Fig 1: Altered hepatic bile acid profiles and bile acid biosynthesis by theabrownin intervention in HFD-induced obesity mice. (A) Altered hepatic BA profiles induced by oral administration of theabrownin for eight weeks in HFD-induced obesity mice (n = 8 per group). (B) Altered hepatic percentage of non-12OH BAs and 12-OH BAs in HFD-induced obesity mice after theabrownin intervention for eight weeks. (C) The expressions of CYP7A1 (n = 3 per group) and CYP7B1 (n = 3 per group) in the liver were detected by western blot. (D) Schematic diagram of the classical and alternative bile acid biosynthesis pathways. Data are presented as mean ± SEM. Differences between data were assessed using the Mann-Whitney U test. Statistical significance of BA data was corrected by the Benjamini-Hochberg method, # p < 0.01, * p < 0.05.
Fig 2: Single Intravenous Administration of HsCYP7B1 or MmCyp7b1 mRNA(A) Cyp7b1−/− mice received a single i.v. injection of 40 μg mRNA LNP (either non-translating mRNA [vehicle, black], MmCyp7b1 [dark green], or HsCYP7B1 mRNA [light green]). 48 h after administration, mice were sacrificed and tissues were processed for further analyses (n = 4 mice per group). (B–D) Oxysterol analysis of (B) liver, (C) serum, and (D) brain samples (square, male; circle, female; green section, wild-type values; ns, not significant; one-way ANOVA, *p < 0.05, **p < 0.01, ****p < 0.0001, error bars show SD).
Fig 3: UDCA treatment attenuated obesity induced by high fat diet. (a) UDCA improved the metabolic profile: changes of body weight and serum paraments. Data are presented as the mean ± SD. n = 5 per group. *p<0•05 and **p<0•01 (unpaired Student's t-test), HFD group vs N group; #p <0•05 and ##p<0•01 (unpaired Student's t-test), HFD+UDCA group vs HFD group. (b) Heatmap of serum bile acids profile of normal diet group (N), high fat diet group (H) and high fat diet + UDCA group (HU). The gradient colours in the heatmap depicted the z-scale value of serum bile acids concentration. (c) The mean percentage of 12-OH bile acids and non-12 bile acids in serum from all the samples of the three group (n = 5 per group). (d) The principal component analysis (PCA) analysis of serum bile acids. n = 5 per group. N: normal diet group; H: high fat diet group; HU: high fat diet group+0•5%UDCA group. (e) Relative mRNA levels of Cyp7a1, Cyp8b1, Cyp27a1 and Cyp7b1 in liver detected by q-PCR assay. All data are presented as the mean ± SD. n = 4 per group. *p<0•05 (unpaired Student's t-test). (f) The expression of CYP8B1 and CYP7B1 in liver of three group mice were detected by western blot. Data are presented as the mean ± SD. n = 3 per group. *p<0•05 (unpaired Student's t-test).
Fig 4: Establishing an In Vivo System for mRNA Administration(A) Wild-type BALB/c mice were dosed with a single i.v. injection of 20 μg of LNP encapsulated with mRNA encoding the reporter PpLuc or PBS as a control. Luciferase expression in various tissues was quantified via bioluminescence 6 h post-injection (n = 4 mice per group). (B–D) Mass spectrometric analysis of oxysterols (25-HC, 27-HC, 3β-HCA) in (B) liver, (C) serum, and (D) brain of Cyp7b1−/− mice (KO, red) and Cyp7b1+/+ mice (WT, gray) (n = 3 mice per group; unpaired one-tailed t test, **p < 0.01, ***p < 0.001, ****p < 0.0001, error bars show SD).
Fig 5: Repeat Intravenous Injection of HsCYP7B1 mRNA(A) Cyp7b1−/− mice received repeat i.v. injections of 40 μg mRNA LNP (either non-translating mRNA [vehicle, black] or HsCYP7B1 mRNA [treated, green]) with an interval of 5 days for a total of four injections. Mice were sacrificed 2 days after the last injection (in total, 17 days) (n = 6 per group). (B–D) Oxysterol analysis (25-HC, 27-HC, 3β-HCA) in (B) liver, (C) serum, and (D) brain samples (square, male; circle, female; green section; range of wild-type values, unpaired one-tailed t test; ns, not significant; **p < 0.01, ***p < 0.001, ****p < 0.0001, error bars show SD). (E) Western blot of CYP7B1 expression in liver of treated animals (B.1–B.6) 2 days after the last injection of HsCYP7B1 mRNA (F) ALT and AST concentrations in serum samples of treated (green) and vehicle (black) animals 2 days after the last injection compared to values in untreated wild-type (WT, gray) and knockout (KO, red) mice (gray section, range of KO values).
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