Fig 1: Gut inflammation and microbial dysbiosis precedes NASH-fibrosis in Foz/Foz mice. (A–C) Plasma from systemic blood as well as portal vein blood was isolated and subjected to enzyme-linked immunosorbent assay. Levels of (A) LPS and (B) LBP in systemic circulation and (C) levels of LPS in portal circulation. Total RNA was isolated from DSI and colon and was subjected to qRT-PCR for (D) Cldn2, (E) Occludin, and (F) Zo1 (relative to Tbp). (G–J) DNA was extracted from cecum contents and the microbiome diversity was analyzed by 16s rRNA sequencing. (G) RPCA β-diversity ordination of the indicated samples after WD feeding, showing distinct separation of the WD and NC-fed group. Arrows represent microbes of the highest magnitude contributing to sample differences. (H) RPCA β-diversity ordination of Foz+WD and WT+WD samples at week 0, weeks 1–2, and week 12; n = sample number in each group; q= PERMANOVA (Permuted Analysis Of Variance) statistics. (I) Log ratios of the top and bottom 5% of microbes by Foz+WD differential rank. (J) Log ratios of Firmicutes to Bacteroidetes annotated microbes. Bar graph data are plotted as means ± SEM while (I and J) box-and-whisker data are plotted with median (middle line), 25th–75th percentiles (box), and minimum–maximum percentiles (whiskers); (A and B) unpaired t test, (C, D–F) ordinary 1-way analysis of variance, (I and J) Mann–Whitney test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001.
Fig 2: Pre-symptomatic Tg mice display impairments of IEB integrity and permeability.a Representative blots and (b) densitometric analysis of ZO-1 and Occ expression assessed by Western blot assay in colonic tissues from nTg and Tg mice at 3, 6, and 9 months of age. In the quantitative analysis protein levels were normalized using Ponceau staining and expressed as percentage of the mean of nTg age-matched controls. All values represent the means ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, significant difference. One-way ANOVA followed by Tukey post hoc test. c Circulating LBP in nTg and Tg mice at 3, 6, and 9 months of age. All values represent the means ± SEM. ***P < 0.001, significant difference versus nTg age-matched controls), aaaP < 0.001 and aaP < 0.01. One-way ANOVA followed by Tukey post hoc test. n = 3–5/group. α-S α-synuclein, LBP lipopolysaccharide-binding protein, nTg non-transgenic, Tg transgenic, Occ occludin, ZO-1 zonulin-1.
Fig 3: Dietary switch to normal chow attenuates fibrosis progression.Foz/Foz mice that continued on WD or switched to normal chow after 12 weeks of WD (regression) were analyzed. (A and B) Luminex analyses of liver lysates for the indicated cytokines and chemokines. (C) Plasma LBP and LPS levels in the indicated groups. Total RNA isolated from (D) DSI and (E) colon region of the intestine was subjected to qRT-PCR for Cldn2, Ocln, and Zo1. Data are expressed as the means ± SEM; 1-way analysis of variance. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. (F) Pairwise PERMANOVA (Permuted Analysis of Variance) of RPCA β-diversity plot by diet-genotype combination. Left: -log10 q-value; right: pseudo–F-statistic. (G) RPCA β-diversity ordination of all samples after 0 weeks. Samples are colored by diet–genotype combination. Arrows represent microbes of the highest magnitude contributing to sample differences. (H) 16s rRNA sequencing of cecal contents was analyzed for the log ratios of Firmicutes to Bacteroidetes annotated microbes in different groups as indicated. Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and minimum–maximum percentiles (whiskers). Mann–Whitney test. KC, Keratinocyte chemoattractant; Reg, Regression.
Supplier Page from Abcam for Mouse LBP ELISA Kit