Protein-educated FMT improves liver function.

(A) Study design- In separate setups, FMT was given to ALD mice (3 alternate days) from donors fed one of three diets—a standard diet, a vegetable protein diet or an egg protein diet—followed by collection of tissues and blood seven days after FMT. (B) Overall, FMT reduced serum AST, ALT and bilirubin levels, with an enhanced reduction in Veg-FMT (all p<0.001). (C) Representative micrographs of liver histology by hematoxylin and eosin (H&E) and Masson’s trichome (MT) staining and immunohistochemistry for smooth muscle actin protein (α-sma) showing decreased hepatic steatosis (p<0.001) and fibrosis (MT and α-sma positive area, p=0.01in both) in protein-educated FMT with statistical analysis. (D) Compared with egg-FMT, Veg-FMT led to greater reductions in hepatic Il6 (p=0.04), Tnfα (p=0.06), Tlr4 (p=0.05), (E) Acta2 (p=0.001) and Tgfβ(p<0.001) and increased IL10 (0.02) mRNA expression. (F) Hepatic TGs were significantly reduced in protein-educated FMT (Veg-FMT: 1.74FC, p<0.001; Egg-FMT: 1.47FC,p=0.009). The data are presented as the means ± SEMs (standard error of the mean). *p < 0.05, **p < 0.01, ***p < 0.001 represent intergroup statistics; #p < 0.05, ##p < 0.01, ###p < 0.001 represent the ALD group (one-way ANOVA followed by Tukey’s multiple comparison test).

Protein-educated FMT restores intestinal barrier integrity.

(A) mRNA expression of the ileal tight junction proteins Zo-1 (p=0.04), Cldn3(p=0.01), and Ocln (p<0.001) was significantly reduced after Veg-FMT compared to ALD. (B) Statistical analysis revealed that the protein expression of the tight junction protein Zo-1 was markedly lower after Veg-FMT than after egg-FMT (1.27FC, p=0.004). (C) mRNA expression of the antimicrobial peptides Reg3ß (p=0.009), Reg3Γ (p=0.006) and (D) Muc2 (p=0.05) also decreased after veg-protein educated FMT. (E) Plasma endotoxin levels (p=0.02) were also significantly lower after veg-protein FMT. (F) Serum endotoxin and Muc2 gene expression were significantly negatively correlated (r2=-0.86, p<0.001; Spearman’s correlation). The data are presented as the means ± SEMs (standard error of the mean). *p < 0.05, **p < 0.01, ***p < 0.001 represent intergroup statistics; #p < 0.05, ##p < 0.01, ###p < 0.001 with respect to the ALD group (A-E, one-way ANOVA followed by Tukey’s multiple comparison test).

Favorable gut microbial variations were observed after protein-FMT.

(A) PCoA plot showing significant (p=0.001, PERMANOVA) differences in microbial composition post-FMT among the different treatment groups. (B) Comparison of the relative abundances of bacterial genera revealed increases in Akkermansia, Lachnopiraceae NK4A136 and Parasutterella abundance post-FMT. (C) Tables showing differentially altered taxa present commonly and uniquely in the FMT treatment groups. (D) Bidirectional bar plot showing selected functional pathways predicted by PICRUSt in veg-FMT (E) Correlations between serum injury biomarkers and differential bacterial taxa identified by Veg-FMT. Pearson’s correlation values are displayed in circles. Red represents a positive correlation, whereas blue represents a negative correlation. Correlation according to the scale given at the bottom of the plot.

Fecal microbiota transplantation alters the hepatic proteome.

(A) Heatmap showing differentially expressed proteins whose expression was upregulated or downregulated in different groups. (B) Venn diagram showing the number of common and unique enriched pathways among abst, Std-FMT, Veg-FMT and egg-FMT, with the (C) table showing pathway names. (D) The PPAR signaling pathway was significantly enriched (p=8.3x10-06) in the Veg-FMT group. (E) Hepatic PPARα mRNA expression was greater in the Veg-FMT group (p<0.001) than in the Egg-FMT group. (F) mRNA expression (top panel; Cpt1 (p=0.003), Fabp1 (p=0.005) and peroxisomal Acox1 (p=0.02)) and protein expression (bottom panel; Cpt1a (p=0.007), Acox1 (p= 0.05), Acadm (p= 0.04) and Fabp1 (p= 0.02)) of PPARα target genes involved in fatty acid beta-oxidation increased after Veg-FMT. The data are presented as the means ± SEMs (standard error of the mean). *p < 0.05, **p < 0.01, ***p < 0.001 represent intergroup statistics; #p < 0.05, ##p < 0.01, ###p < 0.001 represent the ALD group (one-way ANOVA followed by Tukey’s multiple comparison test).

Veg-FMT alleviates hepatic injury through PPARα activation.

(A) Study design showing PPARα inhibitor administration in ALD mice with Veg-FMT. At 7 days after FMT, the tissues and blood were collected for further analyses. (B) mRNA (p<0.001) and (C) protein expression of PPARα decreased significantly (p=0.01). (D) Serum AST(p=0.003), ALT(p=0.002), bilirubin(p=0.04) and hepatic triglyceride levels (p=0.03) significantly increased after PPARα inhibition. (E) Liver histology images showing increased steatosis (p<0.001) by H&E and fibrosis(p<0.001) by MT staining after inhibition copared to veg-FMT. (F) Plasma endotoxin levels (p=0.001) were also increased after inhibition. The data are presented as the means ± SEMs (standard error of the mean). *p < 0.05, **p < 0.01, ***p < 0.001 represent intergroup statistics; #p < 0.05, ##p < 0.01, ###p < 0.001 with respect to the ALD group (one-way ANOVA followed by Tukey’s multiple comparison test).

Caproic acid supplementation mitigates ethanol-induced steatosis by enhancing fatty acid β-oxidation in Huh7 cells.

(A) Study design: Huh7 cells were treated with ethanol or caproic acid alone or in combination. A PPARα inhibitor was also given with caproic acid. Injury was assessed after 24 hours. (B) mRNA expressions of pro-inflammatory reduced (IL6, p=0.002; TNFα, p=0.006; IL1β, p<0.001; Nfkb, p<0.001) and anti-inflammatory markers (IL10, p=0.005) increased after Abst+CA supplementation and these changes reversed in presence of PPARα inhibitor. (C) Cytopathology showing higher lipid accumulation in ethanol treated cells using oil-red-o staining and significantly reduced after CA supplementation (p=0.02). CA supplementation effects were reduced in the presence of PPARα inhibitor (p=0.01). (D) TG levels were significantly decreased after CA supplementation (p=0.05) and effects were reversed after PPARα inhibition. (E) mRNA expression of lipid droplet marker (Plin2) increased in ethanol treatment and reduced CA supplementation (p=0.002), but reversed in presence of PPARα inhibitor. (F) Mitofuel flexibility assay showed increased β-oxidation dependency after CA supplementation (p<0.001), but it was reduced in the presence of the inhibitor (p<0.001). The data are presented as the means ± SEMs (standard error of the mean). *p < 0.05, **p < 0.01, ***p < 0.001 represent intergroup statistics; #p < 0.05, ##p < 0.01, ###p < 0.001 with respect to the ALD and control groups. &p < 0.05, &&p < 0.01, &&&p < 0.001 with respect to ALD (one-way ANOVA followed by Tukey’s multiple comparison test).

Caproic acid supplementation alleviated ethanol-induced liver injury through PPARα upregulation in ALD mice.

(A) Study design- ALD mice treated with caproic acid with or without a PPARα inhibitor. Injury was assessed after 1 week. (B) Serum AST (p=0.02), ALT (p<0.001) and bilirubin (p=0.006) levels were significantly reduced after CA supplementation. (C) Hepatic TGs were significantly reduced after CA supplementation (p=0.02) and these changes were reversed in the presence of PPARα inhibitor (D) Liver histology showing reduced steatosis (examined by H&E and Oil Red O, p<0.001 in both) and fibrosis (p=0.007) by MT staining after CA supplementation. (E) mRNA expression of PPARα target genes involved in fatty acid β-oxidation increased (PPARα, p<0.001; Cpt1, p=0.01; Acox1, p=0.008; Fabp1, p=0.002) and (F) lipogenesis-related mRNA expression decreased (Srebp1c, Scd1 and Fasn, p<0.001 in all) after CA supplementation. These beneficial effects of CA were reversed in the presence of a PPARα inhibitor. The data are presented as the means ± SEMs (standard error of the mean). *p < 0.05, **p < 0.01, ***p < 0.001 represent intergroup statistics; #p < 0.05, ##p < 0.01, ###p < 0.001 with respect to the ALD and control groups. &p < 0.05, &&p < 0.01, &&&p < 0.001 with respect to ALD (one-way ANOVA followed by Tukey’s multiple comparison test).