Hexadecanamide alleviates experimental colitis in mice and modifies the gut microbiome

Abstract

Background

Hexadecanamide (HEX) has been recognized for its significant anti-inflammatory properties. However, its specific role and underlying mechanisms in the context of colitis remain poorly understood.

Results

Herein, we first determined the effect of oral HEX on DSS-induced colitis in mice. Our results showed that HEX alleviated DSS-induced colitis in mice, which was related to the improvement of intestinal barrier integrity and the reduction of colonic inflammatory responses. Interestingly, HEX suppressed the initiation of DSS-induced ferroptosis. In detail, HEX inhibited autophagy and ferritinophagy, which subsequently blocked lipid peroxidation. 16S rRNA sequencing revealed that HEX intervention regulated the gut microbial composition, characterized by an increased relative abundance of Actinobacteriota and Patescibacteria and a decreased the relative abundance of Firmicutes. To validate these findings, fecal microbiota transplantation (FMT) was performed in DSS-treated mice. The microbiota derived from HEX-treated mice exhibited greater efficacy in alleviating colitis compared to that from control-treated mice, as evidenced by prominent anti-inflammatory effects and colonic barrier repair, and consistent alterations in the gut microbial community, which were further confirmed by FMT.

Conclusions

Overall, our findings suggest that HEX markedly ameliorates DSS-induced colitis by limiting inflammation, improving barrier integrity and regulating gut microbial composition. These results highlight the critical role of HEX in maintaining intestinal homeostasis and suggest its potential as a novel preventive and therapeutic strategy.

Graphical Abstract

This study demonstrates that HEX significantly alleviates DSS-induced colitis by inhibiting ferroptosis, improving gut inflammation, enhancing gut barrier integrity, and modulating the composition of the gut microbiota. Specifically, HEX suppresses autophagy and ferritinophagy, which thereby reduce lipid peroxidation. Moreover, the composition of the gut microbiota was altered following HEX treatment, as evidenced by the increased abundance of Romboutsia and Bifidobacterium compared to the DSS-only treatment. This figure was generated using FigDraw.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12915-026-02530-w.

Background

Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), is an acute and chronic inflammation of the intestine, caused by a complex interplay of genetic, immunological, environmental, lifestyle and dietary factors [1]. The global prevalence of these diseases has been steadily increasing [2]. Recent evidence suggests the gut microbiota is closely associated with IBD [3, 4]. The complex intestinal symbiotic community, referred to as the gut microbiota, has been found to be one of the most crucial regulators influencing physiological homeostasis and disease progression in the host [5]. Disruption of gut microbiota homeostasis, termed gut dysbiosis [6], is involved in the initiation, development, and outcome of many diseases, including IBD [7], cancer [6], and obesity [8]. A study has demonstrated that dietary changes-induced gut dysbiosis then caused IBD, manifested as damage to the intestinal barrier and increased intestinal inflammation [9]. Furthermore, our previous studies have supported that gut dysbiosis could disrupt the mucosal barrier and mediate intestinal inflammation [5, 10], with gut microbiota-derived metabolites playing a central role in this process [11]. Additionally, numerous studies have demonstrated that metabolites from the gut microbiota influence the risk of IBD [1, 12]; however, it remains unclear whether it is mediated by changes in microbiota composition.

Hexadecanamide (HEX) was identified as a major metabolite that is significantly reduced in the rumen and milk of dairy cows suffering from intestinal diseases [13]. HEX is a high-melting-point amide non-ionic surfactant that exists in many animals and plants, including the hippocampus nucleus of normal mice, charcoal Kerandang in non-edible parts, Garcinia kola, and Ganoderma lucidum extracts. Moreover, our previous study demonstrated that HEX can alleviate colonic inflammation and improve intestinal barrier dysfunction caused by ruminal microbiota transplantation from diseased cows [13]. Moreover, it has been reported that HEX possesses anti-allergic, antioxidant, and neuroprotective effects [14–17]. However, it is unclear whether HEX can resist DSS-induced colitis or play a role in IBD. Of note, Codium fragile, which contains HEX as one of its components, could regulate superoxide dismutase (SOD) activity, glutathione (GSH) levels and malondialdehyde (MDA) contents, implying that HEX may intervene in ferroptosis [18]. Although other studies have found that there is a close connection among the gut microbiota, ferroptosis and IBD [19, 20], it is still uncertain whether HEX can ameliorate IBD through the modulation of the gut microbiota and ferroptosis.

Ferroptosis is an iron-dependent, non-apoptotic, regulated cell death process, characterized by the elevation of intracellular iron levels and lipid peroxidation, as well as impaired antioxidant production [21]. Among these features, the depletion of GSH and the increase in the mRNA for prostaglandin endoperoxidase 2 (PTGS2) are commonly observed phenomena [22]. Glutathione peroxidase 4 (GPX4) mainly removes lethal lipid peroxides by using GSH as a substrate. The xc-system, mainly composed of solute carrier family 7 member 11 (SLC7A11) and recombinant solute carrier family 3 member 2 (SLC3A2), is responsible for the exchange of extracellular cystine and intracellular glutamate. Cystine is converted into cysteine for the synthesis of glutathione within the cell [23]. An increasing number of studies have shown that ferroptosis is closely related to autophagy and ferritinophagy [21, 24]. In fact, the autophagosome accumulated when ferroptosis occurred and autophagy components promoted ferroptosis [24]. Reducing the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4), SLC7A11, or GPX4 can alleviate the activation of autophagy, thereby reducing cellular damage. Moreover, autophagy leads to the degradation of the iron storage proteins ferritin within cells, resulting in an imbalance in iron content through the nuclear receptor co-activator 4 (NCOA4)-mediated autophagy pathway, which is known as ferritinophagy [25]. Ferritinophagy is an autophagic process and causes ferroptosis by promoting iron accumulation and reactive oxygen species (ROS) overproduction [26]. Recent studies have proven that ferroptosis is involved in the pathophysiology of IBD and dysfunctional autophagy and dysbiosis contribute to IBD [27–29]. Moreover, Li et al. also found that the inhibition of ferroptosis remarkably alleviated colitis symptoms and reduced inflammation in mice [30], indicating that ferroptosis may be a potential therapeutic target for colitis.

In this study, we investigated the anti-inflammatory potential of HEX and its capacity to modulate the gut microbiota. HEX was found to reshape the gut microbiota composition and mitigate DSS-induced colitis by inhibiting ferroptosis and maintaining intestinal barrier integrity. The underlying mechanism involved in autophagy-activated ferritinophagy, which mediated lipid peroxidation initiation caused by DSS; however, the autophagy, ferritinophagy and lipid peroxidation were restrained by HEX. Our findings clarify the protective role of HEX in DSS-induced colitis, which may facilitate the development of therapeutic and preventive strategies for IBD and other inflammatory diseases.

Results

Oral HEX administration mitigates DSS-induced colitis

To study the alleviating effect of HEX on DSS-induced colitis, mice were orally administered HEX for 14 consecutive days, while experimental colitis was induced in mice by administering 3% DSS in water for the following 7 days, followed by 3 days of daily drinking water (Fig. 1A). We found that HEX intervention significantly alleviated DSS-induced colitis, as indicated by the markedly reduced disease activity index (DAI, the combined score of body weight loss, stool consistency, and rectal bleeding) score, recovery of body weight, and relieved colon shortening compared with the DSS-treated group (Fig. 1B-E). Histological analysis further showed that the mice treated with DSS had structural destruction of intestinal villi, massive infiltration of inflammatory cells, and swelling of the mesentery, whereas HEX-treated mice displayed preserved colon architecture with minimal inflammatory lesions (Fig. 1F and I). In addition, HEX treatment markedly reversed the accumulation of CD3⁺ T cells and Ly6G⁺ neutrophils induced by DSS treatment in mice (Fig. 1G-H). Consistently, lower levels of inflammatory markers during colitis, including MPO activity (Fig. 1J), and TNF-α (Fig. 1K) and IL-1β (Fig. 1L), were detected after HEX treatment compared with DSS-treated mice. Collectively, these results suggest that clinical colitis symptoms and colonic damage were alleviated by oral HEX administration.

Fig. 1.

HEX improves DSS-induced colitis. A Diagram illustrating the establishment of a mouse model with colitis and HEX treatment in this study. B The DAI scores of mice throughout the duration of the study (n = 10). C The change in body weight of mice during the experiment (n = 10). D, E Representative images of the colon (D) and the length of the colon (E) in each group (n = 3). F Representative images of H&E-stained colon (scale bar: 50 μm). G, H Representative immunohistochemical images of the colon stained with CD3 (G) and Ly6G (H) antibodies staining (scale bar, 50 μm). I The inflammatory score of H&E-stained colon (n = 5). J-L Proinflammatory indices including MPO activity, TNF-α and IL-1β of the colon (n = 5). Data are expressed as line graph (B-C) or means ± SD (E and I-L). The two-way ANOVA (B-C) and one-way ANOVA (E and I-L) were performed for statistical analysis. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistical significance

Oral HEX inhibits ferroptosis and improves colonic barrier function

To confirm the role of HEX in ferroptosis, Fe²⁺, MDA, GSH, in the colon were assessed. We observed that DSS suppressed concentrations of GSH, and DSS increased levels of Fe²⁺ and MDA in mice with colitis relative to those in the control or HEX-only treatment group (Fig. 2A-C); however, HEX increased GSH expression, and decreased Fe²⁺ and MDA levels compared with those induced by DSS (Fig. 2A-C). PTGS2 and GPX4 are markers of ferroptosis, and the decreased PTGS2 expression and increased GPX4 expression were observed in the DSS + HEX group compared with the DSS group (Fig. 2D, E). Moreover, we also found that, compared with the DSS group, HEX treatment upregulated GPX4 and SLC7A11 levels and downregulated PTGS2 and ACSL4 levels at the protein level in mice (Fig. 2F). Furthermore, we used the ferroptosis inducer RSL3 and found that HEX significantly inhibited the pathological damage, MPO activity, and inflammatory cytokine levels induced by RSL3 (Fig. 2G-K). Similarly, we demonstrated that HEX reversed the increase in PTGS2 and ACSL4 levels and the decrease in GPX4 and SLC7A11 levels caused by RSL3 (Fig. 2L-N). These results suggest that HEX can indeed significantly inhibit ferroptosis induced by DSS or RSL3.

Fig. 2.

HEX inhibits DSS-induced ferroptosis. A-C Concentration of Fe.²⁺ (A), MDA (B) and GSH (C) in the colon from each group (n = 5). D, E The relative mRNA levels of PTGS2 (D) and GPX4 (E) were determined using qPCR. F Representative western blot images of colon PTGS2, GPX4, ACSL4 and SLC7A11 from different groups. G. Representative colonic H&E-stained sections from the indicated mice (scale bar: 50 μm). H Histological scores of the colon from different groups. I-K The levels of inflammatory parameters, including MPO activity (I), TNF-α (J), and IL-1β (K) were detected. L, M The relative mRNA levels of ferroptosis-associated genes from the indicated groups were detected by qPCR. N The relative intensities of PTGS2, GPX4, ACSL4 and SLC7A11 were measured. O Representative protein levels of SQSTM1 and LC3 in the colon of mice from each group. P Representative western blot images of NCOA4 and FTH proteins in the colon tissues from the indicated groups. Data are presented as mean ± SD followed by Tukey’s test (A-F and H-P). ns, no significance, *p < 0.05, **p < 0.01, and ***p < 0.001 suggest significant statistical difference by one-way ANOVA (A-F and H-P)

Autophagy is an evolutionarily conserved degradation process that is important for maintaining the homeostasis of tissues and organisms by degrading misfolded proteins and damaged organelles [31–33]. Autophagy is closely related to DSS-induced colitis, and DSS downregulated autophagy-related genes, including atg5, atg7,atg12, and atg13. However, whether HEX regulates DSS-induced autophagy has not yet been elucidated. Therefore, we detected the levels of biomarkers of autophagy, including SQSTM1 and LC3. These results suggested that DSS markedly decreased the level of SQSTM1 and increased the level of LC3 compared with the control or HEX-only treatment (Fig. 2O). HEX treatment effectively reversed the upregulation of autophagy caused by DSS (Fig. 2O). Furthermore, to explore the mechanisms by which autophagy regulates DSS-induced ferroptosis, we assessed the role of ferritinophagy in the process. As expected, HEX treatment reversed the decrease in NCOA4 and FTH levels caused by DSS (Fig. 2P). On the basis of these data, it was concluded that the NCOA4-mediated ferritinophagy modulates ferroptosis triggered by DSS in the colon, and HEX may ameliorate DSS-induced colitis by inhibiting ferritinophagy and ferroptosis.

To assess the effect of oral HEX on the colonic mucosal barrier, the levels of tight junction (TJ) proteins were measured. We found that DSS significantly reduced the levels of colonic TJ proteins, ZO-1, occludin, and claudin-3 levels compared with the control (Additional file 1: Fig. S1A), indicating that DSS disrupted the integrity of the intestinal barrier. Notably, these decreases in the TJ proteins, ZO-1, occludin, and claudin-3 caused by DSS were reversed after HEX treatment (Additional file 1: Fig. S1A). Then, mucin-secreting goblet cells in the colonic epithelium were measured using Alcian blue staining and immunohistochemistry. Oral HEX administration markedly increased the thickness of the colonic epithelial mucosa (Additional file 1: Fig. S1B, C). Consistently, HEX-treated mice had increased mucin-2 levels in the colon compared with DSS-only treatment (Additional file 1: Fig. S1B-C). Collectively, these findings suggest that oral HEX attenuated DSS-damaged mucosal barrier function.

HEX ameliorates DSS-induced gut microbiota dysbiosis

Next, we further explored the impact of HEX on the gut microbiota composition of DSS-treated mice via 16S rRNA gene sequencing. Principal coordinate analysis (PCoA) identified a marked separation in the gut microbiota composition among C, H, D and HD groups based on the Adonis test method and the Bray Curtis distance (R = 0.21712 P = 0.001, Fig. 3A). The D group had a lower alpha diversity index than the C and HD groups, although the differences were not statistically significant (Fig. 3B). The numbers of shared observed operational taxonomic units (OTUs) in each group were 225 (C and H groups), 43 (H and D groups), 79 (D and HD groups), 106 (C, H and D groups), 43 (C, D and HD groups), 55 (H, D and HD groups), and 363 (C, H, D and HD groups) (Fig. 3C). At the phylum level, DSS increased Firmicutes abundance and decreased Patescibacteria and Actinobacteriota abundance, while the HEX + DSS treatment reversed the abundance of taxa changed by DSS (Fig. 3D). At the genus level in the control treatment and HEX-only treatment, the sequences representative of Candidatus_Saccharimonas, Lactobacillus and Clostridia_UCG014 dominated the fecal microbial community (Fig. 3E). DSS treatment reduced the abundance of some bacteria, such as Romboutsia and Bifidobacterium, while increasing the abundance of Clostridia_UCG014 and Enterorhabdus when compared with DSS + HEX treatment (Fig. 3E). LEfSe showed that oral HEX administration to healthy mice led to an enrichment of ten bacterial genera, including Lachnospiraceae_UCG-001 and Prevotellaceae_UCG-001, while ten taxa were enriched in normal controls (Fig. 3F). Moreover, three bacterial genera including Bifidobacterium and Romboutsia were significantly enriched in mice with colitis treated with HEX pretreatment, but another seven genera were enriched in the DSS-only group (Fig. 3F). Notably, the increase in fecal Ruminococcaceae caused by DSS treatment seemed to be reduced by HEX supplementation, and this was consistent with the changes in this taxon in healthy mice (Fig. 3G). These results indicate that HEX treatment changes the gut microbiota composition.

Fig. 3.

HEX improves DSS-induced gut microbial dysbiosis. A PCoA score plots of fecal samples in the C, H, D and HD groups based on the Bray Curtis distance (n = 6). B Alpha indices of the gut microbiota from the C, H, D, and HD groups. C. Venn diagram of the gut microbiota (n = 6). D, E The relative abundance of fecal bacterial phyla (D) and genera (E) presented in the gut community. F Analysis of differences in the microbial taxa among C group, H group, D group and HD group was performed using LEfSe (LDA score [log 10] > 2.5). G Relative abundance of gut Ruminococcaceae (n = 6). Data are presented as box plots (B and G). Data are presented as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistical significance based on one-way ANOVA. ns, no significance (B and G)

FMT from HEX-treated donor mice reshapes the gut microbiota and alleviates colitis caused by DSS

The gut microbiota coevolved with the intestinal immune system of the host, which influences the development of intestinal inflammation by regulating the immune response [34]. To assess whether HEX mitigated colitis caused by DSS treatment by affecting the gut microbiota, fresh feces from the control group and only HEX-only group were collected and transplanted into DSS-treated mice (Fig. 4A). As before, decreased DAI and increased body weight and colon length were shown in mice in the HEX-FMT + DSS group relative to the Ctrl-FMT + DSS group (Fig. 4B-E). Consistently, Ctrl-FMT + DSS treatment was found to cause obvious colon injury, while HEX-FMT + DSS treatment markedly reversed colon damage (Fig. 4F-G). In addition, increased the accumulation of CD3⁺T cells and Ly6G⁺ neutrophils in the colon of mice with colitis treated by Ctrl-FMT + DSS; these changes were reversed by HEX-FMT + DSS treatment (Fig. 4H-I). Notably, decreased levels of MPO, TNF-α and IL-1β in the colon were shown in mice with colitis treated with HEX-FMT compared with the other group (Fig. 4J-L). To further explore whether HEX ameliorates DSS-induced colitis by maintaining microbiota homeostasis, we next detected the gut microbiota composition in the FMT experiment using 16S rRNA gene sequencing. PCoA score plots for the fecal samples from recipient mice showed that the mice in the Ctrl-FMT + DSS group had distinct microbial structures from those of the HEX-FMT + DSS group (Fig. 5A). Alpha diversity analysis found that mice in the HEX-FMT + DSS group had significantly increased species diversity compared with those in the Ctrl-FMT + DSS group (Fig. 5B-C). At the phylum level, Firmicutes, Bacteroidota, Patescibacteria and Actinobacteriota were the primary phyla in both groups (Fig. 5D). Moreover, the relative abundance of Bacteroidota, Patescibacteria and Actinobacteriota increased in the HEX-FMT + DSS group, while that of Firmicutes was reduced compared with that in the Ctrl-FMT + DSS group (Fig. 5D). FMT from the HEX group obviously increased the relative abundance of norank_f__Muribaculaceae, Candidatus_Saccharimonas, Corynebacterium, Clostridia_UCG-014 and Turicibacter at the genus level (Fig. 5E). LEfSe identified eleven different bacterial genera enriched in the HEX-FMT + DSS group, including Brachybacterium and Micrococcaceae, while only seven bacterial genera were enriched in the Ctrl-FMT + DSS group (Fig. 5G). Altogether, these results suggest that FMT from the HEX-treated group mirrors donor gut microbial change in recipient mice, which contributes to protection against DSS-induced colitis.

Fig. 4.

HEX-FMT treatment ameliorates DSS-induced experimental colitis better than Ctrl-FMT treatment. A Illustration of FMT. Mice were orally gavaged with ABX to reduce the gut microbiota and then subjected to FMT from the Control and HEX treatment groups for three weeks. B The kinetics of DAI scores during this study (n = 10). C Daily body weight changes during the duration of this study (n = 10). D Macroscopic images of the colon from each group. E A statistical analysis of the lengths of the colon from different groups (n = 5). F, G Representative H&E-stained images of the colon (scale bars, 50 μm) (F) and histological scores of the colon (G) (n = 5). H, I Colonic T cells and neutrophils detected by immunohistochemistry stained with CD3 and Ly6G antibody staining (scale bars, 50 μm) (n = 5). J-L Colonic MPO activity (J), inflammatory cytokines TNF-α (K), and IL-1β (L) in different groups (n = 5). Data are expressed as the mean ± SD (B-C, E, G, and J-L), and one-way ANOVA was performed (B-C, E, G, and J-L). *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant difference. ns, no significance

Fig. 5.

FMT from HEX-treated mice alters the gut microbiota composition. A PCoA score plots indicating the separation of gut microbiota structure in Ctrl-FMT + DSS and HEX-FMT + DSS groups (R² = 0.1968, P = 0.007000) based on the unweighted UniFrac distance (n = 6). B, C The ace index (B) and Shannon index (C) indicated that the HEX-FMT + DSS group had increased alpha diversity relative to the Ctrl-FMT + DSS group (n = 6). D, E Relative abundance of gut microbiota from feces at the phylum (D) and genus levels (E) in the indicated groups. F LEfSe showed different bacterial taxa that were enriched in each group (LDA score [log 10] > 2). Data are expressed using boxplots (B and C), and the two-tailed unpaired Student’s t test was performed. *p < 0.05, **p < 0.01 indicates significance

FMT from HEX-treated donor mice alleviates ferroptosis and the colonic barrier damage caused by DSS

To investigate the potential mechanism by which FMT from HEX-treated donor mice alters gut microbiota and alleviates intestinal inflammation, the levels of ferroptosis and ferritinophagy were detected in recipient mice. Increased levels of Fe²⁺ and MDA, and decreased levels of GSH in the colon of recipient mice were shown in the Ctrl-FMT + DSS group compared with the HEX-FMT + DSS group (Fig. 6A-C). Similarly, decreased PTGS2 and increased GPX4 were found in the HEX-FMT + DSS group compared with the Ctrl-FMT + DSS group (Fig. 6D-E). As well, FMT from the Ctrl-treated group distinctly upregulated PTGS2 and ACSL4 protein levels and downregulated GPX4 and SLC7A11 levels in mice, whereas FMT from the HEX-treated group reversed these changes (Fig. 6F). Meanwhile, microbiota from HEX-dosed mice resisted autophagy, manifested as changed autophagy markers, including decreased LC3 protein levels and increased SQSTM1 protein levels relative to the Ctrl-FMT + DSS group (Fig. 6G). Interestingly, mice in the HEX-FMT + DSS group had increased concentrations of NCOA4 and FTH compared with the Ctrl-FMT + DSS group, suggesting that gut microbiota from HEX-treated donor mice inhibited ferritinophagy activation (Fig. 6H). Notably, mice in the Ctrl-FMT + DSS group had reduced levels of mucin and the TJ proteins, ZO-1, Occludin, and Claudin-3, and these decreases were reversed by HEX-FMT + DSS treatment (Fig. 6I-L). Collectively, these results suggest that gut microbiota from HEX-dosed mice protect against ferroptosis, autophagy, ferritinophagy, and colonic barrier damage.

Fig. 6.

HEX-FMT + DSS treatment alleviates ferroptosis and colonic damage better than Ctrl-FMT + DSS treatment. A-C Levels of Fe²⁺ (A), MDA (B) and GSH (C) in the colon (n = 5). D, E The relative mRNA expression levels of PTGS2 (D) and GPX4 (E) were measured by qPCR. F Representative western blot images of PTGS2, GPX4, ACSL4, and SLC7A11 proteins in the colon from the indicated mice. G Representative images of SQSTM1 and LC3 from the differently treated mice. H Representative images of FTH and NCOA4 were shown by western boltting. I Representative western blot images of TJ proteins in the colon tissues from the indicated groups. J, K The alcian blue (AB) staining was performed to examine the mucus layer of the colon from different mice, and the mucin-2 level was assessed by immunohistochemistry (scale bars, 50 μm). L The mucin-2 positive-stained cells were shown in brown. Student’s t test (A-I and L) was performed, and values are presented as the mean ± SD (A-I and L). *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant difference from each group. ns, no significance

Discussion

Mounting evidence suggests gut microbiota-derived metabolites can alleviate inflammatory diseases and have beneficial activities, including maintaining intestinal immune homeostasis, anti-inflammatory effects, and barrier renovation [10, 11]. HEX has been identified as a potential therapeutic agent for infectious and inflammatory diseases [13]. This study aimed to explore the protective role of HEX in DSS-induced colitis. We found that HEX modulated gut microbiota composition and ameliorated DSS-induced colitis through inhibiting ferroptosis and repairing intestinal barrier integrity. As well, we observed that gut microbiota induced by HEX played a crucial role in the attenuation of DSS-induced experimental colitis, as demonstrated by the more markedly alleviated effect from HEX-FMT than that from Ctrl-FMT.

To investigate the protective effect of HEX on colitis, experimental colitis of male mice was induced by 3% DSS and the mice were treated with oral HEX for 7 days in advance of DSS treatment and for 7 days during DSS treatment. As expected, oral HEX alleviated DSS-induced colitis, as supported by decreased DAI indices and reduced colonic histological damage. Although the male mice were used in our study, females often exhibit significantly different responses to colitis and microbiome-targeted therapies. These differences are not only immunological but also influenced by gender-specific changes in the composition of the gut microbiota, metabolic profile, and hormonal regulation of epithelial cells and immune responses. Therefore, we do not rule out the importance of females in colitis research.

MPO activity is a marker of the accumulation of neutrophils, which directly reflects the level of neutrophil aggregation in the colon of mice [35, 36]. Moreover, proinflammatory cytokines are involved in the pathological process of colitis, mainly including TNF-α and IL-1β [37]. We also found that oral HEX significantly mitigated colonic inflammation as indicated by the decrease in MPO activity and inflammatory cytokines including TNF-α and IL-1β in the colon in agreement with our previous study [13]. The accumulation of immune cells, producers of inflammatory cytokines, also reflects the severity of the disease to some extent. Consistent with previous studies [38, 39], DSS induced increased numbers of immune cells including F4/80⁺ macrophages, CD45⁺ leukocytes, CD3⁺ T cells and Ly6G⁺ neutrophils in the colon of mice; however, oral delivery of HEX decreased the accumulation of these cells. Increased inflammation derived from the gut can disrupt the colonic barrier integrity [10]; in turn, the leakage of the barrier contributes to the subsequent development of diseases by allowing the increase in gut-derived microbiota or metabolites [40, 41]. Indeed, DSS induced damage to integrity and barrier function as evidenced by decreased colonic TJ levels and mucus content, while therapeutic oral HEX significantly reversed these changes, consistent with our previous findings [13]. Considering that HEX has intestinal barrier repair capability, we hypothesized that those reversed results were due to the changes in gut microbiota composition.

The specific alterations in gut microbiota composition were associated with a severe inflammatory response, and the immune system can be improved and balanced by inducing favorable changes in gut microbial composition [42]. Herein, oral HEX significantly impacted the gut microbiota composition. PCoA results showed that the gut microbiota structure was markedly separated by oral HEX. Interestingly, we found that HEX-FMT treatment markedly increased the community richness in mice with colitis, suggesting potentially beneficial effects of HEX in the gut microenvironment for animals or humans in the future. HEX-treated mice with colitis had decreased colonic inflammation and a repaired intestinal barrier, contributing to the improvement of colitis caused by DSS. Moreover, HEX-FMT-treated mice with colitis had increased Actinobacteriota, which maintains intestinal homeostasis and promote host health [43]. Bifidobacterium bifidum strain BB1, a type of bacterium belonging to the Actinobacteriota phylum, possesses unique bacterial characteristics [44]. It can rapidly and continuously enhance the barrier function of the intestinal TJ in a manner dependent on TLR-2 and p38 kinase, and protect the body from the effects of DSS-induced colitis [44]. Similarly, Parabacteroides distasonis exhibits anti-inflammatory properties by reducing the release of IL-8, thereby regulating immune responses and resisting DSS-induced colitis [45]. At the genus level, the relative abundances of Romboutsia and Turicibacter were altered in colitic mice following HEX or HEX-FMT treatment. Romboutsia and Turicibacter are gram-positive bacteria correlated with the alleviation effect of colitis and intestinal inflammation [46, 47]. Romboutsia may regulate the expression of genes related to intestinal inflammation by producing short-chain fatty acids [48]. Conversely, Turicibacter is closely associated with gut microbiota dysbiosis and exacerbated inflammation, which might be due to differences in the modeling time and interfering factors [49]. Moreover, HEX treatment promoted the accumulation of Bifidobacterium in mice with colitis. An increasing number of studies showed that Bifidobacterium is effective in improving intestinal inflammation, restoring colonic barrier integrity and increasing the abundance of gut microbiota [50, 51], indicating that the increased bacterial genus might be an important factor for the attenuated effect of HEX on colitis. These outcomes suggested that oral HEX regulated the composition of gut microbiota and contributed to a stable gut microenvironment, which might play a central role in these HEX-induced reversals.

Next, we further explored the potential mechanism by which HEX and HEX-FMT treatments alleviated DSS-induced colitis. Our previous study has already demonstrated that HEX reduced the secretion of inflammatory cytokines and inhibited the activation of the NF-κB signaling pathway [13]. Inflammatory cytokines such as IL-17 can alleviate cytotoxicity, GSH depletion, and MDA accumulation [52]. Moreover, inhibiting the NF-κB axis can significantly mitigate RSL3-induced ferroptosis both in vivo and in vitro, suggesting that HEX may exert a protective effect on colitis by regulating ferroptosis [53]. Ferroptosis was recognized as a novel therapeutic target for colitis and other inflammatory diseases in the gut [54]. Ferroptosis is a novel form of cell death characterized by massive iron accumulation and lipid peroxidation [22], and it is involved in diseases, such as cancer [55], mastitis [22] and Alzheimer's disease [56]. Consistent with these studies [22, 55, 56], ferroptosis was also involved in the pathological process of colitis caused by DSS, manifesting as increased levels of Fe²⁺ and MDA as well as decreased GSH production; however, HEX reversed these changes, reminding us that ferroptosis might play a vital role in HEX’s alleviating effect on colitis in mice. Furthermore, we found that HEX inhibited the activation of autophagy, the promoter of ferroptosis [22], thereby regulating the physiological process of colitis caused by DSS. In agreement with this, a previous study has shown that dysfunctional autophagy is associated with IBD initiation and progression, leading to exacerbation of inflammation and impairment of immune regulation [57]. Ferritinophagy is a type of autophagy, and it can promote the initiation and development of ferroptosis [26]. It is unclear whether ferritinophagy is involved in DSS-induced colitis and HEX is a ferritinophagy or even ferroptosis inhibitor so far. Our results revealed that DSS treatment activated ferritinophagy by reducing NCOA4 and FTH levels, but HEX blocked the occurrence of ferritinophagy through upregulating of these indices, suggesting that HEX might be an inhibitor of ferritinophagy or ferroptosis. As well, Huafeng Liu et al. Found that selenium nanoparticles alleviated renal injury by inhibiting ferritinophagy in line with our research [58]. Based on these results, we believe that HEX mitigated DSS-induced colitis mainly by regulating the initiation and progression of ferroptosis.

Conclusions

Collectively, this study reveals that HEX ameliorates DSS-induced colitis by regulating gut microbiota and inhibiting ferroptosis. To our knowledge, this is the first study to demonstrate that HEX plays a protective role in colitis, thereby providing a novel direction for the prevention and treatment of colitis and other intestinal diseases.

Methods

Mouse and treatment

A total of 90 specific pathogen free (SPF)-grade BALB/c mice (90 male, 21–24 g, 6–8 weeks old) were purchased from Liaoning Changsheng Biotechnology Co., Ltd (Benxi, China). Mice were kept in a standard environment with adequate food and water supplied in individually ventilated cages of the same size, with wood shavings as the bedding material. Also, all mice were housed in a specific SPF facility with a 12-h light and dark cycle, and the temperature and humidity were kept at 22 ± 3℃ and 35 ± 5%, respectively.

The whole animals experimental design for this study included a HEX (Yuanye Biotechnology Co., Ltd., Shanghai, China) supplementation experiment, a FMT experiment and a RSL3 supplementation experiment. For the HEX supplementation experiment, sixty mice were randomly divided into four groups: Control (C), HEX (5 mg/kg) (H), DSS (3% DSS) (D), HEX + DSS (HD). HEX was dissolved in 0.1% sodium carboxymethyl cellulose at 50 ℃ (0.6 g/mL). Three percent DSS (Yuanye Biotechnology Co., Ltd., Shanghai, China) was dissolved in tap water for subsequent experiments. HEX was administered to mice via oral gavage for 14 consecutive days (once a day) and DSS was administered to mice (7 days after HEX treatment) by drinking water for 7 days, followed by 3 days of daily drinking water. For the FMT experiment, thirty mice were used and randomly divided into two groups: Ctrl-FMT + DSS group: mice underwent fecal microbiota transplantation (FMT) for three weeks using mice in the control group as donors, and DSS was administered to mice (11 days after FMT) by drinking water for 7 days, followed by 3 days of daily drinking water; HEX-FMT + DSS group: mice underwent FMT for three weeks using mice in the HEX group as recipients, and DSS was administered to mice (11 days after FMT) by drinking water for 7 days, followed by 3 days of daily drinking water. FMT was performed according to previous studies [59]. Briefly, 0.5 g of fresh fecal from each mouse in the control group and HEX group was weighed and mixed together as a single donor. The prepared fecal samples was homogenized and centrifuged (1000 g × 2 min × 4 °C), and then supernatants were collected under sterile conditions for use. All recipient mice were treated with antibiotics (200 mg/kg of ampicillin (Sigma Aldrich, USA), metronidazole (Sigma Aldrich, USA), and neomycin (Sigma Aldrich, USA) and 100 mg/kg vancomycin (Sigma Aldrich, USA)) daily through oral gavage for 5 days to reduce commensal microbiota. Next, antibiotics were removed for 1 day, and then mice were gavaged with 300 μL of fecal microbial supernatant from the control or HEX donors for 3 consecutive days and then once every two days for three weeks. After the end of FMT (11 days after FMT), DSS was administered to mice for 7 consecutive days, followed by 3 days of daily drinking water. For the RSL3 (MedChemExpress, USA) supplementation experiment, the mice were intraperitoneally injected with RSL3 for 2 days (the last two days of the HEX treatment, 5 mg/kg, dissolved in 2% DMSO + 40% PEG300 + 2% Tween80 + H₂O).

Body weight was recorded daily for the entire duration of the study. Disease Activity Index (DAI) was assessed based on stool consistency (0, normal; 2, loose stools; 4, diarrhea), visible blood in feces (0, none; 2, slight bleeding; 4, gross bleeding), and body weight loss (0, normal; 1, 1–5%; 2, 5–10%; 3, 10–15%; 4, more than 15% body weight loss) [60]. The mice were anesthetized with using urethan (100 mg/kg) intraperitoneally and were sacrificed by cervical dislocation, and the length of the colon was measured, and all tissues including fecal samples and colon were collected under sterile conditions and stored at −80 ℃ for future analysis.

Histological analysis

Histological scores based on hematoxylin and eosin (H&E) staining were performed to evaluate the severity of damage in the colon in the HEX and DSS-treated mouse model. In brief, the colonic tissues used for histological analysis were treated with 4% paraformaldehyde for more than 48 h. Then, these colonic tissues were embedded in paraffin to prepare 4 μm sections. The prepared paraffin sections were dewaxed using xylene and different gradient concentrations of alcohol and stained with H&E. Subsequently, the stained sections were observed and analyzed via optical microscopy (Olympus, Tokyo, Japan) as previously described [13].

Proinflammatory cytokine assay

To assess the proinflammatory cytokine levels in the colon, the levels of tumor necrosis factor (TNF)-α (Cat. No. 430915) and interleukin (IL)−1β (Cat. No.432615) were detected using ELISA kits. Briefly, 10% colonic tissue homogenate was prepared using PBS. Then, the prepared homogenate was centrifuged at 12,000 rpm for 10 min and the supernatants were collected for proinflammatory cytokine assay according to the manufacturer’s instructions (Biolegend, USA).

Myeloperoxidase (MPO) activity assay

The colonic tissues from HEX-, DSS-, Ctrl-FMT + DSS-, and HEX-FMT + DSS-treated mice were weighed and homogenized (0.1 mg/mL) using MPO buffer, and MPO activity was determined by the MPO assay kit (A044-1–1, Nanjing Jianchen, China) at OD 460 nm according to the manufacturer’s instructions.

Measurement of GSH

The colonic samples from donor and recipient mice were weighed and homogenized (0.2 mg/mL) using GSH buffer and centrifuged at 3500 g for 10 min. The prepared supernatants were collected for total glutathione detection using a GSH detection kit (A006-2, Nanjing Jianchen, China) according to the manufacturer’s instructions.

Measurement of MDA

The concentrations of MDA in the colon were measured with a MDA detection kit (A003-4–1, Nanjing Jianchen, China) according to the manufacturer’s instructions. In brief, the colonic supernatants were prepared with MDA buffer, and the level of MDA was determined at OD 530 nm.

Measurement of Fe²⁺

To measure the Fe²⁺ levels, 10% tissue homogenates were prepared using Fe²⁺ buffer and centrifuged at 12,000 g for 10 min. The prepared supernatants were detected using a Fe²⁺ assay kit (Elabscience, China) according to the manufacturer’s instructions.

RNA extraction and RT-PCR

The total RNA of colonic tissues was extracted by Trizol (Invitrogen, CA, USA) as previously described [11]. In brief, the RNA extracted from the colon was extracted using 1 mL of Trizol solution and then treated with chloroform, isopropanol, and 75% anhydrous ethanol under RNase-free conditions. Subsequently, the RNA was reverse-transcribed into cDNA using TransStart Top Green qPCR SuperMix (Beijing Tiangen Biotechnology Co., Ltd.). The prepared cDNA was used together with specific primers to undergo a reaction in the Step One Plus instrument (Applied Biosystems, Foster City, California, USA) using FastStart Universal SYBR Green Master Mix (Roche, Basel, Switzerland, ROX). Specific primer sequences were as follows: PTGS2 (sense 5’-TGAGCAACTATTCCAAACCAGC-3’, antisense 5’-GCACGTAGTCTTCGATCACTATC-3’), GPX4 (sense 5’-GCCTGGATAAGTACAGGGGTT-3’, antisense 5’-CATGCAGATCGACTAGCTGAG-3’), GAPDH (sense 5’-AGGTCGGTGTGAACGGATTTG-3’, antisense 5’-TGTAGACCATGTAGTTGAGGTCA-3’), GAPDH was used as endogenous control.

Immunohistochemistry

Immunohistochemistry was performed to evaluate the role of HEX in DSS-induced intestinal injury in mice. The prepared colon tissues were dewaxed and rehydrated as mentioned above. Subsequently, the dewaxed colon sections were subjected to antigen retrieval using citrate buffer (pH 6.0). The prepared sections were further incubated with endogenous peroxidase blocker (SAP (Mouse/rabbit) IHC kit, MXB, China, KIT-7710) at room temperature for 40 min and then incubated with normal nonimmune goat serum (SAP (Mouse/rabbit) IHC kit, MXB, China, KIT-7710) for 40 min at room temperature. The sections were further treated with mucin-2 (Affinity Biosciences, Beijing, China), CD3 (Cell Signaling Technology, DF, USA) and Ly6G (Cell Signaling Technology, DF, USA) antibodies at 4 ℃ overnight. Next, the sections were treated with secondary antibody (goat anti-rabbit IgG) (SAP (Mouse/rabbit) IHC kit, MXB, China, KIT-7710) at room temperature for 30 min. Then, the slices were incubated and incubated with horseradish peroxidase (HRP) (SAP (Mouse/rabbit) IHC kit, MXB, China) for 20 min at room temperature and washed with PBS. After incubation with DAB color-developing fluid (SAP (Mouse/rabbit) IHC Kit, MXB, China), the sections were stained with hematoxylin for 5 min. Subsequently, the sections were dehydrated with xylene and alcohol. Finally, the slices were fixed with neutral resin and observed under an optical microscopy (Olympus, Tokyo, Japan).

Western immunoblotting

Total proteins from colon tissue samples were extracted using protein extraction reagent (Thermo Fisher Scientific, USA, 78,510), and the protein concentration was detected using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA, 23,227). The targeted proteins were separated by 10% or 15% SDS-PAGE and were then transferred onto 0.45 μm PVDF membranes. After blocking with 5% skim milk for 3 h at room temperature, the membranes were incubated with specific antibodies, including β-actin (1:1000; #AF7018, Affinity Biosciences, OH, USA), ZO-1 (1:1000; #AF5145, Affinity Biosciences, OH, USA), Occludin (1:1000; #DF7504, Affinity Biosciences, OH, USA), Claudin-3 (1:1000; #AF0129, Affinity Biosciences, OH, USA), PTGS2 (1:1000; #AF7003, Affinity Biosciences, OH, USA), GPX4 (1:1000; bs-3884R, Bioss, Beijing, China), NCOA4 (1:1000; YT0302, Immunoway DE, USA), FTH1 (1:1000; bs-5907R, Bioss, Beijing, China), LC3 (1:1000; #AF5402, Affinity Biosciences, OH, USA), SQSTM1 (1:1000; #AF5384, Affinity Biosciences, OH, USA), SLC7A11 (1:1000; #DF12509, Affinity Biosciences, OH, USA), ACSL4 (1:1000; #DF12141, Affinity Biosciences, OH, USA) were used to measure target proteins. The prepared PVDF membranes were then incubated with Goat anti-rabbit or Goat anti-mouse IgG (1:10,000). All prepared western blots were analyzed using Image-Pro Plus 6.0 (Media Cybernetics). β-actin was used as an internal reference protein in this experiment.

Alcian blue staining

All colonic samples used for alcian blue staining were prepared into 4 μm slices as mentioned above. After dewaxing in xylene and different concentrations of alcohols, the prepared sections were stained using an Alcian blue staining assay kit (G1560; CAS: 75,881–23-1; Solarbio, Beijing, China) according to the manufacturer’s instructions.

16S rRNA sequencing

All fecal pellets from mice were aseptically collected from each group. Microbial genomic DNA was extracted from both matrices following our previously described protocol. Briefly, DNA was isolated using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA), and DNA yield/purity were evaluated with a NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, USA). The V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified by PCR using primers 338 F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Amplicons were resolved on 2% agarose gels, excised, and recovered with the AxyPrep DNA Gel Recovery Kit (Axygen). The recovered products were further purified using the PCR Clean-Up Kit (YuHua, Shanghai, China) according to the manufacturer’s instructions, and quantified with a Qubit 4.0 fluorometer (Thermo Fisher Scientific, USA). Purified amplicons were pooled at equimolar amounts and subjected to paired-end sequencing on an Illumina NextSeq 2000 platform (Illumina, San Diego, CA, USA) using standard workflows provided by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).

Alpha diversity was used to analyze the complexity of species diversity for a sample via six indices, including coverage, Chao, ace, Shannon, Simpson, and sobs. Beta diversity analysis, including Principal coordinates analysis (PCoA), was performed to assesse the microbial structures in the fecal samples from different groups. Linear discriminant analysis effect size (LEfSe) was performed to identify the features most likely to explain the difference among donor and recipient mice treatment groups [61].

Statistical analysis

GraphPad Prism version 8.0 (Manufacturer, La Jolla, CA, USA) was used for statistical analysis, and all values are expressed as the means ± SD or represented as a boxplot. Significant differences between the two groups were examined by the two-tailed paired Student’s t test (parametric). Comparisons of more than two groups were performed using one-way analysis of variance (ANOVA) and Mann–Whitney U test followed by Tukey’s test. For all one-way ANOVAs, Mann–Whitney U test, post hoc tests were only performed if F achieved p < 0.05 and no significant variance existed in homogeneity. Statistical significance was indicated by p < 0.05.

Abbreviations

CD

Crohn’s disease

FMT

Fecal microbiota transplantation

GSH

Glutathione

HEX

Hexadecanamide

H&E

Hematoxylin and Eosin

IBD

Inflammatory bowel disease

IL-1β

Interleukin 1β

LEfSe

Linear discriminant analysis effect size

MDA

Malondialdehyde

NCOA4

Nuclear receptor co-activator 4

PCoA

Principal coordinate analysis

ROS

Reactive oxygen species

SOD

Superoxide dismutase

SPF

Specific pathogen free

TNF-α

Tumor necrosis factor α

TJ

Tight junction

UC

Ulcerative colitis

Authors’ contributions

W.C.B., H.J.Y., L.J.B., and L.J. designed the research. L.J.B. and L.J. performed the majority of experiments and data analysis. Y.Y., Y.H.Z. and K.Y.W. performed some of the animal experiments. L.J.B. wrote the original manuscript. L.J.B., L.J., Y.Y., Y.H.Z., K.Y.W., R.P.S., Y.L., Y.H., S.S, N.S.Z, X.Y.H, Y.H.F, and C.J.Z revised the manuscript. All authors read and approved the final manuscript.

Funding

Not applicable.

Data availability

All data generated during the study are included in this article. The 16S rRNA gene sequencing data in the present study are available in the NCBI Sequence Read Archive (SRA) repository under accession number PRJNA1162725.

Declarations

Ethics approval and consent to participate

The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin University (SY202512026).

Competing interests

The authors declare no competing interests.