Journal: Gut Microbes

Article Title: Gut microbes mediate the synergistic effects of dietary cholesterol and saturated fat in driving fibrosing MASH

doi: 10.1080/19490976.2026.2668121

Figure Lengend Snippet: Time- and diet-dependent remodeling of fecal bile acid (BA) composition by cholesterol and saturated fat in SPF mice. (A–D) Hepatic expression of genes involved in bile acid (BA) biosynthesis and sensing shown as fold-change relative to LF-fed SPF mice at 8 weeks, normalized to glyceraldehyde 3-phosphate dehydrogenase ( Gapdh ) and determined via the 2 –∆∆Ct method. Cytochrome P450 family 7 subfamily A member 1 ( Cyp7a1 ) (A); Cytochrome P450 family 27 subfamily A member 1 ( Cyp27a1 ) (B); Farnesoid x receptor ( Fxr ) (C); and Takeda G protein-coupled receptor 5 ( Tgr5 ) (D). (E) Fecal BA concentrations in SPF mice. The data represent the means ± SEMs and were analyzed via three-way ANOVA (factors: cholesterol, fat, and time), followed by Tukey's multiple comparisons. Bars with the same letter are not significantly different ( p > 0.05). (F and G) Principal component analysis (PCA) plots of fecal BA from SPF mice at 8 weeks (F) and 24 weeks (G). The dots represent individual mice, and the open circles represent centroids with lines connecting individual dots, which were analyzed via two-way ADONIS (factors: cholesterol and fat). (H, I) PC loadings of individual BA after 8 weeks (H) and 24 weeks (I). (J–O) Quantification of BA in the feces of SPF mice at 8 and 24 weeks significantly impacted by a main effect of dietary cholesterol (J), the main effects of dietary cholesterol and saturated fat (K), the interaction between dietary cholesterol and saturated fat (L), the interaction between dietary cholesterol and time (M), the main effect of time (N), or not impacted by any factor (O). Data represent the means ± SEMs and were analyzed via three-way ANOVA (factors: cholesterol, fat, and time), followed by Tukey's multiple comparisons. Bars with the same letter are not significantly different ( p > 0.05). n = 4–6/group for panels A–E (see Table S4 for outliers). n = 4/group for panels F–O (subset of mice selected for BA analyses). Low-fat (LF); low-fat + very high-cholesterol (LFVHC); high-fat (HF); high-fat + very high-cholesterol (HFVHC); cholic acid (CA); Muricholic acid (MCA); glycocholic acid (GCA); taurocholic acid (TCA); taurochenodeoxycholic acid (TDCA); ursocholic acid (UCA); deoxycholic acid (DCA); lithocholic acid (LCA); glycochenodeoxycholic acid (GCDCA); chenodeoxycholic acid (CDCA); ursodeoxycholic acid (UDCA).

Article Snippet: In separate experiments, deoxycholic acid (Thermo Fisher Scientific, Waltham, MA) was added to the growth media at the indicated concentrations.

Techniques: Expressing

Journal: Gut Microbes

Article Title: Gut microbes mediate the synergistic effects of dietary cholesterol and saturated fat in driving fibrosing MASH

doi: 10.1080/19490976.2026.2668121

Figure Lengend Snippet: Microbiota-dependent gut factors from HFVHC-fed SPF mice induce pro-fibrotic and pro-inflammatory activation of hepatic stellate cells (HSC) in vitro . (A) Experimental schematic created on Biorender.com; cecal contents were pooled from 3 randomly selected mice/group after 24 weeks on the diet, homogenized, centrifuged, and sterile-filtered, followed by bile acid quantification. LX-2 HSC cells were exposed to homogenates at 10% media (v/v) for 4 hours, followed by qRT-PCR. (B and C) Gene expression of fibrosis-related (B) and inflammation-related (C) HSC activation markers. (D) Concentration of deoxycholic acid (DCA) in cecal homogenates. (E) LX-2 HSC cells were treated with the indicated concentrations of DCA or vehicle for 4 hours, followed by qRT-PCR. Alpha-smooth muscle actin ( ACTA2 ). The data represent the means ± SEM. (B–D) Data were analyzed via three-way ANOVA (factors: time, fat, and microbe) followed by Tukey's multiple comparisons within a timepoint. (E) Data were analyzed via one-way ANOVA followed by Tukey's multiple comparisons. Bars with the same letter are not significantly different ( p > 0.05). The gene expression values (C, E) were normalized to those of GAPDH , determined via the 2 –∆∆Ct method and expressed relative to those of the SPF-LF (B, C) or vehicle (E) groups. n = 3‒5/group, representative of 3 independent experiments. Low-fat (LF); low-fat + very high-cholesterol (LFVHC); high-fat (HF); high-fat + very high-cholesterol (HFVHC); collagen type I alpha I chain ( COL1A1 ), monocyte chemoattractant protein-1 ( MCP1 ), interleukin-6 ( IL-6 ), transforming growth factor-beta receptor type 2 ( TGFβR2 ), tumor necrosis factor-alpha ( TNFα ), interleukin 1-beta ( IL-1β ).

Article Snippet: In separate experiments, deoxycholic acid (Thermo Fisher Scientific, Waltham, MA) was added to the growth media at the indicated concentrations.

Techniques: Activation Assay, In Vitro, Sterility, Quantitative RT-PCR, Gene Expression, Concentration Assay