IκBζ regulates the development of nonalcoholic fatty liver disease through the attenuation of hepatic steatosis in mice

Figures

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  Figure 1. Alb-Cre; Nfkbizfl/fl mice accelerate the development of NAFLD. (A) C57BL/6 mice were fed an NCD or CDAHFD for 4 weeks. mRNA induction of Nfkbiz in the liver was assessed using real-time RT-PCR and was expressed as the fold increase in the Nfkbiz/Hprt1 ratio. Values are expressed as the mean ± SD (n = 3). (B) mRNA expressions of NFKBIZ in the human normal liver, moderate steatosis, and steatohepatitis with bridging fibrosis were assessed using real-time RT-PCR. Values are expressed as the mean ± SD (triplicate experiment from one sample). (C) Nfkbizfl/fl and Alb-Cre; Nfkbizfl/fl mice were fed a CDAHFD or NCD for 8 weeks. mRNA induction of Nfkbiz in the liver was assessed using real-time RT-PCR. Values are expressed as the mean ± SD (n = 3–12 in each point). (D) Representative macroimages of the liver obtained from Nfkbizfl/fl and Alb-Cre; Nfkbizfl/fl mice fed an NCD or CDAHFD for 8 weeks. (E) Representative image of hematoxylin–eosin-stained liver sections obtained from Nfkbizfl/fl and Alb-Cre; Nfkbizfl/fl mice fed an NCD or CDAHFD for 8 weeks. Bar = 100 µm. (F) NAFLD activity score (NAS) was performed by a blinded researcher. The scores of hepatocellular steatosis (Steatosis),…

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  Figure 2. Deficiency of IκBζ in hepatocytes increases liver apoptotic cells in mouse model of NAFLD. Nfkbizfl/fl and Alb-Cre; Nfkbizfl/fl mice were fed an NCD or CDAHFD. (A) TUNEL staining of the liver section was observed using a confocal microscope (Leica TCS SP8; Leica Microsystems). Representative image of TUNEL staining of liver sections from mice fed an NCD or CDAHFD for 8 weeks. Green, apoptotic cells; Blue, DAPI. Bar = 50 µm. (B) The mean number of apoptotic cells in one section at high magnification from mice fed CDAHFD for 2, 4, or 8 weeks or NCD (white bar, Nfkbizfl/fl; black bar, Alb-Cre; Nfkbizfl/fl). Values are expressed as the mean ± SD (four independent fields per mouse, three mice per experiment). (C, D, E, F) mRNA expression of Bax (C), Bak (D), Bcl-xl (E), and Bcl-2 (F) in the liver was assessed using real-time RT-PCR and was expressed as the fold increase in the Nfkbiz/Hprt1 ratio (white bar, Nfkbizfl/fl; gray bar, Alb-Cre; Nfkbizfl/fl). Values are expressed as the mean ± SD (n = 3–12). CDAHFD, choline-deficient, L-amino acid-defined, high-fat diet; NCD, normal chow diet.*P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 3. The deficiency of IκBζ in hepatocytes fails to modify mRNA expression of the proteins related to immune responses in mice model of NAFLD. Nfkbizfl/fl and Alb-Cre; Nfkbizfl/fl mice were fed an NCD or CDAHFD for 8 weeks. Liver RNA was isolated from mice at 2 and 8 weeks after the CDAHFD challenge or at 8 weeks after NCD. mRNA expressions of Ikbkb (A), Nfkb1 (B), Nfkb2 (C), Ccl2 (D), Ccl3 (E), Il18 (F), Il1b (G), Il6 (H), and Tnf (I) were analyzed by TaqMan Array Mouse Immune Panel, according to the manufacturer’s instructions (white bar, Nfkbizfl/fl; gray bar, Alb-Cre; Nfkbizfl/fl). Values are expressed as the mean ± SD (n = 3–4). *P < 0.05. CDAHFD, choline-deficient, L-amino acid-defined, high-fat diet; NCD, normal chow diet.

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  Figure 4. Fibrotic changes in the liver in the mice model of NAFLD. Nfkbizfl/fl and Alb-Cre; Nfkbizfl/fl mice were fed an NCD or CDAHFD for 8 weeks. (A) Representative image of Sirius Red staining of the liver obtained from Nfkbizfl/fl and Alb-Cre; Nfkbizfl/fl mice fed an NCD or CDAHFD for 8 weeks. Bar = 100 µm. (B) Sirius Red-positive area quantified using BZ-X 700 imaging software (Keyence). The fibrosis area was expressed as the percentage of the Sirius Red-positive area. Values are expressed as the mean ± SD (four independent fields per mouse, three mice per experiment). (C–G) mRNA expressions of Tgfb1 (C), Acta1 (D), Pdgfb (E), Timp1 (F), and Col1a1 (G) were analyzed by real-time RT-PCR and expressed as the fold increase in the Nfkbiz/Hprt1 ratio (white bar, Nfkbizfl/fl; gray bar, Alb-Cre; Nfkbizfl/fl). Values are expressed as the mean ± SD (n = 3–12). *P < 0.05. CDAHFD, choline-deficient, L-amino acid-defined, high-fat diet; NCD, normal chow diet.

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  Figure 5. Deficiency of IκBζ in hepatocytes increases liver steatosis cells in the mouse model of NAFLD. Nfkbizfl/fl and Alb-Cre; Nfkbizfl/fl mice were fed an NCD or CDAHFD for 2 weeks. (A) Representative image of Oil Red O staining of liver sections from mice fed an NCD or CDAHFD for 2 weeks. Bar = 100 µm. (B) Oil Red O staining area of liver specimens quantified using BZ-X 700 imaging software (Keyence). The Oil red O staining area was expressed as the percentage of the Oil Red O area. Values are expressed as the mean ± SD (n = 4). (C, D) Plasma lipoprotein fractions were analyzed by high-performance liquid chromatography. (C) Representative image of the chromatogram (violet line, cholesterol; blue line, triacylglycerol). CM, chylomicron; VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein. (D) VLDL-cholesterol and VLDL-triacylglycerol in plasma were quantified (white bar, Nfkbizfl/fl; gray bar, Alb-Cre; Nfkbizfl/fl). Values are expressed as the mean ± SD (n = 3–5). *P < 0.05, **P < 0.01. CDAHFD, choline-deficient, L-amino acid-defined, high-fat diet; NCD, normal chow diet.

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  Figure 6. Overexpression of Nfkbiz in liver hepatocytes by AAV8 vector ameliorates hepatic steatosis in vivo. (A) Schematic representation of the AAV8 vector used in this study. Nfkbiz (D), a deletion mutant of IκBζ lacking trans-activating domain; Nfkbiz (L), full-length IκBζ; ITR, internal terminal repeat; ANK, ankyrin repeat. (B) C57BL/6 mice (7 weeks old) were intravenously treated with 3 × 1011 vg of AAV8 vector harboring Luciferase, Nfkbiz (D), or Nfkbiz (L). The expression of Nfkbiz in the liver obtained at 4 weeks after vector injection was assessed to measure the mRNA of Nfkbiz using real-time PCR and was expressed as the fold increase in the Nfkbiz/Hprt1 ratio. Values are expressed as the mean ± SD (n = 4–5). (C–E) At 4 weeks after AAV injection, the mice were then fed an NCD or CDAHFD for 4 weeks. (C) Representative morphology of the liver. (D) Representative image of hematoxylin–eosin staining of the liver section. Bar = 100 µm. (E) Steatosis area in the section quantified with BZ-X 700 imaging software (Keyence). The steatosis area was expressed as a percentage of the fat droplet area. Values are expressed as the mean ± SD (four independent fields per mouse, three mice per…

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  Figure 7. Microarray analysis in the liver of mice treated with the AAV8 vector harboring Nfkbiz. C57BL/6 mice (7 weeks old) were intravenously treated with 3 × 1011 vg of the AAV8 vector harboring Luciferase or Nfkbiz (L). The CDAHFD challenge started at 4 weeks after vector injection. Liver RNA was isolated from mice at 4 weeks after the challenge. The expressions of transcripts in the liver were analyzed by Mouse Genome 430 2.0 Array (n = 3 in each group). The gene expressions in the liver of mice treated with the AAV vector expressing Nfkbiz (L) were compared with those treated with the AAV vector expressing Luciferase. (A) Volcano plots showing the global transcriptional changes among the groups. All genes on the Mouse Genome 430 2.0 Array were plotted. The log-fold changes in Nfkbiz (L) versus Luciferase group are represented on the X-axis. Statistically upregulated and downregulated genes are expressed in red and green, respectively (> fivefold, P < 0.01). (B) The proportion of upregulated and downregulated genes. (C) Overview of triacylglycerol metabolism in the liver. Transcripts selected for the analysis are shown in red italics. (D) Transcripts related to triacylglycerol…