Additional file 1 of Therapeutic potential of berberine in attenuating cholestatic liver injury: insights from a PSC mouse model

Additional file 1: Fig. S1. Impact of BBR on body weight and serum albumin levels in FVB Mdr2-/- mice and cholestatic liver injury in C57/BL6 Mdr2-/- mice. Mdr2-/- mice with FVB background (Control) and Mdr2-/- mice with C57BL/6 background (Control BL) were treated with vehicle or BBR (50 mg/kg) via oral gavage once daily for 8 weeks, respectively. a Body weight change during the BBR treatment period of 8 weeks in FVB Mdr2-/- mice. b Serum albumin levels in FVB Mdr2-/- mice. c Liver functional enzyme levels in C57/BL6 Mdr2-/- mice. d Representative images of hematoxylin and eosin (H&E) staining of the liver slides (scale bar, 50 µm for 20x, 20 µm for 40× magnification) in Mdr2-/- BL mice. Data are expressed as the mean ± standard error of the mean (SEM). Statistical significance relative to Control BL: *p < 0.05 (n=9-12). Fig. S2. Comparative analysis of differentially expressed genes (DEGs) in experimental groups. a Hierarchical clustering heatmaps for DEGs in FVBWT, Mdr2-/- and Mdr2-/- mice treated with BBR. RNA-seq data were normalized using a Z-score for tag counts, with red and blue colors representing high and low gene expression, respectively. b Volcano plots for the Mdr2-/- vs. WT group comparison. Red dots represent upregulated genes, green dots represent downregulated genes, and black dots represent genes not differentially expressed. c Venn diagram illustrating the overlap of DEGs between the two comparisons: Mdr2-/- vs. WT and BBR-treated Mdr2-/- vs. Mdr2-/- Control. In Mdr2-/- vs. WT, there were a total of 1937 DEGs, including 1260 upregulated and 677 down-regulated genes. In BBR-treated Mdr2-/- vs. Mdr2-/- Control, there were a total of 587 DEGs, comprising 300 upregulated and 287 down-regulated genes. A total of 373 DEGs were common between the two comparisons. Fig. S3. Ingenuity pathway analysis (IPA) in experimental groups. The DEG data set with FC ≥2 and p-value <0.05 was used for IPA analysis. The top 10 activated pathways in Mdr2-/- control mice compared to WT mice and the top 10 inhibited pathways in BBR-treated Mdr2-/- mice compared to Mdr2-/- control mice are shown. Fig. S4. Impact of BBR on hepatic fibrosis. a Representative images of liver sections stained with Picro-Sirius Red and CK19 IHC (scale bar, 100 µm for 10× magnification) and processed images for quantification. b Hepatic hydroxyproline levels. Data are expressed as the mean ± SEM. Statistical significance relative to control: *p < 0.05 (n=9-12). Fig. S5. Impact of BBR on genes associated with hepatic fibrosis in Mdr2-/- mice. a Representative heatmap of key genes involved in hepatic fibrosis in the liver, comparing the BBR-treated group with the control group. The RNA-seq data were normalized using a Z-score for tag counts, with red and blue colors denoting up- and down-regulated gene expression, respectively. b Relative mRNA expression levels of fibrosis-related genes (Pai1,Col12a1, Sox9, Egr1, Egr2, Egr3, Hbegf, Cyr61, and P4ha1), normalized against HPRT1 as an internal control. Data are expressed as the mean ± SEM. Statistical significance relative to control: *p < 0.05, **p < 0.01, ***p < 0.001(n=9-12). Fig. S6. Impact of BBR on genes associated with inflammation in Mdr2-/- mice. Representative heatmap depicting the expression of key genes involved in hepatic inflammation, comparing the liver tissues of Mdr2-/- mice treated with BBR to the control group. The RNA-seq data were normalized using a Z-score, with red indicating upregulated gene expression and blue indicating downregulated gene expression. Fig. S7. Impact of BBR on NF-kB signaling pathway. KEGG pathway analysis was performed on RNA-seq data to analyze functionally and map genes involved in the NF-kB signaling pathway. a NF-kB signaling pathway in Mdr2-/- vs. WT. b NF-kB signaling pathway in Mdr2-/- treated with BBR vs. Mdr2-/- Control. Red and green colors indicate upregulated and downregulated gene expression, respectively. Fig. S8. Impact of BBR on MAPK signaling pathway. KEGG pathway analysis was performed on RNA-seq data to analyze functionally and map genes involved in the MAPK signaling pathway. a MAPK signaling pathway in Mdr2-/- vs. WT. b MAPK signaling pathway in Mdr2-/- treated with BBR vs. Mdr2-/- Control. Red and green colors indicate upregulated and downregulated gene expression, respectively. Fig. S9. Impact of BBR on Oxidative phosphorylation pathway. KEGG pathway analysis was performed on RNA-seq data to analyze functionally and map genes involved in the Oxidative phosphorylation pathway. a Oxidative phosphorylation pathway in Mdr2-/- vs. WT. b Oxidative phosphorylation pathway in Mdr2-/- treated with BBR vs. Mdr2-/- Control. Red and green colors indicate up- and down-regulated gene expression, respectively. Fig. S10. Impact of BBR on Protein processing in endoplasmic reticulum. RNA-seq data were performed to analyze functionally and map genes involved in the Protein processing in the endoplasmic reticulum pathway using KEGG. a Protein processing in endoplasmic reticulum pathway in Mdr2-/- Control vs. WT. b Protein processing in endoplasmic reticulum pathway in Mdr2-/- treated with BBR vs. Mdr2-/- Control. Red and green colors indicate up- and down-regulated gene expression, respectively. Fig. S11. Impact of BBR on BA Metabolism. Representative heatmap of key genes involved in bile acid metabolism in the liver of BBR-treated vs. Control Mdr2-/- mice. A Z-score is calculated for the RNA-seq data to normalize tag counts. Red and blue colors indicate up- and down-regulated gene expression, respectively. Fig. S12. Impact of BBR on bile acid homeostasis in Mdr2-/- mice. The small intestine and feces were processed for BA analysis using LC-MS/MS. a BA composition profile in the small intestine is expressed as a percentage of total BA. b Total BA, total primary BA, total conjugated BA, and TCA in the small intestine. c BA composition profile in the feces is expressed as a percentage of total BA. d Total BA, total secondary BA, TCA, and LCA in the feces. Data are expressed as the mean ± SEM. Statistical significance relative to control: *p < 0.05 (n=9-12). Fig. S13. Effect of BBR on inflammation and ER stress in the intestine of Mdr2-/- mice. Relative mRNA levels of key genes involved in inflammation and ER stress in the intestine were determined by real-time RT–PCR and normalized with HPRT1 as an internal control. a The relative mRNA levels of Mcp-1, Cd11b, Il-1β, Vcam-1, Il-1α, and Cxcl1. b Relative mRNA levels of Asbt, Chop and H19. Data are expressed as the mean ± SEM. Statistical significance relative to Control: *p < 0.05 (n=9-12). Fig. S14. Tissue distribution of BBR in Mdr2-/- mice. Mdr2-/- mice were treated with BBR (50 mg/kg, n = 3) by intragastric administration after a 12-h fast. Blood, spleen, brain, lung, heart, kidney, liver, stomach contents, intestine contents, and colon feces were collected at 3, 6, and 9 h post-treatment. The concentrations of BBR in the serum and various tissues were quantified using LC-MS/MS. a BBR concentration in the serum. b BBR concentration in the tissues. Data are expressed as the mean ± standard error of the mean (SEM). Fig. S15. Analysis of Fecal Microbiota Diversity in Mdr2-/- Mice Treated with BBR. Fecal samples of FVB Mdr2-/- mice treated with either 50 mg/kg or 100 mg/kg of BBR for 8 weeks were subjected to 16S rRNA gene sequencing to assess microbiota composition. a Alpha diversity of the fecal microbiota, presented through various metrics: Shannon Index (a), observed Amplicon Sequence Variants (ASVs) (b), Faith’s phylogenetic diversity (c), and evenness (d). b Beta diversity analysis using Principal Coordinates Analysis (PCoA) plots, which illustrate variations in microbial communities. These plots are based on different distance metrics: Bray-Curtis distance (a), Jaccard distance (b), Weighted UniFrac distance (c), and Unweighted UniFrac distance (d), with each plot depicting variations along two principal coordinates that account for most of the variation. Fig. S16. Influence of BBR on the Proportions of Firmicutes and Bacteroidetes in the Gut Microbiota of Mdr2-/- mice. The pie chart shows the relative percentages of the Firmicutes and Bacteroidetes phyla in the gut microbiota of Mdr2-/- mice. Comparative analysis is shown across three groups: control, BBR-treated at 50 mg/kg, and BBR-treated at 100 mg/kg. This visualization highlights the specific shifts in these major bacterial phyla due to BBR treatment