Figures & data
Figure 1. The effect of UA on chronic alcohol-induced liver injury.
(a) Timeline of experiment 1 depicting the alcohol liquid diet with UA treatment; (b) Body weight (n = 10 mice per group); (c) Body weight gain; (d) Food intake; (e) Weight of liver; (f) Index of liver to body weight; (g) Representative images of H&E staining (n = 3 mice per group) and immunochemical staining of F4/80 staining of liver sections (n = 3 mice per group); (h) ALT and AST levels in serum; (i) TG levels in serum; (j) Immunochemical density of F4/80 and immunofluorescence intensity of MUP1 in the liver; (k) The relative mRNA expressions of hepatic inflammation and injury-related genes (n = 6 mice per group); (l&m) Western blots analysis of hepatic inflammation and injury-related signaling (n = 3, representative of three biological replicates for each group). Data presented as mean ± SEM. *p < .05, **p < .01, compared with Ctrl group, #p < .05, ##p < .01, compared with the Alc group. Significant differences between mean values were determined by one-way ANOVA with Tukey’s multiple comparisons test.
Figure 2. UA alleviates alcohol-induced hepatic endoplasmic reticulum stress via upregulating MUP1.
(a) Volcano plot based on the differential genes (Alc UA vs. Alc); (b) GO-based biological process analysis of RNA sequencing data (Alc UA vs. Alc); (c) Enrichment plot of protein processing in endoplasmic reticulum pathway based on gene set enrichment analysis (GSEA) analysis from the liver RNA sequencing data between the Alc and Alc UA groups; (d) Heatmap of differential genes between Alc and Alc UA group based on the RNA sequencing of the liver (n = 5, representative of 5 biological replicates for each group); (e) Representative immunofluorescence images (left) and immunofluorescence intensity (right) of MUP1 in liver (n = 3 mice per group); (f) Western blots analysis of MUP1 and ER-stress related signaling (IRE-1α/XBP1, PERK/ATF4 and ATF6/GRP78/BIP) (n = 3, representative of 3 biological replicates for each group); (G&J) Western blots analysis of MUP1 in HepG2 cells transformed by MUP1 siRNA; (h) HepG2 cells transformed by MUP1 siRNA; (i) AST and ALT levels in HepG2 cells transformed by MUP1 siRNA; (k&l) Western blots analysis of ER-stress related proteins in HepG2 cells transformed by MUP1 siRNA. Data presented as mean ± SEM. *p < .05, **p < .01, compared with Ctrl group, #p < .05, ##p < .01, compared with the Alc group. Significant differences between mean values were determined by one-way ANOVA with Tukey’s multiple comparisons test.
Figure 3. UA restores alcohol-induced intestinal barrier damage and gut microbiome dysbiosis.
(a) Representative images of H&E staining, Alcian blue staining and MUC2 immunofluorescence staining of colon sections (n = 3 mice per group); (b&c) Western blots analysis of tight junction-related proteins; (d) The villi length; (e) MUC2 fluorescence intensity; (f) LPS levels in serum; (g) Venn diagram of OTUs shared among different groups (n = 5 mice per group); (h) Principal coordinate analysis (PCoA) based on unweighted Unifrac distance and permutational manova (adonis) were used to test the difference in gut microbiota composition and diversity between group; (i) The relative abundance of bacteria at the species level. All genera with an average relative abundance below 0.5% were grouped to ‘‘others’’; (j) Heatmap of the abundance of gut microbiota at the genus level; (K) Heatmap of correlation analysis between the KEGG functional data and relative abundance of B. sartorii, P. distasonis and A. muciniphila; (l) The concentrations of SCFAs, including acetic acid, propionic acid and butyric acid in feces under UA treatment. Data presented as mean ± SEM. *p < .05, **p < .01, compared with Ctrl group, #p < .05, ##p < .01, compared with the Alc group. Significant differences between mean values were determined by one-way ANOVA with Tukey’s multiple comparisons test.
Figure 4. UA alleviates ALD in a gut microbiota-dependent manner.
(a) Timeline of animal experiment 2 depicting the alcohol liquid diet of UA and antibiotic treatment; (b) Representative images of H&E staining (n = 3 mice per group) and immunochemical staining of F4/80 (n = 3 mice per group) and immunofluorescence staining of MUP1 of liver sections (n = 3 mice per group); (c) Immunochemical density of F4/80 and immunofluorescence intensity of MUP1 in the liver; (d) ALT and AST levels in serum; (e) TG levels in serum; (f) The concentrations of SCFAs, including acetic acid, propionic acid and butyric acid in feces under UA and antibiotic treatment; (g) The relative mRNA expressions of hepatic inflammation and injury-related genes (n = 6 mice per group); (H) The relative mRNA expressions of hepatic ER-stress related genes (n = 6 mice per group). Data was presented as mean ± SEM. *p < .05, **p < .01, compared with Alc group, #p < .05, ##p < .01, compared with Alc-Antibiotics group, &p < .05, &&p < .01, versus Alc UA group. Significant differences between mean values were determined by two-way ANOVA (UA and antibiotic treatment as two factors) with Tukey’s multiple comparisons test.
Figure 5. Gut microbiota mediates the activation of MUP1 to alleviate ALD.
(a) Timeline of animal experiment 3 depicting the alcohol liquid diet with FMT treatment; (b) Representative images of H&E staining (n = 3 mice per group) and immunochemical staining of F4/80 (n = 3 mice per group) and immunofluorescence staining of MUP1 of liver sections (n = 5 mice per group); (c) Immunochemical density of F4/80 and immunofluorescence intensity of MUP1 in the liver; (d) ALT and AST levels in serum; (e) TG levels in serum; (f) The concentrations of SCFAs, including acetic acid, propionic acid and butyric acid in feces under FMT treatment; (g&h) Western blots analysis of MUP1 and ER-stress related signaling (IRE-1α/XBP1, PERK/ATF4 and ATF6/GRP78/BIP) (n = 3, representative of 3 biological replicates for each group). Data was presented as mean ± SEM. *p < .05, **p < .01, compared with Ctrl FMT-Ctrl group, #p < .05, ##p < .01, compared with the Alc FMT-Alc group. Significant differences between mean values were determined by one-way ANOVA with Tukey’s multiple comparisons test.
Figure 6. B. sartorii, P. distasonis, A. muciniphila, and their derived propionic acid mediate the activation of hepatic MUP1 in ALD.
(a) Timeline of the animal experiment 4 depicting the alcohol liquid diet with B. sartorii, P. distasonis and A. muciniphila intervention; (b) ALT and AST levels in serum; (c) TG levels in serum; (d) Representative images of H&E staining (n = 3 mice per group) and immunohistochemical staining of F4/80 (n = 3 mice per group) and immunofluorescence staining of MUP1 of liver sections (n = 3 mice per group); (e) Immunochemical density of F4/80 and immunofluorescence intensity of MUP1 in the liver; (f) Western blots analysis of MUP1 and ER-stress related signaling (IRE-1α/XBP1,PERK/ATF4 and ATF6/GRP78/BIP) (n = 3, representative of 3 biological replicates for each group); (g) Protein expression of MUP1 and ER-stress related signaling (IRE-1α/XBP1, PERK/ATF4 and ATF6/GRP78/BIP) relative to control; (h) The concentrations of propionic acid in serum; (i) Heatmap of correlation analysis between the UA upregulated transcript genes and the relative abundance of differential species between Alc UA group and Alc group; (j) Heatmap of correlation analysis between MUP1 and SCFAs; (k) The effect of propionic acid on HepG2 cells treated by alcohol; (i) Western blots analysis of expression of MUP1 and GLP1-R in HepG2 cells treated by propionic acid; (m) The concentrations of ALT and ALT in HepG2 cells treated by propionic acid. Data presented as mean ± SEM. *p < .05, **p < .01, compared with Ctrl group, #p < .05, ##p <.01, compared with the Alc group. Significant differences between mean values were determined by one-way ANOVA with Tukey’s multiple comparisons test.
Supplemental material
Supplementary Material clean.docx
Download MS Word (4.8 MB)Data availability statement
The raw data of RNA sequencing in the current study were deposited on GEO, and the accession number is GSE253307. The accession number for the entire 16S rRNA sequencing dataset reported in this manuscript is NCBI Bio Project: PRJNA1065023.
Original data have been deposited to Mendeley Data, the DOI number is 10.17632/84nnnpx8d8.2.