1. Introduction
Ulcerative colitis (UC) is a chronic idiopathic inflammatory bowel disease whose clinical manifestations include recurrent abdominal pain, diarrhea, and mucopurulent bloody stools, which seriously affect the quality of life of patients [
1]. The prevalence of UC has been steadily rising for the past years, resulting in a growing strain on healthcare systems globally [
2]. Nevertheless, the existing clinical treatment for UC is inadequate and plagued by numerous issues, rendering it incapable of satisfying the requirements of patients. Aminosalicylic acid formulations effectively manage mild-to-moderate UC but do not demonstrate efficacy in treating severe UC [
3]. For moderate-to-severe UC, the first-line therapy is oral systemic glucocorticoids. However, these are unsuitable for long-term maintenance therapy due to hormone dependency and significant side effects, including femoral head osteonecrosis and infection [
4]. Patients with moderate-to-severe UC require immunosuppressants and biologics for maintenance. These treatments help relieve UC symptoms; however, immunosuppressants such as mercaptopurine may induce myelosuppression and raise cancer risk [
5,
6]. Therefore, carefully evaluating the use of these medications is crucial. In addition, biologics such as infliximab (IFX), ustekinumab (UST), and vedolizumab (VDZ) can be used for targeted treatment of UC. Nevertheless, recent studies have highlighted a potential risk of loss of response during treatment with biologics and the high cost of biologics, which makes them unaffordable for many families [
7]. These considerations indicate that addressing UC through a singular factor is ineffective, has a poor prognosis, and is financially burdensome. Consequently, the development of a cost-effective, secure, and potent targeted medication with diverse therapeutic properties for managing UC is imperative [
8].
Pterostilbene (PSB), a naturally occurring phenolic compound with a chemical structure resembling that of resveratrol, was first isolated in 1940 from the wood of the rosewood tree. It has since been found to be abundant in various plants, including blueberries and grapes. Studies have shown that PSB has the potential to decrease inflammation, function as an antioxidant, combat cancer, and regulate the immune system [
9,
10]. According to reports, PSB can serve as an adaptive ligand for the aryl hydrocarbon receptor (AHR) and stimulate its activation [
11]. AHR is expressed in intestinal immune and non-immune cells and affects epithelial barrier integrity [
12]. Therefore, PSB may be a potential UC treatment.
However, the weak oral impact of PSB, low targeted effectiveness, rapid plasma clearance, and poor water solubility have limited its clinical usage. To address these issues, a medical mode of delivery is urgently needed. Health-beneficial prebiotics, including fructooligosaccharides, galactose oligosaccharides, and inulin, are solely used by the host gut bacteria and are virtually completely undegraded in the stomach. Hence, they exhibit intrinsic benefits as delivery vehicles specifically tailored for colon targeting [
13]. Research indicates that prebiotics have the potential to mitigate colon inflammation by modulating gut microbiota [
14]. Nonetheless, a singular dose of prebiotics has been shown to be insufficient for gut restoration, necessitating combination therapy with other medications. Therefore, we suggest utilizing PSB-loaded prebiotics to modulate the intestinal microenvironment and reinforce the intestinal barrier through various mechanisms.
This work used microfluidics to uniformly encapsulate the natural polyphenol PSB in sodium alginate (Alg) and resistant starch (RS) prebiotic microcapsules covered with chitosan (CS). On the one hand, the pH-sensitive core-shell structure of PSB-loaded prebiotic microcapsules (PSB@MC) minimized its breakdown in the acidic conditions of the upper gastrointestinal tract, thereby reducing PSB degradation in the stomach
(Fig. 1). On the other hand, PSB@MC rapidly swelled in the environment of the lower gastrointestinal tract, leading to the release of PSB alongside prebiotic dispersion. As a result, PSB exerted its anti-inflammatory and antioxidant effects, thereby alleviating colonic inflammation. Prebiotics, in turn, can be metabolized by probiotics to promote gut homeostasis by regulating gut microbiota and metabolite production. Furthermore, PSB@MC could repair the intestinal mechanical barrier and restore its morphological and functional integrity by activating the AHR/IL-22 pathway. These results suggest that PSB@MC could serve as a novel, secure, and efficient UC therapy option.
2. Materials and Methods
2.1. Materials
PSB was obtained from Meilun Bio (China), Alg was acquired from Alta Aesar (England), and RS was purchased from Yuanyebio (China). CaCl2 was acquired from Sigma-Aldrich (St. Louis, MO, USA); CS, acetic acid, and hydrochloric acid were acquired from Aladdin (China); lumisphere monodisperse fluorescent microspheres were purchased from Tianjin BaseLine Chrom Techn Research Center (China); near-infrared fluorescence-labeled polystyrene microspheres were purchased from Suzhou Nanomicro Technology Co., Ltd. (China); phosphate-buffered saline (PBS) was acquired from Sevier (China); 2,7-dichlorofluorescein diacetate (DCFH-DA) was obtained from Beyotime Biotechnology (China); dextran sulfate sodium salt (DSS) was acquired from MP Biomedicals (USA); malondialdehyde (MDA) and myeloperoxidase (MPO) test kits were provided by Nanjing Jiancheng Bioengineering Institute (China). Deionized water (18.2 MΩ cm; MilliporeSigma, USA) was used to prepare all aqueous solutions.
2.2. PSB@MC preparation
The Alg (2.0%, weight/volume (w/v)) and RS (2.0%, w/v) solutions were thoroughly mixed to form the Alg/RS solution. Subsequently, PSB was evenly distributed throughout the Alg/RS solution. The mixture of Alg/RS solution and PSB was injected into a syringe to create PSB@Alg/RS microcapsules using the microfluidic electrospray technique. The capillary, with an inner diameter ranging from 150 to 270 μm, was positioned 10 cm above the surface of the collecting bath while an electrostatic potential of 8–12 kV was applied. Droplets were collected in a CaCl2 solution (3.0% w/v). After 30 min of adequate Ca2+ cross-linking, the microcapsules were immersed in a CS solution (1.0% w/v) containing 1.0% (w/v) acetic acid for 30 min to form a CS shell. Finally, the microcapsules were rinsed numerous times with deionized water before being freeze-dried for 24 h. Empty microcapsules were formed from a PSB-free Alg/RS solution.
2.3. PSB@MC characterization
Optical (NEXCAM-T20, Nexcope, China) and scanning electron (SEM; SU-8010, Hitachi, Japan) microscopy were used to study the morphology of PSB@MC. Green and red fluorescently labeled polystyrene microspheres were added to the PSB/Alg/RS and CS solutions, respectively, with the polystyrene microspheres making up 2.0% of the volume, v/v. Subsequently, fluorescence images were acquired using a Nikon confocal microscope (Nikon, Japan) to analyze the core-shell architecture of the microspheres.
2.4. Animals
Male C57BL/6 mice were provided by the Xi'an Jiaotong University Experimental Animal Center (China). The animals were housed under a 12-h light-dark cycle at a temperature of 22–25 °C and humidity of 65% ± 5%, with free access to water and food. All animal experiments were performed following the Principles of Laboratory Animal Care and Guidelines of the Laboratory Animal Care Committee of Xi’an Jiaotong University (No: 2021-213).
2.5. In vivo biodistribution
Fluorescently labeled MC and PSB@MC were generated by adding near-infrared (NIR) fluorescently labeled polystyrene microspheres (1.0%, v/v) to the Alg/RS and PSB/Alg/RS solutions for intestinal colonization experiments. Each healthy mouse was then gavaged with ≈200 µL of PSB@MC or MC. Next, at various periods (0, 1, 3, 6, and 12 h), in vivo imaging was conducted using a VISQUE® InVivo Smart-LF System (Vieworks, Republic of Korea), and the average fluorescence intensity for each group was noted. After 12 h of in vivo imaging, the mice were necropsied, and the hearts, livers, spleens, lungs, kidneys, and entire digestive tracts were collected for transient fluorescence imaging. The average fluorescence intensities for each group were recorded, and the fluorescence intensities for each site were used to assess the ability of PSB@MC to target the colon under physiological conditions. In addition, 2.5% DSS was administered to establish a UC model, and the differences in body and tissue distribution of different orally administered drugs in UC mice were assessed according to the same protocol.
2.6. Disease activity index
Weight change was assessed as follows: 0–5% weight loss = 0 points, 5–10% weight loss = 1 point, 10–15% weight loss = 2 points, 15–20% weight loss = 3 points, and weight loss >20% weight loss = 4 points. For fecal consistency, normal feces = 0 points, softened and pasty feces = 1 point, loose and unformed feces = 2 points, and watery stools = 3 points. Regarding rectal bleeding, no blood in the feces = 0 points, positive fecal occult blood or a small amount of blood streaks = 1 point, obvious blood in the feces = 2 points, and severe bloody stools = 3 points. The total disease activity index (DAI) score was obtained by adding the scores of these parameters.
2.7. Protective and therapeutic effects of PSB@MC against UC
Male C57BL/6 mice were randomly allocated to six groups: control, DSS, MC, PSB, PSB@MC, and 5-aminosalicylic acid (5-ASA). To assess the colon-protective effects of PSB@MC, a colitis model was induced by administering water supplemented with 2.5% DSS for 7 days. During the modeling period, 100 mg·kg−1·d−1 each of MC, PSB, PSB@MC, or 5-ASA were administered by gavage every other day. In the treatment model, the mouse colitis model was established using the same method. After establishing the colitis model, mice were administered 100 mg·kg−1·d−1 each of MC, PSB, PSB@MC, or 5-ASA for 5 days to evaluate the therapeutic effect. Daily weight and DAI were recorded during the experiment. The colon and cecum were excised and stored after euthanasia for pathological evaluation, quantitative real-time polymerasechain reaction (qRT-PCR) analysis, and evaluation of other PSB@MC in vivo protective and therapeutic effects.
2.8. 16S ribosomal DNA (rDNA) sequencing
Complete genomic DNA was recovered from samples using a Mag-bind soil DNA kit (Omega Bio-Tek, USA). After collection, DNA purity and concentration were analyzed. Barcoded primers and high-fidelity DNA polymerase were used for PCR amplification. Primers and polymerase were selected based on the V3–V4 variable regions sequenced. A Quant-iT PicoGreen dsDNA test kit (Invitrogen, USA) was used to extract the specific fragment from the PCR product after 2% agarose gel electrophoresis. Based on the electrophoresis results, a fluorescence quantification system equipped with a microplate reader (FLx800; BioTek Instruments, USA) was used to quantify the recovered PCR-amplified products. Thereafter, the products were pooled in appropriate quantities for sequencing each sample. Libraries were constructed using Illumina's TruSeq Nano DNA LT Library Preparation Kit (Illumina, USA). Libraries were evaluated using an Agilent Bioanalyzer 2100 (Agilent Technologies, USA) and Promega QuantiFluor (Promega, USA), ensuring their suitability for sequencing.
2.9. Untargeted metabolomics analysis
A Vanquish UHPLC (Thermo Fisher Scientific, USA) was used for the evaluation. Shanghai Applied Protein Technology Co., Ltd. (China) coupled an Orbitrap mass spectrometer (Thermo Fisher Scientific) to the UHPLC. A Waters ACQUIY UPLC BEH Amide 1.7 µm column (2.1 mm × 100 mm; Waters Corporation, USA) was used for hydrophilic interaction chromatography (HILIC) separation. Mobile phase A comprised 25 mM ammonium hydroxide and acetate in water, while mobile phase B consisted of acetonitrile, used in both the positive and negative electrospray ionization (ESI) modes. After 1.5 min at 98% B, the gradient decreased to 2% over 10.5 min, then rapidly increased from 2 to 98% in 0.1 min after 2 min. A 3-min readjustment followed. The ESI source conditions were as follows: Gas1 and Gas2 at 60 ℃, CUR at 30 ℃, source temperature at 600 ℃, and ion spray voltage floating at ±5500 V. The instrument was configured to capture mass spectrometry (MS)-only data within a range of 80–1200 Da. A resolution of 60 000 allowed for the detection of closely spaced peaks, with data acquisition occurring over 100 ms. Auto MS/MS collection was enabled for mass-to-charge ratios between 70 and 1200 Da. No acquisition period shorter than 4 s was permitted, using a resolution of 30 000 and an accumulation time of 50 ms.
2.10. Statistical analysis
GraphPad Prism 8.3 (GraphPad Software, USA) was used to analyze the data. Group data were analyzed using Student’s t-test. The data are presented as mean ±SD. Statistical significance was considered at *P < 0.05, **P < 0.01, and ***P < 0.001.
3. Results
3.1. Preparation and characterization of PSB@MC
In the synthesis of prebiotic microcapsules loaded with PSB, the primary materials used included Alg, RS, CS, and PSB. Alg is a natural polysaccharide [
15] that can form a gel-like structure through cross-linking with divalent cations, such as Ca
2+. In the preparation of PSB@MC, Alg served as a major component of the inner matrix. Its ability to form a stable gel helps encapsulate PSB and provides a physical barrier. RS is a type of starch that resists digestion in the upper gastrointestinal tract. Along with Alg, it formed the core of the microcapsule. RS can be fermented by the gut microbiota in the colon, which is beneficial for modulating the intestinal microenvironment. It also contributes to the slow-release property of the microcapsules, ensuring that PSB is released gradually in the colon. CS is a cationic polysaccharide that was used to coat the Alg/RS microcapsules. CS has good biocompatibility and can interact with Alg through electrostatic forces, forming a stable core-shell structure. Additionally, CS has antibacterial properties and can enhance the adhesion of microcapsules to the intestinal mucosa, facilitating the retention of PSB in the colon. PSB, the active ingredient, has anti-inflammatory, antioxidant, and potential immunomodulatory effects. However, its poor water solubility, low oral bioavailability, and rapid plasma clearance limit its clinical application. Nevertheless, microcapsule encapsulation can overcome these limitations. As shown in
Fig. 2(a), we first uniformly dispersed PSB in 2.0% Alg and 2.0% RS solutions after feeding them together into the capillary tube. Subsequently, droplets were generated using microfluidic electrospray technology, during which their structure was more porous, facilitating the escape of PSB from the Alg/RS network. Afterward, we collected the generated droplets in a 3.0% CaCl
2 solution and leveraged the rapid cross-linking between Ca
2+ and Alg to rapidly solidify the microcapsules, thus sealing the PSB tightly in the prebiotic microcapsules. Finally, the electrostatic interaction between CS and Alg was utilized to further cover the PSB@Alg/RS microcapsules with a CS shell layer to obtain the final PSB@MC. This additional layer enhances protection against PSB degradation in an acidic environment.
Light microscopy revealed that both empty microcapsules (MC) and PSB-loaded probiotic microcapsules (PSB@MC) exhibited a uniformly-sized spherical-like structure with high dispersion (
Fig. 2(b)). Further observation of microcapsule structure using confocal microscopy showed that both MC and PSB@MC formed a core-shell structure with CS (red) as the shell and Alg/RS (green) as the core (
Fig. 2(c)). Subsequent scanning electron microscopy revealed a smooth MC structure, whereas that of PSB@MC was wrinkled in
Figs. 2 (d) and
(e), a property that could enhance the intestinal adhesion of the microcapsules [
16]. Next, we determined the relationship between the actual drug loading rate (DLR) and the initial PSB concentration in PSB@MC. As shown in Fig. S1(a), when the initial PSB concentration increased from 0.15 to 0.6%, the DLR of PSB@MC increased from 5 to 24%, indicating a positive correlation between the DLR of PSB@MC and initial PSB concentration. In summary, PSB@MC was successfully synthesized.
3.2. Targeting and retention capacity of PSB@MC in the colon
Oral administration is the most straightforward and efficient approach for treating UC. However, to effectively apply this approach in the complex environment of the gastrointestinal tract, medication with targeted release and retention in the colon is necessary. We observed a slow initial release of PSB@MC in the first 2 h (<40.0%) in simulated gastric fluid (SGF), followed by an even slower release over the next 2–6 h. Notably, PSB@MC reached a cumulative drug release of 50% at 5 h. In contrast, simulated intestinal fluid (SIF) and simulated colonic fluid (SCF), PSB@MC exhibited a much quicker release rate of 70.0% in the first 2 h and a total release rate of 100% at 5 h (Fig. S1(b)). Consistent with the drug release pattern, PSB@MC was more susceptible to swelling in SIF and SCF, whereas its structure was relatively stable in SGF (Fig. S1(c)). This pH-sensitive property of PSB@MC provides it with targeted intestinal release capabilities when orally administered. Meanwhile, the folds on the surface of PSB@MC could enhance its adhesion ability, implying that PSB@MC has the potential for prolonged intestinal retention.
To further evaluate the colonic targeting and retention ability of PSB@MC, we gavaged fluorescently labeled MC and PSB@MC to healthy and colitis mice and then imaged different organs at different times after gavage (0, 1, 3, 6, and 12 h). According to
Fig. 3(a) and Fig. S2, PSB@MC was primarily enriched in the colon, with minimal distribution in other organs in both healthy and colitis mice, suggesting effective colon targeting. In addition, 12-h
in vivo imaging and fluorescence showed that the fluorescence intensity of PSB@MC was much higher than that of MC, implying that morphologically wrinkled PSB@MC could remain
in vivo for a longer period than did MC (
Figs. 3(a)–(c)). Meanwhile, imaging and fluorescence intensity studies of the isolated colon showed that PSB@MC was mainly retained in the colon compared with MC, further implying the colonic targeting and retention ability of PSB@MC in
Figs. 3(a),
(d), and
(e).
3.3. Protective effect of PSB@MC against DSS-induced colitis
We established a colitis prevention model and used various parameters to comprehensively assess the protective effect of PSB@MC against DSS-induced acute colitis [
17]. To construct the acute colitis model in mice, we administered drinking water containing 2.5% DSS for seven consecutive days. During the modeling period, 100 mg·kg
−1·d
−1 each of MC, PSB, PSB@MC, or 5-ASA was administered by gavage every other day (
Fig. 4(a)). Protection was assessed using body weight, DAI, colon length, splenic index, fecal status, endoscopy, histology, and inflammatory cytokine levels. We were able to quantify the overall effect of PSB@MC on mice by tracking body weight and DAI. Mice in the DSS group displayed a persistent increase in DAI and a progressive decline in body weight as the disease advanced, indicating a more severe course. In contrast, mice in the control group exhibited continuous body weight gain and no discernible change in DAI. Compared with the DSS group, the four therapy groups exhibited varying degrees of inhibition in weight loss and DAI increase. In particular, the PSB@MC group showed the greatest inhibition, indicating the best colon protection in
Figs. 4(b) and
(c). Next, we further analyzed the splenic index, colon length, defecation, endoscopic changes, and histological alterations in mice. The results showed that mice in the DSS group exhibited mucopurulent blood-like feces, diffuse ulceration of the intestinal mucosa visible under endoscopy, an elevated spleen index, and a shortened colon length, all of which indicated severe colonic inflammation in the mice. However, after receiving MC, PSB, PSB@MC, and 5-ASA by gavage, the mice showed improved defecation, a reduced splenic index, colon length restoration, and varying degrees of amelioration of endoscopically visible ulcers, suggesting colitis relief. The PSB@MC group displayed the most significant degree of recovery in
Figs. 4(d)–(f) and Fig. S3(a). Hematoxylin and eosin (H&E) staining for histologic evaluation showed no significant areas of inflammation in the colon sections of normal mice. However, after induction by DSS, the colonic epithelium and crypts were completely disrupted, and considerable inflammatory cell infiltration was observed, suggesting severe colonic inflammation. We observed that the other treatment groups showed varying degrees of reduction in colonic inflammation severity compared with that in the DSS group. PSB@MC treatment significantly restored colonic crypts and epithelial cell structure, suggesting a strong colon-protective effect against experimental colitis in
Fig. 4(f) and Fig. S3(b). Cytokines play a major role in colitis development. TNF-α, IL-1α, and IL-6 are examples of cytokines that intensify inflammation. The colon homogenates of mice treated with DSS exhibited considerably higher levels of inflammatory cytokines, as demonstrated by the qRT-PCR findings. However, this trend was reversible following pharmacologic intervention such as PSB@MC treatment in
Figs. 4(g)–(i). In conclusion, PSB@MC demonstrated strong protective effects in the experimental colitis model.
3.4. Therapeutic effect of PSB@MC against DSS-induced colitis
We constructed a colitis treatment model to further evaluate the therapeutic effect of PSB@MC. Here, we administered 2.5% DSS for seven consecutive days to establish an acute murine colitis model. Subsequently, we administered each of MC, PSB, PSB@MC, or 5-ASA (100 mg
kg
−1·d
−1) for 5 days (
Fig. 5(a)). At the conclusion of the experiment, the mice were euthanized, and various measurements were taken to comprehensively evaluate the therapeutic effect of PSB@MC against experimental colitis. The DSS-induced colitis model was successfully established as all mice lost weight, had an increased DAI, and had bloody diarrhea after 7 days. After 5 days of treatment with MC, PSB, PSB@MC, or 5-ASA, the mice exhibited improvements in weight loss and DAI, with the PSB@MC group demonstrating the most pronounced improvement (
Figs. 5(b) and
(c)). The assessment of splenic index and colon length also indicated that PSB@MC improved therapeutic effectiveness in
Figs. 5(d)–(f) and Fig. S3(d). H&E staining and endoscopy indicated treatment disparities between the groups. Compared with the other experimental groups, the PSB@MC group showed reduced inflammatory cell infiltration and aggregation, as well as the restoration of the orderly epithelial cell organization (
Fig. 5(f) and Figs. S3(c)- (d)). These results collectively imply that PSB@MC has outstanding anti-colitis treatment benefits.
3.5. Restoration effects of PSB@MC on the intestinal barrier
Treatment of UC requires restoring the intestinal barrier's structural and functional integrity, which is frequently disrupted. The colonic tissues of treatment model mice were evaluated for tight junction (TJ) proteins (e.g., ZO-1, Occludin, and Claudin-1) to determine whether PSB@MC protects the colonic epithelial barrier. Immunofluorescence labeling demonstrated that DSS treatment drastically reduced TJ protein expression and damaged the intestinal barrier. Following treatment with MC, PSB, PSB@MC, or 5-ASA, ZO-1, Occludin, and Claudin-1 in mouse colonic tissues were recovered. The recovery of TJ proteins in mice after treatment with MC and PSB was less effective (
Figs. 6(a)–(f)). PSB@MC and 5-ASA showed stronger TJ protein recovery (
Figs. 6(a)–(f)). Next, we performed Alcian Blue staining on colon tissue sections to further evaluate the effect of PSB@MC on the intestinal mucus barrier [
18]. Staining revealed a decrease in the number of goblet-shaped cells, and their secretory vesicles were reduced in the DSS group compared with those in the control group, reflecting decreased mucus secretion in mice. In contrast, the number of goblet cells in the colonic mucosa of mice in the treatment group was restored, suggesting that mucus barrier function was repaired, with PSB@MC having the most pronounced repair effect (
Fig. 6(g)).
3.6. Anti-inflammatory and antioxidant effects of PSB@MC on colitis
Research has shown that individuals with imbalanced gut microenvironments have a higher likelihood of acquiring intestinal barrier diseases. Inflammatory response and oxidative stress are important factors in the development of these kinds of disorders. Therefore, more research is necessary to ascertain the possible anti-inflammatory and antioxidant characteristics of PSB@MC in therapeutic models.
The production of inflammatory cytokines is an important factor in the development of colitis. Pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 exacerbate the inflammatory response, whereas inflammation-suppressing cytokines such as IL-10 promote the reduction of inflammation. Detection of inflammatory cytokines in colon homogenates using qRT-PCR revealed that pro-inflammatory cytokines, including
TNF-α,
IL-1β, and
IL-6, were significantly elevated, whereas inflammation-suppressing cytokines, such as
IL-10, were decreased in mice induced by DSS. However, this trend was reversed after pharmacologic treatment with PSB@MC (
Figs. 7(a)–(d)). When colonic inflammation occurs, neutrophil infiltration occurs in the inflamed tissues, and myeloperoxidase (MPO) activity may reflect the number of neutrophils in the tissues to some extent. The MPO kit assay revealed that MC, PSB, 5-ASA, and PSB@MC all reduced MPO activity, with PSB@MC having the most significant effect (
Fig. 7(e)). These results suggest that the combined anti-inflammatory properties of PSB@MC are significantly better than those of other drugs. MDA levels can indirectly reflect the degree of cellular oxidative stress. The MDA assay showed that PSB significantly alleviated oxidative stress in the mouse colon compared with that in the DSS group (
Fig. 7(f)). ROS usually accumulate in large quantities at the site of inflammation and cause cellular oxidative damage. Here, ROS were labeled with DCFH-DA and DHE in colon tissues (
Figs. 7(g) and
(h)). MFI results showed that the DSS and MC groups exhibited the strongest fluorescence, suggesting excessive ROS accumulation and severe oxidative damage (Figs. S4 in Appendix A). Fluorescence in the PSB and 5-ASA groups was weaker, suggesting ROS reduction. Fluorescence in the PSB@MC group was the weakest, suggesting the strongest ability to scavenge ROS. These findings indicate that the collective antioxidant characteristics of PSB@MC are notably superior to those of alternative medications. In summary, PSB@MC may restore the intestinal barrier by modifying the intestinal microenvironment through anti-inflammation and antioxidation.
3.7. Modulation of gut microbiota by PSB@MC
The intestinal microbiota is a crucial component of intestinal microecology and significantly impacts the regulation of host metabolism, immunology, and intestinal barrier function [[
19], [
20], [
21]]. We obtained samples of mouse intestinal contents and conducted 16S rDNA amplicon analysis to investigate the influence of PSB@MC on gut microbiota modulation. Species diversity serves as a crucial indicator of the structural and functional attributes of a community, encompassing α- and β-diversity. Shannon's and Simpson's diversity indexes indicated no significant disparity in α-diversity between the control and DSS groups (
Figs. 8(a) and
(b)). Beta diversity is measured using principal coordinate analysis (PCoA), which showed that DSS-treated mice have considerably different gut microbiota profiles than those of control mice (
Fig. 8(c)). Integrating these two approaches revealed changes in the gut microbial profiles of mice receiving DSS. Additionally, the findings of diversity experiments (β and α) showed that the composition and diversity of the gut microbiota in the DSS group differed significantly from those in the PSB@MC group. The gut microbiota of mice in the PSB@MC group was comparable to that of the control group in terms of abundance and composition (
Fig. 8(a)–(c)). This shows that PSB@MC can reduce or reverse DSS-induced gut microbiome alterations in mice. Phylum-level gut microbiota composition was similar in PSB@MC and control groups (
Fig. 8(d)).
Fig. 8(e) displays heat maps illustrating the comparative prevalence of various gut bacteria at the genus level. The taxonomic composition of the samples was represented at each level of classification, resulting in a taxonomic branching map of LEfSe (
Fig. 8(f)). The DSS group had an elevated prevalence of harmful or potentially pathogenic bacteria, including
Escherichia-Shigella,
Aeromonas,
Enterococcus, and
Romboutsia. Conversely, the PSB@MC group had a greater percentage of beneficial microbes, including
Muribaculum,
Muribaculaceae, and
Akkermansia. At the genus level,
Akkermansia,
Muribaculaceae, and
Escherichia-Shigella dominated. After DSS treatment, pathogenic
Escherichia-Shigella increased, whereas probiotic
Muribaculaceae and
Akkermansia declined. In contrast, PSB@MC reversed this trend and restored gut microbiota homeostasis (
Figs. 8(g)–(i)). In conclusion, PSB@MC also restores gut microenvironmental homeostasis by modulating the gut microbiota.
3.8. Regulation of gut microbial metabolites by PSB@MC
Small-molecule metabolites such as short-chain fatty acids (SCFAs), tryptophan (Trp), and bile acids (BAs) facilitate communication between the gut microbiota and the host, which has a major impact on host health [
22,
23]. Metabolomic analysis of gut contents was used to study the impact of PSB@MC on gut microbiota metabolites after composition control. Principal component analysis revealed significant changes in intestinal metabolites in the DSS group mice relative to the other two groups (
Fig. 9(a)). In
Fig. 9(b), 630 distinct metabolites were found in the DSS group compared with those in the control group after further analysis using a volcano plot. Of these, 577 metabolites showed significant downregulation, and 28 showed significant upregulation in expression. In the PSB@MC treatment group, 147 divergent metabolites were identified in comparison to the DSS group, with 21 exhibiting significant upregulation and 105 demonstrating significant downregulation (
Fig. 9(c)). The Venn plot indicated a total of 76 differential metabolites in Group 1 (Control vs. DSS) compared with Group 2 (DSS vs PSB@MC) (
Fig. 9(d)). Concerning differential metabolites, namely fatty acids and their derivatives, BAs and their derivatives, and indoles and their derivatives, we performed a heat map study. Our findings showed that the PSB@MC and control groups exhibited a similar change trend, contrasting with the trend observed in DSS change (
Figs. 9(e)–(g)). By triggering the AHR/IL-22 signaling pathway, indolic acid and its derivatives have been shown to protect the intestinal barrier and reduce colonic inflammation [
24]. Hence, we conducted a more detailed evaluation of the alterations in indole and its derivatives. According to the findings, bufotenine, gramine, α-methyltryptamine, indole-3-carboxylic acid indole-3-acetonitrile, and other indoles and their derivatives exhibited decreased levels after DSS treatment but were restored after PSB@MC treatment (
Figs. 9(h)–(l)). According to the above findings, PSB@MC influences the metabolites produced by the gut microbiota and also affects indole and its derivatives, which may help activate the AHR/IL-22 pathway to restore the intestinal barrier.
3.9. Activation of AHR/IL-22 pathway by PSB@MC
The AHR/IL-22 pathway has emerged as a novel target for UC therapy due to its capacity to facilitate intestinal barrier restoration [
25,
26]. PSB is a highly adaptive ligand for AHR. In addition, our study revealed that PSB@MC can promote the production of indole and its derivatives. Therefore, we hypothesized that PSB@MC could enhance the AHR/IL-22 signaling pathway, which in turn may aid in intestinal barrier restoration. Cyp1a1 and Cyp1b1 are downstream components of the AHR signaling system, and their elevated expression indicates activation of this pathway (
Fig. 10). qRT-PCR revealed that PSB@MC significantly increased the expression of
AHR,
Cyp1a1, and
Cyp1b1 mRNA, implying AHR activation (
Figs. 10(a),
(b), and
(e)). AHR immunofluorescence staining of colon tissues, along with mean fluorescence analysis, similarly demonstrated that oral administration of PSB@MC could activate AHR in intestinal tissues (
Figs. 10(f) and
(g)). The biological effects of IL-22 depend on binding to IL-22RA [
27,
28], after which it can restore the intestinal barrier [
29]. We studied the link between IL-22 and IL-22RA expression in intestinal tissues and found that PSB@MC increased
IL-22 and
IL-22RA expression (
Figs. 10(c) and
(d)). Immunofluorescence staining of IL-22 in colon tissues and mean fluorescence analysis further proved this hypothesis (
Fig. 10(h) and
(i)). Collectively, these results suggest that oral PSB@MC administration could activate the AHR/IL-22 pathway in the intestine.
3.10. Biocompatibility evaluation of PSB@MC
To guarantee the safety of PSB@MC administration, an assessment of its biosafety was conducted. As demonstrated in Fig. S5(a), no notable histologic alterations or cellular damage were observed in the essential organs of mice treated with PSB@MC compared with those of healthy animals. Furthermore, we examined the blood, liver, and renal functions of mice treated with PSB@MC. Our analysis revealed no notable disparities compared with the health status of normal mice (Figs. S5(b)–(d)). Regarding changes in body weight, no discernible differences were observed between the two groups of mice throughout the trial (Fig. S5(e)). These results demonstrate that PSB@MC exhibits favorable biological safety and is suitable for in vivo experimentation.
4. Discussion
The intestinal barrier comprises two parts: functional and physical barriers. Intestinal goblet cell mucus, intestinal epithelium, and microbiota make up the physical barrier [
30]. The intestinal epithelium performs its protective function by forming a barrier mainly through TJ proteins that prevent pathogenic microorganisms and other harmful substances from entering [
31]. Meanwhile, epithelial goblet cells produce mucus, which prevents direct contact between pathogenic microorganisms and the epithelium [
32]. The commensal microbiota compete for nutrition with intestinal pathogenic microorganisms to protect the host [
33]. UC is characterized by inflammation of unknown cause, with its exact causes not yet fully understood. However, disruption of intestinal barrier function, caused by genetic, immunologic, and environmental factors, is thought to be the main cause of UC [
34]. For this reason, repairing the intestinal barrier is essential to treating UC.
Recent studies [
35] have suggested that individuals with disturbances in the intestinal microenvironment are more likely to experience disruptions in intestinal barrier function and structure. Oxidative stress and gut microbiota dysbiosis are important factors contributing to gut microenvironmental disorders. Oxidative stress in the intestinal tract is mainly caused by ROS, and physiologically, cells can tolerate a certain amount of ROS due to the presence of natural antioxidant enzymes in the body [
36]. During the active phase of UC, immune cells such as neutrophils and macrophages infiltrate the intestinal mucosa at the site of inflammation, releasing large amounts of ROS and causing severe oxidative stress. This exacerbates inflammation, intensifies intestinal barrier damage, and accelerates UC progression [
37]. Therefore, ROS clearance is an important part of UC treatment. Gut microbiota dysbiosis can disrupt the homeostasis of the intestinal internal environment by stimulating the release of pro-inflammatory cytokines and increasing the production of endotoxins [
38]. Furthermore, gut microbiota can shape the intestinal microenvironment by secreting small-molecule metabolites, such as SCFAs, Trp, and BAs, which can affect the intestinal barrier [[
39], [
40], [
41]]. Therefore, the combination of modulating the intestinal microenvironment through ROS removal and gut microbiota to restore intestinal microenvironmental homeostasis and repair the intestinal barrier is a new trend in the clinical treatment of UC.
PSB, a dimethyl analog of resveratrol, is structurally similar to resveratrol and, therefore, has comparable antioxidant, anti-inflammatory, and anticancer properties [
42,
43]. Research suggests that dietary PSB supplementation may provide some protection against gut-related diseases [
44]. However, the low oral potency, poor intestinal transit, rapid plasma clearance, and insufficient water solubility of PSB limit its clinical use. An optimal medication delivery system for treating UC involves delivering the drug directly to the inflammatory colon at the highest possible dosage while minimizing exposure of the entire gastrointestinal tract and the rest of the body to the drug. Hence, a more efficient and manageable specific carrier is required to administer PSB in colitis therapy. Prebiotics have a natural advantage as a vehicle for targeting the colon because of their unique properties, including selective catabolism and utilization by beneficial intestinal flora. Prebiotics can stimulate the growth of host-specific gut microbiota, thereby improving intestinal function. Exogenous supplementation of prebiotics in several UC models reportedly inhibits the intestinal growth of harmful bacteria and promotes colonization by beneficial bacteria, which helps restore the patient's gut microbiota homeostasis [
45,
46]. In addition, prebiotics can exert a therapeutic effect on UC by regulating gut microbiota metabolites. Despite the many advantages of prebiotics in regulating gut microbiota, few studies on prebiotics and UC have been conducted. Existing studies are often limited by the lack of placebo controls and small sample sizes, restricting their use to adjunctive therapy for UC. Therefore, we constructed prebiotic microcapsules loaded with PSB. As a class of prebiotic substances that are difficult to enzymatically digest in the upper gastrointestinal tract, Alg, RS, and CS can pass through intact and reach the colon, where they are fermented and utilized by the intestinal microbiota. They can not only achieve the precise targeted delivery of PSB to inflammatory lesions in patients with UC but effectively reduce the systemic bioavailability of the drug and minimize potential adverse reactions. Meanwhile, the pH-responsive structural system constructed by combining the three can maintain structural stability in the acidic gastric environment, protecting PSB from degradation by gastric acid and pepsin. In the alkaline environment of the lower intestinal tract, this structure swells and releases PSB in a controlled manner. In addition, Alg, RS, and CS can be metabolized by the intestinal microbiota. By promoting the proliferation of beneficial bacteria and inhibiting the growth of harmful bacteria, they can restore intestinal microbial community homeostasis that is disrupted under the pathological conditions of UC. Combined with the anti-inflammatory and anti-oxidative stress activities of PSB itself, they jointly achieve comprehensive regulation of the intestinal microecological environment.
Notably, we also found that PSB@MC can activate the AHR/IL-22 pathway to repair the intestinal barrier. However, the specific mechanism underlying AHR activation by PSB@MC remains unclear. Based on our experimental results, we hypothesized the following: 1) PSB@MC releases PSB from the microcapsules after it reaches the colon, and PSB, as a highly adaptive ligand for AHR, can activate AHR to promote IL-22 secretion; 2) PSB@MC can activate AHR by regulating the metabolism of the bacterial colony to produce AHR ligands, such as indole and its derivatives. IL-22 is a downstream target gene of AHR, which is positively correlated with the AHR/IL-22 pathway. After AHR/IL-22 pathway activation, it exerts multiple impacts on immune responses, gut microbiota, and epithelial cell functions. In the immune responses, AHR activation prompts immune cells to secrete IL-22. IL-22 then binds to its receptor, activating the JAK-STAT pathway, which promotes immune cell migration and activation [
47]. It also induces cytokine and chemokine production, regulates immune cell differentiation, and influences Th17-Treg balance, enhancing immune defense yet potentially causing inflammation [
47]. Regarding the gut microbiota, pathway activation induces intestinal epithelial cells to secrete antimicrobial peptides to inhibit bacteria, promotes mucin secretion to thicken the mucus layer, and affects epithelial cell metabolism to adjust metabolite distribution, thus regulating the gut microbiota and maintaining its stability [
48]. For epithelial cell functions, the AHR/IL-22 pathway strengthens the cell barrier by upregulating TJ proteins via signal transducer and activator of transcription 3 (STAT3) signaling. Moreover, it promotes cell repair by activating phosphatidylinositol 3–kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) pathways. Additionally, intestinal epithelial cells secrete immunomodulatory factors to regulate local immunity and cellular activity, maintaining normal intestinal tissue integrity [
49].
Notwithstanding, this study has several limitations. Although PSB@MC showed good efficacy in the treatment of UC, only an acute colitis model was constructed in this study, and the efficacy of PSB@MC in a chronic colitis model still needs to be further explored. Moreover, this study only evaluated the reparative effects of PSB@MC on physical barriers, while its ability to repair functional intestinal barriers remains to be investigated. Future research will aim to refine this work and explore the systemic therapeutic potential of PSB@MC to expand clinical treatment options.
5. Conclusion
In conclusion, PSB@MC, a novel probiotic microcapsule, was successfully developed with advantageous biosafety properties, ensuring targeted delivery of PSB to the colon, protection against PSB degradation in the stomach, and prolonged colon residence. Oral PSB@MC administration in an acute experimental murine model of colitis exhibited both protective and therapeutic effects on the colon. Further studies revealed that PSB@MC possessed excellent anti-inflammatory and antioxidant properties, which could restore intestinal microenvironmental homeostasis and alleviate colonic inflammation by facilitating inflammatory cytokine suppression and ROS scavenging. Meanwhile, PSB@MC could further remodel the intestinal microenvironment by regulating the intestinal flora and its metabolites, repairing the intestinal barrier, and alleviating colonic inflammation. Notably, PSB@MC can activate the AHR/IL-22 pathway, facilitating coordinated intestinal barrier repair. These findings indicate that PSB@MC may represent a promising, safe, and efficacious alternative therapy for UC.
CRediT authorship contribution statement
Huanyu Li: Writing – original draft, Validation, Software, Methodology, Investigation, Formal analysis, Data curation. Ziwei Yang: Writing – review & editing, Investigation. Chuanyu Zhang: Writing – review & editing, Funding acquisition. Xueyong Wei: Supervision, Resources, Project administration, Funding acquisition, Conceptualization. Wenjing Wang: Methodology, Formal analysis, Data curation. Ting Bai: Methodology, Formal analysis, Conceptualization. Zhichao Deng: Investigation, Data curation. Bowen Gao: Data curation. Manli Cui: Data curation. Weixuan Jing: Data curation. Mingzhen Zhang: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. Zhaoxiang Yu: Resources, Methodology, Investigation, Conceptualization. Mingxin Zhang: Writing – review & editing, Methodology, Funding acquisition, Formal analysis, Conceptualization.
Declaration of competing interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mingxin Zhang reports financial support was provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Key Research and Development Program of China (2022YFC2406600) and the Program for Innovation Team of Shaanxi Province (2021TD-23), the Fundamental Research Funds for the Central Universities (xtr052023008), the Young Talent Support Plan of Xi’an Jiaotong University (YX6J001), the Shaanxi Province Key Research and Development Program (2023-YBSF-072 and 2024JC-YBMS-664), and the Xi'an Science and Technology Plan Project (24YXYJ0143).
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