Hepatocyte-Conditional Knockout of Phosphatidylethanolamine Binding Protein 4 Aggravated LPS/D-GalN-Induced Acute Liver Injury via the TLR4/NF-κB Pathway

Abstract

Acute liver injury (ALI) is a disease that seriously threatens human health and life, and a dysregulated inflammation response is one of the main mechanisms of ALI induced by various factors. Phosphatidylethanolamine binding protein 4 (PEBP4) is a secreted protein with multiple biological functions. At present, studies on PEBP4 exist mainly in the field of tumors and rarely in inflammation. This study aimed to explore the potential roles and mechanisms of PEBP4 on lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced ALI. PEBP4 was downregulated after treatment with LPS/D-GalN in wild-type mice. PEBP4 hepatocyte-conditional knockout (CKO) aggravated liver damage and repressed liver functions, including hepatocellular edema, red blood cell infiltration, and increased aspartate aminotransferase (AST)/alanine aminotrans-ferase (ALT) activities. The inflammatory response was promoted through increased neutrophil infiltration, myeloperoxidase (MPO) activities, and cytokine secretions (interleukin-1β, IL-1β; tumor necrosis factor alpha, TNF-α; and cyclooxygenase-2, COX-2) in PEBP4 CKO mice. PEBP4 CKO also induced an apoptotic effect, including increasing the degree of apoptotic hepatocytes, the expressions and activities of caspases, and pro-apoptotic factor Bax while decreasing anti-apoptotic factor Bcl-2. Furthermore, the data demonstrated the levels of Toll-like receptor 4 (TLR4), phosphorylation-inhibitor of nuclear factor kappaB Alpha (p-IκB-α), and nuclear factor kappaB (NF-κB) p65 were upregulated, while the expressions of cytoplasmic IκB-α and NF-κB p65 were downregulated after PEBP4 CKO. More importantly, both the NF-κB inhibitor (Ammonium pyrrolidinedithiocarbamate, PDTC) and a small-molecule inhibitor of TLR4 (TAK-242) could inhibit TLR4/NF-κB signaling activation and reverse the effects of PEBP4 CKO. In summary, the data suggested that hepatocyte-conditional knockout of PEBP4 aggravated LPS/D-GalN-induced ALI, and the effect is partly mediated by activation of the TLR4/NF-κB signaling pathway.

Keywords: NF-κB; PEBP4; TLR4; acute liver injury; apoptosis; inflammation.

Figure 1

PEBP4 hepatocyte-conditional knockout (CKO) mice were constructed and identified. (A): Western blotting was used to detect the expression of PEBP4. (B): The undisturbed PEBP4 gene in WT mice (top panel), schematic of loxP sites introduced into exon 3 of the PEBP4 locus for the generation of PEBP4^flox/flox mice (middle panel), and schematic for the generation of PEBP4CKO mice (bottom panel). (C): The LoxP sites flanking the PEBP4 gene, the Cre transgene and PEBP4 allele were detected by PCR analysis of tail DNA and liver DNA. (D): Agarose gel electrophoresis was employed to examine the gene expression of PEBP4 in the heart, liver, lungs, spleen, and kidneys of WT and CKO mice. (E): PEBP4 protein expression was tested by western blotting in the heart, liver, lungs, spleen, and kidneys of WT and CKO mice. Data are presented as mean ± SD values (n = 3). ^** P < 0.01 compared to WT mice in the control group.

Figure 2

PEBP4 CKO could aggravate LPS/D-GalN–induced ALI. (A): Histological changes of liver tissue (H&E staining, ×400) and the injury scores of HE were quantifies, ↑ for inflammatory cell infiltration, □ for hepatocyte degeneration and red blood cell infiltration. (B): We detected the activities of AST and ALT in serum (n = 6). (C): MPO activities were tested in liver tissue (n = 6). (D, E): The levels of IL-1β and TNF-α in serum were detected by ELISA (n = 6); (F): Western blot was used to examine the expression of COX-2 (n = 3). (G): The apoptotic level of hepatocytes was detected with a TUNEL fluorescence detection kit (the DAPI-stained nuclei were blue and apoptotic nuclei were green, ×400) and positive cells of TUNEL were quantified, ↑ for TUNEL (+) cells. ( H, I). The activities and protein expression of caspase-3, -8, and -9 were detected by the activity test kit (n = 6) and western blotting (n = 3), respectively; (J). The mRNA expression levels of Bax and Bcl-2 were detected by real-time PCR. Data are presented as mean ± SD values. ^NS P > 0.05, ^** P < 0.01, and ^*** P < 0.001 compared to the WT control group; ^### P < 0.001 compared to the CKO control group; and ^Δ P < 0.05 and ^ΔΔ P < 0.01 compared to the WT LPS/D-GalN group.

Figure 3

PEBP4 CKO could activate the TLR4/NF-κB signaling pathway. Western blotting was employed to examine the expressions of TLR4, IκB-α, p-IκB-α, nuclear protein NF-κB p65, and cytoplasmic protein NF-κB p65. Data are presented as mean ± SD values (n = 3). ^NS P > 0.05, ^** P < 0.01, and ^*** P < 0.001 compared to the WT control group; ^### P < 0.001 compared to the CKO control group; and ^Δ P < 0.05 and ^ΔΔ P < 0.01 compared to the WT LPS/D-GalN group.

Figure 4

PDTC and TAK-242 could partially counteract TLR4/NF-κB signaling activation induced by PEBP4 CKO in ALI. (A): Histological changes of liver tissue (H&E staining, ×400) and the injury scores of HE was quantifies, ↑ for inflammatory cell infiltration, □ for hepatocyte degeneration and red blood cell infiltration. (B): Serum ALT/AST activities (n = 6). (C): Liver MPO activities (n = 6). (D, E): The contents of serum TNF-α and IL-1β (n = 6). (F): TUNEL staining results of liver tissue (×400) and positive cells of TUNEL were quantified, ↑ for TUNEL (+) cells. (G): The expressions of cleaved caspase-3/pro–caspase-3 (n = 3); (H): The activities of caspase-3, -8, and -9 in liver tissue (n = 6). (I): The expression levels of TLR4, IκB-α, p-IκB-α, NF-κB p65, and nuclear protein NF-κB p65 were detected by western blotting, and the gray levels were analyzed (n = 3). Data are presented as mean ± SD values. ^Δ P < 0.05, ^ΔΔ P < 0.01, and ^ΔΔΔ P < 0.001 compared to the WT LPS/D-GalN group, ⁺ P < 0.05, ⁺⁺ P < 0.01, and ⁺⁺⁺ P < 0.001 compared to CKO LPS/D-GalN group. NS, no significance.

Figure 5

Schematic of PEBP4-driven TLR4/NF-κB signaling pathway-associated inflammatory and apoptotic talk in LPS/D-GalN-induced ALI. PEBP4 CKO could activate the TLR4/NF-κB signaling pathway, promote NF-κB translocation into the nucleus, and then regulate the transcription of inflammatory factors and apoptotic genes to mediate acute liver injury (ALI) from both inflammation and apoptosis. TLR4 inhibitor (TAK-242) and NF-κB inhibitor (PDTC) could partially reverse these effects.