Exogenous Administration of Low-Dose Lipopolysaccharide Potentiates Liver Fibrosis in a Choline-Deficient l-Amino-Acid-Defined Diet-Induced Murine Steatohepatitis Model - PubMed (original) (raw)
. 2019 Jun 3;20(11):2724.
doi: 10.3390/ijms20112724.
Kosuke Kaji 2, Mitsuteru Kitade 3, Takuya Kubo 4, Masanori Furukawa 5, Soichiro Saikawa 6, Naotaka Shimozato 7, Shinya Sato 8, Kenichiro Seki 9, Hideto Kawaratani 10, Kei Moriya 11, Tadashi Namisaki 12, Hitoshi Yoshiji 13
Affiliations
- PMID: 31163617
- PMCID: PMC6600174
- DOI: 10.3390/ijms20112724
Exogenous Administration of Low-Dose Lipopolysaccharide Potentiates Liver Fibrosis in a Choline-Deficient l-Amino-Acid-Defined Diet-Induced Murine Steatohepatitis Model
Keisuke Nakanishi et al. Int J Mol Sci. 2019.
Abstract
Various rodent models have been proposed for basic research; however, the pathogenesis of human nonalcoholic steatohepatitis (NASH) is difficult to closely mimic. Lipopolysaccharide (LPS) has been reported to play a pivotal role in fibrosis development during NASH progression via activation of toll-like receptor 4 (TLR4) signaling. This study aimed to clarify the impact of low-dose LPS challenge on NASH pathological progression and to establish a novel murine NASH model. C57BL/6J mice were fed a choline-deficient l-amino-acid-defined (CDAA) diet to induce NASH, and low-dose LPS (0.5 mg/kg) was intraperitoneally injected thrice a week. CDAA-fed mice showed hepatic CD14 overexpression, and low-dose LPS challenge enhanced TLR4/NF-κB signaling activation in the liver of CDAA-fed mice. LPS challenge potentiated CDAA-diet-mediated insulin resistance, hepatic steatosis with upregulated lipogenic genes, and F4/80-positive macrophage infiltration with increased proinflammatory cytokines. It is noteworthy that LPS administration extensively boosted pericellular fibrosis with the activation of hepatic stellate cells in CDAA-fed mice. Exogenous LPS administration exacerbated pericellular fibrosis in CDAA-mediated steatohepatitis in mice. These findings suggest a key role for LPS/TLR4 signaling in NASH progression, and the authors therefore propose this as a suitable model to mimic human NASH.
Keywords: NF-κB; choline-deficient l-amino-acid-defined diet; fibrosis; lipopolysaccharide; nonalcoholic steatohepatitis; toll-like receptor.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Figure 1
Activation of hepatic TLR 4/NF-κB signaling pathway by CDAA diet feeding and LPS administration. (A) Schematic of LPS administration to CDAA diet-induced steatohepatitis model. (B,C) Relative mRNA expression levels of Lbp (B) and Cd14 (C) in the liver of experimental mice. (D) Western blots for TLR4 expression and NF-κB phosphorylation in the liver of experimental mice. (E) Quantification of protein expression of TLR4. (F) Quantitative phosphorylation rate of phosphorylated NF-κB/NF-κB. The mRNA expression levels were measured by quantitative RT–PCR (qRT–PCR), and Gapdh was used as internal control for qRT–PCR (B,C). The protein was determined by western blotting, and β-Actin was used as the loading control (D). Quantitative values are indicated as ratios to the values of CSAA-LPS (−) group (B,C,E,F). Data are mean ± SD (n = 6). a: p < 0.05 compared with CSAA-LPS (−), b: p < 0.05 compared with CSAA-LPS (+), c: p < 0.05 compared with CDAA-LPS (−).
Figure 2
Altered characteristics and hepatic steatosis by CDAA diet feeding and LPS administration. (A) Body weight (Bw) in the experimental groups at sacrifice. (B) Ratio of liver weight (Lw) to body weight in the experimental groups at sacrifice. (C) Representative macroscopic appearances and microphotographs of hematoxylin-eosin (H&E) staining in the experimental groups. Scale Bar: 50 μm. (D) Serum levels of alanine aminotransferase (ALT), triglyceride (TG) and total cholesterol (T-Cho) in the experimental groups. Data are mean ± SD (n = 6). * p < 0.05, indicating a significant difference between groups.
Figure 3
Changes in glycemic status and lipid metabolism by CDAA diet feeding and LPS administration. (A) Plasma glucose levels after 0, 15, 30, 60, 120 min for oral glucose tolerance test (OGTT) at the end of experiment. (B) The values of Glucose-AUC (area under the blood concentration-time curve) in the experimental group. The value of AUC was calculated as the area under the curve of plasma glucose level at OGTT. (C) Plasma insulin levels in the experimental groups at sacrifice (120 min. after oral glucose intake). (D) The multiplicated values of plasma glucose and insulin level in the experimental groups at sacrifice (120 min. after oral glucose intake). (E–G) Relative mRNA expression levels of Srebf1, Fas, Acc1 (E), Ppara (F) and Pparg (G) in the liver of experimental mice. The mRNA expression levels were measured by quantitative RT–PCR (qRT–PCR), and Gapdh was used as internal control for qRT–PCR (E–G). Quantitative values are indicated as ratios to the values of CSAA-LPS (−) group (E–G). Data are mean ± SD (n = 6). a: p < 0.05 compared with CSAA-LPS (−), b: p < 0.05 compared with CSAA-LPS (+), c: p < 0.05 compared with CDAA-LPS (−).
Figure 4
Impact of CDAA diet feeding and LPS administration on macrophage infiltration and inflammatory response. (A) Representative microphotographs of liver sections stained with F4/80. Scale bar: 50 μm. (B) Semi-quantitation of F4/80 immuno-positive macrophages in high-power field (HPF) by NIH imageJ software. (C) Relative mRNA expression levels of proinflammatory cytokines, Tnfa, Il1b and Il6 in the liver of experimental mice. The mRNA expression levels were measured by quantitative RT–PCR (qRT–PCR), and Gapdh was used as internal control for qRT–PCR (C). Quantitative values are indicated as ratios to the values of CSAA-LPS (−) group (B,C). Data are mean ± SD (n = 6). a: p < 0.05 compared with CSAA-LPS (−), b: p < 0.05 compared with CSAA-LPS (+), c: p < 0.05 compared with CDAA-LPS (−).
Figure 5
Impact of CDAA diet feeding and LPS administration on liver fibrosis development. (A) Representative microphotographs of liver sections stained with Sirius Red (upper panels) and α-SMA (lower panels). Scale Bar: 50 μm. (B,C) Semi-quantitation of Sirius Red-stained fibrotic area (B) and α-SMA immune-positive area (C) in high-power field (HPF) by NIH imageJ software. (D) Relative mRNA expression levels of fibrosis markers, Acta2, Col1a1 and Tgfb1 in the liver of experimental mice. The mRNA expression levels were measured by quantitative RT–PCR (qRT–PCR), and Gapdh was used as internal control for qRT–PCR (D). Quantitative values are indicated as ratios to the values of CSAA-LPS (−) group (C,D). Data are mean ± SD (n = 6). a: p < 0.05 compared with CSAA-LPS (−), b: p < 0.05 compared with CSAA-LPS (+), c: p < 0.05 compared with CDAA-LPS (−).
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