Deletion of TLR4 attenuates lipopolysaccharide-induced acute liver injury by inhibiting inflammation and apoptosis

Abstract

Septic acute liver injury is one of the leading causes of fatalities in patients with sepsis. Toll-like receptor 4 (TLR4) plays a vital role in response to lipopolysaccharide (LPS) challenge, but the mechanisms underlying TLR4 function in septic injury remains unclear. In this study, we investigated the role of TLR4 in LPS-induced acute liver injury (ALI) in mice with a focus on inflammation and apoptosis. Wild-type (WT) and TLR4-knockout (TLR4^-/-) mice were challenged with LPS (4 mg/kg) for 6 h. TLR4 signaling cascade markers (TLR4, MyD88, and NF-κB), inflammatory markers (TNFα, IL-1β, and IL-6), and apoptotic markers (Bax, Bcl-2, and caspase 3) were evaluated. We showed that LPS challenge markedly increased the levels of serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) and other liver pathological changes in WT mice. In addition, LPS challenge elevated the levels of liver carbonyl proteins and serum inflammatory cytokines, upregulated the expression of TLR4, MyD88, and phosphorylated NF-κB in liver tissues. Moreover, LPS challenge significantly increased hepatocyte apoptosis, caspase 3 activity, and Bax level while suppressing Bcl-2 expression in liver tissues. These pathological changes were greatly attenuated in TLR4^-/- mice. Similar pathological responses were provoked in primary hepatic Kupffer cells isolated from WT and TLR4^-/- mice following LPS (1 μg/mL, 6 h) challenge. In summary, these results demonstrate that silencing of TLR4 attenuates LPS-induced liver injury through inhibition of inflammation and apoptosis via TLR4/MyD88/NF-κB signaling pathway. TLR4 deletion confers hepatoprotection against ALI induced by LPS, possibly by repressing macrophage inflammation and apoptosis.

Keywords: LPS; TLR4; TLR4−/− mice; acute liver injury; apoptosis; inflammation; primary hepatic Kupffer cells; sepsis.

Fig. 1. Effect of TLR4 deficiency on LPS-induced hepatic pathological and biochemical parameters.

Mice were challenged with saline or LPS (4 mg/kg, 6 h). a Liver samples were stained with H&E for histological assessment, and representative images from each group are shown (original magnification, ×200). b Histopathological scores. c Levels of oxidative carbonyl proteins in liver tissues in the groups. Serum levels of ALT (d) and AST (e) were measured at 6 h after intraperitoneal injection of LPS. Mean±SEM, n = 6–8 mice in each group; ^*P < 0.05 between the indicated groups.

Fig. 2. Western blot results showing the expression of TLR4 and its downstream targets and the IHC analysis results showing the level of NF-κB in liver tissue in the indicated groups.

a Representative gel blots showing the levels of TLR4, MyD88, NF-κB, and p-NF-κB (GAPDH as the loading control). b Representative images showing the binding of p-NF-κB antibody and DAPI (original magnification, ×400). c TLR4 expression. d MyD88 expression. e NF-κB phosphorylation (p-NF-κB-to-NF-κB ratio). f Quantitative results showing p-NF-κB-positive cells. Mean±SEM, n = 4–8 mice in each group; ^*P < 0.05 between the indicated groups.

Fig. 3. Impact of TLR4 knockout (TLR4^−/−) on LPS-induced effects on hepatic and serum markers of inflammation.

a Serum IL-6 levels. b Serum IL-1β levels. c Serum TNF-α levels. d Hepatic IL-6 mRNA levels. e Hepatic IL-1β mRNA levels. f Hepatic TNF-α mRNA levels. Mean ± SEM, n = 4–7 mice in each group; ^*P < 0.05 between the indicated groups.

Fig. 4. Impact of TLR4 knockout (TLR4^−/−) on LPS-induced hepatocyte apoptosis.

Apoptosis in hepatic tissues was determined by TUNEL assays, caspase 3 activity assays, and Western blotting, and the results show the expression of apoptosis-associated proteins. a Representative TUNEL staining images for each group (original magnification, ×400). b The number of TUNEL-positive cells in each group. c Caspase 3 activity. d Bcl-2 expression. e Bax expression. Mean ± SEM, n = 6–8 mice in each group; ^*P < 0.05 between the indicated groups.

Fig. 5. Effect of TLR4 knockout (TLR4^−/−) on cell viability in primary hepatic macrophages following LPS exposure (1 μg/mL, 6 h).

a Representative flow cytometry result showing F4/80 and CD11b staining. b The percentages of F4/80⁺ cells and F4/80⁺CD11b⁺ cells. c The viability of Kupffer cells in each group was determined by CCK-8 assays. Mean ± SEM of three independent experiments, ^*P < 0.05 between the indicated groups.

Fig. 6. Effect of TLR4 knockout (TLR4^−/−) on the protein and mRNA levels of factors involved in the TLR4/MyD88/NF-κB pathway in isolated primary hepatic macrophages following LPS exposure (1 μg/mL, 6 h).

a The protein expression of TLR4. b The protein expression of MyD88. c The level of phosphorylated NF-κB. d The mRNA expression of TLR4. e The mRNA expression of MyD88. f The mRNA expression of NF-κB. Insets: Representative gel blots showing the proteins of interest (GAPDH as a loading control). Mean ± SEM, n = three independent experiments, ^*P < 0.05 between the indicated groups.

Fig. 7. Effect of TLR4 knockout (TLR4^−/−) on the protein and mRNA levels of proinflammatory cytokines in isolated primary hepatic macrophages following LPS exposure (1 μg/mL, 6 h).

a IL-6 protein levels. b IL-1β protein levels. c TNF-α protein levels. d IL-6 mRNA levels. e IL-1β mRNA levels. f TNF-α mRNA levels. Mean ± SEM, n = three independent experiments, ^*P < 0.05 between the indicated groups.

Fig. 8. Effect of TLR4 knockout (TLR4^−/−) on isolated primary hepatic macrophage apoptosis following LPS exposure (1 μg/mL, 6 h).

a RAW264.7 macrophage apoptosis was analyzed by flow cytometry. b Quantitative analysis of the total number of apoptotic cells in each group. c Bax protein expression. d Bcl-2 protein expression. Insets: Representative gel blots showing the proteins of interest (GAPDH as a loading control). Mean ± SEM, n = three independent experiments, ^*P < 0.05 between the indicated groups.