https://orcid.org/0000-0003-3577-1713
Figures & data
Figure 1. Guar gum exacerbated DSS-induced acute colitis.
(a) Experimental design employed to investigate the effect of guar gum (GuD) on acute colitis. Four weeks old WT mice (n=6–7 per group) were maintained on control (Con) or GuD for 4-weeks before colitis was induced by switching to DSS (1.4% w/v) containing water for 7 days. (b) Percent change in body weight. (c) Gross colon appearance (d) Spleen weight. (e) Colon length. (f i-iv) Representative images of: H&E (i) and alcian blue (ii)-stained colon sections (original magnification, x100). (iii-iv) Colonic sections displaying immunohistochemical staining for mucin 2 (Muc2) (iii, green), and Lcn2 (iv, red). DAPI was used to visualize nucleus [blue, (original magnification, ×200)]. (g) Histopathological scores from blinded evaluation of H&E-stained colonic sections. (h-j) Colonic and Serum Lcn2 and SAA. (k) Disease severity was assessed by summing scores (0–4) for weight loss, stool consistency, fecal occult blood, and histopathological changes. Values are presented as mean ±SEM. (d–e and g–k) One-way ANOVA, multiple comparisons test. *p< .05, **p< .01, ***p< .001, and ****p<.0001.
Table 1. Control and guar gum diet composition.
Figure 2. Antibiotic-mediated depletion of gut microbiota rescued GuD-fed mice from DSS-induced colitis.
(a) Experimental design showing the timeline for administration of antibiotics mixture. Experimental groups received either regular (no treatment; NT) or antibiotic mixture-containing (Abx) water maintained on a Con or GuD diet and switched to DSS (1.4% w/v) during the last week. (b) Percent change in body weight. (c) Gross colon appearance. (d) Spleen weight. (e) Colon length. (f) Representative H&E and Alcian blue stained images (i-ii, original magnification, x100) of colon, and sections immunostained for Muc2 (iii, green), and Lcn2 (iv, red) (original magnification, ×200)] (g) Histopathological scores derived from blinded evaluation of H&E-stained colonic sections. (h) Colonic Lcn2. Serum (i) Lcn2 and (j) SAA. (k) Disease severity score. Data, presented as mean ± SEM, were combined from two independent experiments. Unpaired t-test. * p < .05, ** p < .01, *** p < .001, and **** p < .0001.
Figure 3. Guar gum fed mice displayed shift in gut microbiota enriching Actinobacteriota.
(a) Experimental layout to study the shift in gut microbiota profile in guar gum (GuD) fed mice. Four-week-old WT mice (n= 5–7 per group) maintained either on Con or GuD diet for 5 weeks. Feces were aseptically collected and used for 16S rRNA sequencing. (b-g) Microbial signature of control and GuD-fed mice. (b) Comparison of microbiota profiles by Principal coordinates analysis (PCoA) plot (c) Alpha diversity calculated by the Shannon index. (d) Relative abundance and distribution of microbial composition at the phylum level. The box plot represents the enrichment of Actinobacteriota following GuD intervention. (e–g) Mean relative abundance of conspicuously altered bacterial taxa at the genus level. The boxplot in each diagram represents the relative abundance plotted as a percentage of the total reads. Values are presented as mean ±SEM. (d–g) Statistical analysis was performed using MaAsLin2 (Multivariate association with linear models) approach. The q-value (Benjamini–Hochberg false discovery rate corrected) < 0.05 was considered as significant.
Figure 4. Guar gum-induced alterations in gut microbiota metabolism was associated with luminal accumulation of intermediate metabolites, including lactate and succinate.
Four-week-old WT mice (n = 7–8 per group) were maintained on either a Con or GuD diet for 5 weeks. Ceca (with cecal contents) were collected in a tube and snap-frozen immediately. About 50 mg of cecal content was used for metabolite analysis by using quantitative1H NMR. The violin plots represent metabolites measured in µmol/g of cecal content. (a) Short chain fatty acids (SCFAs, acetate, propionate, and butyrate) (b) Branched-chain amino acids (BCAAs; valine, leucine, and isoleucine). (c) Lactate (d) Succinate (e) Fumarate. (f–g) Colon samples obtained from Con or GuD fed mice were used to determine (f) mRNA level of Sucnr1, and (g) colonic expression pattern of Sucnr1 via immunohistochemistry. Values are presented as mean ±SEM. (a-c and e-f) Unpaired t-test, (d) Unpaired non-parametric Mann–Whitney test. * p < .05, ** p < .01, and *** p < .001.
Figure 5. Guar gum suppressed the colonic mRNA expression of mucins and mucosal barrier proteins.
Four weeks old WT mice (n= 5–6 per group) maintained on Con or GuD diet for 5 weeks. Colonic mRNA expression of (a–d) mucins Muc1, Muc2, Muc3, and Muc4, (e–l) tight junction proteins occludin (Ocln), zonula occludens-1 (Zo 1), and claudins, and (m) E-cadherin 1. Values are presented as mean ±SEM. (a–m) Unpaired t-test. * p < .05, ** p < .01, and *** p < .001.
Figure 6. Guar gum-fed mice displayed reduced expression of colonic IL-18.
Four weeks old WT mice (n = 4-5 per group) maintained on Con or GuD diet for 5 weeks. After euthanasia, colonic tissue was collected for RNA and protein extraction and for immunohistochemical staining. (a-c) Colonic mRNA expression of iNos, Il-6, and Il-18. (d) Colonic IL-18 assessed by ELISA. (e) Representative images of colonic IL-18 immunostaining (green) (original magnification, ×200)]. Values are presented as mean ±SEM. (a–d) Unpaired t-test. * p < .05, ** p < .01, and *** p < .001.
Figure 7. Exogenous IL-18 administration reduced colitis severity in GuD-fed mice.
(a) Experimental layout to assess the effect of exogenous recombinant IL-18 (rIL-18) on colitis severity. Four-week-old mice (n = 4–5 per group) were maintained on a GuD for 4 weeks and were supplemented with vehicle (PBS) or rIL-18 at the dose of 1 µg/mouse (in PBS) intraperitoneally for 3 consecutive days before switching them to DSS (1.4% w/v)-containing water or water only for 7 days. (b) Percent change in body wt. (c) Gross colon appearance . (d) Spleen wt. (e) Colon length. (f) Colonic Lcn2. (g i-iv) Representative images of colon sections stained with H&E (i) and Alcian blue (ii) (original magnification 100x). Colonic sections immunostained for Muc2 (iii, green) and Lcn2 (iv, red) (original magnification, 200x). (h) Histopathological scores derived from blinded evaluation of H&E-stained colonic sections. (I-J) Serum levels of (i) Lcn2 and (j) SAA. (k) Disease severity score. Values are presented as mean ±SEM. (b) Unpaired t-test, (d–f and h–k) One-way ANOVA, multiple comparisons test. *p< .05, **p< .01, ***p< .001, and ****p<.0001.
Table 2. Composite scoring system for assessing disease severity.
Table 3. Primer sequences.
Supplemental material
Supplemental Material
Download MS Word (871.2 KB)Data availability statement
The microbiota sequencing data that support the findings of this study will be openly available in the European Nucleotide Archive under accession number PRJEB64411.