Pomegranate peel extract ameliorates the severity of experimental autoimmune encephalomyelitis via modulation of gut microbiota

Abstract

Multiple sclerosis (MS) is a CNS autoimmune disease characterized by demyelination and inflammatory infiltration with a high disability rate. Increasing evidence has demonstrated the importance of gut microbiota as an environmental risk factor in MS and its animal model experimental autoimmune encephalomyelitis (EAE). Diet is the main determinant of gut microbiota composition and function, which greatly affects the shaping of microbial structure. Pomegranate peel, a waste product in the production of juice, is rich in health-promoting compounds. However, its individual constituents, immunoregulatory activities, and action associated with bacterial diversity in the gut microbiota are largely unknown. Here, the main nutrient ingredients of pomegranate peel extract (PPE) were identified as phenols, flavonoids, amino acids, carbohydrates, fatty acids, lipids, nucleotides, organic acids, alcohols, and vitamins via metabolomics evaluation. We found, for the first time, oral PPE (100 mg/kg/day) not only effectively relieves EAE, inhibits CNS inflammatory factor infiltration and myelin loss, but also reshapes gut microbiota. Furthermore, recipient EAE mice with fecal transplantation from the PPE-treated donor delayed the disease development significantly. 16S rRNA gene sequencing revealed the increased gut microbiota richness in PPE-treated group. Among them, Lactobacillaceae enriched significantly, while Alcaligenaceae and Acidaminococcacea decreased remarkably. In conclusion, our data demonstrated that gut microbiota mediated the beneficial effects of oral PPE on EAE, and provided new ideas for developing the prebiotic value of pomegranate peel for the treatment of autoimmune diseases.

Keywords: 16s rRNA gene sequencing; experimental autoimmune encephalomyelitis; gut microbiota; multiple sclerosis; pomegranate peel extract.

Figure 1.

Oral PPE treatment remarkably alleviated EAE. C57BL/6 mice were immunized and treated by oral gavage with PBS or PPE (100 mg/kg/day) at d 0 post-immunization (p.i.) (a, n = 7), d 10 p.i. (c, n = 6), and d 16 p.i. (e, n = 5) Arrow indicated treatment time point. (b, d, f) Cumulative scores were the statistical analysis of the total of daily clinical scores from a, c, and e. PBS- or PPE -treated EAE mice described in (a) were sacrificed at d 24 p.i., and spinal cords were isolated for immunohistochemical staining. (g, h) LFB staining and statistical analysis. (i, j) H&E staining and statistical analysis. (k, l) MBP immunofluorescence staining and intensity quantization. Full slice bar = 500 μm, partial slice bar = 200 μm. Data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, determined by two-way ANOVA (a, c, e), or unpaired Student’s t-test (b, d, f, h, j, l). Data are represented by three independent experiments

Figure 2.

PPE treatment reduced CNS inflammation. EAE mice treated with PBS or PPE described in Figure 1(a) were sacrificed at d 24 p.i., and the brain and spinal cord were harvested. (a) Total number of mononuclear cells (MNCs) in CNS. (b–i) The percentages and absolute numbers of CD45⁺, CD45⁺CD11b⁺, CD11c⁺, CD11c⁺CD80⁺, CD11c⁺CD86⁺, CD4⁺ cells were measured by flow cytometry. Data are expressed as the mean ± SEM (n = 3 for each group). **p < 0.01, determined by unpaired Student’s t-test

Figure 3.

Effect of PPE treatment on T cell inflammatory factors in CNS. Tissue extraction and processing are the same as shown in Figure 2. (a–d) Assess the proportion and absolute numbers of IL-17⁺, IFN-γ⁺, IL-10⁺, and Foxp3⁺ cells in CD4⁺ cells by flow cytometry. (e, f) The percentage of these cells in the CD4⁺ cells in the CNS. Data are expressed as the mean ± SEM (n = 3 for each group). **p < 0.01, determined by unpaired Student’s t-test

Figure 4.

PPE treatment inhibited peripheral inflammation and pro-inflammatory cytokine production. As shown in Figure 1(a), mice were treated with PBS or PPE on the day of EAE immunization and sacrificed at d 24 p.i. to collect the spleen. Splenocytes were stimulated with 25 µg/mL MOG_35–55 for 3 day. (a, b) The percentage and statistics of CD11b⁺, CD11b⁺MHCII⁺, CD11c⁺, CD11c⁺CD80⁺, CD11c⁺CD86⁺ were measured by flow cytometry. (c) Cytokine concentration in the cultured splenocyte supernatant was measured by ELISA. Data are expressed as the mean ± SEM (n = 3 for each group). *p < 0.05, determined by unpaired Student’s t-test

Figure 5.

Fecal microbiota transplantation (FMT) from PPE-treated mice has a protective effect on EAE. Feces of EAE mice treated with PBS or PPE were collected, and perform FMT on recipient mice. FMT was administered daily from the day of immunization. (a) Clinical score and (d) body weight of EAE mice transferred with fecal matter from PPE or PBS group (n = 5). (b) Cumulative scores from d 10 to d 12 p.i. of the disease. (c) AUC of mean clinical scores on d 10–12 p.i. (e) Body weight of mice from d 18 to 20 p.i. Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, determined by unpaired Student’s t-test. Data are represented by three independent experiments

Figure 6.

Comparison of gut microbiota characteristics of EAE mice treated with PBS and PPE. 16s rRNA sequencing were performed on stool samples from PBS and PPE treatment groups (n = 7). (a) Total OTU counts of gut microbiota in each group. (b) Venn diagram of the two groups. (c) Partial least-square discriminant analysis (PLS-DA) in PBS (red points) and PPE (blue points) group. Distance algorithm (Bray-Curtis) was used to calculate the significance of differences between two groups samples (p= 0.0001). (d) Chao index (α diversity) of gut microbiota at the OTU level. (e) Family level analysis of significant difference bacterial species between different groups. Comparison proportion of family levels of (f) Lactobacillaceae, (g) Alcaligenaceae and (h) Acidaminococcaceae. (i) Linear discriminant analysis effect size (LEfSe) was used to calculate the microbiota difference between the two groups. Determined by non-parametric factorial Kruskal-Wallis (KW) sum-rank test. Red and blue nodes indicate microbial groups that are significantly enriched in the PBS and PPE groups, and have a significant effect on the differences between groups; The yellow nodes represent that there is no significant difference between the two groups of microbiome, phylum, class, order, family, and genus are represented by circles from inside to outside. Abbreviation of the letters was shown in Supplementary Table S2. Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, determined by Student’s t-test

Figure 7.

Health benefits of oral PPE on relieving the clinical symptoms of EAE and improving the gut microbiota