Microcapsules for the synergistic delivery of probiotics and postbiotics from microfluidics for the treatment of colitis (Part II)

To explore the protective effect of IPA@MC on colitis in vivo, mice were orally administered IPA@MC, IPA, and MC for 5 consecutive days, and then colitis was induced for 7 days using dextran sulfate sodium (DSS) in drinking water, as shown in figure a. Compared to the healthy group, the DSS group showed a significant weight loss after DSS treatment. However, there was no significant difference in weight between the mice receiving IPA@MC and the healthy mice, indicating that IPA@MC significantly protects mice from DSS-induced weight loss (figure b). Additionally, the disease activity index (DAI) trend in the IPA@MC group was significantly lower than that of the other DSS treatment groups (figure c). Besides weight loss, DSS-induced colitis is typically characterized by colon shortening and an increased spleen/body weight ratio. In the study, the colon length in the IPA@MC group was comparable to that of the healthy control group and significantly longer than that of the IPA, MC, and DSS groups (p < 0.05, figure d). Furthermore, the spleen/body weight ratio in the IPA@MC group was significantly lower compared to the IPA and DSS groups (p < 0.05, figure e), further confirming the preventive effect of IPA@MC on colitis in mice. The inflammation levels in colon tissues were examined through hematoxylin-eosin (H&E) staining and immunohistochemical staining for pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factor-alpha (INF-α). The results showed no significant difference in H&E staining images between the healthy control group and the IPA@MC group, contrasting sharply with the severe inflammatory response reflected by the loss of crypt structure, epithelial damage, and immune cell infiltration in the other DSS treatment groups (figure f). Additionally, the expression levels of IL-6 and INF-α in the IPA@MC group were lower than those in the IPA, MC, and DSS treatment groups, indicating that IPA@MC improved inflammation. In summary, these results suggest that IPA@MC exhibits strong preventive and therapeutic effects on DSS-induced colitis in mice compared to the use of IPA or MC alone.

The protective effect of IPA@MC on DSS colitis in mice

a) Schematic diagram of the animal study design. IPA@MC is expected to have a strong protective effect on DSS-induced colitis in mice.b) Weight changes from day 0 to day 7.c) Changes in the disease activity index (DAI) from day 0 to day 7.d) Colon length ande) Spleen/body weight ratio of mice on day 7.f) Representative H&E staining images of colon tissues on day 7.Arrows indicate inflammatory cell infiltration and epithelial erosion. Mice were randomly divided into 5 groups (Control: PBS + regular water; IPA@MC: IPA microcapsule + DSS water; IPA: IPA + DSS water; MC: empty microcapsule + DSS water; DSS: PBS + DSS water).

Given that dietary prebiotics can help regulate colitis ingut health, prebiotics including alginate, RS, and chitosan were selected as carriers for postbiotics in the study. Although the trends of colitis in the IPA and MC groups were lower than those in the DSS group, there was no statistical significance between these groups, indicating that the therapeutic effects of single postbiotics or prebiotics were insufficient. In contrast, the IPA@MC group showed significant differences in weight loss, DAI, colon length, and spleen/body weight ratio compared to the other DSS treatment groups, suggesting that the synergistic effect between prebiotics and postbiotics in IPA@MC provides a more effective strategy for the prevention and treatment of colitis. This result may be attributed to the synergistic effect between prebiotics and postbiotics in IPA@MC. Prebiotics are primarily indigestible oligosaccharides that can be utilized by specific beneficial bacteria. Therefore, gut bacteria metabolize these dietary components to produce beneficial metabolites (postbiotics) to maintain a healthy gut microbiota. Conversely, it also indicates that known postbiotics can increase beneficial bacteria and inhibit the growth of harmful bacteria, further enhancing the fermentation of prebiotics in the gut. The increased abundance of beneficial bacteria fed by prebiotics can then produce higher levels of postbiotics to maintain a healthy gut.postbioticsSubsequently, the gut bacterial composition at the phylum, order, and genus levels was analyzed to study the taxonomic changes in colitis mice after various treatments. As shown in figure c, Bacteroidetes, Firmicutes, and Proteobacteria were the main bacterial phyla detected in all groups. Notably, compared to the DSS group, the ratio of Bacteroidetes to Firmicutes in the IPA@MC group was similar to that of the healthy group, with an increased abundance of Firmicutes and a decreased abundance of Bacteroidetes (figure d). Additionally, compared to the healthy control group, the DSS group exhibited taxonomic changes at the order level, including an increased abundance of Bacteroidales and a decreased abundance of Campylobacterales, Clostridiales, and Enterobacterales. Consistent with the changes at the phylum level, the mice in the IPA@MC group were similar to healthy mice at the order level, while almost no changes were observed in the DSS-induced IPA or MC mice compared to the DSS group (figure e). Next, further changes in the gut bacterial profile at the genus level were measured (figure f). Only moderate changes in the top bacterial genera were observed among the groups. This result is consistent with previous studies indicating that lower abundance bacterial groups may play an important role in disease occurrence.

Using IPA@MC to modulate gut microbiota in colitis mice

a) Chao1 richness across all groups. T-tests were used to test the significance between groups.

b) Principal coordinate analysis (PCoA) of Bray-Curtis distances, showing the stratification of gut microbiota in the healthy control group compared to the IPA@MC and DSS-colitis groups. Statistical significance was determined by permutational multivariate analysis of variance (PERMANOVA). In all groups, c) relative abundance of gut bacteria at the phylum level. d) Ratio of Bacteroidetes to Firmicutes levels in all groups. In all groups, e) relative abundance of gut bacteria at the order and f) genus levels. Mice were randomly divided into 5 groups (Control: PBS + regular water; IPA@MC: IPA microcapsule + DSS water; IPA: IPA + DSS water; MC: empty microcapsule + DSS water; DSS: PBS + DSS water).b) Bray-Curtis 距离的主坐标分析 (PCoA)，显示IPA@MC 和 DSS-结肠炎组的健康对照组肠道微生物群的分层。通过置换多变量方差分析（PERMANOVA）确定统计显著性。在所有组中 c) 门水平中肠道细菌的相对丰度。d)所有组中拟杆菌门和厚壁菌门水平的比率。在所有组中，e) 顺序和 f) 属水平中肠道细菌的相对丰度。将小鼠随机分为5组（对照：PBS+普通水；IPA@MC：IPA微胶囊+DSS水；IPA：IPA+DSS水；MC：空微胶囊+DSS水；DSS：PBS+DSS水）。

The consumption of bacterial groups that produce SCFA is also related to DSS-induced colitis and other intestinal diseases. It was further explored whether PA@MC could increase the abundance of bacteria that produce postbiotics after oral administration. Therefore, the differences in gut bacterial genera among all groups were compared using linear discriminant analysis effect size (LEfSe). A total of 17 different gut bacterial genera were observed across all groups (Figure a). Among the differential taxa, the DSS group showed a reduced relative abundance of SCFA-producing bacteria, including Faecalibacterium and Roseburia, compared to healthy mice (Figures b, c). Interestingly, IPA@MC treatment significantly increased the relative abundance of SCFA-producing bacteria in colitis mice. However, no significant differences in the abundance of these genera were observed between the DSS group and the groups treated with single IPA or MC. This result confirms that the synergistic effect of prebiotics and postbiotics provided by IPA@MC can enhance the recovery of a healthy gut microbiome compared to the MC group that only received prebiotics or the IPA group that only received postbiotics.

IPA@MC alters gut bacteria and rebuilds healthy ecological communities.

a) The gut microbiota of mice treated with IPA@MC changed, indicating that the gut microbial profile was highly similar to that of healthy mice. b,c) The relative abundance of SCFA-producing bacterial genera (Faecalibacterium and Roseburia) increased in mice treated with IPA@MC. Differential taxa were identified using linear discriminant analysis effect size (LEfSe). d) Compared to DSS group mice, the total SCFA levels in cecal samples from IPA@MC treated mice increased. e) The number of correlations between gut bacterial genera. Statistical significance was determined using the chi-square test. f) The heatmap shows that the ecological correlations between gut bacterial genera in the IPA@MC group were higher than in the DSS group. Mice were randomly divided into 5 groups (Control: PBS + regular water; IPA@MC: IPA microcapsules + DSS water; IPA: IPA + DSS water; MC: empty microcapsules + DSS water; DSS: PBS + DSS water).

Consistent with the changes in the abundance of SCFA-producing bacterial genera after IPA@MC treatment, it was found that the total SCFA levels in cecal samples from the IPA@MC group increased compared to the DSS group (Figure d). However, the correlation between SCFA levels and the abundance of Faecalibacterium and Roseburia did not reach statistical significance. Further studies with larger sample sizes and more in-depth metagenomic analyses may provide evidence of the expression of SCFA and the taxa that produce SCFA after IPA@MC treatment. Additionally, we analyzed the correlations between these bacterial genera to understand the ecological changes among all treatment groups. It was found that the number of correlations between bacterial genera was lower in DSS-induced colitis mice compared to healthy mice (Figures e, f). In contrast, the significant correlations in the IPA@MC group were higher than in the DSS group, indicating that IPA@MC is beneficial for rebuilding a healthy ecological community of the colitis gut microbiome. The above results suggest that IPA@MC can reverse gut microbiome dysbiosis, thereby having a significant therapeutic effect on colitis.

Note: The article is translated from 'Prebiotics and Postbiotics Synergistic Delivery Microcapsules from Microfluidics for Treating Colitis.'

For academic reference only.