The probiotic Lp082 was fed with DSS to mice to evaluate the potential preventive effects of this strain on UC development. Various tissue samples, including immune organs, serum, proximal colon, fecal, cecal contents, distal colon, and other tissues, were collected to evaluate physiological indexes, inflammatory cytokines, pathological indicators, shotgun metagenomic sequencing, SCFAs analysis, and RNA sequencing (Fig. 1a).
The intake of Lp082 alleviated physiological lesions in DSS-induced colitis mice
DSS group, Lp082 group, and SASP group-under the same DSS conditions showed similar changes on days 1–7, i.e., water intake, food intake, and body weight decreased significantly on the 6th day, the 2nd and 3rd day, respectively, and DAI scores increased significantly on day 3 (p < 0.05) (Fig. 1b). After SASP and Lp082 intake, water and food intake increased from day 8, body weight increased from day 11, and DAI scores decreased from day 9, but the DSS group maintained the original trend on these variables (Fig. 1b).
The mice in the control group were mentally active and responsive, while the mice in the DSS group developed symptoms, such as slow response, arched back, and easy panic as the disease worsened (Fig. S1 b). More importantly, the immune organ index of mice in the DSS group was significantly increased (p < 0.05) (Fig. 1c), and the spleen became larger and blacker (Fig. S1 c), shortened colon, hyperemia, and swelling of intestinal contents (Fig. 1d), and feces were not formed (Fig. S1 a). It is important to note that after ingesting SASP and Lp082, the mice no longer had blood in the stool (Fig. S1 a), the mental state went back to normal activities (Fig. S1 b), the immune organ index decreased significantly (Fig. 1c), and the length of the colon increased significantly (p < 0.05) (Fig. 1d).
The above pathological indexes in Lp082 group were better than those in SASP group, suggesting that Lp082 has a better remission effect on UC.
Effects of Lp082 on inflammatory cytokines in mice with colitis
To further assess the damage in colons, the pro-inflammatory cytokines TNF-, IL-1β, IFN-α, IL-6, and MPO and anti-inflammatory cytokine IL-10 in the serum of mice were quantified. It was found that compared with the control group, the pro-inflammatory cytokines were significantly increased, and the anti-inflammatory cytokines were significantly decreased in the DSS group (p < 0.05), while the opposite was in the Lp082 and SASP group (Fig. 1e).
Effects of Lp082 on pathological lesions in DSS-induced colitis mice
The intake of Lp082 significantly decreased colon histopathology score and intestinal wall thickness (p < 0.05). The result of H&E staining paraffin section indicated that colonic tissue underwent severe damage in DSS group, including neutrophil infiltrate deep into the serosal layer (green arrow), crypt disappear (black arrows), goblet cell loss (red arrow), and inflammatory cell aggregation (blue arrow) (Fig. 2a). These were consistent with the significantly increased colon histopathology score in DSS group (p < 0.05) (Fig. 2b). However, the intake of Lp082 and SASP significantly improved the above situation, including increased the number of crypts (orange arrow) and goblet cells (yellow arrow), alleviated inflammatory cell foci, alleviated neutrophil infiltration, and promoted the tight junctions of intestinal glands (gray arrow) (Fig. 2a). Moreover, a better recovery in the Lp082 group than in the SASP group was consistent with a lower histopathological score in the Lp082 group (Figure. 2b). In addition, the intestinal wall was thicker in the DSS group, thinner in the SASP group, and much thinner in the Lp082 group (Fig. 2c). On the other hand, immunofluorescence results showed that the MUC-2 protein (green fluorescence) and ZO-1 protein (red fluorescence) contents were higher in the control group, almost disappeared in the DSS group, and significantly recovered in the Lp082 and SASP groups (p < 0.05), and even increased more than SASP in Lp082 group (Fig. 2d-e). These results were consistent with the surface density results of the two proteins (Fig. 2f-g).
Effects of Lp082 on gut microbiota
Lp082 improved the α diversity and optimized the β diversity of cecal microbiota in mice. On days 1–7 of the study, the Shannon index in DSS, Lp082, and SASP groups, under the same DSS condition, was significantly decreased with the same level (Fig. 3a) but significantly increased after the intake of Lp082 (p < 0.05) (Fig. 3a). The three groups (M_B, M_C, M_D) ( under the same DSS molding condition) and control group (M_A) were significantly separated on day 7 (p < 0.05) (Fig. 3b). However, on day 15, the DSS group was still significantly separated from the control group (T_B), while the distance between Lp082 group (T_C), SASP group (T_D), and control group (T_A) was significantly reduced (p values < 0.05), and the distance between Lp082 group and control group was closer, the above results were consistent with the PCoA distance results (Fig. 3c).
Potential colitis pathogenic bacteria such as Helicobacter hepaticus increased significantly in DSS group but decreased in Lp082 group (p < 0.05), while the potential beneficial bacteria, such as Lactobacillus plantarum, Bifidobacterium pseudolongum, Akkermansia muciniphila, Bacteroides ovatus, Parabacteroides distasonis, Lactobacillus reuteri, Anaerotruncus sp G3 2012 significantly decreased in DSS group but significantly increased in Lp082 group (p < 0.05) (Fig. 3d).
The regulatory role of SCFAs
Some of the increased beneficial bacteria in Lp082 group may produce SCFAs. So, we further explored the gut microbiota metabolic pathway associated with SCFAs and found that 2 related pathways in the Lp082 group were activated: the fermentation of pyruvate to propionate I and the fermentation of pyruvate to acetate and lactate II (Fig. 4a). These two pathways directly promote the production of acetate and propionate, which was consistent with the GC-MS results of SCFAs, i.e., the contents of acetic acid, propionic acid, butyric acid were significantly decreased in the DSS group but significantly increased in the Lp082 group (p < 0.05) (Fig. 4b).
To further understand the role of SCFAs, we performed a correlation analysis and found that Helicobacter hepatica enriched in DSS group and 7 potential probiotics enriched in Lp082 group were negatively and positively correlated with acetic acid, propionic acid, and butyric acid, respectively (Fig. 4c). These SCFAs were all negatively associated with the pro-inflammatory factors TNF-α, IL-1β, IFN-γ, IL-6, MPO but positively associated with the inflammatory suppressor IL-10 (Fig. 4d).
Comparative study on the transcriptome of intestinal epithelial cells in each group
The volcanic map showed that Lp082 significantly affected gene expression distribution (Fig. 5a-f). To further explore the impact of these differentially expressed genes (DEGs), we analyzed the pathways involved in DEGs.
The results of GO analysis showed that the DEGs of the DSS group and the control group were mainly involved in biological processes such as the humoral immune response, activation of an immune response, negative regulation of hemostasis; and cellular component such as blood microparticle, membrane attack complex; and molecular functions such as lipid binding, lipopolysaccharide-binding, thrombospondin receptor activity (Fig. 6a). On the other hand, the DEG of the Lp082 and DSS groups was mainly involved in biological processes such as blood coagulation, fibrin clot formation, regulation of humoral immune markers, regulation of inflammatory cytokines; and cellular components such as Golgi lumen, endoplasmic reticulum, and molecular functions such as endopeptidase activity and peptidase activity (Fig. 6b).
Considering that in the Lp082, the up-regulated DEGs were far more than down-regulated DEGs (Fig. 5a-f) and the DEGs have the largest proportion of participation in biological processes (Fig. 6a-c), we further conducted GO-BP analysis on significantly up-regulated DEGs. The results of GO-BP analysis showed that compared to control group, up-regulated DEGs in DSS group were mainly enriched in the 6 inflammation-related GO-BP. Among those, the genes IL-1β and IL-1α were both involved in the IL-1β production and TNF production, the oncogene Ereg were involved in the IL-1β production, the genes IL-1β and IL-1rn, oncogene Fga were all involved in positive regulation of nuclear factor kappa-B (NF-κB) transcription factor activity, the oncogene Ldlr, Dgat2, and Mfsd2a were all involved in the regulation of toll-like receptor 4 signaling pathway, the pro-oncogenes Cdc7, Dbf4 were all involved in the acute inflammatory response, the anti-tumour gene Syk and the inflammatory genes Nlrp3 as well as Syk were all involved in the pro-inflammatory factor IL-6 production (Fig. 6d). Compared to DSS group, the up-regulated genes in Lp082 group were mainly enriched in the 6 anti-inflammatory-related GO-BP. Among them, the gene Isg15, which exerted both its antiviral and anti-inflammatory effects in innate immunity, and the gene Prg2, which played an important role in wound healing, were involved in the anti-inflammatory factors IL-10 production (Fig. 6e).
The results of KEGG analysis showed that the DEGs in DSS and control groups were mainly enriched in systemic lupus erythematosus, Staphylococcus aureus infection, Viral carcinogenesis, Pathways in cancer, TNF signaling pathway, Cellular senescence, and mitogen-activated protein kinase (MAPK) signaling pathway (Fig. S2a). However, the DEG in both Lp082 and DSS groups, SASP and DSS groups, and SASP and Lp082 groups were mainly enriched in the following five pathways: Complement and coagulation cascades, Platelet activation, Autophagy - animal, Phagosome and N-Glycan biosynthesis (Fig. S2b-S2d). Besides, the DEGs in Lp082 and DSS groups, as well as SASP and DSS groups were involved in protein processing in the endoplasmic reticulum and metabolic pathways (Fig. S2b-S2c).
The results of gut mucosal barrier analysis showed that gene expression of MUC-2, ZO-1, ZO-2, occludin was significantly reduced in the DSS group but significantly increased in the Lp082 and SASP groups (p values < 0.05), and the gene expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM,) claudin-1, and claudin-2 increased significantly in the DSS group but decreased significantly in the Lp082 and SASP groups (p values < 0.05) (Fig. 6g-j). It is worth mentioning that MUC-2 is an essential component of gut mucosa; ICAM-1 and VCAM induce gut mucosal lesions; ZO-1, ZO-2, and occludin promote tight junctions of gut epithelial cells; claudin-1 and claudin-2 increase intestinal permeability and aggravate inflammation.
Results of gene analysis related to NF-κB pathway showed that Lp082 also inhibited the mRNA expression of NF-κB1, NF-κB2, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), Toll-4, and RelA. These genes are signaling molecules in the NF-κB signaling pathway (Fig. 6g-j).
The potential mechanism of Lp082 alleviated the DSS-induced colitis
After confirming that probiotics can help relieve UC, we explored their potential mechanisms of action. For that, correlation and interaction between Lp082 and mouse symbiotic gut microbiome were elucidated by conducting Pearson correlation analysis, and the results are summarized in 3 parts of Fig. 7a.
The upper right portion of Fig. 7a shows that Lp082 was positively correlated with six strains that are positively correlated with two metabolic pathways: pyruvate fermentation to propanoate I, Pyruvate fermentation to acetate and lactate II; and these two metabolic pathways are positively correlated with acetic acid and propionic acid; and these two acids are negatively correlated with pro-inflammatory factors such as TNF-α, IL-1β, IFN-γ, IL-6, and MPO, but positively correlated with anti-inflammatory cytokine IL-10. The pro-inflammatory factors were negatively correlated with MUC-2, ZO-1, body weight, colon length while positively correlated with histologic scores, DAI Score, spleen coefficient, but anti-inflammatory cytokine was just the opposite (Fig. 7a).
The circle in the bottom half of Fig. 7a shows that Lp082 reduces inflammation by regulating DEG and the GO-BP they participate in. Lp082 was negatively correlated with the genes Nlrp3, Syk and Cdc7, Dbf4 and IL-1β, IL-1α, P2ry2 and IL-1β, IL-1α, Ereg and Ldlr, Mfsd2a, respectively, which were involved in IL-6 production, acute response, TNF production, IL-1β production, and toll-like receptor 4 (TLR4) signaling pathway. Lp082 was positively correlated with Abcc2 and Prg2 Isg15, which were positively correlated with drug transport, drug metabolic process, and IL-10 production, respectively (Fig. 7a).
The upper left of Fig. 7a shows that Lp082 was negatively correlated with NF-κB2, NF-κB1, COX-2, Rela, Toll4, iNOS, all of which were positively correlated with NF-κB, indicating that Lp082 inhibited the NF-κB signaling pathway by down-regulating the genes of NF-κB signaling molecules, thus relieving inflammation (Fig. 7a).
In a further comprehensive analysis of the data, we found that Lp082 improves the intestinal mucosal barrier by optimizing the following 4 barriers. First, Lp082 improved the biological barrier by improving the gut microbiota diversity and optimizing species composition, increasing the bacteria that produce SCFAs, enhancing the metabolic pathway of SCFAs and the content of SCFAs. Second, Lp082 improved the chemical barrier by increasing the content of goblet cells, MUC-2, reducing the ICAM-1 and VCAM content. Third, Lp082 improved the mechanical barrier by increasing the mRNA expression of ZO-1, ZO-2, occludin, decreasing the mRNA expression of claudin-1, claudin-2. Fourth, Lp082 improved the immune barrier by reducing the content of IL-1β, IL-6, TNF-α, MPO, IFN-γ and increasing the content of IL-10, transforming growth factor-beta1 (TGF-β1), transforming growth factor-beta2 (TGF-β2) (Fig. 7b).