEGs) that showed significant expression variations between upregulated and downregulated genes. The enhanced DEGs were significantly enriched in pathways such as the tight junction, phagosome, endocytosis, and Wnt signaling pathways. Conversely, the downregulated DEGs were predominantly enriched in pathways such as autophagy, cellular senescence, and the MAPK signaling pathway. These results contribute to a comprehensive understanding of the fundamental mechanisms of action of NMN on cIECs and illustrate the potential significance of NMN in their prevention and treatment.

Oxidative stress is a type of physiological stress that happens when the antioxidant system fails to keep up with the generation of ROS [44]. MDA is a byproduct of lipid peroxidation that makes cells toxic and is used as a biomarker to measure the level of oxidative stress in an organism. This imbalance leads to disturbances in gene expression regulation, alterations in cellular redox status, and damage to macromolecules. GSH, CAT, SOD [45], and ATP are considered the principal defensive mechanisms against reactive oxygen species associated with oxidative damage. SOD, CAT, GSH and ATP levels were considerably increased in cIECs treated with NMN in this study. NMN-induced increases in NAD+ levels improve ATP synthesis, change the cellular redox state, and activate NAD+-dependent proteins such SIRT1, which modify chromatin structure and deacetylate histones [46,47]. However, when compared to the control group, the NMN treatment considerably decreased ROS and MDA levels. The formation of ROS in colonic intestinal epithelial cells (cIECs) and other cell types is one of the primary ways mitochondrial complexes I, III and IV contribute to the development and management of oxidative stress. The electron transport chain (ETC), located in the inner mitochondrial membrane, is made up of five protein complexes (Complexes I-V). Complexes I, III and V are necessary for electron transport and ATP synthesis, but they are also important for the production of ROS [48]. In the current investigation, Complexes I, III and V were significantly upregulated in cIECs following NMN therapy. All of these results point to the protective effects of NMN against oxidative stress and the improvement of cellular energy status, both of which are essential for preserving the integrity and functionality of intestinal epithelial cells. To completely clarify the various applications of NMN in canine and potentially human health, more research, particularly clinical trials are required.

Histone deacetylases (HDACs) and histone demethylases (KDMs) are examples of epigenetic modifiers that alter chromatin structure to regulate gene expression [49]. HDACs remove acetyl groups from histone tails, which frequently causes chromatin condensation and transcriptional suppression [50]. KDMs remove methyl groups from histone tails, which can either activate or repress gene expression depending on the context [51]. In this study, we measured acetylated marks of HDACs by qPCR in NMN-treated cells, revealing significant downregulation of Hdac2, Hdac6 and Hdac8 in comparison to the Ctrl. Additionally, the methylation marks of KDMs, including Kdm5a, Kdm5b and Kdm5c, were considerably elevated in the NMN-treated cells in comparison to the Ctrl group. Recent studies indicate that HDAC1 and HDAC2 are functionally prevalent during fat deposition. To stop liver cells from storing too much fat, a study found that decreasing HDAC2 activity made the GLP-1 receptor (GLP-1R) gene more active on the surface of liver cells and started the AMPK-ACC signaling pathway [52]. Suppressing either HDAC1 or HDAC2 individually has minimal impact on adipocyte differentiation; however, the concurrent silencing of both can reduce fat accumulation in mouse embryonic fibroblasts throughout their differentiation into adipocytes [53]. HDAC6 is mostly expressed in the cytoplasm; many physiological processes, including ER stress, apoptosis, and autophagy, which are essential for malignancies, inflammation, and liver disorders, are impacted by aberrant expression of HDAC6 [54,55]. According to reports, in the AKI animal model, HDAC6 activation and rhabdomyolysis (RM) cause oxidative stress, apoptosis, inflammation, and macrophage infiltration of renal tubular epithelial cells [56]. According to a recent study, HDAC8 may have a new function in metabolism and obesity. By upregulating the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α), inhibiting HDAC8 improved mitochondrial biosynthesis [57]. The KDM5 family of histone demethylases, which includes KDM5A, KDM5B and KDM5C, is essential for controlling gene expression because it removes methyl groups from histone H3’s lysine 4 (H3K4me) [58]. This epigenetic change influences a variety of cellular activities, including oxidative stress and inflammation, which are frequently linked in many clinical conditions such as cancer, neurological disorders, and chronic kidney disease. Understanding the roles of KDM5A, KDM5B and KDM5C in oxidative stress and inflammation provides intriguing therapy approaches for a wide range of disorders. These findings demonstrated that NMN-treated cells can affect health by controlling the KDM5 family and HDACs in cIECs.

NR4A1, a nuclear receptor, has been found to reduce intestinal inflammation [59]. NR4A1 increases IL-10 expression, inhibits NFκB signaling, and reduces pro-inflammatory factors in intestinal epithelial cells [60,61]. Additionally, cellular senescence is closely related to gut health [62], and NR4A1 can maintain overall health by alleviating cellular senescence [63]. Mitogen-Activated Protein Kinase 13, or MAPK13, regulates stress kinases and inflammatory reactions in epithelial cells. Research suggests that MAPK13 regulates epithelial stem cells and may have a role in illness and structural reorganization after epithelial injury [26]. MicroRNA-351-5p has been discovered to target MAPK13 in the context of intestinal ischemia/reperfusion (II/R) injury, indicating its role in inflammation and apoptosis during such events. For example, dioscin has been shown to minimize II/R damage by changing the inflammation and apoptosis in IEC-6 cells produced by miR-351-5p/MAPK13 [64]. Furthermore, blocking MAPK13 has been examined as a therapy method for mucus formation and lung inflammation, underlining its role in inflammatory processes [65]. EDN-1 is known to be involved in oxidative stress and inflammation. In more general biological contexts, EDN-1 often acts as a potent vasoconstrictor and a facilitator of inflammatory processes, which often involve oxidative stress, although this is not extensively addressed for cIECs in the available research. TNFAIP6 (Tumor Necrosis Factor Alpha-Induced Protein 6) is associated with anti-inflammatory responses. The resistance of bladder cancer to temozolomide has been linked to TNFAIP6-mediated post-translational modifications, highlighting its broader impact on cellular processes that may be indirectly associated with inflammation in the tumor microenvironment [66]. TNF-α increases its expression, and it is essential for a number of inflammatory processes [67]. It is conceivable that NMN affects TNFAIP6 expression or activity because TNF-α can stimulate TNFAIP6 expression and NMN has anti-inflammatory properties. The cytokine TNFSF15 (Tumor Necrosis Factor Superfamily Member 15) controls inflammation and keeps blood vessels stable. TNFSF15 activates T cells and makes Th1 cytokines while stopping endothelial cells from growing and endothelial progenitor cells from changing into other types of cells [68]. The association of risk alleles with inflammatory bowel disorders (IBDs) underscores this gene’s role in intestinal inflammatory responses [69]. SLC7A11 (Solute Carrier Family 7 Member 11) is an important element of the cystine/glutamate antiporter system. It helps extracellular cystine enter into cells, which is necessary for making cysteine and eventually GSH, a very important antioxidant that the body creates on its own [70,71]. Cancer cells frequently exhibit high levels of SLC7A11 expression, which increases their antioxidant capacity and helps them survive. SLC7A11 activity is closely related to ferroptosis, a type of regulated cell death marked by iron-dependent lipid peroxidation, since SLC7A11 suppression causes cystine deprivation, glutathione depletion, and increased oxidative stress, all of which cause ferroptosis [72]. CPT1C (Carnitine Palmitoyltransferase 1C) and ACOX2 (Acyl-CoA Oxidase 2) are involved in lipid metabolism, which is closely related to oxidative stress. Cell cycle control is the main function of CCNA1 and CCNE1 (Cyclin E1). Cell cycle regulators can indirectly affect inflammation and oxidative stress in cIECs through their roles in cellular proliferation, repair mechanisms, and responses to cellular damage, even though their direct involvement in these processes is not specifically described in the materials presented. In the present study, NMN-treatment decreased the MAPK13, EDN1, TNFAIP6, TNFSF15 and SLC7A11, while ACOX2, CPT1C, CCNA1 and CCNE1 were increased significantly compared to Ctrl. A crucial component of NMN’s therapeutic potential in cIECs seems to be finding a balance between lowering inflammatory signals and improving metabolic and regeneration processes.

The steroid receptor coactivator (SRC) family members, such as SRCs interact with p300/CBP by binding CBP [73]. As a result, these SRCs are frequently found within the p300/CBP epigenetic regulatory complex. SRC1 in the central nervous system is very important for motor learning, the growth of neural stem cells, and the plasticity of neurons [74]. The transcription coactivator SRC1 regulates energy expenditure in the stomach, liver, and adipose tissue [75]. The paralogous proteins P300 (E1A binding protein) and CBP (CREB binding protein) are important for controlling gene expression at the transcriptional level [76]. Recent research examining the therapeutic applications of P300-specific inhibitors in cancer and autoimmune illnesses have revealed their potential [77]. In particular, Treg cell proliferation and function are impacted by inhibitors targeting the P300 bromodomain, offering a way to improve effector responses to ROS [78]. The activity of RNA polymerase II (Pol II), which is essential for eukaryotic gene transcription, is strictly controlled, especially by phosphorylating its C-terminal domain (CTD) [79]. Serines at positions 2 (Ser2) and 5 (Ser5) are important phosphorylation sites in the YSPTSPS heptapeptide sequence, which is repeated numerous times in the CTD [80]. In the context of inflammation and oxidative stress within cIECs, these phosphorylation events, especially those involving Ser5-Pol II and Ser2-Pol II, are essential for coordinating different levels of transcription and co-transcriptional RNA processing [81]. In the present study, NMN-treatment decreased EDN-1 binding enrichments at the target loci of NR4A1, SCR1, P300, Pol II and Ser5-Pol II compared to Ctrl in cIECs. According to this research, NMN may affect how these factors bind to the regulatory areas of the EDN-1 gene, hence modulating its expression. This could have wider anti-inflammatory, anti-oxidant, or cytoprotective effects on cIECs.

Acetyl and non-acetyl histone acylation are often associated with active gene transcription [13]. The production of histone acetyl/acylation states is probably hierarchical. This is shown by the fact that several histone acetyl/acyl-transferases are often grouped into big complexes that include histone acylation reader modules [82]. Histone methylation is an important kind of post-translational alteration that occurs frequently in living things. Among the methylations that have been studied more thoroughly are those in histone H3 Lys 4 (H3K4), H3K9, H3K27, H3K36, H3K79 and H4K20 [83]. The chromatin regions of transcriptionally active genes typically include H3K4, H3K36 and H3K79, which have activating functions. H3K9, H3K27 and H4K20 are mostly associated with the silencing of gene expression and frequently function as repressive signals [84]. In this study, we detected histone modifications in NMN-treated cells using ChIP-qPCR at the target loci of EDN-1 compared to the Ctrl group. NMN treatment significantly decreased the levels of H3K23ac and H3K27ac, while increasing H3K27me3 at the EDN-1 locus. Therefore, a decrease in EDN-1 gene expression is strongly correlated with the observed decrease in H3K23ac and H3K27ac, as well as an increase in H3K27me3 at EDN-1 loci. The persistence of H3K9ac and H3K4me3 has indicated that the shift to repression is a selective silencing mechanism that primarily affects the H3K27 modification pathway rather than a global shutdown of all active marks at the locus. This suggests that the machinery that sustains H3K4me3 and H3K9ac may function independently or be less susceptible to the signals that cause H3K27 changes at this particular locus. For instance, H3K4me3 marks are typically shielded from de novo DNA methylation and are frequently preserved at the promoters of actively transcribed or poised genes [85]. H3K27ac and H3K27me3, for instance, have been found to alter gene regulation reciprocally; an increase in H3K27me3 frequently results in gene repression, whereas an increase in H3K27ac usually encourages gene activation [86,87,88]. The simultaneous increase in repressive marks (H3K27me3) and decrease in activating marks (H3K23ac, H3K27ac) clearly implies that chromatin remodeling is actively suppressing EDN-1 expression [89]. The fact that H3K27me3-rich genomic regions can act as silencers to suppress gene expression through chromatin interactions provides more evidence for this. As a result of these epigenetic changes, EDN-1 expression would be downregulated, which would lessen its pro-inflammatory and pro-oxidative effects downstream. This is especially important for cIECs, in which oxidative stress and inflammation are major causes of intestinal damage and dysfunction.

Canine in vitro gastrointestinal models are frequently employed as substitutes for human gastrointestinal models due to the numerous similarities between the digestive systems of dogs and humans [90]. Consequently, the results of this study are significant for elucidating the impact of NMN on canine intestinal health. One of the study’s limitations is that the experiments were conducted in vitro. While research on cIECs in vitro offers a controlled environment for analyzing particular biochemical pathways, extrapolating these results to an entire organism needs careful consideration of the complex in vivo environment. The obtained results are greatly impacted by the physiological context created by NMN bioavailability, gut microbiota metabolism, enterohepatic circulation, immune system interactions, and systemic hormonal control. Future research should employ in vivo models to validate our findings and examine the long-term effects of NMN on canine intestinal health. Moreover, the precise NMN concentration employed in this study might not correspond to levels of dietary intake, suggesting that dose–response studies are necessary to ascertain the ideal therapeutic dosage.

5. Conclusions

This study provides important insights into the molecular mechanisms by which NMN affects cIECs. Improved cellular energetics and reduced oxidative stress, as evidenced by increased antioxidant capacity and mitochondrial complex activity (ATP, SOD, CAT, GSH, complexes I, III, V), likely promote intestinal integrity. The epigenetic impact of NMN is demonstrated by the downregulation of inflammatory mediators such as EDN-1, which is mediated by NR4A1 and certain histone modifications. These results highlight NMN’s potential as a therapeutic agent to promote gut health and prevent canine age-related deterioration. To translate these findings into practical applications, subsequent studies should investigate in vivo effects and optimal dosages, opening the door for NMN-based therapeutics to promote canine health span and minimize inflammatory or age-related intestinal disorders.