Fig 2: Proposed mechanisms for the preventive effects of the decreased tissue n-6/n-3 ratio on the CPT-11-induced gut toxicities. Diagram illustrating that elevated tissue omega-3 PUFA status with a reduced tissue n-6/n-3 PUFA ratio alters gut microbiome composition and functions, exerts anti-inflammatory and mucosal protective effects, and additionally, reduces the abundance of GUSB-producing bacteria, GUSB activity and potentially the conversion of inactive SN-38G to toxic SN-38. These alterations together with other gut-microbiota-independent mechanisms reduce mucosal injuries, mucosal inflammation, goblet cell dysfunction, impairment in the gut barrier, and systemic endotoxemia, resulting in the prevention of CPT-11-induced gut toxicities (bodyweight loss, late-onset diarrhea, and bloody diarrhea, and death).

Fig 3: Dietary n-3 PUFA supplementation reduces CPT-11-induced alterations in the gut microbiome. (A) Principal coordinates analysis (PCOA) plot showing the results of Bray–Curtis distance-based analysis of beta diversity metrics. (B) Violin plot with lines at the median (dashed lines) and quartiles (complete lines) showing the differences in the Pielou’s evenness index (a-diversity). (C) Microbe–microbe interactions network [SparCC correlation analysis (CO+CPT-11 vs. FO+CPT-11)]. Each node (*, GUSB-producing taxa; #, healthy gut making taxa) represents a taxon (colored based on the phylum level and sized based on the number of connections to that taxon). Two taxa are connected by an edge (co-occurrences: red; anti-occurrences: blue; p-value < 0.05 and correlation threshold 0.3; size reflects the magnitude). (D) Random Forests classification of taxa (genus level) of CPT-11 treated CO and FO groups. (E) Composition summary showing the phyla detected in the CPT-11 treated CO and FO groups. The numbers above each bar show the relative abundance (RA) of the Proteobacteria phylum. (F–I) RA of differentially abundant bacterial groups such as Enterobacteriaceae (F) with representative colonic luminal contents MacConkey agar culture plate photos (G) showing the difference (H) in the growth of Escherichia Coli and Bifidobacterium (I), which is not detectable in CO group. (J) qPCR results showing the RA of beta-glucuronidase (GUSB)-producing bacteria. (K) The difference in GUSB activity was measured at baseline (BL) and days (d) 6 using stool samples and at days 11 using cecal contents. (L) Representative pictures are showing GUSB expression measured at proximal colon using the immunohistochemical technique. (M) RA of GUSB (K01195) gene predicted using PICRUSt2. Data are shown as mean ± standard error of the mean. Data with different superscript letters are significantly different (p < 0.05) according to the nonparametric Mann–Whitney test (* p < 0.05, ** p < 0.01) or ordinary two-way (K) ANOVA followed by Sidak’s multiple comparisons tests. Scale bar for images in (J) panel: 2000 µm.

Fig 4: Decreased tissue n-6/n-3 ratio prevents CPT-11-induced alterations in the gut microbiome. (A) Principal coordinates analysis (PCOA) plot showing the results of Bray–Curtis distance-based analysis of beta diversity metrics. (B) Violin plot with lines at the median (dashed lines) and quartiles (complete lines) showing the differences in the Pielou’s evenness index. (C) Microbe–microbe interactions network [SparCC correlation analysis (WT+CPT-11 vs. FAT-1+CPT-11)]. Each node (*, GUSB-producing taxa; #, healthy gut making taxa) represents a taxon (colored based on the phylum level and sized based on the number of connections to that taxon). Two taxa are connected by an edge (co-occurrences: red; anti-occurrences: blue; p-value < 0.05 and correlation threshold 0.3; size reflects the magnitude). (D) Random Forests classification of taxa (genus level) in the vehicle (W and F1) or CPT-11 (WC and F1C) treated groups. (E) Phyla detected in the control and CPT-11 treated WT and FAT-1 mice. The numbers above each group show the relative abundance (RA) of the Proteobacteria phylum. (F–K) RA of differentially abundant (ANCOM test by QIIME2) bacterial groups such as Enterobacteriaceae (F) with representative colonic luminal contents MacConkey agar culture plate photos (G) showing the difference (H) in the growth of Escherichia Coli (pink colonies), Enterococcus (I), Bifidobacterium (J) and Akkermansia (K). (L) RA of beta-glucuronidase (GUSB)-producing bacteria measured using qPCR. (M) The difference in GUSB activity was measured at baseline (BL) and days (d) 6 using stool samples and at days 11 using cecal contents. (N) Immunohistochemical staining-based GUSB gene expression patterns in the proximal colon. (O) RA of GUSB (K01195) gene predicted using PICRUSt2. Data are shown as mean ± standard error of the mean. Data with different superscript letters are significantly different (p < 0.05) according to the Kruskal–Wallis test (B) or Mann–Whitney test, or ordinary two-way (M) ANOVA followed by Sidak’s multiple comparisons test. Scale bar for images in (J) panel: 2000 µm.