From Microbiota to Defense: The Preventive Effect and Mechanism of Total Flavonoids from Sea Buckthorn Leaves in DSS-Induced Colitis

TL;DR

This study shows that flavonoids from sea buckthorn leaves can prevent colitis in mice by improving gut health and reducing inflammation.

Contribution

The study reveals a novel preventive mechanism of sea buckthorn flavonoids in colitis through microbiota and metabolite regulation.

Findings

Flavonoids reduced colonic damage and improved intestinal barrier function in mice.

They suppressed inflammation by lowering TNF-α and IL-6 levels.

Flavonoids increased beneficial gut bacteria and short-chain fatty acid production.

Abstract

Objectives: The main purpose of this study was to evaluate the potential preventive effect of Total Flavonoids from Sea Buckthorn Leaves (Fla) on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice from an integrated perspective of “gut microbiota–host interaction,” and to elucidate its regulatory mechanism within the microbiota–metabolite–barrier–immune axis. Methods: A DSS-induced UC mouse model was established, and mice were randomly assigned into normal control, model, mesalazine, and Fla low, middle, and high–dose groups. Disease severity, colonic barrier integrity, inflammatory cytokines, gut microbiota composition, and short-chain fatty acid levels were evaluated using histopathological, molecular biological, and metabolomic analyses. Result: Fla significantly ameliorated colonic damage and other pathological symptoms. It enhanced intestinal barrier integrity by upregulating ZO-1, E-cadherin, and MUC2 expression and suppressed inflammation by reducing TNF-α and IL-6 levels. Furthermore, Fla reshaped gut microbiota, increasing beneficial Akkermansia abundance, and elevated short-chain fatty acid (SCFA) production, with the most pronounced effects observed at the middle dose. Conclusions: In summary, in this preventive model, Fla synergistically enhances intestinal barrier function and suppresses excessive local inflammatory responses by reshaping the gut microbiota and promoting SCFA production, ultimately alleviating UC. This study provides scientific evidence for the high-value utilization of sea buckthorn leaves (a by-product) and offers new insights for developing microbiome-based preventive strategies for UC.

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1. Introduction

Ulcerative Colitis (UC) is a chronic gastrointestinal disease characterized by complicated etiology and a propensity for relapse. The inflammation typically begins in the rectum and extends continuously to the proximal colon, primarily affecting the colonic mucosa and submucosa [1,2]. Its main clinical features include diarrhea, bloody stools, and abdominal pain. In severe cases, it can lead to various complications such as colonic perforation, hemorrhage, and systemic toxic reactions, significantly impairing patients’ quality of life and increasing the risk of colorectal cancer [3]. In recent years, with changes in lifestyle and antibiotic overuse, the global incidence of UC has continued to rise, making it a significant public health burden. However, the pathological mechanisms of UC are not exactly elucidated, and it is generally believed to result from interactions among genetic, environmental, immune, intestinal barrier, and microbial factors [4,5]. Increasing research is focusing on the core perspective of “gut microbiota–host interaction,” where disordered microbial metabolic function and the resultant imbalance in immune-barrier homeostasis are considered key drivers of UC onset and progression.

A healthy gut microbiota is fundamental to host health, and its disruption is central to UC pathogenesis. UC patients typically present marked gut microbiota dysbiosis, characterized by diminished microbial diversity, decreased abundance of beneficial bacteria, and proliferation of potential pathogens [6,7]. This microbial imbalance compromises intestinal barrier integrity through multiple mechanisms: a reduction in beneficial bacteria weakens the positive regulation of tight junction proteins such as Zonula Occludens (ZO-1), E-cadherin and mucin 2 (MUC2), while an increase in pathogenic bacteria may directly attack epithelial cells and degrade key junction proteins. This cascade of events increases intestinal permeability, facilitating the translocation of bacteria and their metabolites, which, in turn, activates abnormal immune responses. In terms of immunoregulation, microbiota dysbiosis also triggers excessive immune activation. The interplay between regulatory T cells (Tregs) and T helper 17 cells (Th17) is central to the maintenance of immune homeostasis in the gut. In UC patients, microbiota dysbiosis disrupts the Treg/Th17 balance, leading to enhanced immune system activation, overproduction of inflammatory mediators such as Tumor Necrosis Factor-alpha (TNF-α), Interleukin-6 (IL-6), and insufficient secretion of anti-inflammatory factors such as Interleukin-10 (IL-10), ultimately resulting in chronic inflammation and damage to colonic tissue [8,9,10].

Notably, many protective effects of gut microbiota are conveyed by short-chain fatty acids (SCFAs)—key metabolites generated during dietary fiber fermentation. SCFAs (mainly including Acetic acid, Propanoic acid, and Butanoic acid) serve as a critical link between microbiota and host health, regulating intestinal barrier function and immune responses through G protein-coupled receptors (FFAR2, FFAR3, GPR109A) and histone deacetylase (HDAC) inhibition [11]. Butyrate, in particular, serves as the principal energy substrate for colonic epithelial cells, stimulates tight junction protein expression and mucin secretion, and exerts anti-inflammatory effects through GPR109A activation in macrophages [12,13].Thus, the gut microbiota and SCFAs form a closely linked causal chain in UC pathology: initial microbiota dysbiosis leads to a reduction in SCFA-producing bacteria, consequently causing a decrease in SCFAs (particularly butyrate) levels; SCFA deficiency then weakens their restorative effects on the intestinal barrier and their regulatory functions on the immune response, ultimately manifesting as barrier damage and immune dysregulation. The resulting aggravated intestinal inflammatory environment further deteriorates the microbiota composition, forming a positive feedback loop that continuously drives disease progression.

Clinically, drugs such as 5-aminosalicylates, glucocorticoids, and immunosuppressants are commonly used as first-line treatments. However, their application is often limited due to insufficient efficacy, drug resistance, and significant adverse effects [14,15,16]. Consequently, exploring efficient and low-toxicity novel therapeutic strategies from natural products has become a research hotspot. Flavonoids, widely present in plants, have attracted considerable attention due to their notable pharmacological activities, including anti-inflammatory, antioxidant, immunomodulatory, and intestinal barrier-protecting effects [17,18]. Sea buckthorn (Hippophae rhamnoides L.), a plant used for both medicinal and culinary purposes, has fruits rich in vitamins and flavonoids and is widely utilized in functional foods and nutraceuticals [19]. In contrast, sea buckthorn leaves, often discarded as by-products during processing, possess an even higher total flavonoid content than the fruits, indicating significant development potential [20]. We hypothesize that Total Flavonoids from Sea Buckthorn Leaves (Fla) may act as a “gut microbiota modulator,” potentially alleviating UC by promoting the proliferation of beneficial microbiota, leveraging SCFAs as a pivotal point, and consequently coordinating barrier repair and immune balance from the top down. However, their potential in UC prevention and treatment, and particularly whether their mechanism of action involves the systematic regulation of the aforementioned “microbiota- metabolite -barrier-immune” axis, warrants further in-depth exploration.

Against this backdrop, we put forward the following hypothesis: Fla can alleviate UC pathological progression by reshaping the gut microbiota structure, promoting SCFA production, thereby contributing to the enhancement of intestinal barrier function and suppression of excessive inflammatory responses. To test this hypothesis, we employed a DSS-induced murine UC model, combined with multi-dimensional techniques including phenotypic observation, histopathology, molecular biology, and microbiology. Our goal was to clarify the underlying mechanism of Fla in alleviating UC from a gut microbiota–host interaction perspective, potentially opening new avenues for nutritional intervention and drug development for UC.

2. Materials and Methods

2.1. Experimental Materials

Total flavonoids from sea buckthorn leaves (Fla) were provided by Zhejiang Beiduokang Health Industry Co., Ltd. (Hangzhou, China). The extract was prepared from the leaves of Hippophae rhamnoides L. (harvested from Gansu Province, China) using a standardized process. Briefly, dried leaves were extracted with 70% ethanol (solid-to-liquid ratio 1:20) under ultrasonication at 25 °C. The extraction was performed in triplicate. The combined extracts were concentrated and further purified to obtain a flavonoid-enriched powder. (Batch No.: SBLF-20240801) Total flavonoids (Fla) refers to the sum of major flavonoid constituents quantified by HPLC (isorhamnetin, quercetin, kaempferol, luteolin, and proanthocyanidin), expressed as % w/w of the extract. The detailed phytochemical composition is provided in Supplementary Table S1. HPLC analysis was performed using an Agilent ZORBAX SB-C18 column with a gradient of acetonitrile and 0.1% formic acid in water (detection at 360 nm). Quantification was achieved via external standard calibration. As this was a research-grade batch for experimental use, it was not assessed for residual solvents, heavy metals, or endotoxins. The extract was stored at 4 °C until use.

2.2. Animals and Experimental Design

Male mice of the C57BL/6J strain (6–8 weeks old, body weight 20 ± 5 g) were provided by GemPharmatech Co., Ltd. (Nanjing, China; Production License No.: SCXK (Su) 2023-0009). A one-week acclimatization phase was implemented at the Experimental Animal Center of Zhejiang Academy of Agricultural Sciences, where animals were housed under environmentally controlled conditions (22–24 °C, 12 h light/dark cycle) with standard chow and water provided ad libitum. Mice were allocated to experimental groups (n = 6) using a random number table method. Investigators involved in daily monitoring and outcome assessments were blinded to group assignments. The entire animal experiment was carried out with the approval of the local Animal Ethics Committee (Approval No.: 25ZALAS06). The ARRIVE guidelines have been followed.

During the 14-day study, mice were randomly allocated to six experimental groups (n = 6): normal control group (NC); DSS-induced UC model group (DSS); mesalazine-treated group (Mes); low-dose Fla intervention group (Fla-Low); middle-dose Fla intervention group (Fla-Mid); and high-dose Fla intervention group (Fla-High). From Day 1 to Day 7: The NC, DSS, and Mes groups were administered 0.1 mL of physiological saline per day via gavage, while the Fla intervention groups received equivalent volumes of Fla at 50, 100, and 200 mg/kg/day, respectively. From Day 8 to Day 14: The NC group was given 0.1 mL of saline solution every day. The DSS group received 0.1 mL of physiological saline daily and free access to 3% (w/v) DSS (molecular weight 36,000–50,000 Da, MP Biomedicals, LLC, Irvine, CA, USA) in drinking water. Fresh DSS solution was prepared every other day. The Mes group received mesalazine (200 mg/kg/day in an equivalent volume) and free access to 3% DSS. The Fla intervention groups received Fla at 50, 100, and 200 mg/kg/day (in equivalent volumes), respectively, along with free access to 3% DSS.

2.3. Sampling Procedure

Body weight and stool characteristics were monitored daily throughout the experiment. Fresh fecal samples were collected daily at 4:00 PM for occult blood detection using the o-toluidine method. The disease activity index (DAI) was calculated as follows: DAI score = (score for weight loss + score for stool consistency + score for occult blood). The grading criteria for DAI scoring are described in Supplementary Table S2. At the endpoint (Day 14), fecal samples were collected prior to euthanasia for subsequent short-chain fatty acids (SCFAs) analysis. At the end of the experiment, after a 12 h fast (with free access to water), we administered an intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg) to anesthetize all mice. Whole blood was collected via cardiac puncture. The entire colon, cecum, and spleen were carefully dissected. Cecal contents were collected and immediately stored at −80 °C for downstream experiments.

2.4. Colon Histopathology

To assess the degree of inflammation and structural damage in colonic tissue—the gold standard for histopathological evaluation—sections were stained with hematoxylin and eosin (H&E). Distal colon tissues were preserved in 4% paraformaldehyde for 24 h for fixation. Following fixation, tissues were dehydrated through a graded ethanol series, embedded in paraffin, and sectioned at a thickness of 4–5 μm. The sections were then stained with hematoxylin (to visualize cell nuclei) and eosin (to visualize cytoplasm and extracellular matrix). Pathological changes in the colonic tissues were observed and evaluated under a high-power optical microscope. Histopathological scoring was performed by two independent, blinded investigators according to the criteria detailed in Supplementary Table S3.

2.5. ELISA

Whole blood was collected via cardiac puncture and maintained at ambient temperature for 1 h. The serum was collected as the supernatant after a 15 min centrifugation at 3000 rpm and 4 °C. The levels of TNF-α, IL-6, and IL-10 in the mouse serum were measured using commercial ELISA kits obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China) (Cat. No.: SEKM-0034 for TNF-α, SEKM-0007 for IL-6, and SEKM-0010 for IL-10). All samples were measured in duplicate, and the mean values were used for statistical analysis.

2.6. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

Total RNA was extracted from colon tissues using the TransZol Up Plus RNA Kit (ER501, TransGen Biotech, Hangzhou, China). cDNA synthesis was performed using a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (AT314, TransGen Biotech). RT-qPCR analysis was carried out using PerfectStart Green qPCR SuperMix (AQ601, TransGen Biotech). The amplification efficiency for each primer pair was validated prior to the study and ranged between 90% and 110%. Each reaction was performed in technical triplicate. Relative transcription levels were calculated using the 2 ^(−ΔΔCt) method using GAPDH as an internal reference. All RT-qPCR primers are detailed in Supplementary Table S4, and these primers were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China).

2.7. Western Blot Analysis

Total protein was extracted from colon tissues using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with a protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). Protein concentration was determined using a BCA protein assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Protein concentrations of all samples were adjusted to a uniform concentration using RIPA buffer, mixed with 5× Loading Buffer, and denatured by boiling in a metal bath at 100 °C for 10 min. A total of 30 μg protein per sample was loaded, and electrophoresis was stopped after the protein bands had separated sufficiently. Proteins were then transferred from the gel onto a 0.22 μm PVDF membrane using a wet transfer system. After transfer, the membrane was blocked with 5% skim milk (in TBST) at room temperature for 1 h. Following blocking, the membrane was incubated with specific primary antibodies at 4 °C overnight: ZO-1 (Biodragon, Beijing, China; dilution 1:1000), MUC2 (ABclonal, Wuhan, China; dilution 1:1000), E-cadherin (ABclonal, Wuhan, China; dilution 1:1000), and GAPDH (ABclonal, Wuhan, China; dilution 1:50,000). Following overnight antibody recovery, the membrane was rinsed with TBST to eliminate unbound primary antibodies, then incubated with the corresponding secondary antibodies for 1 h at room temperature before additional washes. For visualization, ECL chemiluminescence substrate was applied to the membrane, and images were captured using a chemiluminescence imaging analysis system. Band intensity was quantified using ImageJ software (version 1.54p, National Institutes of Health, Bethesda, MD, USA) and normalized to the corresponding GAPDH intensity for each sample.

2.8. Short-Chain Fatty Acids (SCFAs)

Fecal samples (0.1 g) were homogenized in phosphate-buffered saline (PBS; 1:10, w/v), vortexed thoroughly, centrifuged at 12,000× g for 10 min at 4 °C, and the supernatant was acidified with metaphosphoric acid, then filtered through a 0.22 μm aqueous phase membrane. To minimize volatilization of SCFAs, samples were processed promptly under low-temperature conditions. SCFAs were analyzed using a GC9720II gas chromatograph (Fuli Analytical Instrument Co., Wenling, China) equipped with a flame ionization detector (FID) and an FFAP capillary column (30 m × 0.25 mm × 0.25 μm). The injector temperature was 220 °C, the detector temperature was 250 °C, and the injection volume was 1.0 μL with a split ratio of 5:1. The oven temperature program ranged from 75 °C to 220 °C. High-purity nitrogen was used as carrier gas at 2.5 mL/min. Crotonic acid was used as an internal standard for quantification. SCFA concentrations were determined using the internal standard method with calibration curves and normalized to fecal sample weight. Quantification was performed using a 6-point calibration curve (0.1–10 µmol/mL, R^2^ > 0.995); quality control standards were run daily to ensure linearity and instrument stability. Results were expressed as μmol/g wet feces.

2.9. 16S rRNA Sequencing and Gut Microbiota Analysis

Genomic DNA was extracted from cecal contents. The V3-V4 region of the bacterial 16S rRNA gene was amplified with primers 338F/806R and sequenced on an Illumina NovaSeq platform (paired-end). Bioinformatic analysis was performed using the BMKCloud platform. Raw sequences were quality-filtered, denoised, and clustered into amplicon sequence variants (ASVs) using the DADA2 pipeline. Taxonomic assignment was performed against the SILVA 138 database. The ASV table was rarefied for subsequent analysis. The sample size (n = 6 per group) was based on common practice in rodent intervention studies. Sequencing depth averaged 65,000–75,000 reads per sample. Differential abundance was analyzed by LEfSe (LDA > 3.0, α = 0.05). The raw sequencing data and complete sample metadata have been deposited in the NCBI SRA under BioProject accession number PRJNA1371858.

2.10. Statistical Analysis

Data are presented as mean ± SEM. Normality was assessed using the Shapiro–Wilk test. For comparisons across multiple groups, one-way ANOVA with Tukey’s post hoc test was applied for normally distributed data; Kruskal–Wallis with Dunn’s post hoc test was used when normality assumptions were violated. Specific analyses for microbiome data (e.g., LEfSe) utilized non-parametric methods. All experiments were performed once with n = 6 biological replicates per group. Statistical analyses were performed using GraphPad Prism 9.5.0 (GraphPad Software, San Diego, CA, USA). Results with p-values below 0.05 were deemed statistically significant.

3. Results

3.1. Fla Ameliorated Pathological Symptoms and Damage in UC Mice

In this experiment, we established a DSS-induced mouse UC model, employing the clinically prevalent drug mesalazine (Mes) as a positive control. Figure 1A illustrates the experimental workflow for this animal study. Figure 1B,C demonstrate changes in mouse body weight and DAI. As shown, compared to the NC group, mice in the DSS group exhibited a significant decrease in body weight and a marked increase in DAI. The Mes group exhibited a pronounced ameliorative effect, significantly restoring both body weight and DAI in UC mice. The Fla-Mid and Fla-High groups also showed significant improvement, although the effects were slightly less pronounced than in the Mes group. Furthermore, as depicted in Figure 1D,E, DSS-induced colonic shortening was markedly improved in the Mes, Fla-Mid, and Fla-High groups. Notably, the Fla-Mid group showed a restoration of colonic length that was comparable to that achieved with Mes. Analysis of spleen indices in UC mice revealed that the spleen index in the DSS group was significantly elevated compared to the NC group, while both Mes and Fla-Mid treatments resulted in significant reductions (Figure 1F). H&E staining results are shown in Figure 1H. The DSS group exhibited marked inflammatory cell infiltration, destruction of crypt architecture, and reduction in goblet cells. As quantified in Figure 1G, the histological score was significantly increased in the DSS group compared to the NC group. These pathological features were significantly alleviated following Mes and Fla-Mid treatment, with both the histological score and inflammatory cell infiltration showing a marked reduction (Figure 1G,H). These findings indicate that Fla effectively mitigates pathological symptoms in UC mice, with Fla-Mid demonstrating the most pronounced preventive effect.

3.2. Fla Modulates Tight Junction Protein Levels in the Colonic Epithelium in UC Mice

This study employed qRT-PCR to assess the expression levels of key tight junction-associated genes in colonic tissue. As depicted in Figure 2A–C, compared with the NC group, the DSS group exhibited significantly downregulated expression of ZO-1, MUC2, and E-cadherin genes. Following Fla intervention, expression levels of these tight junction-associated genes exhibited varying degrees of recovery.

To further verify these results at the protein level, we selected the Fla-Mid group for Western blot analysis (Figure 2D). Consistent with the mRNA data, the protein expression levels of ZO-1, MUC2, and E-cadherin were significantly lower in the DSS group than in the NC group. Compared with the DSS group, protein expression levels of ZO-1, MUC2, and E-cadherin were significantly elevated in both the Mes and Fla-Mid groups, consistent with the trends observed in gene expression.

These results collectively demonstrate that Fla, particularly at the middle dose, can strengthen the intestinal epithelial structure from a molecular standpoint by effectively upregulating the expression of key barrier proteins such as ZO-1, MUC2, and E-cadherin, thereby protecting intestinal barrier function.

3.3. Fla Alleviated Colonic Inflammation by Suppressing Pro-Inflammatory Cytokine Expression

The levels of inflammatory gene expression in colonic tissue and key serum inflammatory cytokines are shown in Figure 3. DSS induction triggered a robust local inflammatory response in the colon, markedly increasing TNF-α and IL-6 expression levels while significantly reducing IL-10 expression. Compared to the DSS group, the Fla-Mid and Fla-High groups markedly attenuated the excessive expression of TNF-α and IL-6 in the colon. Although Fla demonstrated limited efficacy in reducing serum TNF-α levels and IL-10 levels were not significantly altered, the middle and high-dose Fla groups markedly mitigated the abnormal elevation of IL-6 in systemic circulation. These findings indicate that Fla modulates immune homeostasis in UC mice by regulating pro-inflammatory factor expression.

3.4. Fla Restored Short-Chain Fatty Acids Metabolic Homeostasis in UC Mice

This study employed GC-FID to determine SCFA levels in mouse feces, with results presented in Figure 4. Experimental findings indicate that, compared with the NC group, the DSS group exhibited markedly decreased levels of acetic acid and propionic acid in feces. Other SCFAs, including isobutyric acid, butanoic acid, isovaleric acid, and valeric acid, also showed a downward trend. After Fla intervention, all dose groups reversed the decrease in SCFA levels to varying degrees. Notably, the middle dose of Fla (Fla-Mid group) exhibited the most significant and comprehensive effect, significantly increasing the levels of all six measured SCFAs compared to the DSS group. Cluster thermal analysis demonstrated that the SCFA content in the DSS group was significantly lower than that in the NC group. The Fla-Mid group was characterized by a distinct SCFA profile, marked by elevated levels of multiple SCFAs. These results indicate that Fla, particularly at the middle dose, can effectively ameliorate the SCFA metabolic deficiency in UC mice. These findings suggest a potential association between Fla treatment and SCFA levels, which may contribute to intestinal mucosal health.

3.5. Fla Alleviates Dysbiosis of the Gut Microbiota in UC Mice

Further, we selected the Fla-Mid group, which showed the most pronounced preventive efficacy across multiple indicators (e.g., SCFAs, colon length, histopathology), as the representative dose for in-depth microbiota analysis. To assess the impact of DSS and Fla intervention on the gut microbiota, cecal contents from mice in the NC, DSS, Mes, and Fla-Mid groups (n = 6 per group) were subjected to 16S rRNA gene sequencing. Firstly, regarding microbial diversity, DSS induction significantly reduced the alpha diversity of the gut microbiota. The Chao1 index was markedly lower in the DSS group compared to the NC group, indicating severe impairment in species richness and evenness of the gut microbiota in UC mice and significantly increased following Fla intervention (Figure 5A). Principal coordinates analysis (PCoA) plot demonstrated clear structural separation of gut microbiota, with significant differences between groups confirmed by PERMANOVA (R^2^ = 0.486, p = 0.001). Specifically, the DSS group clustered apart from both the NC and Mes groups, confirming the overall dysbiosis caused by DSS (Figure 5B). Notably, the Fla-Mid group exhibited higher alpha diversity indices than the DSS group. In the PCoA plot, its distribution was closer to the NC and Mes groups and distinctly separated from the DSS group. These results indicate that middle-dose Fla intervention effectively reversed the loss of microbial diversity induced by DSS and promoted a shift in the overall microbiota structure towards a healthier state.

Secondly, in terms of specific microbial composition, we observed trends with potential biological significance (Figure 5C,D). At the phylum level, compared to the NC group, DSS induction led to structural disruption of the gut microbiota, characterized by a decreasing trend in the average relative abundance of Firmicutes and increasing trends in the average relative abundances of Bacteroidota and Proteobacteria. This resulted in a decreased Firmicutes/Bacteroidota (F/B) ratio. While these changes did not reach statistical significance and should be interpreted with caution, the directionality of the trends observed in the Fla intervention group—showing an F/B ratio numerically closer to the NC and Mes groups than to the DSS group—may suggest a potential modulatory effect that merits further investigation in future studies with larger sample sizes. At the genus level, we analyzed key bacterial genera associated with intestinal health. In the Fla group, the abundances of Akkermansia and Lachnospiraceae_NK4A136_group were significantly increased, while the abundance of Ileibacterium was significantly decreased. This specific pattern of microbial shifts reflects a dysbiotic profile commonly associated with an intestinal inflammatory state. LEfSe analysis further clarified inter-group differences in species composition (Figure 5E). The DSS group exhibited indicative taxa, including potential pathogens such as Escherichia-Shigella, Mucispirillum, and Parasutterella. In contrast, the Fla intervention group also possessed its own unique indicative taxa, primarily beneficial bacteria such as Akkermansia, Lachnospiraceae_NK4A136_group, and Intestinimonas.

3.6. Interplay Among the Gut Microbiota, Metabolites, and Host Physiology

We employed Spearman correlation analysis to reveal the associations among the gut microbiota, SCFAs, and physiological indicators. As shown in Figure 6A, Akkermansia, Lachnospiraceae_NK4A136_group, and Lachnospiraceae_UCG_006 showed significant positive correlations with SCFAs, whereas Bacteroides and Ileibacterium exhibited significant negative correlations with SCFAs. Figure 6B demonstrates that Lachnospiraceae_UCG_006, Ligilactobacillus, and Limosilactobacillus exhibited a significant positive correlation with body weight, colon length, ZO-1, MUC2, E-cadherin, and IL-10, and significantly negatively correlated with the DAI score, spleen index, TNF-α, and IL-6. Figure 6C reveals that SCFAs exhibited a significant positive correlation with body weight, colon length, ZO-1, MUC2, E-cadherin and IL-10, and significantly negatively correlated with the DAI score, spleen index, TNF-α, and IL-6. As shown in the correlation network (Figure 6D), tight associations exist between the host’s physiological state, gut microbial communities, and metabolites.

4. Discussion

Currently, the evolution of therapeutic strategies for UC demonstrates a trend shifting from single anti-inflammatory approaches towards systemic, multi-target interventions. Within this context, natural products are increasingly becoming important sources for drug discovery due to their multi-component, multi-target characteristics and favorable biosafety profiles. Numerous studies have confirmed that natural active ingredients like flavonoids and polysaccharides can exert therapeutic effects through multiple pathways, including modulating the gut microecology, enhancing epithelial barrier function, and balancing immune responses. Sea buckthorn leaves, an underutilized resource rich in flavonoids, represent a promising candidate for such multi-target interventions.

Through analysis of multi-level indicators, including clinical phenotypes, molecular biology, and microbiology, we found that Fla exerts preventive effects via multi-target mechanisms, with the middle dose group (Fla-Mid) showing optimal efficacy in most indicators. It is important to acknowledge a key limitation of the present study. Our experimental design, in which Fla was administered both prior to and concurrently with DSS exposure, primarily models a preventive or prophylactic scenario rather than a treatment of established colitis. Clinically, UC patients typically present after symptom onset, and the efficacy of Fla as a treatment for active colitis remains to be determined. Future studies employing therapeutic administration protocols (initiating treatment after disease establishment) are warranted to fully elucidate the clinical translational potential of Fla. Nevertheless, our findings provide valuable insights into the preventive mechanisms and support the potential use of Fla as a dietary supplement for high-risk populations or as an adjunct preventive strategy.

The pathological progression of UC is not confined to the local intestine but often involves systemic manifestations [21]. The amelioration of core symptoms (weight loss, DAI, colon shortening) by Fla indicates its comprehensive therapeutic potential. Particularly noteworthy is the significant splenomegaly, a hallmark phenotype in UC mice reflecting systemic immune system overactivation [22,23]. The effect of Fla in improving the spleen index suggests its potential to modulate systemic immune responses, thereby alleviating UC-associated immune organ abnormalities. Improvements in histopathology further confirmed its ability to promote mucosal repair and suppress inflammatory infiltration, aligning closely with the clinical treatment goal of mucosal healing. These improvements provide an important phenotypic foundation for subsequent in-depth exploration of its molecular mechanisms and also suggest that Fla may exert preventive effects through synergistic actions across multiple systems and targets.

The intestinal barrier acts as the frontline defense in preserving gut homeostasis and preventing the translocation of harmful substances [24,25]. This study confirmed, from gene to protein levels, that Fla effectively preserves intestinal barrier integrity in UC mice. At the gene expression level, the inflammatory environment induced by DSS significantly disrupted the normal expression of barrier-related genes, with mRNA levels of ZO-1, MUC2, and E-cadherin all being significantly downregulated. Fla intervention demonstrated precise regulatory characteristics: its universal promotion of ZO-1 expression reflects reinforcement of the basic tight junction structure; its selective upregulation of MUC2 expression (Fla-High group) indicates targeted repair of the chemical barrier; and its significant restoration of E-cadherin expression (Fla-Mid group) shows particular attention to interepithelial adhesion junctions. Notably, middle dose Fla exhibited the most balanced barrier repair profile overall. At the protein level, we further validated the promoting effect of middle-dose Fla on barrier protein expression. Western blot results confirmed marked up-regulation of ZO-1, MUC2, and E-cadherin, highly consistent with the changes at the gene level, confirming the reliability of its barrier-protective effect at the functional protein level. The synergistic repair of these structures collectively builds a complete defense line against intestinal harmful substances, establishing a robust structural foundation for the subsequent alleviation of inflammation.

The imbalance of the intestinal immune response is a core pathological link in UC [26]. Cytokines produced by immune cells can promote or inhibit inflammation, thereby influencing the onset and progression of UC. The significant suppression of TNF-α and IL-6 by Fla indicates its effective intervention in key pro-inflammatory pathways (e.g., NF-κB, STAT3) that are central to UC pathology [27,28,29,30]. Although the increase in IL-10 did not reach statistical significance in our study, the observed trend aligns with the activation of an anti-inflammatory JAK-STAT pathway [31,32], suggesting a modulatory direction. This selective modulation pattern can effectively control inflammation while potentially avoiding the adverse effects associated with broad immunosuppression, highlighting the multi-target regulatory advantage of Fla as a natural product.

SCFAs, as the main metabolites produced by gut microbiota fermenting dietary fiber, play a central role in maintaining intestinal homeostasis [33,34]. This study showed that DSS-induced colitis led to a comprehensive depletion of SCFAs, particularly significant reductions in Acetic acid and Propanoic acid, reflecting severe impairment of gut microbial metabolic function. Fla intervention, especially the middle dose, significantly reversed the declining trend of all six major SCFAs. This comprehensive metabolic remodeling holds significant physiological importance. Following the Fla intervention, a simultaneous elevation of multiple SCFAs was observed. Particularly noteworthy is the restoration of Butanoic acid levels, which not only provides energy for intestinal epithelial cells but also directly participates in gene expression regulation and immune cell function modulation through pathways such as suppressing histone deacetylases and activating G protein-coupled receptors [35], closely related to the observed improvements in barrier function and inflammation alleviation.

This study, through 16S rRNA sequencing, revealed the specific regulatory effect of Fla on the gut microbiota. Firstly, Fla effectively reversed the DSS-induced microbial dysbiosis, restoring community diversity and a healthy structure. More importantly, Fla did not simply increase microbial diversity but was associated with shifts in the microbiota structure toward a specific functional phenotype. Notably, the abundance of Akkermansia—a core species for maintaining mucus layer homeostasis and immune regulation—was significantly increased [36,37,38]. Concurrently, SCFA-producing bacteria such as Lachnospiraceae_NK4A136_group were also enriched [39]. In contrast, genera associated with inflammation, such as Ileibacterium, were suppressed [40]. These findings are consistent with a potential association between Fla treatment (particularly at the middle dose) and enrichment of beneficial bacteria like Akkermansia as well as elevated SCFA levels, concomitant with improvements in barrier function and reduced inflammation. However, whether these associations represent a causal chain or parallel consequences of Fla’s effects remains to be determined through experimental interventions such as fecal microbiota transplantation or SCFA receptor antagonism. This study reveals a preliminary dose-dependent efficacy of Fla in alleviating colitis, with the middle-dose group (Fla-Mid) exhibiting the most balanced preventive effects across the majority of phenotypic, biochemical, and inflammatory indicators measured. This pattern suggests a potential “optimal bioactive concentration window,” broadly consistent with the hormesis theory, where both insufficient low doses and excessive high doses may compromise therapeutic efficacy [41]. However, this theory was not experimentally validated in the present study, and alternative explanations (e.g., non-monotonic dose responses unrelated to hormesis) cannot be excluded. Nevertheless, as an exploratory investigation, the dose selection was empirically guided by phenotypic improvements (DAI, colon length, spleen index, and histopathology), and subsequent mechanistic analyses were focused on the Fla-Mid (Western blot and 16S rRNA). While this approach enabled in-depth characterization of the most effective dose, it inherently precludes establishing formal dose–response curves for specific protein targets, signaling pathways, and microbial taxa across the full dose spectrum. Future studies incorporating comprehensive dose-series mechanistic assays and pharmacokinetic profiling are therefore warranted to validate these preliminary findings, delineate precise concentration thresholds, and precisely define the preventive strategies window and mechanisms of Fla.

Furthermore, Several methodological considerations should be noted. First, while mice were co-housed within treatment groups, potential cage effects on the gut microbiota cannot be entirely ruled out, although randomization aimed to mitigate this. Second, the sample size of n = 6 per group, while chosen based on common practice in the field and the 3R principles, may provide limited statistical power for detecting subtle effects, particularly in high-variability analyses such as those involving specific microbial taxa. Results should be interpreted as exploratory and require validation in larger cohorts.

It must be noted that the exploration of the dose effect in this study remains at the stage of phenomenological description; the mechanisms discussed above are largely reasonable speculations based on existing theories. The exact molecular mechanisms—for instance, whether they involve dose-dependent bidirectional regulation of key signaling pathways such as Nrf2, AMPK, or NF-κB—have not been validated. This represents a limitation of the present study but also points to an important direction for future research. Elucidating the cellular and molecular mechanisms behind this optimal dose will not only help determine the precise dosage range for the clinical application of Fla but also provide a valuable model for understanding the universal principle of the “optimal effect” of complex natural products.

Finally, from a translational perspective, the extraction process for Fla utilizes food-grade ethanol and established unit operations (ultrasonication, concentration), which are amenable to industrial scale-up in a batch mode. While this study provides the foundational bio-efficacy data, future techno-economic analysis (TEA) and life-cycle assessment (LCA) will be crucial to fully evaluate the environmental footprint and economic viability of large-scale production. To further optimize the process, comparative analyses with other emerging green extraction technologies for efficiency and scalability will be valuable [42]. These integrated assessments will guide the realistic development of sea buckthorn leaf flavonoids as a nutraceutical or functional food ingredient for gut health.

5. Conclusions

In summary, this study, conducted using a preventive model, provides preliminary evidence of Fla administration being associated with amelioration of DSS-induced colitis in mice, concomitant with alterations in gut microbiota composition, increased SCFA levels, enhanced intestinal barrier function, and reduced local inflammatory responses. These associations generate hypotheses regarding a potential modulatory role of the gut microbiota–metabolite–barrier–immune axis, though causality remains unproven. The middle dose (100 mg/kg) appeared most effective among those tested, but formal dose-optimization and mechanistic validation are warranted. This work generates hypotheses for further investigation of sea buckthorn leaf flavonoids as potential dietary supplements for high-risk populations or adjunct preventive strategies for UC, pending validation in therapeutic models and clinical studies.