Apolipoprotein A5 alleviates LPS/D-GalN-induced fulminant liver failure in mice by inhibiting TLR4-mediated NF-κB pathway

Abstract

Background: Fulminant liver failure (FHF) is a serious clinical problem and liver transplantation is the major intervention. But the overall survival rate of FHF is low owing to the donated organ shortage. Apolipoprotein A-V (ApoA5) is a regulator of triglyceride metabolism and has been reported to act as a predictor for remnant liver growth after preoperative portal vein embolization and liver surgery. This study aimed to investigate the therapeutic effect of ApoA5 on lipopolysaccharide/D-galactosamine (LPS/D-GalN)-induced fulminant liver failure in mice.

Methods: FHF mouse model was established using LPS/D-GalN and ApoA5 plasmid was injected by tail vein prior to LPS/D-GalN treatment. The expressions of ApoA5, toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B p65 (NF-κBp65) were assessed by real-time PCR and western blotting. Serum alanine aminotransferase (ALT) and tumor necrosis factor-α (TNF-α) levels were measured using automatic biochemical analyzer. Histological assessment and immunohistochemical (IHC) staining were conducted. Survival rate after LPS/D-GalN administration was also determined with Kaplan-Meier curve. Meanwhile, the expression of ApoA5 in injured huh7 cells was tested. Cell apoptosis analysis was performed after huh7 cells were transfected with ApoA5 plasmid and stimulated with LPS.

Results: The expressions of ApoA5 decreased both in injured huh7 cells and FHF mice. ApoA5 overexpression reduced cell death rate using flow cytometry. ApoA5 not only decreased the serum ALT and TNF-α levels but also attenuated hepatic damage in hematoxylin-eosin (HE)-stained liver section. The protein expressions of TLR4, MyD88 and NF-κBp65 were inhibited when ApoA5 overexpressed. But the inhibitory effect would weaken with the increasing concentration of LPS in spite of ApoA5 overexpression. Besides, ApoA5 improved liver injury in a dose-dependent manner and the survival rate in FHF mice increased with increasing concentration of ApoA5.

Conclusion: ApoA5 had a protective effect against LPS/D-GalN-induced fulminant liver failure in mice within a certain range by inhibiting TLR4-mediated NF-κB pathway.

Keywords: Apolipoprotein A-V; Endotoxemia; Fulminant liver failure; Lipopolysaccharide; TLR4.

Fig. 1

Dynamic change of ApoA5 protein levels in huh7 cells treated with different concentration of LPS (0 μg/mL, 5 μg/mL, 10 μg/mL) (a), and in the mice with LPS/d-GalN administration for different period of time (0 h, 4 h and 8 h) (b)

Fig. 2

ApoA5 overexpression attenuated hepatic damage. A The protein expression level of ApoA5 in huh7 cells tranfected with pEGF-N1 ApoA5; B cell apoptosis analysis using Flow cytometry; C histology of liver sections stained with HE; D serum levels of ALT and TNF-α in the NS injected (group A), pEGF-N1 vector injected (group B) and pEGF-N1-ApoA injected (group C); E survival rate within 12 h after LPS/d-GalN administration in three groups

Fig. 3

ApoA5 regulated key molecules expressions involved in TLR4 signaling pathway. a The protein expression levels of ApoA5 and TLR4 in huh7 cells transfected with pEGF-N1-ApoA5; b relative mRNA levels of molecules involved in TLR4 signaling pathway (ApoA5, TLR4 and MyD88); c effect of ApoA5 on the protein levels of ApoA5, TLR4 and MyD88 and NF-κBp65 after LPS/d-GalN administration by Western blot analysis; d assessment of immunohistochemistry for key molecules (ApoA5, TLR4 and MyD88) in liver tissue

Fig. 4

Both ApoA5 and LPS affect the severity of liver injury in a dose-dependent manner. a Liver histological damage was alleviated in a ApoA5-dose-dependent manner; b survival rate analysis in the mice pretreated with increasing concentrations of ApoA5; c the protein levels of ApoA5, TLR4 and MyD88 and NF-κB p65 in the mice confronted with d-GalN and increasing concentrations of LPS; d survival rate analysis in mice after injection of d-GalN and different concentrations of LPS