Docosahexaenoic acid alleviates DSS-induced colitis by regulating the gut microbiota and restoring the gut barrier

Docosahexaenoic acid alleviates DSS-induced colitis by regulating the gut microbiota and restoring the gut barrier | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Docosahexaenoic acid alleviates DSS-induced colitis by regulating the gut microbiota and restoring the gut barrier Sumin Wang, Shiping Hu, Yan Yan, Lingyi Wu, Li Tang, Yiyang Hu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9355144/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Growing evidence suggests that docosahexaenoic acid (DHA), a long-chain omega-3 polyunsaturated fatty acid, possesses anti-inflammatory properties potentially beneficial for treating inflammatory diseases. However, the precise molecular mechanisms underlying the therapeutic role of DHA in inflammatory bowel disease (IBD) remain incompletely understood. This study investigated the ameliorative effects of DHA on dextran sodium sulfate (DSS)-induced colitis in mice. We found that DHA significantly ameliorated colitis symptoms, intestinal barrier disruption, and colonic inflammation. Moreover, 16S rDNA sequencing revealed that DHA mitigated gut microbiota dysbiosis. Furthermore, antibiotic cocktail (ABX ) treatment and fecal microbiota transplantation (FMT) demonstrated that the therapeutic potential of DHA depends on the intestinal microbiota. Therefore, our findings provide evidence that DHA could serve as a potential dietary supplement for preventing and treating ulcerative colitis (UC). docosahexaenoic acid colitis gut microbiota gut barrier dietary supplement polyunsaturated fatty acid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Inflammatory bowel disease (IBD) comprises chronic, inflammatory disorders, primarily ulcerative colitis (UC) and Crohn's disease (CD). The incidence of IBD has increased over the years, particularly in developing countries [ 33 ]. However, current IBD treatments are often associated with severe side effects and limited efficacy [ 24 ]. Consequently, there is an urgent need to identify alternative therapies and explore novel, safe, and effective nutritional or dietary interventions for IBD management. UC is a multifactorial disease arising from the interplay of genetic, environmental, microbiotic, and mucosal immunomodulatory factors [ 23 ]. Mounting evidence has implicated gut microbiota dysbiosis as a key contributor to IBD pathogenesis [ 35 ]. Studies have shown that IBD patients exhibit gut dysbiosis characterized by reduced biodiversity and compositional imbalance. Specifically, compared with healthy controls, IBD patients display increased abundances of Proteobacteria and Actinobacteria and decreased abundances of Firmicutes and Bacteroidetes [ 17 ]. Furthermore, fecal microbiota transplantation (FMT) has emerged as an effective and relatively safe therapy for patients with refractory or recurrent IBD[ 36 ] , [ 16 ]. Therefore, targeting the intestinal microbiota represents a promising approach to IBD management. The intestinal mucosal barrier comprises mechanical, immune, biological, and chemical components that act synergistically to prevent the infiltration of pathogenic microorganisms [ 29 ]. Barrier impairment is a hallmark feature of UC[ 32 ]. Microbiota imbalances can dysregulate intestinal immunity, compromising epithelial integrity and repair, thereby promoting intestinal inflammation. Aberrant activation of both innate and adaptive immunity characterizes UC pathology [ 15 ]. Activated immune cells proliferate and release proinflammatory effectors, including TNF-α, IL-17A, and other mediators, which further damage the barrier and drive UC progression [ 22 ]. Conversely, certain immune cells, such as regulatory T cells (Tregs), produce the protective cytokine IL-10, which has anti-inflammatory effects [ 8 ]. DHA is an essential polyunsaturated fatty acid for mammals, since endogenous synthesis is minimal, and it must be obtained from marine or plant sources [ 18 ]. Numerous human and rodent studies have demonstrated that dietary DHA supplementation ameliorates cognitive decline in elderly individuals[ 38 ] and Alzheimer's disease patients [ 7 ]. Additionally, the evidence suggests that DHA possesses anti-inflammatory properties with potential benefits for treating inflammation-related diseases, including rheumatoid arthritis [ 10 ], cancer [ 44 ], cardiovascular disease [ 42 ], and IBD [ 26 ]. Clinical studies have indicated an inverse correlation between the intake of DHA and the risk of developing IBD, including both UC and CD [ 25 ]. However, the precise molecular mechanisms underlying the therapeutic effects of DHA in IBD remain incompletely understood and warrant further investigation. This study aimed to elucidate the mechanisms by which DHA alleviates UC and to investigate its modulatory effects on the intestinal microbiota, mucosal barrier, and colonic immune homeostasis. Our findings provide new insights into the potential utility of DHA for treating IBD and related gastrointestinal disorders. Materials and methods Dextran sulfate sodium (DSS)-induced colitis Following a 7-day acclimatization period, all the mice were randomly divided into four groups. Colitis was then induced using a previously reported method with minor modifications[ 21 ]. Briefly, the mice were given 2.5% DSS (MP Biomedicals, UK) in their drinking water for 7 consecutive days, followed by 5 days of normal water. For the mice in the DHA + DSS group, DHA (35 mg/kg) was suspended in corn oil and orally administered to the mice from the initiation of the experiment until its completion. Disease activity index (DAI)-related parameters, such as body weight loss, stool consistency, and hematochezia, were monitored and recorded daily. On Day 12, the mice were humanely euthanized, and the colon lengths were measured. Fecal samples were collected for subsequent analysis of the gut microbiota. Antibiotic cocktail (ABX) experiments and fecal microbiota transplantation (FMT) To eradicate the intestinal microbiota in mice, a broad-spectrum ABX consisting of ampicillin (200 mg/kg), neomycin sulfate (200 mg/kg), metronidazole (200 mg/kg), and vancomycin (100 mg/kg) was administered orally once daily for 5 days. All antibiotics were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). FMT was performed using a modified previously described method. Briefly, colitis was induced in the donor mice, which were given either drinking water or DHA as previously described. Recipient mice were given ABX for 5 days to deplete their gut microbiota. Afterward, fresh stools from the donor mice were collected, suspended in PBS, and administered to the recipient mice via oral gavage daily for 5 days. Histopathology Distal colon samples were collected and fixed in a 10% neutral formalin solution for 24 h. After fixation, the samples were embedded in paraffin. Sections (5 µm thick) were prepared from the embedded samples and subjected to hematoxylin and eosin (H&E) staining. The histological scores were determined on the basis of established criteria [ 6 ], which consider the extent of epithelial damage and the depth of inflammatory infiltration. FITC-dextran intestinal permeability assay As previously reported, intestinal permeability was assessed using fluorescein isothiocyanate (FITC)-dextran [ 11 ]. Briefly, the mice were fasted for 3 h before oral administration of 0.6 mg/g body weight of FITC-dextran (Sigma–Aldrich). Serum samples were collected after 4 h, and fluorescence measurements were performed at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a Varioskan Flash Multimode Reader (Thermo Scientific). RNA extraction and quantitative real-time PCR RNA extraction and quantitative real-time PCR RNA was isolated from the colon tissues using RNAiso Plus reagent (Takara Biotechnology, Dalian, China). cDNA was subsequently synthesized from 1 µg RNA using a PrimeScript RT reagent kit (Takara, Japan). Quantitative real-time polymerase chain reaction (qRT–PCR) was performed on an ABI 7300 HT Fast Real-Time Cycler (Applied Biosystems, USA) using a TB Green Premix Ex Taq II kit (Takara, Japan). The mRNA expression of the target genes was normalized to that of the reference gene, Actin, and quantified using the 2-ΔΔCt method. The primers used for the qRT–PCR analysis were synthesized by Sangon Biotech Limited (Shanghai, China), and their sequences are provided in Supplementary Table 1. Western blotting Colon tissue samples were homogenized in RIPA buffer supplemented with PMSF protease inhibitors for efficient protein extraction. The protein concentration in the resulting lysates was determined using a BCA protein assay kit according to the manufacturer's recommended protocol (Beyotime Biotechnology, Shanghai, China). Equivalent amounts of protein were then electrophoresed on a 10% SDS–PAGE gel and transferred to an Immobilon PVDF membrane. To prevent nonspecific binding, the membrane was incubated with 5% nonfat dry milk in TBST for 1 h at room temperature. After the blocking step, the membrane was probed with primary antibodies against ZO-1, occludin, or β-actin at a dilution of 1:1000 and incubated overnight at 4°C. After thorough washing, the membrane was further incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the protein bands were visualized using an enhanced chemiluminescence (ECL) substrate (Thermo Scientific). DNA extraction and 16S rRNA gene sequencing Total DNA was extracted from each fecal sample using a TIANamp stool DNA kit (TIANGEN, Beijing, China). The quality and concentration of the extracted DNA were evaluated using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and 1.0% agarose gel electrophoresis. To amplify the V3-V4 hypervariable regions of the bacterial 16S-rDNA gene, we utilized the following primers: 341F (5'-ACTCCTACGGGRSGCAGCAG-3') and 806R (5'-GGACTACVV GGGTATCTAATC-3'). After purification with a GeneJET gel extraction kit (Thermo Fisher Scientific, USA), the PCR products were sequenced using an Illumina NovaSeq 6000 platform (Novogene, Tianjin, China). Immunohistochemistry Immunohistochemical analysis was performed according to a previously published protocol[ 3 ]. Briefly, 5-µm sections of paraffin-embedded tissues were deparaffinized in xylene and rehydrated using ethanol. Antigen retrieval was accomplished by heating the sections in 10 mM sodium citrate buffer (pH 6.0) in a microwave for 10 min, and then cooling them to room temperature. Subsequently, the sections were blocked with a solution of 3% H2O2 and goat serum and then incubated overnight at 4°C with primary antibodies specific to occludin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or zonula occludens (ZO)-1 (Santa Cruz Biotechnology). After incubation, the sections were washed three times with PBS for 5 min each and subsequently stained using 3,3'-diaminobenzidine-tetrahydrochloride (DAB). Finally, all the samples were examined under a microscope. DNA extraction and 16S rRNA gene sequencing Total DNA was extracted from each fecal sample using a TIANamp stool DNA kit (TIANGEN, Beijing, China). The quality and concentration of the extracted DNA were evaluated using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and 1.0% agarose gel electrophoresis. To amplify the V3-V4 hypervariable regions of the bacterial 16S-rDNA gene, we utilized the following primers: 341F (5'-ACTCCTACGGGRSGCAGCAG-3') and 806R (5'-GGACTACVV GGGTATCTAATC-3'). After purification with a GeneJET gel extraction kit (Thermo Fisher Scientific, USA), the PCR products were sequenced using an Illumina NovaSeq 6000 platform (Novogene, Tianjin, China). Bioinformatic analysis The QIIME2 platform (version 2020.2) was used to analyze the raw sequencing data. To ensure the accuracy and reliability of the data obtained, the reads were subjected to denoising and quality filtering using the DADA2 approach. The data were subsequently clustered into operational taxonomic units (OTUs) using classify-sklearn against the Greengenes database (version 13.8) with a similarity threshold of 97%. The Chao1, Shannon, and ACE indices were used to assess α diversity, while weighted and unweighted UniFrac distances were used to assess β diversity. Flow cytometric analysis Mouse colons were isolated and subjected to a series of procedures. First, the colons were resected and longitudinally opened, followed by washing with cold PBS. Afterward, the colons were incubated in 10 mL Hank's balanced salt solution (HBSS; Lonza, Basel, Switzerland), which was supplemented with 1 mM dithiothreitol (DTT) and 5 mM ethylenediaminetetraacetic acid (EDTA), at 37°C for 30 minutes with constant shaking at 300 rpm on an orbital shaker. After being washed, the colons were carefully minced and cut into smaller pieces before being digested with 15 mL of Hank's balanced salt solution (HBSS), which contained 10% fetal bovine serum (FBS), 1.5 mg/mL type-VI collagenase, and 40 µg/mL DNase I. The digestion process was conducted at 37°C for 40 min under continuous agitation at 300 rpm. The obtained single-cell suspensions were passed through 70-µm cell filters to remove any remaining tissue debris. The filtered suspensions were then subjected to centrifugation at 1300 rpm for 5 min at 4°C. The cells were then stained with a live/dead stain and several antibodies, including PerCP5.5-conjugated anti-mouse CD45, Brilliant Violet 510-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD25, and AF647-conjugated anti-mouse Foxp3, according to the manufacturer's instructions. The stained cells were subsequently analyzed on a Beckman Gallios flow cytometer, and the resulting data were analyzed using FlowJo software. Statistical analysis All experimental data are expressed as the means ± standard errors of the mean (SEMs). The data were analyzed using unpaired and independent Student's t tests for comparisons of two groups, and one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test was used to compare multiple groups. * p < 0.05 i ndicates a significant difference, **p < 0.01 indicates an extremely significant difference, and NS indicates no significance. Results DHA administration significantly alleviates DSS-induced colitis To investigate the potential anti-inflammatory effects of DHA, we employed a widely used acute colitis mouse model induced by DSS, which mimics several key features of human UC (Fig. 1 A). Throughout the establishment of the model, the mice experienced weight loss and progressively increased diarrhea accompanied by bloody and mucous stools. DHA administration significantly ameliorated DSS-induced colitis, as shown by reduced weight loss (Fig. 1 B), mortality (Fig. 1 C), and DAI scores (Fig. 1 D). Notably, the administration of DHA effectively reversed colon shortening, a notable characteristic of DSS-induced colitis (Fig. 1 E–F). Additionally, histopathological analysis of colon sections revealed that the glandular structure was disrupted, the number of goblet cells was reduced, and substantial infiltration of inflammatory cells was observed in the DSS group, whereas DHA treatment alleviated these changes and mitigated histopathological scores (Fig. 1 G-H). Moreover, the administration of DHA led to the downregulation of proinflammatory cytokine expression (Fig. 1 I–K). Collectively, these findings strongly suggest that DHA supplementation significantly alleviates the symptoms associated with DSS-induced colitis, highlighting its potential as a therapeutic intervention. DHA amelioration of DSS-induced colitis is dependent on the gut microbiota Intestinal microbiota dysbiosis has been linked to the development and progression of UC. Recent studies have suggested that DHA can increase the abundance of probiotics. To examine the potential role of the gut microbiota in mediating the therapeutic effects of DHA on DSS-induced colitis, the gut microbiota was depleted using antibiotics prior to colitis induction (Fig. 2 A). Our results show that antibiotic depletion of the microbiota significantly reduced the therapeutic effect of DHA. The protective effect of DHA was lost when the gut flora was exhausted, as evidenced by comparable weight loss (Fig. 2 B), mortality rates (Fig. 2 C), DAI scores (Fig. 2 D), colonic lengths (Fig. 2 E and F) and colonic histological changes (Fig. 2 G and H) between the ABX(DSS) group and the ABX(DSS + DHA) group. Collectively, these data suggest that the protective effect of DHA on DSS-induced colitis depends on the presence of a healthy gut microbiota. Anticolitis effects are transferable via the gut microbiota To determine whether the attenuation of DSS-induced colitis by DHA was mediated by alterations in the microbiota, we performed FMT. Intestinal microbiota-depleted mice were transplanted with the gut microbiota of either DSS-treated (FMT(DSS)) or DHA-treated (FMT(DSS + DHA)) donor mice (Fig. 3 A). The results demonstrated that recipients of FMT from DSS + DHA-treated donors exhibited reduced colitis symptoms compared to recipients of FMT from DSS-treated donors. The FMT(DSS + DHA) group showed improvements in various parameters, including reduced weight loss (Fig. 3 B), an increased survival rate (Fig. 3 C), a lower disease activity index (DAI) score (Fig. 3 D), and a longer colon length (Fig. 3 E and F). Histological analysis of colon tissue from FMT(DSS + DHA) recipient mice revealed less inflammation, less histological damage, and lower histology scores than those from FMT(DSS) recipient mice did (Fig. 3 G and H). Collectively, these findings indicate that DHA-induced changes in the microbiota during DSS treatment are beneficial to mice with colitis. DHA administration modulates the composition and diversity of the gut microbiota To assess the effect of DHA on the gut microbiota composition, we performed high-throughput gene sequencing of fecal bacterial 16S rRNA genes isolated from mice in the DSS group and the DSS + DHA group. Principal coordinate analysis (PCoA) based on the Bray–Curtis metric and Jaccard distance revealed a distinct separation in the DSS + DHA group compared with the DSS group, indicating greater microbial community richness and diversity (Fig. 4 A). Principal coordinate analysis (PCoA) based on Bray–Curtis and Jaccard distances revealed distinct separations in the gut microbiota structure between the two groups (Fig. 4 B). We assessed compositional differences in the composition of the gut flora. Taxonomic classification at the phylum level revealed Bacteroidetes and Firmicutes as the dominant orders in both the DSS and DSS + DHA groups. However, the Bacteroidetes/Firmicutes ratio significantly increased in the DSS + DHA group (Fig. 4 C). Similarly, at the genus leve, DHA treatment significantly increased the abundance of Bifidobacterium and Ruminococcus_torques_group but significantly ecreased the abundance of Ileibacterium and Ruminococcaceae_UCG_014, as represented in Fig. 4 D. To visualize compositional differences in gut microbiota composition between the two groups, a heatmap was generated on the basis of operational taxonomic unit (OTU) abundance at the genus level. As shown in Fig. 4 E, the abundance of the genus Bifidobacterium was relatively high in the DSS + DHA group, whereas the abundance of the genus Ruminococcaceae_UCG_014 was significantly enriched in the DSS group. Furthermore, to identify the predominant bacteria affected by DHA treatment, we conducted linear discriminant analysis effect size (LEfSe) analysis, which revealed Ruminococcaceae_UCG_010, Desulfovibrio, and Lactobacillus as the dominant bacteria in DSS-treated mice, whereas Bifidobacterium and Tyzzerella emerged as the predominant bacteria in mice receiving DHA treatment (Fig. 4 F, G). Our findings suggest that DHA treatment significantly altered the diversity and composition of the gut microbiota in mice with DSS-induced colitis. Taken together, our findings shed light on the profound impact of DHA treatment on the diversity and composition of the gut microbiota in mice afflicted with DSS-induced colitis. DHA ameliorates DSS-induced intestinal barrier damage via the gut microbiota Disturbances in the composition of the gut microbiota may disrupt gut barrier function, resulting in various pathological conditions. To evaluate the effect of DHA on intestinal barrier integrity, we performed experiments. First, we evaluated the intestinal permeability of the mice by measuring the concentration of fluorescein isothiocyanate-conjugated dextran (FITC-dextran) in the serum following oral administration. As shown in Fig. 5 A, compared with DSS + DHA mice, mice with DSS-induced colitis exhibited significantly increased serum levels of FITC-dextran. The protein expression of the tight junction markers occludin and ZO-1 was significantly greater in the colons of the DHA-treated mice than in those of the untreated colitis mice (Fig. 5 B). Immunohistochemistry confirmed that the expression and localization of ZO-1 and occludin were significantly greater in the DHA treatment group than in the control group (Fig. 5 C, D). In summary, these findings indicate that DHA administration alleviates intestinal permeability and upregulates the expression of tight junction proteins, ultimately contributing to reduced inflammation. To further confirm the pivotal role of the gut microbiota in the therapeutic efficacy of DHA, we performed antibiotic (ABX) and fecal microbiota transplantation (FMT) experiments to investigate the effects of DHA on intestinal barrier function. Our results indicate that ABX-treated mice did not exhibit changes in intestinal permeability or the expression of epithelial tight junction (TJ) proteins in response to DHA treatment (Fig. 5 E–H). In contrast, FMT from DHA-treated mice restored intestinal barrier integrity in mice with colitis, as evidenced by the assessment of TJ protein expression and intestinal permeability (Fig. 5 I–L). These findings underscore the essential role of the gut microbiota in mediating the beneficial effects of DHA on intestinal barrier function in mice with colitis. DHA treatment increased the frequency of Tregs in mice with DSS-induced colitis in a microbiota-dependent manner Compared with no treatment, DHA treatment significantly increased the frequency of colonic Treg cells (characterized by CD25⁺Foxp3⁺ expression) (4.24% vs. 2.66%; P < 0.05; Fig. 6 A). However, antibiotic-mediated microbiota depletion abrogated this effect, as Treg percentages showed no significant difference between the ABX(DSS) and ABX(DSS + DHA) groups (3.64% vs. 3.52%; P = 0.5875; Fig. 6 B). Conversely, recipients of FMT from DHA-treated donors exhibited higher Treg frequency than those receiving FMT from DSS-treated donors (5.23% vs. 1.69%; P < 0.05; Fig. 6 C). These data demonstrate that DHA supplementation expands colonic Treg cell populations via microbiota-dependent mechanisms. Discussion In this study, we evaluated the therapeutic effects of DHA on DSS-induced colitis and explored the underlying mechanisms. Our results demonstrate that DHA treatment significantly alleviated DSS-induced colitis, as evidenced by reduced weight loss, mortality, and DAI scores; increased colon length; improved histology scores; and decreased proinflammatory cytokine levels. ABX and FMT experiments confirmed that these beneficial effects were dependent on the gut microbiota. Moreover, DHA administration increased gut microbial diversity and altered its composition. DHA treatment also enhanced intestinal barrier integrity and increased the proportion of anti-inflammatory colonic Treg cells, collectively suppressing inflammatory and immune responses. These findings support the potential efficacy of DHA supplementation for mitigating DSS-induced colitis. Omega-3 polyunsaturated fatty acids (PUFAs) are considered essential fatty acids because they cannot be endogenously synthesized by the body and must be acquired through dietary sources. A large body of evidence [ 1 ]suggests that n-3 PUFAs have anti-inflammatory effects[ 28 ], particularly in the context of IBD[ 5 ]. Studies have indicated that increased consumption of n-6 polyunsaturated fatty acids (PUFAs) and decreased intake of n-3 PUFAs are associated with an increased risk of developing UC[ 34 ]. For example, a prospective cohort study revealed that n-6 PUFA intake was positively correlated with the risk of UC, whereas n-3 PUFA intake was negatively correlated[ 19 ]. Similarly, a long-term follow-up study revealed a negative association between the consumption of n-3 PUFAs and the risk of UC[ 2 ]. However, the specific role of DHA in the development of colitis remains unclear. Therefore, the effects of DHA on UC should be investigated, and the underlying mechanisms involved should be elucidated. In this study, we evaluated the therapeutic potential of oral DHA administration in mice with DSS-induced colitis. Consistent with these findings, our results revealed a significant protective effect of DHA treatment against DSS-induced colitis, as indicated by attenuated weight loss, improved survival rates, reduced disease activity index (DAI) scores, ameliorated mucosal injury, and decreased secretion of inflammatory cytokines. Emerging studies have shown significant differences in the composition of the gut microbiota between people with UC and healthy individuals[ 30 ]. Reduced microbial diversity and dysbiosis between probiotic and harmful bacteria play crucial roles in the pathogenesis of UC[ 41 ]. In addition, alterations in the microbiota lead to changes in its metabolites, which have a major impact on gut health[ 14 ]. Therefore, modulating the gut microbiota is a promising therapeutic strategy for patients with UC. Indeed, studies have shown that restoring microbial balance can alleviate DSS-induced colitis [ 12 ]in mice[ 13 ]. As the gut microbiome has been recognized as a critical player in the development and progression of IBD[ 35 ], it is important to investigate whether the gut microbiome contributes to the anticolitis effects of DHA. ABX experiments have revealed that microbiota depletion abolished the protective effects of DHA, as the severity of colitis was comparable between DHA-treated and untreated ABX mice. Subsequently, FMT from DHA-treated donors ameliorated colitis in recipient mice, confirming a microbiota-dependent mechanism. Taken together, our results suggest that DHA treatment ameliorates DSS-induced colitis in a gut microbiota-dependent manner. We further analyzed the α-diversity of the intestinal flora using Chao1 and Shannon indices and found that DHA supplementation significantly increased gut microbiota diversity. Our PCoA results revealed that DHA administration significantly altered the biological community structure in mice with colitis. Additionally, LEfSe analysis revealed that Bifidobacterium was significantly enriched in the DHA-treated mice. Previous research has shown that Bifidobacterium administration can relieve colitis. Yao et al. reported that Bifidobacterium longum administration can alleviate colitis in mice and may be used as an alternative or adjunctive treatment for IBD patients[ 43 ]. Cui et al. reported that Bifidobacterium bifidum administration ameliorated DSS-induced colitis by enhancing the intestinal barrier and anti-inflammation, potentially via the AhR pathway[ 9 ]. Our findings, together with those of previous reports[ 43 ][ 9 ], suggest that DHA mitigates DSS-induced dysbiosis by increasing microbial diversity and enriching beneficial taxa such as Bifidobacterium. Dysfunction of the intestinal epithelial barrier is a critical factor in the pathogenesis of UC[ 27 ]. Impairment of the intestinal mucosal barrier is the initiating event in colitis, leading to an increase in intestinal permeability and the infiltration of antigens, toxins, and pathogens into the mucosal tissue, ultimately leading to inflammation[ 31 ]. Therefore, preserving TJ protein expression and function to reduce epithelial permeability is essential for IBD treatment [ 40 ]. TJs are important structures for maintaining the mechanical barrier and permeability of the intestinal epithelium, and tight junction proteins include claudin-1, occludin, ZO-1, and others [ 39 ] [ 37 ]. We found that DHA treatment reduced intestinal permeability in DSS-treated mice (Fig. 5 A). Furthermore, DHA restored ZO-1 and occludin protein expression (Fig. 5 B, C, D) in the DSS-induced colitis model, suggesting that DHA may help restore the integrity of the intestinal mucosal barrier. These results indicate that DHA effectively restored intestinal barrier function in mice with experimental colitis. Maintaining a balance between pro- and anti-inflammatory mechanisms is critical for intestinal immune homeostasis, which can be influenced by the gut microbiota and metabolites[ 20 ]. Treg cells play a crucial role in preserving intestinal homeostasis by restraining the activity of other effector T cells and suppressing the secretion of proinflammatory cytokines, thus limiting the progression of inflammation in the colon[ 8 ]. Alterations in the number, phenotype, and suppressive function of Treg cells may contribute to the development of IBD[ 4 ]. We observed a significant increase in the frequency of colonic Treg cells following DHA treatment (Fig. 6 A). After being administered a broad-spectrum antibiotic cocktail, the ABX(DSS) group of mice exhibited similar percentages of colonic Treg cells to those in the ABX(DSS + DHA) group. Additionally, the FMT(DSS + DHA) group, which received FMT from the These findings demonstrate that DHA enhances Treg cell responses in a gut microbiota-dependent manner. Furthermore, DHA suppressed DSS-induced increases in proinflammatory cytokines (IL-1β, IL-6, TNF-α) and reversed the decrease in anti-inflammatory IL-10 expression (Fig. 1 I–K). These results indicate that the therapeutic effect of DHA involves modulating cytokine balance, suppressing proinflammatory activity and promoting anti-inflammatory responses. Conclusions In conclusion, our findings demonstrate that DHA alleviates experimental colitis in mice by modulating the gut microbiota, preserving intestinal barrier integrity, and regulating mucosal immunity, primarily through microbiota-dependent mechanisms(Fig. 7 ). Moreover, these results suggest that DHA holds promise as a candidate drug for UC therapy. Our future research will focus on identifying specific bacterial mediators of the effects of DHA and strategies to optimize its therapeutic efficacy. Declarations Institutional Review Board Statement This study was conducted with prior approval from the Animal Care and Use Committee of Third Military Medical University (Chongqing, China, approval code:20265512) and adhered to the principles and guidelines outlined in the Guide for the Care and Use of Laboratory Animals, ensuring the ethical and responsible utilization of animals in experimental research. Conflicts of Interest The author(s) stated that this study was carried out without any commercial or financial affiliations that might be perceived as a potential conflict of interest. Funding This research was supported by the Youth Project of the China National Natural Science Foundation under Grant No. 82300680. Author Contribution WN Wang conceived and designed this study;Y Yan performed the animal studies. LY Wu performed the molecular biology experiments. L Tang performed the high-throughput 16S-rRNA data analysis. SM Wang provided technical support. SM Wang and Sp Hu drafted and edited the manuscript. YY Hu and SP Hu supervised the study and revised the manuscript. All the authors contributed to the article and approved the submitted version. Data Availability Statement All data reported in this article will be shared by the lead contact upon request. This article does not report original code. References Ajabnoor SM, Thorpe G, Abdelhamid A, Hooper L (2021) Long-term effects of increasing omega-3, omega-6 and total polyunsaturated fats on inflammatory bowel disease and markers of inflammation: a systematic review and meta-analysis of randomized controlled trials. 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Gut 68:2142–2151 Salim SY, Söderholm JD (2011) Importance of disrupted intestinal barrier in inflammatory bowel diseases. Inflamm Bowel Dis 17:362–381 Sc N, Hy S, N H, Fe U, Ei WT, R B, Jcy PSG, Fkl W, Jjy C, Gg S K (2017) Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies. Lancet (London Engl. 10.1016/S0140-6736(17)32448-0 Scaioli E, Liverani E, Belluzzi A (2017) The Imbalance between n-6/n-3 Polyunsaturated Fatty Acids and Inflammatory Bowel Disease: A Comprehensive Review and Future Therapeutic Perspectives. Int J Mol Sci 18:2619 Shan Y, Lee M, Chang EB (2022) The Gut Microbiome and Inflammatory Bowel Diseases. Annu Rev Med 73:455–468 Sood A, Singh A, Mahajan R, Midha V, Mehta V, Gupta YK, Narang V, Kaur K (2020) Acceptability, tolerability, and safety of fecal microbiota transplantation in patients with active ulcerative colitis (AT&S Study). J Gastroenterol Hepatol 35:418–424 Suzuki T (2013) Regulation of intestinal epithelial permeability by tight junctions. Cell Mol Life Sci 70:631–659 Weiser MJ, Butt CM, Mohajeri MH (2016) Docosahexaenoic Acid and Cognition throughout the Lifespan. Nutrients 8:99 Wt K, L Z, Ma O, Cb SMGS, C G Jr T (2021) The Tight Junction Protein ZO-1 Is Dispensable for Barrier Function but Critical for Effective Mucosal Repair. Gastroenterology. 10.1053/j.gastro.2021.08.047 Bj YM, Jm C R (2014) Mucosal barrier, bacteria and inflammatory bowel disease: possibilities for therapy. Digestive diseases (Basel, Switzerland). 10.1159/000358156 Z YH, Y YMWYSLL X, K Q, Z H, M Y, F L, Q Y (2021) The Communication Between Intestinal Microbiota and Ulcerative Colitis: An Exploration of Pathogenesis, Animal Models, and Potential Therapeutic Strategies. Front Med. 10.3389/fmed.2021.766126 Yamagata K (2017) Docosahexaenoic acid regulates vascular endothelial cell function and prevents cardiovascular disease. Lipids Health Dis 16:118 Yao S, Zhao Z, Wang W, Liu X (2021) Bifidobacterium Longum: Protection against Inflammatory Bowel Disease. J Immunol Res 2021:8030297 Yum H-W, Na H-K, Surh Y-J (2016) Anti-inflammatory effects of docosahexaenoic acid: Implications for its cancer chemopreventive potential. Semin Cancer Biol 40–41:141–159 Additional Declarations No competing interests reported. Supplementary Files Supplemental.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 17 Apr, 2026 Editor assigned by journal 17 Apr, 2026 Submission checks completed at journal 08 Apr, 2026 First submitted to journal 08 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9355144","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":627802918,"identity":"fc2f6237-8fe1-42a1-bf8a-76a8524ce460","order_by":0,"name":"Sumin Wang","email":"","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Sumin","middleName":"","lastName":"Wang","suffix":""},{"id":627802919,"identity":"8e6e0ad8-3582-4eeb-9833-d03cd83b22ea","order_by":1,"name":"Shiping Hu","email":"","orcid":"","institution":"Chinese PLA 983 Hospital of joint Logistics Support Force","correspondingAuthor":false,"prefix":"","firstName":"Shiping","middleName":"","lastName":"Hu","suffix":""},{"id":627802920,"identity":"2348e37a-a0d9-4015-91c4-f8b94a7be463","order_by":2,"name":"Yan Yan","email":"","orcid":"","institution":"Xinqiao Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Yan","suffix":""},{"id":627802921,"identity":"dcfbae07-f0af-4d21-8456-88915581aa88","order_by":3,"name":"Lingyi Wu","email":"","orcid":"","institution":"Xinqiao Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lingyi","middleName":"","lastName":"Wu","suffix":""},{"id":627802923,"identity":"00c2cd5c-d7df-425c-88f9-2583b923b4b4","order_by":4,"name":"Li Tang","email":"","orcid":"","institution":"Xinqiao Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Tang","suffix":""},{"id":627802928,"identity":"71e443c2-6266-42c6-858b-ad9e2bfefc0a","order_by":5,"name":"Yiyang Hu","email":"","orcid":"","institution":"The General Hospital of Western Theater Command","correspondingAuthor":false,"prefix":"","firstName":"Yiyang","middleName":"","lastName":"Hu","suffix":""},{"id":627802931,"identity":"e0d386c6-3462-49d3-a199-aab0f8fe094e","order_by":6,"name":"Wanneng Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYDADfijN2EC0FkmQ0gMkaTE4QKwWg+Nnjz34uKM2cfPxw9ukPzDYyG44wPzsAV4tZ/LSDWeeOZ647UxamcQBhjTjDQfYzA3waTE7kGMmzdt2LHfbDR4zoJbDiRsO8LBJ4NVy/o2Z9F+gls0zwFr+E6HlBtAWxraa3A0SYC0HCGuxv/HGTLK37UD9jDNpxRZnDJKNZx5mM8OrRbI/x0ziZ1udMX/74Y03KirsZPuONz/DqwUKDoMIAzBiYCZCPRDUQbWMglEwCkbBKMACAJkjTioH6ojLAAAAAElFTkSuQmCC","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Wanneng","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-04-08 09:57:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9355144/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9355144/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107738662,"identity":"fa355b32-0575-4d20-83a2-061a52be4e9c","added_by":"auto","created_at":"2026-04-24 14:31:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1802150,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDHA supplementation ameliorated DSS-induced colitis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eExperimental design illustrated by a schematic diagram. \u003cstrong\u003e(B) \u003c/strong\u003eBody weights. \u003cstrong\u003e(C)\u003c/strong\u003e Survival curve. \u003cstrong\u003e(D) \u003c/strong\u003eDAI scores. \u003cstrong\u003e(E) \u003c/strong\u003eRepresentative pictures of the measurements of colon length. \u003cstrong\u003e(F) \u003c/strong\u003eColon lengths. \u003cstrong\u003e(G)\u003c/strong\u003e Representative microscopy images of H\u0026amp;E staining (40× and 100× magnification);\u003cstrong\u003e (H)\u003c/strong\u003e Histology scores. \u003cstrong\u003e(I-K)\u003c/strong\u003e mRNA expression of IL-1β, IL-6, and TNF-α. Data (A–K; n = 8 mice/group) are presented as the means ± SDs. P values were calculated by two-way ANOVA (\u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Onlinefig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9355144/v1/3727db65319f5477dc63b5b7.png"},{"id":107738666,"identity":"c7644c82-6d81-4b7a-ba10-289c8b6ec25c","added_by":"auto","created_at":"2026-04-24 14:31:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1422647,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe protective effects of DHA against DSS-induced colitis disappeared after gut microbiota depletion.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Design of the ABX experiment.\u003cstrong\u003e (B)\u003c/strong\u003e Body weights. \u003cstrong\u003e(C)\u003c/strong\u003eSurvival curve. \u003cstrong\u003e(D) \u003c/strong\u003eDAI scores.\u003cstrong\u003e (E)\u003c/strong\u003e Representative pictures of the measurements of colon length; \u003cstrong\u003e(F)\u003c/strong\u003e Colon lengths. \u003cstrong\u003e(G)\u003c/strong\u003eRepresentative microscopy images of H\u0026amp;E staining (40× and 100× magnification); \u003cstrong\u003e(H)\u003c/strong\u003e Histology scores. \u003cstrong\u003e(A–H)\u003c/strong\u003e n = 8 mice per group; data are presented as the means ± SDs. P values were calculated by an unpaired t test (\u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Onlinefig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9355144/v1/3a9a1993dea8ee41e83c59c5.png"},{"id":107738663,"identity":"957aa3c5-51bc-49f6-9075-b42de17794ed","added_by":"auto","created_at":"2026-04-24 14:31:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1341169,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFecal microbial transplantation mitigated DSS-induced experimental colitis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eDesign of the FMT experiment. \u003cstrong\u003e(B)\u003c/strong\u003e Body weights. \u003cstrong\u003e(C)\u003c/strong\u003e Survival curve. \u003cstrong\u003e(D)\u003c/strong\u003e DAI scores. \u003cstrong\u003e(E)\u003c/strong\u003eRepresentative pictures of the measurements of colon length; \u003cstrong\u003e(F)\u003c/strong\u003e Colon lengths. \u003cstrong\u003e(G)\u003c/strong\u003e Representative microscopy images of H\u0026amp;E staining (40× and 100× magnification); (H) Histology scores. \u003cstrong\u003e(A-H)\u003c/strong\u003e n = 7 mice/group. Data are presented as the means ± SDs. P values were calculated by an unpaired t test (\u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Onlinefig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9355144/v1/523003056e7239f533c564ac.png"},{"id":107868853,"identity":"cb930dc4-5d63-470d-b4d2-0cbaea442e1a","added_by":"auto","created_at":"2026-04-27 07:34:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":796884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;DHA treatment significantly altered the diversity and composition of the gut microbiota.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eα diversity. \u003cstrong\u003e(B) \u003c/strong\u003eβ diversity. \u003cstrong\u003e(C)\u003c/strong\u003e Bar plots of the phyla in the DSS and DSS+DHA groups. \u003cstrong\u003e(D)\u003c/strong\u003e Bar plots of the genera in the DSS and DSS+DHA groups. \u003cstrong\u003e(E)\u003c/strong\u003eHeatmap of the selected most differentially abundant features at the genus level. \u003cstrong\u003e(F) \u003c/strong\u003eLinear discriminant analysis (LDA) score plot of differentially abundant taxonomic features between the DSS and DSS+LC groups (LDA score for discriminative features \u0026gt; 3.5).\u003cstrong\u003e (G) \u003c/strong\u003eTaxonomic cladogram produced from LEfSe analysis. Green indicates taxa enriched in the DSS groups; orange indicates taxa enriched in the DSS+DHA groups.\u003c/p\u003e","description":"","filename":"Onlinefig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9355144/v1/b2d4512d341e74e13f9e1c95.png"},{"id":107738669,"identity":"7cba5afb-52cf-495b-8c48-291cbfd8ab3c","added_by":"auto","created_at":"2026-04-24 14:31:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3382302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDHA improves DSS-induced intestinal barrier disruption through targeting of the gut microbiota.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e FITC-dextran serum concentrations. \u003cstrong\u003e(B)\u003c/strong\u003e The colonic protein expression of occludin and ZO-1 in each group were measured by Western blotting. \u003cstrong\u003e(C)\u003c/strong\u003e Representative images of immunohistochemical staining for ZO-1 and occludin in colon samples from the DSS and DSS+DHA groups. \u003cstrong\u003e(D)\u003c/strong\u003eThe positive protein integral optical density was determined using ImageJ software. \u003cstrong\u003e(E)\u003c/strong\u003e FITC-dextran serum concentrations.\u003cstrong\u003e (F)\u003c/strong\u003e The colonic protein expression of occludin and ZO-1 in each group were measured by Western blotting. \u003cstrong\u003e(G) \u003c/strong\u003eRepresentative images of immunohistochemical staining for ZO-1 and occludin in colon samples from the ABX(DSS) and ABX(DSS+DHA) groups. \u003cstrong\u003e(H)\u003c/strong\u003eThe positive protein integral optical density was determined using ImageJ software. \u003cstrong\u003e(I) \u003c/strong\u003eFITC-dextran serum concentrations. \u003cstrong\u003e(J) \u003c/strong\u003eThe colonic protein expression of occludin and ZO-1 in each group were measured by Western blotting. \u003cstrong\u003e(K) \u003c/strong\u003eRepresentative images of immunohistochemical staining for ZO-1 and occludin in colon samples from the FMT(DSS) and FMT(DSS+DHA) groups. \u003cstrong\u003e(L) \u003c/strong\u003eIntegrated density of positive staining was determined using ImageJ software.\u003cstrong\u003e (A–L)\u003c/strong\u003e n = 3 mice/group. Data are presented as the means ± SDs. P values were calculated by an unpaired t test (\u003cem\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Onlinefig5.png","url":"https://assets-eu.researchsquare.com/files/rs-9355144/v1/fe87819765bf61049e28d0e3.png"},{"id":107738677,"identity":"781d9d9f-b99c-48d1-a874-e258d4819063","added_by":"auto","created_at":"2026-04-24 14:31:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1071610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDHA treatment increased the frequency of Tregs in mice with DSS-induced colitis in a microbiota-dependent manner.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eRepresentative flow cytometry plots of Treg cells (CD25+Foxp3+CD4+) in the colonic LP from DSS- and DSS+DHA-treated mice and bar charts of the percentage of Treg cells. \u003cstrong\u003e(B)\u003c/strong\u003e Representative flow cytometry plots of Treg cells (CD25+Foxp3+CD4+) in the colonic LP from ABX(DSS) and ABX(DSS+DHA) group mice and bar charts of the percentage of Treg cells. \u003cstrong\u003e(C) \u003c/strong\u003eRepresentative flow cytometry plots of Treg cells (CD25+Foxp3+CD4+) in the colonic LP from FMT(DSS) and FMT(DSS+DHA) group mice and bar charts of the percentage of Treg cells. (A-C) n = 3 mice/group. Data are presented as the means ± SDs. P values were calculated by an unpaired t test (\u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Onlinefig6.png","url":"https://assets-eu.researchsquare.com/files/rs-9355144/v1/71a417b95c60da13cb0028a4.png"},{"id":107738664,"identity":"22dbff9a-6929-420e-a75c-7c318ade5998","added_by":"auto","created_at":"2026-04-24 14:31:42","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":211236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of the protective effects of DHA during ulcerative colitis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMechanisms include maintaining the balance of the microbiota, promoting the expression of tight junction proteins and suppressing inflammatory cell infiltration.\u003c/p\u003e","description":"","filename":"fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9355144/v1/960a23d11bdf05497351660e.jpg"},{"id":108490840,"identity":"124e485d-839f-47e9-b52b-cbdf49719b6c","added_by":"auto","created_at":"2026-05-05 09:49:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2356099,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9355144/v1/1246c41c-b641-495c-95cd-1567d4bfce01.pdf"},{"id":107738661,"identity":"61fe4e0d-f5da-4a14-a41f-e63d37f78b09","added_by":"auto","created_at":"2026-04-24 14:31:42","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16945,"visible":true,"origin":"","legend":"","description":"","filename":"Supplemental.docx","url":"https://assets-eu.researchsquare.com/files/rs-9355144/v1/644f98e3a0dfaeb8e3cfdaec.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Docosahexaenoic acid alleviates DSS-induced colitis by regulating the gut microbiota and restoring the gut barrier","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInflammatory bowel disease (IBD) comprises chronic, inflammatory disorders, primarily ulcerative colitis (UC) and Crohn's disease (CD). The incidence of IBD has increased over the years, particularly in developing countries [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, current IBD treatments are often associated with severe side effects and limited efficacy [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Consequently, there is an urgent need to identify alternative therapies and explore novel, safe, and effective nutritional or dietary interventions for IBD management.\u003c/p\u003e \u003cp\u003eUC is a multifactorial disease arising from the interplay of genetic, environmental, microbiotic, and mucosal immunomodulatory factors [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Mounting evidence has implicated gut microbiota dysbiosis as a key contributor to IBD pathogenesis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Studies have shown that IBD patients exhibit gut dysbiosis characterized by reduced biodiversity and compositional imbalance. Specifically, compared with healthy controls, IBD patients display increased abundances of Proteobacteria and Actinobacteria and decreased abundances of Firmicutes and Bacteroidetes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, fecal microbiota transplantation (FMT) has emerged as an effective and relatively safe therapy for patients with refractory or recurrent IBD[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, targeting the intestinal microbiota represents a promising approach to IBD management.\u003c/p\u003e \u003cp\u003eThe intestinal mucosal barrier comprises mechanical, immune, biological, and chemical components that act synergistically to prevent the infiltration of pathogenic microorganisms [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Barrier impairment is a hallmark feature of UC[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Microbiota imbalances can dysregulate intestinal immunity, compromising epithelial integrity and repair, thereby promoting intestinal inflammation. Aberrant activation of both innate and adaptive immunity characterizes UC pathology [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Activated immune cells proliferate and release proinflammatory effectors, including TNF-α, IL-17A, and other mediators, which further damage the barrier and drive UC progression [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Conversely, certain immune cells, such as regulatory T cells (Tregs), produce the protective cytokine IL-10, which has anti-inflammatory effects [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDHA is an essential polyunsaturated fatty acid for mammals, since endogenous synthesis is minimal, and it must be obtained from marine or plant sources [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Numerous human and rodent studies have demonstrated that dietary DHA supplementation ameliorates cognitive decline in elderly individuals[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and Alzheimer's disease patients [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, the evidence suggests that DHA possesses anti-inflammatory properties with potential benefits for treating inflammation-related diseases, including rheumatoid arthritis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], cancer [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], cardiovascular disease [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and IBD [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Clinical studies have indicated an inverse correlation between the intake of DHA and the risk of developing IBD, including both UC and CD [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the precise molecular mechanisms underlying the therapeutic effects of DHA in IBD remain incompletely understood and warrant further investigation.\u003c/p\u003e \u003cp\u003eThis study aimed to elucidate the mechanisms by which DHA alleviates UC and to investigate its modulatory effects on the intestinal microbiota, mucosal barrier, and colonic immune homeostasis. Our findings provide new insights into the potential utility of DHA for treating IBD and related gastrointestinal disorders.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDextran sulfate sodium (DSS)-induced colitis\u003c/h2\u003e \u003cp\u003eFollowing a 7-day acclimatization period, all the mice were randomly divided into four groups. Colitis was then induced using a previously reported method with minor modifications[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Briefly, the mice were given 2.5% DSS (MP Biomedicals, UK) in their drinking water for 7 consecutive days, followed by 5 days of normal water. For the mice in the DHA\u0026thinsp;+\u0026thinsp;DSS group, DHA (35 mg/kg) was suspended in corn oil and orally administered to the mice from the initiation of the experiment until its completion. Disease activity index (DAI)-related parameters, such as body weight loss, stool consistency, and hematochezia, were monitored and recorded daily. On Day 12, the mice were humanely euthanized, and the colon lengths were measured. Fecal samples were collected for subsequent analysis of the gut microbiota.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntibiotic cocktail (ABX) experiments and fecal microbiota transplantation (FMT)\u003c/h3\u003e\n\u003cp\u003eTo eradicate the intestinal microbiota in mice, a broad-spectrum ABX consisting of ampicillin (200 mg/kg), neomycin sulfate (200 mg/kg), metronidazole (200 mg/kg), and vancomycin (100 mg/kg) was administered orally once daily for 5 days. All antibiotics were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). FMT was performed using a modified previously described method. Briefly, colitis was induced in the donor mice, which were given either drinking water or DHA as previously described. Recipient mice were given ABX for 5 days to deplete their gut microbiota. Afterward, fresh stools from the donor mice were collected, suspended in PBS, and administered to the recipient mice via oral gavage daily for 5 days.\u003c/p\u003e\n\u003ch3\u003eHistopathology\u003c/h3\u003e\n\u003cp\u003eDistal colon samples were collected and fixed in a 10% neutral formalin solution for 24 h. After fixation, the samples were embedded in paraffin. Sections (5 \u0026micro;m thick) were prepared from the embedded samples and subjected to hematoxylin and eosin (H\u0026amp;E) staining. The histological scores were determined on the basis of established criteria [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], which consider the extent of epithelial damage and the depth of inflammatory infiltration.\u003c/p\u003e\n\u003ch3\u003eFITC-dextran intestinal permeability assay\u003c/h3\u003e\n\u003cp\u003eAs previously reported, intestinal permeability was assessed using fluorescein isothiocyanate (FITC)-dextran [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Briefly, the mice were fasted for 3 h before oral administration of 0.6 mg/g body weight of FITC-dextran (Sigma\u0026ndash;Aldrich). Serum samples were collected after 4 h, and fluorescence measurements were performed at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a Varioskan Flash Multimode Reader (Thermo Scientific).\u003c/p\u003e\n\u003ch3\u003eRNA extraction and quantitative real-time PCR\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eRNA extraction and quantitative real-time PCR\u003c/div\u003e \u003cp\u003eRNA was isolated from the colon tissues using RNAiso Plus reagent (Takara Biotechnology, Dalian, China). cDNA was subsequently synthesized from 1 \u0026micro;g RNA using a PrimeScript RT reagent kit (Takara, Japan). Quantitative real-time polymerase chain reaction (qRT\u0026ndash;PCR) was performed on an ABI 7300 HT Fast Real-Time Cycler (Applied Biosystems, USA) using a TB Green Premix Ex Taq II kit (Takara, Japan). The mRNA expression of the target genes was normalized to that of the reference gene, Actin, and quantified using the 2-ΔΔCt method. The primers used for the qRT\u0026ndash;PCR analysis were synthesized by Sangon Biotech Limited (Shanghai, China), and their sequences are provided in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eColon tissue samples were homogenized in RIPA buffer supplemented with PMSF protease inhibitors for efficient protein extraction. The protein concentration in the resulting lysates was determined using a BCA protein assay kit according to the manufacturer's recommended protocol (Beyotime Biotechnology, Shanghai, China). Equivalent amounts of protein were then electrophoresed on a 10% SDS\u0026ndash;PAGE gel and transferred to an Immobilon PVDF membrane. To prevent nonspecific binding, the membrane was incubated with 5% nonfat dry milk in TBST for 1 h at room temperature. After the blocking step, the membrane was probed with primary antibodies against ZO-1, occludin, or β-actin at a dilution of 1:1000 and incubated overnight at 4\u0026deg;C. After thorough washing, the membrane was further incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the protein bands were visualized using an enhanced chemiluminescence (ECL) substrate (Thermo Scientific).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA extraction and 16S rRNA gene sequencing\u003c/h3\u003e\n\u003cp\u003eTotal DNA was extracted from each fecal sample using a TIANamp stool DNA kit (TIANGEN, Beijing, China). The quality and concentration of the extracted DNA were evaluated using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and 1.0% agarose gel electrophoresis. To amplify the V3-V4 hypervariable regions of the bacterial 16S-rDNA gene, we utilized the following primers: 341F (5'-ACTCCTACGGGRSGCAGCAG-3') and 806R (5'-GGACTACVV GGGTATCTAATC-3'). After purification with a GeneJET gel extraction kit (Thermo Fisher Scientific, USA), the PCR products were sequenced using an Illumina NovaSeq 6000 platform (Novogene, Tianjin, China).\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eImmunohistochemical analysis was performed according to a previously published protocol[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Briefly, 5-\u0026micro;m sections of paraffin-embedded tissues were deparaffinized in xylene and rehydrated using ethanol. Antigen retrieval was accomplished by heating the sections in 10 mM sodium citrate buffer (pH 6.0) in a microwave for 10 min, and then cooling them to room temperature. Subsequently, the sections were blocked with a solution of 3% H2O2 and goat serum and then incubated overnight at 4\u0026deg;C with primary antibodies specific to occludin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or zonula occludens (ZO)-1 (Santa Cruz Biotechnology). After incubation, the sections were washed three times with PBS for 5 min each and subsequently stained using 3,3'-diaminobenzidine-tetrahydrochloride (DAB). Finally, all the samples were examined under a microscope.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and 16S rRNA gene sequencing\u003c/h2\u003e \u003cp\u003eTotal DNA was extracted from each fecal sample using a TIANamp stool DNA kit (TIANGEN, Beijing, China). The quality and concentration of the extracted DNA were evaluated using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and 1.0% agarose gel electrophoresis. To amplify the V3-V4 hypervariable regions of the bacterial 16S-rDNA gene, we utilized the following primers: 341F (5'-ACTCCTACGGGRSGCAGCAG-3') and 806R (5'-GGACTACVV GGGTATCTAATC-3'). After purification with a GeneJET gel extraction kit (Thermo Fisher Scientific, USA), the PCR products were sequenced using an Illumina NovaSeq 6000 platform (Novogene, Tianjin, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic analysis\u003c/h2\u003e \u003cp\u003eThe QIIME2 platform (version 2020.2) was used to analyze the raw sequencing data. To ensure the accuracy and reliability of the data obtained, the reads were subjected to denoising and quality filtering using the DADA2 approach. The data were subsequently clustered into operational taxonomic units (OTUs) using classify-sklearn against the Greengenes database (version 13.8) with a similarity threshold of 97%. The Chao1, Shannon, and ACE indices were used to assess α diversity, while weighted and unweighted UniFrac distances were used to assess β diversity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometric analysis\u003c/h2\u003e \u003cp\u003eMouse colons were isolated and subjected to a series of procedures. First, the colons were resected and longitudinally opened, followed by washing with cold PBS. Afterward, the colons were incubated in 10 mL Hank's balanced salt solution (HBSS; Lonza, Basel, Switzerland), which was supplemented with 1 mM dithiothreitol (DTT) and 5 mM ethylenediaminetetraacetic acid (EDTA), at 37\u0026deg;C for 30 minutes with constant shaking at 300 rpm on an orbital shaker. After being washed, the colons were carefully minced and cut into smaller pieces before being digested with 15 mL of Hank's balanced salt solution (HBSS), which contained 10% fetal bovine serum (FBS), 1.5 mg/mL type-VI collagenase, and 40 \u0026micro;g/mL DNase I. The digestion process was conducted at 37\u0026deg;C for 40 min under continuous agitation at 300 rpm. The obtained single-cell suspensions were passed through 70-\u0026micro;m cell filters to remove any remaining tissue debris. The filtered suspensions were then subjected to centrifugation at 1300 rpm for 5 min at 4\u0026deg;C. The cells were then stained with a live/dead stain and several antibodies, including PerCP5.5-conjugated anti-mouse CD45, Brilliant Violet 510-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD25, and AF647-conjugated anti-mouse Foxp3, according to the manufacturer's instructions. The stained cells were subsequently analyzed on a Beckman Gallios flow cytometer, and the resulting data were analyzed using FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experimental data are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors of the mean (SEMs). The data were analyzed using unpaired and independent Student's t tests for comparisons of two groups, and one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test was used to compare multiple groups. *\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05 i\u003c/em\u003endicates a significant difference, \u003cem\u003e**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e indicates an extremely significant difference, and NS indicates no significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDHA administration significantly alleviates DSS-induced colitis\u003c/h2\u003e \u003cp\u003eTo investigate the potential anti-inflammatory effects of DHA, we employed a widely used acute colitis mouse model induced by DSS, which mimics several key features of human UC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Throughout the establishment of the model, the mice experienced weight loss and progressively increased diarrhea accompanied by bloody and mucous stools. DHA administration significantly ameliorated DSS-induced colitis, as shown by reduced weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), mortality (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), and DAI scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Notably, the administration of DHA effectively reversed colon shortening, a notable characteristic of DSS-induced colitis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u0026ndash;F). Additionally, histopathological analysis of colon sections revealed that the glandular structure was disrupted, the number of goblet cells was reduced, and substantial infiltration of inflammatory cells was observed in the DSS group, whereas DHA treatment alleviated these changes and mitigated histopathological scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H). Moreover, the administration of DHA led to the downregulation of proinflammatory cytokine expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI\u0026ndash;K). Collectively, these findings strongly suggest that DHA supplementation significantly alleviates the symptoms associated with DSS-induced colitis, highlighting its potential as a therapeutic intervention.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDHA amelioration of DSS-induced colitis is dependent on the gut microbiota\u003c/h2\u003e \u003cp\u003eIntestinal microbiota dysbiosis has been linked to the development and progression of UC. Recent studies have suggested that DHA can increase the abundance of probiotics. To examine the potential role of the gut microbiota in mediating the therapeutic effects of DHA on DSS-induced colitis, the gut microbiota was depleted using antibiotics prior to colitis induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Our results show that antibiotic depletion of the microbiota significantly reduced the therapeutic effect of DHA. The protective effect of DHA was lost when the gut flora was exhausted, as evidenced by comparable weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), mortality rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), DAI scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), colonic lengths (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and F) and colonic histological changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and H) between the ABX(DSS) group and the ABX(DSS\u0026thinsp;+\u0026thinsp;DHA) group. Collectively, these data suggest that the protective effect of DHA on DSS-induced colitis depends on the presence of a healthy gut microbiota.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAnticolitis effects are transferable via the gut microbiota\u003c/h2\u003e \u003cp\u003eTo determine whether the attenuation of DSS-induced colitis by DHA was mediated by alterations in the microbiota, we performed FMT. Intestinal microbiota-depleted mice were transplanted with the gut microbiota of either DSS-treated (FMT(DSS)) or DHA-treated (FMT(DSS\u0026thinsp;+\u0026thinsp;DHA)) donor mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The results demonstrated that recipients of FMT from DSS\u0026thinsp;+\u0026thinsp;DHA-treated donors exhibited reduced colitis symptoms compared to recipients of FMT from DSS-treated donors. The FMT(DSS\u0026thinsp;+\u0026thinsp;DHA) group showed improvements in various parameters, including reduced weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), an increased survival rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), a lower disease activity index (DAI) score (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), and a longer colon length (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). Histological analysis of colon tissue from FMT(DSS\u0026thinsp;+\u0026thinsp;DHA) recipient mice revealed less inflammation, less histological damage, and lower histology scores than those from FMT(DSS) recipient mice did (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and H). Collectively, these findings indicate that DHA-induced changes in the microbiota during DSS treatment are beneficial to mice with colitis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDHA administration modulates the composition and diversity of the gut microbiota\u003c/h2\u003e \u003cp\u003eTo assess the effect of DHA on the gut microbiota composition, we performed high-throughput gene sequencing of fecal bacterial 16S rRNA genes isolated from mice in the DSS group and the DSS\u0026thinsp;+\u0026thinsp;DHA group. Principal coordinate analysis (PCoA) based on the Bray\u0026ndash;Curtis metric and Jaccard distance revealed a distinct separation in the DSS\u0026thinsp;+\u0026thinsp;DHA group compared with the DSS group, indicating greater microbial community richness and diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Principal coordinate analysis (PCoA) based on Bray\u0026ndash;Curtis and Jaccard distances revealed distinct separations in the gut microbiota structure between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). We assessed compositional differences in the composition of the gut flora. Taxonomic classification at the phylum level revealed Bacteroidetes and Firmicutes as the dominant orders in both the DSS and DSS\u0026thinsp;+\u0026thinsp;DHA groups. However, the Bacteroidetes/Firmicutes ratio significantly increased in the DSS\u0026thinsp;+\u0026thinsp;DHA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Similarly, at the genus leve, DHA treatment significantly increased the abundance of Bifidobacterium and Ruminococcus_torques_group but significantly ecreased the abundance of Ileibacterium and Ruminococcaceae_UCG_014, as represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD. To visualize compositional differences in gut microbiota composition between the two groups, a heatmap was generated on the basis of operational taxonomic unit (OTU) abundance at the genus level. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, the abundance of the genus Bifidobacterium was relatively high in the DSS\u0026thinsp;+\u0026thinsp;DHA group, whereas the abundance of the genus Ruminococcaceae_UCG_014 was significantly enriched in the DSS group. Furthermore, to identify the predominant bacteria affected by DHA treatment, we conducted linear discriminant analysis effect size (LEfSe) analysis, which revealed Ruminococcaceae_UCG_010, Desulfovibrio, and Lactobacillus as the dominant bacteria in DSS-treated mice, whereas Bifidobacterium and Tyzzerella emerged as the predominant bacteria in mice receiving DHA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G). Our findings suggest that DHA treatment significantly altered the diversity and composition of the gut microbiota in mice with DSS-induced colitis. Taken together, our findings shed light on the profound impact of DHA treatment on the diversity and composition of the gut microbiota in mice afflicted with DSS-induced colitis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDHA ameliorates DSS-induced intestinal barrier damage via the gut microbiota\u003c/h2\u003e \u003cp\u003eDisturbances in the composition of the gut microbiota may disrupt gut barrier function, resulting in various pathological conditions. To evaluate the effect of DHA on intestinal barrier integrity, we performed experiments. First, we evaluated the intestinal permeability of the mice by measuring the concentration of fluorescein isothiocyanate-conjugated dextran (FITC-dextran) in the serum following oral administration. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, compared with DSS\u0026thinsp;+\u0026thinsp;DHA mice, mice with DSS-induced colitis exhibited significantly increased serum levels of FITC-dextran. The protein expression of the tight junction markers occludin and ZO-1 was significantly greater in the colons of the DHA-treated mice than in those of the untreated colitis mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Immunohistochemistry confirmed that the expression and localization of ZO-1 and occludin were significantly greater in the DHA treatment group than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). In summary, these findings indicate that DHA administration alleviates intestinal permeability and upregulates the expression of tight junction proteins, ultimately contributing to reduced inflammation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further confirm the pivotal role of the gut microbiota in the therapeutic efficacy of DHA, we performed antibiotic (ABX) and fecal microbiota transplantation (FMT) experiments to investigate the effects of DHA on intestinal barrier function. Our results indicate that ABX-treated mice did not exhibit changes in intestinal permeability or the expression of epithelial tight junction (TJ) proteins in response to DHA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE\u0026ndash;H). In contrast, FMT from DHA-treated mice restored intestinal barrier integrity in mice with colitis, as evidenced by the assessment of TJ protein expression and intestinal permeability (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI\u0026ndash;L). These findings underscore the essential role of the gut microbiota in mediating the beneficial effects of DHA on intestinal barrier function in mice with colitis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDHA treatment increased the frequency of Tregs in mice with DSS-induced colitis in a microbiota-dependent manner\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCompared with no treatment, DHA treatment significantly increased the frequency of colonic Treg cells (characterized by CD25⁺Foxp3⁺ expression) (4.24% vs. 2.66%; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). However, antibiotic-mediated microbiota depletion abrogated this effect, as Treg percentages showed no significant difference between the ABX(DSS) and ABX(DSS\u0026thinsp;+\u0026thinsp;DHA) groups (3.64% vs. 3.52%; P\u0026thinsp;=\u0026thinsp;0.5875; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Conversely, recipients of FMT from DHA-treated donors exhibited higher Treg frequency than those receiving FMT from DSS-treated donors (5.23% vs. 1.69%; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). These data demonstrate that DHA supplementation expands colonic Treg cell populations via microbiota-dependent mechanisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we evaluated the therapeutic effects of DHA on DSS-induced colitis and explored the underlying mechanisms. Our results demonstrate that DHA treatment significantly alleviated DSS-induced colitis, as evidenced by reduced weight loss, mortality, and DAI scores; increased colon length; improved histology scores; and decreased proinflammatory cytokine levels. ABX and FMT experiments confirmed that these beneficial effects were dependent on the gut microbiota. Moreover, DHA administration increased gut microbial diversity and altered its composition. DHA treatment also enhanced intestinal barrier integrity and increased the proportion of anti-inflammatory colonic Treg cells, collectively suppressing inflammatory and immune responses. These findings support the potential efficacy of DHA supplementation for mitigating DSS-induced colitis.\u003c/p\u003e \u003cp\u003eOmega-3 polyunsaturated fatty acids (PUFAs) are considered essential fatty acids because they cannot be endogenously synthesized by the body and must be acquired through dietary sources. A large body of evidence [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]suggests that n-3 PUFAs have anti-inflammatory effects[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], particularly in the context of IBD[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Studies have indicated that increased consumption of n-6 polyunsaturated fatty acids (PUFAs) and decreased intake of n-3 PUFAs are associated with an increased risk of developing UC[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. For example, a prospective cohort study revealed that n-6 PUFA intake was positively correlated with the risk of UC, whereas n-3 PUFA intake was negatively correlated[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Similarly, a long-term follow-up study revealed a negative association between the consumption of n-3 PUFAs and the risk of UC[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the specific role of DHA in the development of colitis remains unclear. Therefore, the effects of DHA on UC should be investigated, and the underlying mechanisms involved should be elucidated. In this study, we evaluated the therapeutic potential of oral DHA administration in mice with DSS-induced colitis. Consistent with these findings, our results revealed a significant protective effect of DHA treatment against DSS-induced colitis, as indicated by attenuated weight loss, improved survival rates, reduced disease activity index (DAI) scores, ameliorated mucosal injury, and decreased secretion of inflammatory cytokines.\u003c/p\u003e \u003cp\u003eEmerging studies have shown significant differences in the composition of the gut microbiota between people with UC and healthy individuals[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Reduced microbial diversity and dysbiosis between probiotic and harmful bacteria play crucial roles in the pathogenesis of UC[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In addition, alterations in the microbiota lead to changes in its metabolites, which have a major impact on gut health[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, modulating the gut microbiota is a promising therapeutic strategy for patients with UC. Indeed, studies have shown that restoring microbial balance can alleviate DSS-induced colitis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]in mice[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. As the gut microbiome has been recognized as a critical player in the development and progression of IBD[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], it is important to investigate whether the gut microbiome contributes to the anticolitis effects of DHA. ABX experiments have revealed that microbiota depletion abolished the protective effects of DHA, as the severity of colitis was comparable between DHA-treated and untreated ABX mice. Subsequently, FMT from DHA-treated donors ameliorated colitis in recipient mice, confirming a microbiota-dependent mechanism. Taken together, our results suggest that DHA treatment ameliorates DSS-induced colitis in a gut microbiota-dependent manner.\u003c/p\u003e \u003cp\u003eWe further analyzed the α-diversity of the intestinal flora using Chao1 and Shannon indices and found that DHA supplementation significantly increased gut microbiota diversity. Our PCoA results revealed that DHA administration significantly altered the biological community structure in mice with colitis. Additionally, LEfSe analysis revealed that Bifidobacterium was significantly enriched in the DHA-treated mice. Previous research has shown that Bifidobacterium administration can relieve colitis. Yao et al. reported that Bifidobacterium longum administration can alleviate colitis in mice and may be used as an alternative or adjunctive treatment for IBD patients[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Cui et al. reported that Bifidobacterium bifidum administration ameliorated DSS-induced colitis by enhancing the intestinal barrier and anti-inflammation, potentially via the AhR pathway[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Our findings, together with those of previous reports[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e][\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], suggest that DHA mitigates DSS-induced dysbiosis by increasing microbial diversity and enriching beneficial taxa such as Bifidobacterium.\u003c/p\u003e \u003cp\u003eDysfunction of the intestinal epithelial barrier is a critical factor in the pathogenesis of UC[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Impairment of the intestinal mucosal barrier is the initiating event in colitis, leading to an increase in intestinal permeability and the infiltration of antigens, toxins, and pathogens into the mucosal tissue, ultimately leading to inflammation[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, preserving TJ protein expression and function to reduce epithelial permeability is essential for IBD treatment [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. TJs are important structures for maintaining the mechanical barrier and permeability of the intestinal epithelium, and tight junction proteins include claudin-1, occludin, ZO-1, and others [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. We found that DHA treatment reduced intestinal permeability in DSS-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Furthermore, DHA restored ZO-1 and occludin protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C, D) in the DSS-induced colitis model, suggesting that DHA may help restore the integrity of the intestinal mucosal barrier. These results indicate that DHA effectively restored intestinal barrier function in mice with experimental colitis.\u003c/p\u003e \u003cp\u003eMaintaining a balance between pro- and anti-inflammatory mechanisms is critical for intestinal immune homeostasis, which can be influenced by the gut microbiota and metabolites[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Treg cells play a crucial role in preserving intestinal homeostasis by restraining the activity of other effector T cells and suppressing the secretion of proinflammatory cytokines, thus limiting the progression of inflammation in the colon[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Alterations in the number, phenotype, and suppressive function of Treg cells may contribute to the development of IBD[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. We observed a significant increase in the frequency of colonic Treg cells following DHA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). After being administered a broad-spectrum antibiotic cocktail, the ABX(DSS) group of mice exhibited similar percentages of colonic Treg cells to those in the ABX(DSS\u0026thinsp;+\u0026thinsp;DHA) group. Additionally, the FMT(DSS\u0026thinsp;+\u0026thinsp;DHA) group, which received FMT from the These findings demonstrate that DHA enhances Treg cell responses in a gut microbiota-dependent manner. Furthermore, DHA suppressed DSS-induced increases in proinflammatory cytokines (IL-1β, IL-6, TNF-α) and reversed the decrease in anti-inflammatory IL-10 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI\u0026ndash;K). These results indicate that the therapeutic effect of DHA involves modulating cytokine balance, suppressing proinflammatory activity and promoting anti-inflammatory responses.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our findings demonstrate that DHA alleviates experimental colitis in mice by modulating the gut microbiota, preserving intestinal barrier integrity, and regulating mucosal immunity, primarily through microbiota-dependent mechanisms(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Moreover, these results suggest that DHA holds promise as a candidate drug for UC therapy. Our future research will focus on identifying specific bacterial mediators of the effects of DHA and strategies to optimize its therapeutic efficacy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eInstitutional Review Board Statement\u003c/h2\u003e \u003cp\u003e This study was conducted with prior approval from the Animal Care and Use Committee of Third Military Medical University (Chongqing, China, approval code:20265512) and adhered to the principles and guidelines outlined in the Guide for the Care and Use of Laboratory Animals, ensuring the ethical and responsible utilization of animals in experimental research.\u003c/p\u003e \u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eThe author(s) stated that this study was carried out without any commercial or financial affiliations that might be perceived as a potential conflict of interest.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by the Youth Project of the China National Natural Science Foundation under Grant No. 82300680.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWN Wang conceived and designed this study;Y Yan performed the animal studies. LY Wu performed the molecular biology experiments. L Tang performed the high-throughput 16S-rRNA data analysis. SM Wang provided technical support. SM Wang and Sp Hu drafted and edited the manuscript. YY Hu and SP Hu supervised the study and revised the manuscript. All the authors contributed to the article and approved the submitted version.\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e \u003cp\u003eAll data reported in this article will be shared by the lead contact upon request. This article does not report original code.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAjabnoor SM, Thorpe G, Abdelhamid A, Hooper L (2021) Long-term effects of increasing omega-3, omega-6 and total polyunsaturated fats on inflammatory bowel disease and markers of inflammation: a systematic review and meta-analysis of randomized controlled trials. 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Semin Cancer Biol 40\u0026ndash;41:141\u0026ndash;159\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"docosahexaenoic acid, colitis, gut microbiota, gut barrier, dietary supplement, polyunsaturated fatty acid","lastPublishedDoi":"10.21203/rs.3.rs-9355144/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9355144/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGrowing evidence suggests that docosahexaenoic acid (DHA), a long-chain omega-3 polyunsaturated fatty acid, possesses anti-inflammatory properties potentially beneficial for treating inflammatory diseases. However, the precise molecular mechanisms underlying the therapeutic role of DHA in inflammatory bowel disease (IBD) remain incompletely understood. This study investigated the ameliorative effects of DHA on dextran sodium sulfate (DSS)-induced colitis in mice. We found that DHA significantly ameliorated colitis symptoms, intestinal barrier disruption, and colonic inflammation. Moreover, 16S rDNA sequencing revealed that DHA mitigated gut microbiota dysbiosis. Furthermore, antibiotic cocktail (ABX ) treatment and fecal microbiota transplantation (FMT) demonstrated that the therapeutic potential of DHA depends on the intestinal microbiota. Therefore, our findings provide evidence that DHA could serve as a potential dietary supplement for preventing and treating ulcerative colitis (UC).\u003c/p\u003e","manuscriptTitle":"Docosahexaenoic acid alleviates DSS-induced colitis by regulating the gut microbiota and restoring the gut barrier","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-24 14:31:33","doi":"10.21203/rs.3.rs-9355144/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-17T13:21:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-17T13:20:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-09T01:31:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Microbiology","date":"2026-04-08T09:47:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"074ebb06-5ffc-43ec-8c01-e8848a0ce492","owner":[],"postedDate":"April 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-24T14:31:34+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-24 14:31:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9355144","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9355144","identity":"rs-9355144","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}