Effectiveness of probiotic- and fish oil-loaded water-in-oil-in-water (W₁/O/W₂) emulsions at alleviating ulcerative colitis

Abstract

Ulcerative colitis (UC) is a common chronic inflammatory disease that causes serious harm to human health. Probiotics have the effect of improving UC. This study evaluated the preventative potential of water-in-oil-in-water (W₁/O/W₂) emulsions containing both probiotics and fish oil on UC and associated anxiety-like behavior using a mice model. UC model was established in mice by administering dextran sulfate sodium salt (DSS). Free probiotics, probiotic-loaded emulsions, or fish oil and probiotic co-loaded emulsions were then orally administered to the mice. Various bioassays, histological studies, 16s rDNA gene sequencing, and behavioral experiments were conducted to assess changes in the intestinal environment, microbiota, and anxiety-like behavior of the mice. The fish oil and probiotic co-loaded emulsions significantly reduced the inflammatory response by enhancing tight junction protein secretion (ZO-1, Occludin, and Claudin-1), inhibiting pro-inflammatory factors (TNF-α, and IL-1β), and promoting short-chain fatty acids (SCFAs) production. These emulsions also modified the gut microbiota by promoting beneficial bacteria and suppressing pathogenic bacteria, thereby restoring a balanced gut microbiota. Notably, the emulsions containing both probiotics and fish oil also ameliorated anxiety-like behavior in the mice. The co-delivery of probiotics and fish oil using W₁/O/W₂ emulsions has shown significant promise in relieving UC and its associated anxiety-like behavior. These findings provide novel insights into the development of advanced therapeutic strategies for treating UC.

---

1. Introduction

Crohn's disease and ulcerative colitis (UC) are types of inflammatory bowel disease that are often caused by immune system abnormalities. Typical symptoms of inflammatory bowel disease include chronic diarrhoea, abdominal pain, rectal bleeding, and fever.^1,2 UC is a chronic and recurrent inflammatory disease of the intestinal tract, primarily affecting the colonic mucosa. Currently, about 30–50 individuals per 100 000 population are affected by UC globally.² Additionally, there was evidence indicating that individuals with UC had a 2–3 times higher occurrence of anxiety and depression compared to the general population.^3,4 The current treatment methods for UC include medication and surgical treatment. Drugs such as antibiotics, aminosalicylates, and corticosteroids are commonly used to reduce inflammation and promote intestinal barrier repair. However, their non-specific anti-inflammatory properties can lead to undesirable side effects when used long-term.⁵

Probiotics are living microorganisms that can provide health benefits to the host when ingested in sufficient quantities.⁶ They play a crucial role in regulating microbial composition, maintaining gut microbiota balance, and improving human health and well-being. Currently, probiotic-based therapies have emerged as important topics of study in food and biomedical research.⁷ Probiotics can improve host health and prevent diseases by competing with pathogenic bacteria for limited adhesion sites and nutrients in the intestinal tract, as well as by producing antimicrobial substances that inhibit the growth and colonization of pathogens.^8,9 Some studies have also shown that habitual supplementation of fish oil rich in the polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can reduce the risk of developing IBD,¹⁰ as well as improving gut health by enhancing anti-inflammatory effects and modulating the gut microflora.^11,12 Many researchers are trying to understand the mechanisms by which probiotics and/or fish oil improve gut inflammation.

The regional immune mechanism of the gut is determined by its structure, function, and microenvironment.¹³ However, the gut microbiota plays a vital role in altering the structure and function of the immune system, reshaping the immune microenvironment, thereby limiting the progression of several diseases.¹³ Intestinal inflammation triggers an increase in intestinal epithelial permeability, leading to the entry of pathogens into the bloodstream that migrate into other organs. The host's intestinal tract contains beneficial and pathogenic bacteria, and only when these two categories of bacteria are balanced in terms of type and quantity can the intestine maintain a healthy state. Once the balance of the gut microbiota is disrupted, it may impact digestion, absorption, detoxification, and immune functions.^14,15 Studies have shown that a combination of lactic acid bacteria (Lactobacillus acidophilus, Lactobacillus helveticus, and Lactobacillus plantarum) could alleviate dextran sulfate sodium salt (DSS) induced UC by modulating the gut microbiota and repairing the intestinal barrier.¹⁶ Moreover, iron-enriched probiotics have been shown to enhance the expression of tight junction proteins in mice with UC, effectively reducing the secretion of pro-inflammatory factors and the occurrence of oxidative stress.¹⁷

Probiotics can improve host gut health by modulating the gut microbiota, immune response, and intestinal mechanical barrier.^18,19 However, the viability and activity of orally administered probiotics are significantly reduced as they pass through the gastrointestinal tract due to environmental stresses, such as gastric acids, digestive enzymes, and bile salts.⁸ For this reason, oral delivery systems have been developed to protect probiotics from the harsh gastrointestinal environment, thereby ensuring their viability upon reaching the colon.^20–22 For example, researchers have shown that encapsulating Lactobacillus Plantarum in oil-in-water (O/W) high internal phase emulsions enhanced probiotic activity after gastrointestinal digestion.²³ This study demonstrated that emulsion-based delivery systems have the potential to improve probiotic viability and activity. Researchers have also shown that the co-encapsulation of probiotics with other substances can enhance the activity, stability, and efficacy of both.⁸ In our recent research, co-encapsulation of probiotics and fish oil was shown to significantly promote the growth of probiotics and enhance their activity and adhesion in the large intestine.²⁴

In this study, we utilized water-in-oil-in-water (W₁/O/W₂) emulsions to co-encapsulate probiotics and fish oil. These emulsions were used because they contain internal water and oil domains, which allows both hydrophilic and hydrophobic substances to be simultaneously incorporated.²⁵ Initially, W₁/O emulsions were prepared using polyglycerol polyricinoleate (PGPR) as a hydrophobic emulsifier. Then, W₁/O emulsions were homogenized with water (W₂) containing soy protein isolate (SPI) and sodium alginate (SA) to form W₁/O/W₂ emulsions, where the SPI and SA acted as emulsifiers and stabilizers, respectively. The probiotics were loaded into the internal water phase (W₁) of these emulsions, while the fish oil was loaded into the oil phase (O).

A UC mice model was established to investigate the potential of the probiotic- and fish oil-loaded W₁/O/W₂ emulsions to reduce UC. The impact of this treatment using the co-encapsulated system on colon tissue health, immune expression, short-chain fatty acids (SCFAs) production, gut microbiota composition, and anxiety of mice with UC was investigated. The results of this study may lead to new food-based treatments that can alleviate the adverse effects of inflammatory bowel disease on human health and well-being.

2. Experimental section

2.1. Materials and reagents

Lactobacillus acidophilus was purchased from the China Center of Industrial Culture Collection. Sodium alginate (SA) was obtained from BSZH Scientific Inc. (Beijing, China). Fish oil from menhaden (EPA: 13.5%, DHA: 11.5%) was obtained from Sigma-Aldrich. Soy protein isolate (SPI) was obtained from Ruida Henghui Technology Development Company (Beijing, China). Polyglycerol polyricinoleate (PGPR) was obtained from Qingyuan Food Additives Company (Beijing, China). Medium-chain triglycerides (MCTs) were provided by Yixiu Bogu Biotechnology Company (Beijing, China). Fecal occult blood reagent was produced by Beisuo Biotechnology Co., Ltd (Zhuhai, China). Alcian blue staining reagent kit was obtained from Meixuan Biotechnology Company (Shanghai, China). EZNA fecal DNA extraction kit was obtained from Omega Bio-Tek (Norcross, GA USA). Mouse interleukin-1β (IL-1β) kit and mouse tumor necrosis factor α (TNF-α) kit were purchased from Ruixin Biotechnology Company (Quanzhou, China).

2.2. Preparation of probiotic- and fish oil-loaded W₁/O/W₂ emulsions

The preparation of W₁/O/W₂ emulsions was performed according to the method we reported previously.²⁴ Specifically, 5% v/v of fish oil was dissolved in MCTs, and then 4% v/v of PGPR was added, and the oil phase (O) was thoroughly mixed. Then, L. acidophilus was dispersed in 0.15 M NaCl solution to obtain probiotic suspension as the internal aqueous phase (W₁). Subsequently, this internal aqueous phase (10% v/v) was slowly added dropwise into oil phase (90% v/v), and the mixture was sheared with a high-shear mixer at 6000 rpm for 2 min to prepare W₁/O emulsions. Then, 60% v/v of this W₁/O emulsion was mixed with 40% v/v of aqueous phase (W₂) containing SPI (1.5 wt%) and SA (0.75 wt%). The resulting mixture was then sheared at 6000 rpm for 2 min using a high-shear mixer to obtain SPI-SA stabilized W₁/O/W₂ emulsions. In some cases, the fish oil was omitted from the oil phase to prepare only probiotic-loaded W₁/O/W₂ emulsions. In other cases, the probiotics were omitted to prepare fish oil-loaded W₁/O/W₂ emulsions.

2.3. Animals and experimental groups

Eight-week-old male C57BL/6J mice were used in the experiments, which were purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). The mice were housed in an environment with a temperature of 25 ± 2 °C and a humidity of 50 ± 5%, with free access to food and water. The housing conditions were controlled on a 12-hour light–dark cycle. The animal experiments followed the guidelines outlined in “Guidelines for the Care and Use of Laboratory Animals (ISBN-10: 0-309-15394-4, 8th edition)”, and the experiments were approved by the Experimental Animal Management and Ethics Review Committee of Northwest A&F University. The standard feed (AIN93M) was purchased from Nantong Trofie Feed Technology Co., Ltd (Nantong, Jiangsu, China).

In this study, mice were randomly divided into six groups (n = 13): Control group (Con); UC model group (DSS); Free probiotic group (Free P); Encapsulated probiotic group (P); Encapsulated fish oil group (FO); and co-encapsulated probiotic and fish oil group (P + FO). A single dose of each sample (0.1 mL per 10 g) was administered by oral gavage of mice. All mice were fed a standard diet and drank water freely. The specific treatments were as follows (Fig. 1):

1. Control group (Con): Mice were orally administered with empty vehicle emulsions (no fish oil and no probiotic).

2. UC model group (DSS): Mice were orally administered with empty vehicle emulsions, and during the test period, 2.5% DSS aqueous solution replaced the drinking water.

3. Free probiotics group (Free P): Mice were orally administered with free probiotics, and during the test period, 2.5% DSS aqueous solution replaced the drinking water.

4. Encapsulated probiotic group (P): Mice were orally administered with an emulsion containing encapsulated probiotics, and during the test period, 2.5% DSS aqueous solution replaced the drinking water.

5. Encapsulated fish oil group (FO): Mice were orally administered with an emulsion containing encapsulated fish oil, and during the test period, 2.5% DSS aqueous solution replaced the drinking water.

6. Co-encapsulated probiotic and fish oil group (P + FO): Mice were orally administered with an emulsion containing both probiotics and fish oil, and during the test period, 2.5% DSS aqueous solution replaced the drinking water.

Fig. 1 Timeline of intervention in mice. Mice were administered the different control and test samples by oral gavage. In all cases, except the control, the mice were administered with DSS to induce colitis.

2.4. Disease activity index and survival rate during test period

During the seven days of the mice drinking DSS water, the body weight of each group was measured and recorded daily to analyze the weight change. The shape of the mice's feces was observed and scored daily. Occult blood in the feces was tested according to the method described in the occult blood reagent instruction manual (Piramidon semi-quantitative test). The disease activity index (DAI) was scored according to Table 1.

Table 1 DAI scoring reference table

Score	Weight loss	Stool shape	Occult blood
0	None	Normal	Negative, no occult blood
1	1–5	Normal	Weakly positive, occult blood
2	6–10	Semi-loose stool	Positive, occult blood
3	10–15	Semi-loose stool	Positive, visible bleeding
4	>15	Loose stool, sticky	Positive, heavy bleeding

---

During the test period, the survival of the mice was recorded, and the survival rate was calculated using the formula:

2.5. Collection of mouse feces

After the successful establishment of the UC mice model, the feces of each group of mice were collected for gut microbiota analysis and SCFAs analysis. The collected feces from each group of mice were placed in sterile centrifuge tubes and then immediately frozen in liquid nitrogen before being transferred to a −80 °C freezer for storage and subsequent analysis.

2.6. Histopathological and morphological observations

After the mice were sacrificed, the colon tissues were fixed in 4% paraformaldehyde, then soaked in 75%–100% ethanol for dehydration, transparently treated with xylene solution, buried in paraffin, and then sliced.

Hematoxylin and eosin staining (H&E staining). Tissue sections were dewaxed and soaked in xylene, then the tissue sections were placed in a gradually decreasing concentration of ethanol and rehydrated in succession. The tissue sections were stained with hematoxylin, differentiated by acetic acid, and turned blue by dilute ammonia, then dyed with eosin dye solution. Finally, the tissue sections were dehydrated with gradient ethanol, transparent with xylene, and sealed. The morphology was observed by an optical microscope. Refer to Table 2 for histological evaluation of colitis.

Table 2 Principles for histological evaluation of colitis

Score	Tissue injury	Infiltration of inflammatory cells in lamina propria
0	None	Seldom
1	Local epithelial injury	Neutrophil increase
2	Mucosal destruction and ulcers	Inflammatory cell clusters in the submucosa
3	Extensive injury, deep into the intestinal wall	Abundant neutrophil infiltration in the submucosa

---

Alcian blue staining. The goblet cells are usually observed with Alcian blue dye. First, the colon tissue sections were dewaxed, rehydrated, and rinsed. After staining with Alcian blue dye for 5–10 min, the sections were rinsed under running water; treated with periodic acid solution for 10 min, and rinsed with distilled water again; then the tissue sections were stained with Schiff's reagent for 15–30 min. Sections were stained with Mayer hematoxylin solution for about 1 min.  Finally, sections were dehydrated, treated with transparency, sealed, observed, and photographed using an optical microscope.

Immunohistochemistry (IHC). The tissue sections were dewaxed, rehydrated and permeated, and then antigen retrieval was carried out. It is then treated with 3% H₂O₂ to eliminate endogenous peroxidase. A sealing solution containing goat serum was added to the tissue sections and incubated for 20 min. The sealed tissue sections were incubated at 4 °C overnight with the primary antibody, and then incubated with the secondary antibody and the working solution labeled Streptomycin by horseradish enzyme for 20 min each. After diaminobenzidine (DAB) treatment, the tissue sections were counterstained with hematoxylin, then differentiated with acetic acid, dehydrated with gradient ethanol, treated with xylene for transparency, and finally sealed with neutral resin, observed and photographed using an optical microscope.

2.7. Pro-inflammatory cytokine protein content of colonic tissue

The contents of TNF-α and IL-1β in the colonic tissue of mice were determined using an ELISA kit according to the manufacturer's instructions.

2.8. SCFAs content in feces

A known mass of fecal sample collected from the mice (0.15 g) was placed into a centrifuge tube. Next, 1.0 mL of ultrapure water was added, and the mixture was vortexed for 10 min to ensure uniformity. Subsequently, 0.15 mL of 50% H₂SO₄ solution and 1.6 mL of ether were added. The mixture then underwent vigorous shaking for 20 min on a shaking bed, followed by centrifugation at 12 000 rpm for 10 min. The upper layer was filtered through a 0.22 μm filter, and 1.0 mL was collected and dried with nitrogen to 0.2 mL and then transferred to a vial for gas chromatography analysis for SCFAs content.

The gas chromatograph utilized a DB-FFAP column (30 m × 0.25 mm × 0.25 μm) with a hydrogen flame detector. The injection temperature was 250 °C, and the detector temperature was 270 °C. The injection volume was 2 μL, with nitrogen as the carrier gas. The split ratio of injection was 10 : 1, with a flow rate of 2 mL min⁻¹. The temperature program was set as follows: 50 °C for 10 min; 50 °C to 120 °C at 15 °C min⁻¹; 120 °C to 170 °C at 5 °C min⁻¹; 170 °C to 220 °C at 15 °C min⁻¹; 220 °C for 5 min.

2.9. Determination of intestinal microbiota composition

The contents of the colon were quickly collected under sterile conditions after the mice were sacrificed. DNA was extracted from the samples using the EZNA Stool DNA kit. The highly variable V3–V4 region of 16S rDNA was amplified by PCR and quantified on a QuantiFluor-ST fluorometer using primers 341_F: 5′-CCTACGGGNGGCWGCAG-3′ and 802 R: 5′-TACNVGGGTATCTAATCC-3′. Following previously published databases, the samples were sequenced on the Illumina MiSeq platform. In the microbial community analysis, raw reads were merged, trimmed, and chimeras were removed, then zero-radius operational taxonomic units (zOTU) were constructed by implementing UNOISE in Vsearch (v2.6.0). The Greengenes (v13.8) 16S rRNA gene database was used as the reference for annotation. Differences between groups were analyzed by partial least-squares discriminant analysis (PLS-DA) using the mixOmics R package (v3.2.1). Microbes with linear discriminant analysis (LDA) score greater than 3 were analyzed.

2.10. Assessment of anxiety of UC mice

Elevated plus-maze test. An elevated plus-maze test was used that had two open and two closed arms measuring 30 cm × 8 cm, raised 70 cm above the ground. A mouse was placed in the central area and the number of times and duration that a mouse entered the open and closed arms within 5 minutes were recorded. Finally, the percentage of time and the percentage of number of times the mouse entered the open arm were calculated.

Marble burying test. A cuboid container (460 mm × 300 mm × 160 mm) was filled with sawdust bedding approximately 5 cm thick. Four rows of five 15 mm diameter marbles (totaling 20 marbles) were placed on top of the sawdust bedding. The mouse was then placed in the container, and the number of marbles buried by the mouse in 15 min (burial considered if over 2/3 covered) was recorded.

Tail suspension test. The mouse's tail was suspended from a rack for 6 min, and the time the mouse remained still was recorded.

2.11. Statistical analysis

All experiments were conducted in triplicate to calculate mean values and standard deviations. One-way analysis of variance (ANOVA) was performed using SPSS software. The Duncan's test was used to calculate significant differences between means (p < 0.05).

3. Results and discussion

3.1. Health status of mice during modeling

The onset of UC is often accompanied by weight loss. By regularly measuring weight changes in mice, it is possible to track how the disease progresses.² Therefore, during the DSS intervention experiment, the weight of the mice should be checked daily, and the changes in the weight of the mice should be recorded and analyzed. In contrast to the control group, the body weight of the other 5 groups of mice decreased over time (Fig. 2A). Weight data collected on the seventh day showed a significant reduction in mouse weight for all groups receiving the DSS treatment (Fig. 2B). This weight loss may be because DSS induces colitis in mice, thereby leading to diarrhea and dehydration. Our results are consistent with the symptoms observed in mice in other studies using DSS-induced colitis.²⁶ The body weight of the mice in the Free P, FO, and P groups closely resembled the DSS group, while the weight of the P + FO group was significantly higher than for the other groups. This result suggests that the emulsions containing both fish oil and probiotics have hindered the progression of UC.

Fig. 2 (A) Changes in body weight of mice during modeling; (B) body weight of mice on day 7; (C) survival rate of mice during modeling; (D) survival rate of mice at the end; (E) DAI score of mice during modeling; and (F) DAI score on the 7th day of the model (n = 11). Different lowercase letters in the same figure indicate significant differences (P < 0.05).

In rare cases, when UC becomes exceptionally severe, it may pose a threat to the patient's life.²⁷ So, we assessed the survival rates of mice in the different groups (Fig. 2C and D). Compared to the control group, the DSS group's survival rate was only around 85%, indicating that DSS caused severe damage to the mice. In a previous study, it was reported that C57BL/6 mice developed UC after 7 days of intervention with 2.5% DSS, with a survival rate of 75% in the DSS group.²⁸ These results indicated that 2.5% DSS was used to simulate acute UC in mice for 7 days, which caused serious damage to mice. The FO group's survival rate (92%) was higher than that of the DSS group, which suggested that fish oil provided some protection against colitis, but that some fatalities still occurred. The survival rates of the remaining intervention groups (Free P, P, P + FO groups) and control group were 100%. These three groups all received probiotic-containing formulations, highlighting the effectiveness of probiotics in reducing DSS-induced colitis mortality.

The DAI is a key gauge for assessing mouse colitis severity.²⁹ Compared to the control group, the DAI score of the DSS group was significantly higher (Fig. 2E), indicating that the mice in this group had severe colitis. On the 7th day of the DSS intervention, the Free P, P, and FO groups exhibited slightly lower DAI scores than the DSS group (Fig. 2F), with no significant difference between them. Previous studies have shown that Lactobacillus acidophilus had an ameliorative effect on acute, chronic, and antibiotic-associated diarrhea.³⁰ Other researchers have also induced colitis using DSS and found that supplementation with fish oil containing 180 mg EPA and 120 mg DHA for one month significantly alleviated the symptoms of colitis-related inflammation (diarrhea, blood in stools, and weight loss).³¹ The above studies showed that both probiotics and fish oil improved colitis in mice to some extent. In this study, it is possible that due to the low number of probiotics reaching the colon in the Free P group, the weak adhesion ability of probiotics in the intestine in the P group, and the low content of fish oil in the FO group and the short intervention time, the above intervention groups showed less reduction in DAI score and weaker improvement in UC compared to the P + FO group. Moreover, the P + FO group's score was significantly lower than the DSS group and was closest to the control group's score, indicating that the mice in this group remained relatively healthy. Therefore, the W₁/O/W₂ emulsions containing both probiotics and fish oil were most effective at combating the adverse effects of DSS-induced colitis. Other researchers have also reported that colitis-afflicted mice treated with a combination of probiotics and other bioactive substances (modified apple polysaccharides) demonstrated lower DAI score than those treated solely with probiotics.³²

3.2. Impact of probiotic- and fish oil-loaded W₁/O/W₂ emulsions on colon length in mice with UC

The decrease in body weight, increase of DAI score, and rise in mortality observed in mice in the DSS group during the preliminary experiment confirmed the successful establishment of the UC model. Colon shortening is one of the common symptoms in UC patients, so the measurement of colon length after sacrificing mice is widely used as an objective assessment parameter to evaluate the severity and therapeutic efficacy of treatments using the UC model. As shown in Fig. 3A, the colon of mice in the DSS group clearly exhibited shortening, bleeding, swelling, and darkening. As indicated in Fig. 3B, the average colon length of the control group mice was 7.6 cm, while that of the DSS group mice (5.9 cm) was significantly shorter. The colon length of the Free P group (6.3 cm) and the FO group (6.3 cm) mice did not significantly differ from the DSS group, suggesting that free probiotics had little effect on the colon length of mice with UC. This could be because the free probiotics are largely inactivated when passing through the gastrointestinal tract and thus cannot exert a beneficial biological regulation effect.²⁵ The results also showed that fish oil had little effect on colon length in UC mice. This was possibly due to the relatively low content of fish oil used, with each mouse (26 g weight) ingesting only around 7 μL of fish oil every day. Previous studies have shown that ingesting 20 mg of fish oil per mouse per day could significantly improve symptoms associated with UC.³³ Therefore, due to the relatively low amount of fish oil used in the present study, the FO group did not significantly improve the shortening of the colon in mice. However, the P group (6.6 cm) and P + FO group (7.2 cm) mice had significantly longer colon lengths than the DSS group, suggesting that encapsulated probiotics could survive in sufficiently large numbers in the intestine and thus inhibit UC. The colon length of the P + FO group was slightly different from the control group, but this difference was not significant. This result suggests that the W₁/O/W₂ emulsions containing both probiotics and fish oil could significantly reduce DSS-induced colon shortening.

Fig. 3 (A) Representative images of mice colon; (B) colon length statistics of mice (n = 11). Different lowercase letters in the same figure denote significant differences (p < 0.05).

3.3. Impact of probiotic- and fish oil-loaded W₁/O/W₂ emulsions on colon tissue in mice with UC

Damage to colon tissue is a common symptom observed in patients with intestinal inflammation. The compromised integrity of the intestinal barrier can lead to increased intestinal permeability, which reduces its ability to prevent the invasion of pathogenic bacteria.^34,35 The degree of inflammation can be judged by observing the health of the colon tissue using H&E staining. As shown in Fig. 4A, compared to the control group, mice in the DSS group exhibited thickened intestinal walls, the crypts were absent, and a large number of inflammatory cells infiltrated the mucosal layer. However, the Free P, P, and FO groups showed irregular crypt surfaces, partial inflammatory cell infiltration, and a slight increase in intestinal wall thickness. By comparing these three groups, the probiotic-administered groups (Free P group and P group) exhibited thinner intestinal walls than the FO group, and the colon histological score of the FO group was slightly higher than for the other two (Fig. 4B). This result suggests that probiotics are more effective in delaying UC development than fish oil. In addition, the crypts of the P group were prominent and numerous, suggesting that the colons of these mice were healthier than those in the Free P group. Encapsulated probiotics demonstrated higher efficacy in suppressing DSS-induced colon inflammation than free probiotics. Previous studies have shown that lipid-encapsulated probiotics (Escherichia coli Nissle 1917) can notably ameliorate colon tissue damage.³⁶ However, the colon of the P + FO group mice did not show thickening of the intestinal wall, and the structure and number of crypts were prominent. Notably, the histological score of the P + FO group closely resembled that of the control group. This suggests that there was a synergistic effect between the probiotics and fish oil, making the intestinal condition of this group closest to the healthy control group. Thus, the W₁/O/W₂ emulsions containing both probiotics and fish oil were most effective at mitigating the barrier damage in the colon of mice with UC.

Fig. 4 (A) Representative H&E staining of colon tissue; (B) colitis histological score; (C) representative images of goblet cell stained in colon tissue; (D) proportion of goblet cell regions in colon tissue (n = 11). Different lowercase letters in the same figure denote significant differences (p < 0.05).

Goblet cells primarily synthesize and secrete mucins (such as MUC-2), forming a protective mucus layer on intestinal epithelial cell surfaces to safeguard the intestine's integrity.³⁷ Prior research has confirmed the significant role of probiotics (Bifidobacterium bifidum ATCC 29521) in safeguarding goblet cells in colitis-afflicted mice.³⁸ Goblet cells staining outcomes for each mouse group's colon are shown in Fig. 4C. Both the Free P group and FO group showed a significant reduction in goblet cells, a typical phenomenon in UC. The content of goblet cells in the P group and P + FO group did not significantly differ from the control group (Fig. 4D). This implies that encapsulated probiotics can counteract DSS-induced goblet cell decrease, which should promote the maintenance of intestinal barrier integrity. This effect was primarily attributed to the protection of the probiotics by the W₁/O/W₂ emulsion in the digestive tract, thereby enabling a more significant number of viable probiotics to reach the colon, where they could have their beneficial effects.

3.4. Impact of probiotic- and fish oil-loaded W₁/O/W₂ emulsions on tight junction proteins in colon tissue of mice with UC

Tight junction proteins in the gut physical barrier, which mainly includes the ZO family, Occludin, and Claudins, establish tight connections between intestinal epithelial cells, providing a sealed environment for intestinal tissue and protecting it from the intestinal cavity. Claudins mainly regulate paracellular permeability, with Claudin-1 playing a sealing role.³⁹ ZO-1 binds to Claudin-1 and acts as a scaffold to anchor transmembrane proteins to the actin cytoskeleton.⁴⁰ Immunohistochemical images of these proteins in the mouse colon are shown in Fig. 5. Compared with the control group, the secretion of ZO-1, Occludin, and Claudin-1 in the DSS and FO groups decreased. There were no significant difference in protein content for the DSS and FO groups, which indicated that the fish oil alone could not mitigate DSS-induced tight junction proteins reduction. Free P group mice exhibited higher Occludin and Claudin-1 secretion than the DSS group. This might be because many free probiotics die after digestion, but there are still a few surviving probiotics that regulate intestinal health. However, the P group and P + FO group could suppress the reduction in the secretion of ZO-1, Occludin, and Claudin-1 caused by DSS. The amount of tight junction proteins secreted in the intestines of these two groups of mice did not significantly differ from the control group (Fig. 5B, D and F). This may be because the probiotics encapsulated within the W₁/O/W₂ emulsions still had a relatively high viability after reaching the intestine. Consequently, they could promote the secretion of tight junction proteins to maintain the integrity of the intestinal barrier. Our result is consistent with previous studies that have shown that taking probiotics is beneficial to the integrity of the intestinal mucosa, thus improving the function of the intestinal barrier.⁴¹

Fig. 5 (A, C, E) Representative images of immunohistochemical staining of ZO-1, Occludin, and Claudin-1 in the mouse colon. (B, D, F) Area ratio of the ZO-1, Occludin, and Claudin-1 protein regions in the mouse colon to the colon wall region. n = 11. Different lowercase letters in the same figure denote significant differences (p < 0.05).

3.5. Impact of probiotic- and fish oil-loaded W₁/O/W₂ emulsions on expression of pro-inflammatory factor in colon tissue of mice with UC

In a healthy intestine, probiotics and pathogenic bacteria coexist, and there is a dynamic balance between them. When the intestinal tissue is damaged, the weakened physical barrier of the intestine leads to the invasion of pathogens into the intestinal tissue, triggering the secretion of pro-inflammatory factors (like TNF-α and IL-1β) and initiating inflammation. It has been reported that probiotics can effectively reduce pro-inflammatory factor levels.⁴² Expression of TNF-α and IL-1β in mouse colon tissue was therefore assessed using ELISA kits. As shown in Fig. 6, DSS treatment triggered a strong inflammatory response in mouse colon tissue, with a significant increase in the protein expression of pro-inflammatory factors TNF-α and IL-1β. TNF-α is an activator of the NF-kB signaling pathway and may promote the increase of other inflammatory cytokines.⁴³ IL-1β can increase intestinal permeability, promote the activation of dendritic cells and macrophages, and induce other inflammatory factors to participate in inflammation.⁴⁴ The results for the Free P group and FO group did not significantly differ from the DSS group, indicating that using free probiotics alone or using fish oil alone had no inhibitory effect on the inflammatory response induced by DSS. In contrast, the encapsulated probiotics (P group and P + FO group) significantly decreased TNF-α secretion (Fig. 6A) and IL-1β levels (Fig. 6B). This phenomenon may be attributed to the protective effect of the emulsion system, which shields probiotics from the harsh gastrointestinal environment, thereby ensuring that more viable probiotics reach the colon. Additionally, the presence of fish oil may enhance the adhesion of the probiotics within the colon, making it easier for them to attach to the intestinal wall, thereby providing a more stable foundation for probiotic action.²⁵

Fig. 6 Protein levels of inflammatory cytokines TNF-α (A) and IL-1β (B) in the colon of mice (n = 11). Different lowercase letters in the same figure denote significant differences (p < 0.05).

3.6. Effects of probiotic- and fish oil-loaded W₁/O/W₂ emulsions on SCFAs in feces of mice with UC

SCFAs are metabolites of microbial degradation of dietary fibers in the gut, which include acetate, propionate, butyrate, isobutyrate, and valeric acid. They play a significant regulatory role in regulating the intestinal microenvironment, immune response, and microbial structure.⁴⁵Fig. 7 shows the SCFAs concentrations in the feces of mice in different groups. DSS significantly reduced the content of all the SCFAs in the feces, which would be detrimental to gut health. The contents of various SCFAs in the FO group were slightly higher than the DSS group but not significantly. Thus, W₁/O/W₂ emulsions containing only fish oil had no significant effect on SCFAs production in mouse feces. In the Free P group, the concentrations of propionate and isobutyrate were significantly higher than that of the DSS group, and there was no significant difference in the other three. This suggests that the free probiotics had a modest beneficial effect. The reason for this relatively small effect is that the majority of free probiotics lost their activity when exposed to gastrointestinal conditions, thereby reducing their ability to produce large amounts of SCFAs. The probiotics in P group and P + FO group were protected by being encapsulated within the internal aqueous phase of the W₁/O/W₂ emulsions. Consequently, a higher fraction of viable probiotics could safely reach the intestine and promote the production of SCFAs. As a result, the SCFAs concentrations for these two groups were relatively close to those of the control group. In addition, the concentrations of propionate and isobutyrate in the P + FO group were higher than that in the P group. This effect may be because the fish oil in the emulsions promoted the growth and reproduction of the encapsulated Lactobacillus acidophilus, thereby increasing the concentrations of SCFAs produced by the degradation of large molecular carbohydrates in the intestine. Thus, the W₁/O/W₂ emulsions containing both fish oil and probiotics enhanced SCFAs production, which was attributed to their impact on microbial metabolism. At the same time, SCFAs lower the pH in the intestine, thereby creating a microenvironment that is unfavorable for pathogenic bacteria but favorable for probiotics.⁴⁶ It has been reported that Bifidobacterium can produce acetate to inhibit intestinal pathogens.⁴⁷ This effect may account for the reduced inflammation observed in the mice in the P + FO group.

Fig. 7 SCFAs concentrations in mouse feces. (A) Acetate; (B) propionate; (C) isobutyrate; (D) butyrate; (E) valeric acid (n = 6). Different lowcase letters in the same figure denote significant differences (p < 0.05).

3.7. Effects of probiotic- and fish oil-loaded W₁/O/W₂ emulsions on microbiota in colon tissue of mice with UC

UC patients often exhibit intestinal microbiome dysregulation, which is mainly manifested by reduced diversity and altered composition of the intestinal microbiome.⁴⁸ Identifying characteristic microbiota can therefore provide a better understanding of the pathogenesis of UC.⁴⁹ Changes in the intestinal microbiota of mice with UC can be observed by analyzing operational taxonomic units (OTUs) of intestinal bacteria (Fig. 8). The species accumulation curve indicated that the number of new OTUs observed was relatively small with increasing sample size, suggesting that the sampling of the mice samples used in this experiment was sufficient (Fig. 8A). The petal diagram clearly showed that there were differences in the microbial species in the intestines of each group of mice (Fig. 8B). The OTUs quantity of intestinal microbiota structure in the DSS mice (212) was significantly lower than that in the control mice (305). This result suggests that DSS can severely disrupt the structure of the intestinal microbiota, thereby causing a lack of various gut bacteria in the mice. The Free P group (213), P group (213), and FO group (215) only had 1–3 more than the DSS group. However, the P + FO group (224) had appreciably more, suggesting that the co-encapsulated probiotics and fish oil could inhibit the reduction in the diversity of gut microbiota caused by DSS. Furthermore, the shannon and chao indices, which also represent the diversity of the gut microbiota, followed a similar trend as the petal diagram (Fig. 8C and D). DSS significantly reduced gut microbiota diversity in the mice, but the extent of this reduction was the least in the P + FO group. Again, this effect can be attributed to the fact that more viable probiotics reached the intestine when they were encapsulated and that the probiotics and fish oil may have exhibited a synergistic effect.

Fig. 8 Analysis of intestinal microbial diversity in mice with UC. (A) Species accumulation curve; (B) Operational taxonomic units petal diagram; (C) Shannon index of microbial diversity; (D) Chao index of microbial diversity; (E) Partial least-squares discriminant analysis (n = 11). Different lowcase letters in the same figure denote significant differences (p < 0.05).

The PLS-DA analysis clearly showed differences in microbiota between each group of mice (Fig. 8E). DSS significantly altered the composition of the gut microbiota in the mice. The Free P group, P group, FO group, and DSS group were relatively close together, indicating a high similarity in their microbiota structures. In contrast, the position of the P + FO group was appreciably different from that of the other treatments. The fact that the P group and P + FO group were considerably different suggests that fish oil enhanced the probiotics’ ability to regulate the gut microbiota. This may have been due to its ability to promote the growth and proliferation of the probiotics and to improve probiotics adhesion to the gut walls.

The α-diversity of the gut microbiota and LDA are often used to analyze differences in microbiota between groups. As shown in Fig. 9A and B, this study screened out specific differential bacteria with LDA values greater than 3. The dominant bacteria in the DSS group are pathogenic bacteria such as Peptococcus, Escherichia, Enterobacteriaceae, and Gammaproteobacteria, indicating that DSS disrupts the balance of the gut microbiota, allowing pathogenic bacteria to dominate, thereby leading to UC. The dominant bacteria in the Free P group were also pathogenic bacteria such as Parasutterella, Burkholderiales, and Sutterellaceae. This is because the majority of free probiotics lost their activity after digestion, unable to compete for adhesion sites and nutrient activity with the abundant pathogenic bacteria in the UC intestinal environment, thereby allowing a large number of pathogenic bacteria to survive in the gut of these mice.⁸ The P group mainly included Akkermansia, Proteus, and Verrucomicrobiaeae, which are considered to be beneficial, harmful, and neutral bacteria, respectively. This suggests that these mice in the P group had a more balanced gut microbiota than those in the DSS group, which was probably because a greater fraction of viable probiotics reached the intestine after encapsulation. Consequently, these probiotics can more effectively suppress the proliferation of the pathogenic bacteria that cause inflammation. The dominant bacteria in the FO group were beneficial bacteria such as Parabacteroides, Flavonifractor, and Clostridium. This result suggests that fish oil promoted the survival of beneficial bacteria in the gut. As reported in previous studies, fish oil was known to have a beneficial effect on colitis.⁵⁰ The dominant bacteria in the P + FO group included propionic acid-producing and butyric acid-producing Rikenellaceae, and Butyricimonas, which are considered to be beneficial bacteria. This result corresponded to the high propionate and butyrate content of P + FO group in Fig. 7C. The predominance of probiotics in the P + FO group may be due to the following reasons: fish oil promotes the adhesion of Lactobacillus acidophilus in the intestine, competing with other pathogenic bacteria for limited growth sites and promoting the elimination of pathogenic bacteria.⁹ In addition, the probiotics capable of producing SCFAs in the P + FO group proliferate, thereby increasing the production of SCFAs. These SCFAs can promote the growth and reproduction of probiotics. Moreover, the SCFAs reduce the pH within the intestinal environment, thereby creating a more acidic environment in the colon that promotes the growth of probiotics but inhibits the growth of pathogenic bacteria.⁵¹

Fig. 9 (A) α-Diversity of the gut microbiota in mice; (B) Linear discriminant analysis of gut microbiota in mice (n = 11). Different lowcase letters in the same figure denote significant differences (p < 0.05).

3.8. Effects of probiotic- and fish oil-loaded W₁/O/W₂ emulsions on anxiety-like behavior in mice with UC

Many studies have shown that the incidences of depression and anxiety increase in patients with intestinal diseases.^52,53 Moreover, studies with mice have reported that these adverse mental conditions are reduced after the colon in healed.²⁹ This anxiety may be due to the overactivation of microglial cells, which causes persistent inflammation in the central system.⁵³ Elevated plus-maze, marble burying, and tail suspension tests are widely used to measure the anxiety responses of laboratory animals.^54–56 Therefore, we used these behavioral experiments to explore the effects of the different treatments on the anxiety of the mice (Fig. 10). The marble burying test's schematic is depicted in Fig. 10A. The DSS group's mice buried significantly more beads within a specific time compared to the control group (Fig. 10B), indicating that DSS could cause significant anxiety-like behavior. Both the Free P and FO groups exhibited bead-burying tendencies second only to the DSS group, suggesting that free probiotics and fish oil could only partially alleviate anxiety behavior. The number of beads buried by the mice in the P group was significantly less than in the Free P group, indicating that the encapsulated probiotics had a higher anxiety-inhibiting effect than the free probiotics. Additionally, no significant difference was found in the bead-burying behavior of the P + FO group and the control group, implying that the co-encapsulated probiotics and fish oil could synergistically alleviate DSS-induced anxiety. The tail suspension experiment showed that the immobility time of mice in the P + FO group was similar to that of the control group, while the other four groups were significantly longer than these two groups (Fig. 10C). These results there also show that the mental state of the mice in the P + FO group was better than that in the Free P, FO, and P groups. The elevated plus-maze test schematic is depicted in Fig. 10D. The proportion of times and time the mice in the P + FO group entered the open arms were similar to the control group (Fig. 10E and F), indicating that the mice were in a relatively active state.

Fig. 10 Anxiety-like behavior associated with UC. (A) Schematic diagram of the marble burying experiment; (B) number of beads buried by mice; (C) length of silence time in mice during the tail suspension experiment; (D) schematic diagram of the elevated plus-maze; (E) the proportion of the frequency of entries into open arms by mice; (F) the proportion of time spent in open arms by mice (n = 6). Different lowercase letters in the same figure denote significant differences (p < 0.05).

In summary, the behavior test results reveal that the W₁/O/W₂ emulsions containing both probiotics and fish oil could prevent anxiety-like behavior associated with DSS-induced colitis.

4. Conclusions

In this study, we established a UC mice model to investigate the preventative effects of free probiotics, encapsulated probiotics, encapsulated fish oil, and co-encapsulated probiotics and fish oil on UC and associated anxiety-like behavior in mice. The probiotics were encapsulated within the internal water phase of W₁/O/W₂ emulsions, while the fish oil was encapsulated within the oil phase. The results showed that administration of co-encapsulated probiotics and fish oil significantly ameliorated the secretion of tight junction proteins (ZO-1, Occludin, and Claudin-1), protected intestinal epithelial structure, inhibited pro-inflammatory cytokines (TNF-α and IL-1β), reduced inflammation, enhanced SCFAs production, and modulated gut microbiota. The co-encapsulated probiotics and fish oil not only mitigated the pathological changes induced by DSS but also significantly relieved the anxiety-like behavior observed in UC mice. This suggests that the co-encapsulated system has considerable potential for improving gut and mental health in those suffering from UC.

In conclusion, our study has shed light on the significant promise of co-encapsulated probiotics and fish oil for the treatment of UC and associated behavior disorders. These findings set the stage for further research in this direction, with a focus on the long-term effects and potential clinical applications of this combined therapy.

Author contributions

Q. G. and Z. J., conceptualized the project and wrote the paper; K. L. and Y. L., performed experiments; X. Y., analyzed date; D. J. M., writing – review & editing; C. M., interpreted results of experiments; F. L. project administration and drafted the manuscript. All authors read and approved the final manuscript.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China.

References

1. Y. Lin, H. Liu, L. Bu, C. Chen and X. Ye, Review of the effects and mechanism of curcumin in the treatment of inflammatory bowel disease, Front. Pharmacol., 2022, 13, 908077.
2. R. Ungaro, S. Mehandru, P. B. Allen, L. Peyrin-Biroulet and J.-F. Colombel, Ulcerative colitis, Lancet, 2017, 389, 1756–1770.
3. C. N. Bernstein, C. A. Hitchon, R. Walld, J. M. Bolton, J. Sareen, J. R. Walker, L. A. Graff, S. B. Patten, A. Singer, L. M. Lix, R. El-Gabalawy, A. Katz, J. D. Fisk, R. A. Marrie and C. T. D. B. Managi, Increased burden of psychiatric disorders in inflammatory bowel disease, Inflamm. Bowel Dis., 2019, 25, 360–368.
4. C. D. Moulton, P. Pavlidis, C. Norton, S. Norton, C. Pariante, B. Hayee and N. Powell, Depressive symptoms in inflammatory bowel disease: an extraintestinal manifestation of inflammation?, Clin. Exp. Immunol., 2019, 197, 308–318.
5. I. Curkovic, M. Egbring and G. A. Kullak-Ublick, Risks of inflammatory bowel disease treatment with glucocorticosteroids and aminosalicylates, Dig. Dis., 2013, 31, 368–373.
6. C. Hill, F. Guarner, G. Reid, G. R. Gibson, D. J. Merenstein, B. Pot, L. Morelli, R. B. Canani, H. J. Flint and S. Salminen, Expert consensus document: The international scientific association for probiotics and prebiotics consensus statement on the scope and appropriate use of the term probiotic, Nat. Rev. Gastroenterol. Hepatol., 2014, 11, 506–514.
7. J. Yang, G. Zhang, X. Yang, M. Peng, S. Ge, S. Tan, Z. Wen, Y. Wang, S. Wu, Y. Liang, J. An, K. Zhang, J. Liu, J. Shi and Z. Zhang, An oral “Super probiotics” with versatile self-assembly adventitia for enhanced intestinal colonization by autonomous regulating the pathological microenvironment, Chem. Eng. J., 2022, 446, 137204.
8. Q. Gu, Y. Yin, X. Yan, X. Liu, F. Liu and D. J. McClements, Encapsulation of multiple probiotics, synbiotics, or nutrabiotics for improved health effects: A review, Adv. Colloid Interface Sci., 2022, 309, 102781.
9. J. Plaza-Diaz, F. Javier Ruiz-Ojeda, M. Gil-Campos and A. Gil, Mechanisms of action of probiotics, Adv. Nutr., 2019, 10, S49–S66.
10. X. Huang, Y. Li, P. Zhuang, X. Liu, Y. Zhang, P. Zhang and J. Jiao, Habitual fish oil supplementation and risk of incident inflammatory bowel diseases: A prospective population-based study, Front. Nutr., 2022, 9, 905162.
11. P. C. Calder, Omega-3 fatty acids and inflammatory processes: from molecules to man, Biochem. Soc. Trans., 2017, 45, 1105–1115.
12. L. Costantini, R. Molinari, B. Farinon and N. Merendino, Impact of omega-3 fatty acids on the gut microbiota, Int. J. Mol. Sci., 2017, 18, 2645.
13. B. Zhou, Y. Yuan, S. Zhang, C. Guo, X. Li, G. Li, W. Xiong and Z. Zeng, Intestinal flora and disease mutually shape the regional immune system in the intestinal tract, Front. Immunol., 2020, 11, 575.
14. J. Chen, K. Wright, J. M. Davis, P. Jeraldo, E. V. Marietta, J. Murray, H. Nelson, E. L. Matteson and V. Taneja, An expansion of rare lineage intestinal microbes characterizes rheumatoid arthritis, Genome Med., 2016, 8, 43.
15. Z. Jiang, M. Li, D. J. McClements, X. Liu and F. Liu, Recent advances in the design and fabrication of probiotic delivery systems to target intestinal inflammation, Food hydrocolloids, 2022, 125, 107438.
16. J. Shi, Q. Xie, Y. Yue, Q. Chen, L. Zhao, S. E. Evivie, B. Li and G. Huo, Gut microbiota modulation and anti-inflammatory properties of mixed lactobacilli in dextran sodium sulfate-induced colitis in mice, Food Funct., 2021, 12, 5130–5143.
17. N. Zhao, J.-M. Liu, F.-E. Yang, X.-M. Ji, C.-Y. Li, S.-W. Lv and S. Wang, A novel mediation strategy of DSS-induced colitis in mice based on an iron-enriched probiotic and in vivo bioluminescence tracing, J. Agric. Food Chem., 2020, 68, 12028–12038.
18. P. A. Bron, P. van Baarlen and M. Kleerebezem, Emerging molecular insights into the interaction between probiotics and the host intestinal mucosa, Nat. Rev. Microbiol., 2012, 10, 66–78.
19. Y. Chen, B. Yang, J. Zhao, R. P. Ross, C. Stanton, H. Zhang and W. Chen, Exploiting lactic acid bacteria for colorectal cancer: a recent update, Crit. Rev. Food Sci. Nutr., 2022, 1–17.
20. Y.-X. Zhu, Y. You, Z. Chen, D. Xu, W. Yue, X. Ma, J. Jiang, W. Wu, H. Lin and J. Shi, Inorganic nanosheet-shielded probiotics: A self-adaptable oral delivery system for intestinal disease treatment, Nano Lett., 2023, 23, 4683–4692.
21. R. Wang, K. Guo, W. Zhang, Y. He, K. Yang, Q. Chen, L. Yang, Z. Di, J. Qiu, P. Lei, Y. Gu, Z. Luo, X. Xu, Z. Xu, X. Feng, S. Li, Z. Yu and H. Xu, Poly-gamma-glutamic acid microgel-encapsulated probiotics with gastric acid resistance and smart inflammatory factor targeted delivery performance to ameliorate colitis, Adv. Funct. Mater., 2022, 32, 2113034.
22. X. He, W. Yang and X. Qin, Ultrasound-assisted multilayer Pickering emulsion fabricated by WPI-EGCG covalent conjugates for encapsulating probiotics in colon-targeted release, Ultrason. Sonochem., 2023, 97, 106450.
23. X.-S. Qin, Q.-Y. Gao and Z.-G. Luo, Enhancing the storage and gastrointestinal passage viability of probiotic powder (Lactobacillus Plantarum) through encapsulation with pickering high internal phase emulsions stabilized with WPI-EGCG covalent conjugate nanoparticles, Food hydrocolloids, 2021, 116, 106658.
24. Z. Jiang, J. Tian, X. Bai, D. J. McClements, C. Ma, X. Liu and F. Liu, Improving probiotic survival using water-in-oil-in-water (W₁/O/W₂) emulsions: Role of fish oil in inner phase and sodium alginate in outer phase, Food Chem., 2023, 417, 135889.
25. Y. Dai, X. Lu, R. Li, Y. Cao, W. Zhou, J. Li and B. Zheng, Fabrication and characterization of W/O/W emulgels by sipunculus nudus salt-soluble proteins: Co-encapsulation of vitamin C and β-carotene, Foods, 2022, 11, 2720.
26. Y. Chen, B. Yang, C. Stanton, R. P. Ross, J. Zhao, H. Zhang and W. Chen, Bifidobacterium pseudocatenulatum ameliorates DSS-induced colitis by maintaining intestinal mechanical barrier, blocking proinflammatory cytokines, inhibiting TLR4/NF-κB signaling, and altering gut microbiota, J. Agric. Food Chem., 2021, 69, 1496–1512.
27. A. Kohyama, K. Watanabe, A. Sugita, K. Futami, H. Ikeuchi, K.-i. Takahashi, Y. Suzuki and K. Fukushima, Ulcerative colitis-related severe enteritis: an infrequent but serious complication after colectomy, J. Gastroenterol., 2021, 56, 240–249.
28. Y. Wu, L. Ran, Y. Yang, X. Gao, M. Peng, S. Liu, L. Sun, J. Wan, Y. Wang, K. Yang, M. Yin and W. Chunyu, Deferasirox alleviates DSS-induced ulcerative colitis in mice by inhibiting ferroptosis and improving intestinal microbiota, Life Sci., 2023, 314, 121312.
29. X. Zhang, Q. Zou, B. Zhao, J. Zhang, W. Zhao, Y. Li, R. Liu, X. Liu and Z. Liu, Effects of alternate-day fasting, time-restricted fasting and intermittent energy restriction DSS-induced on colitis and behavioral disorders, Redox Biol., 2020, 32, 101535.
30. J. M. Remes Troche, E. Coss Adame, M. A. Valdovinos Diaz, O. Gomez Escudero, M. E. Icaza Chavez, J. Antonio Chavez-Barrera, F. Zarate Mondragon, J. A. R. Velarde Velasco, G. R. Aceves Tavares, M. A. Lira Pedrin, E. Cerda Contreras, R. I. Carmona Sanchez, H. Guerra Lopez and R. Solana Ortiz, Lactobacillus acidophilus LB: A useful pharmabiotic for the treatment of digestive disorders, Ther. Adv. Gastroenterol., 2020, 13, 1756284820971201.
31. M. Sharma, R. Kaur, K. Kaushik and N. Kaushal, Redox modulatory protective effects of ω-3 fatty acids rich fish oil against experimental colitis, Toxicol. Mech. Methods, 2019, 29, 244–254.
32. S. Mohanta, S. K. Singh, B. Kumar, M. Gulati, R. Kumar, A. K. Yadav, S. Wadhwa, J. Jyoti, S. Som, K. Dua and N. K. Pandey, Efficacy of co-administration of modified apple polysaccharide and probiotics in guar gum-Eudragit S100 based mesalamine mini tablets: A novel approach in treating ulcerative colitis, Int. J. Biol. Macromol., 2019, 126, 427–435.
33. C. Silvestri, E. Pagano, S. Lacroix, T. Venneri, C. Cristiano, A. Calignano, O. A. Parisi, A. A. Izzo, V. Di Marzo and F. Borrelli, Fish oil, cannabidiol and the gut microbiota: An investigation in a murine model of colitis, Front. Pharmacol., 2020, 11, 585096.
34. L.-Z. Shu, Y.-D. Ding, Q.-M. Xue, W. Cai and H. Deng, Direct and indirect effects of pathogenic bacteria on the integrity of intestinal barrier, Ther. Adv. Gastroenterol., 2023, 16, 17562848231176427.
35. Y.-R. Tan, S.-Y. Shen, H.-Q. Shen, P.-F. Yi, B.-D. Fu and L.-Y. Peng, The role of endoplasmic reticulum stress in regulation of intestinal barrier and inflammatory bowel disease, Exp. Cell Res., 2023, 424, 113472.
36. H. Zhao, Y. Du, L. Liu, Y. Du, K. Cui, P. Yu, L. Li, Y. Zhu, W. Jiang, Z. Li, H. Tang and W. Ma, Oral nanozyme-engineered probiotics for the treatment of ulcerative colitis, J. Mater. Chem. B, 2022, 10, 4002–4011.
37. M. E. V. Johansson, J. M. H. Larsson and G. C. Hansson, The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host-microbial interactions, Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 4659–4665.
38. A. U. Din, A. Hassan, Y. Zhu, K. Zhang, Y. Wang, T. Li, Y. Wang and G. Wang, Inhibitory effect of Bifidobacterium bifidum ATCC 29521 on colitis and its mechanism, J. Nutr. Biochem., 2020, 79, 108353.
39. C.-S. Alvarez, J. Badia, M. Bosch, R. Gimenez and L. Baldoma, Outer membrane vesicles and soluble factors released by probiotic Escherichia coil Nissle 1917 and commensal ECOR63 enhance barrier function by regulating expression of tight junction proteins in intestinal epithelial cells, Front. Microbiol., 2016, 7, 1981.
40. L. Shen, C. R. Weber, D. R. Raleigh, D. Yu and J. R. Turner, Tight junction pore and leak pathways: A dynamic duo, Annu. Rev. Physiol., 2011, 73, 283.
41. Y. Chen, L. Zhang, G. Hong, C. Huang, W. Qian, T. Bai, J. Song, Y. Song and X. Hou, Probiotic mixtures with aerobic constituent promoted the recovery of multi-barriers in DSS-induced chronic colitis, Life Sci., 2020, 240, 117089.
42. A. Vincenzi, M. I. Goettert and C. F. Volken de Souza, An evaluation of the effects of probiotics on tumoral necrosis factor (TNF-α) signaling and gene expression, Cytokine Growth Factor Rev., 2021, 57, 27–38.
43. Y. Kagoya, A. Yoshimi, S. Arai, K. Kataoka, M. Nakagawa, K. Kumano and M. Kurokawa, NF-κB/TNF-α positive feedback loop with active proteasome machinery supports myeloid leukemia initiating cell capacity, Blood, 2012, 120, 654.
44. R. Al-Sadi, S. Guo, K. Dokladny, M. A. Smith, D. Ye, A. Kaza, D. M. Watterson and T. Y. Ma, Mechanism of interleukin-1β induced-increase in mouse intestinal permeability in vivo, J. Interferon Cytokine Res., 2012, 32, 474–484.
45. A. Rauf, A. A. Khalil, U.-U. Rahman, A. Khalid, S. Naz, M. A. Shariati, M. Rebezov, E. Z. Urtecho, R. D. D. G. de Albuquerque, S. Anwar, A. Alamri, R. K. Saini and K. R. R. Rengasamy, Recent advances in the therapeutic application of short-chain fatty acids (SCFAs): An updated review, Crit. Rev. Food Sci. Nutr., 2022, 62, 6034–6054.
46. G. T. Macfarlane and S. Macfarlane, Bacteria, Colonic fermentation, and gastrointestinal health, J. AOAC Int., 2012, 95, 50–60.
47. S. Fukuda, H. Toh, K. Hase, K. Oshima, Y. Nakanishi, K. Yoshimura, T. Tobe, J. M. Clarke, D. L. Topping, T. Suzuki, T. D. Taylor, K. Itoh, J. Kikuchi, H. Morita, M. Hattori and H. Ohno, Bifidobacteria can protect from enteropathogenic infection through production of acetate, Nature, 2011, 469, 543–U791.
48. Z. Dai, X. Ma, R. Yang, H. Wang, D. Xu, J. Yang, X. Guo, S. Meng, R. Xu, Y. Li, Y. Xu, K. Li and X. Lin, Intestinal flora alterations in patients with ulcerative colitis and their association with inflammation, Exp. Ther. Med., 2021, 22, 1322.
49. D. Pu, Z. Zhang and B. Feng, Alterations and potential applications of gut microbiota in biological therapy for inflammatory bowel diseases, Front. Pharmacol., 2022, 13, 906419.
50. E. Yorulmaz, H. Yorulmaz, E. S. Gokmen, S. Altinay, S. H. Kucuk, O. Zengi, D. S. Celik and D. Sit, Therapeutic effectiveness of rectally administered fish oil and mesalazine in trinitrobenzenesulfonic acid-induced colitis, Biomed. Pharmacother., 2019, 118, 109247.
51. D. Li, X. Wang, J. Wang, M. Wang, J. Zhou, S. Liu, J. Zhao, J. Li and H. Wang, Structural characterization of different starch-fatty acid complexes and their effects on human intestinal microflora, J. Food Sci., 2023, 88, 3562–3576.
52. B. Xia, X. Liu, X. Li, Y. Wang, D. Wang, R. Kou, L. Zhang, R. Shi, J. Ye, X. Bo, Q. Liu, B. Zhao and X. Liu, Sesamol ameliorates dextran sulfate sodium-induced depression-like and anxiety-like behaviors in colitis mice: the potential involvement of the gut-brain axis, Food Funct., 2022, 13, 2865–2883.
53. E. Dempsey, A. Abautret-Daly, N. G. Docherty, C. Medina and A. Harkin, Persistent central inflammation and region specific cellular activation accompany depression- and anxiety-like behaviours during the resolution phase of experimental colitis, Brain, Behav., Immun., 2019, 80, 616–632.
54. H. Shoji and T. Miyakawa, Effects of test experience, closed-arm wall color, and illumination level on behavior and plasma corticosterone response in an elevated plus maze in male C57BL/6J mice: a challenge against conventional interpretation of the test, Mol. Brain, 2021, 14, 34.
55. P. V. Dixit, R. Sahu and D. K. Mishra, Marble-burying behavior test as a murine model of compulsive-like behavior, J. Pharmacol. Toxicol. Methods, 2020, 102, 106676.
56. Y.-J. Shin, D.-Y. Lee, J. Y. Kim, K. Heo, J.-J. Shim, J.-L. Lee and D.-H. Kim, Effect of fermented red ginseng on gut microbiota dysbiosis- or immobilization stress-induced anxiety, depression, and colitis in mice, J. Ginseng Res., 2023, 47, 255–264.

---

Footnote

† These authors contributed equally to this work.

---