TLR4 promotes liver inflammation by activating the JNK pathway

W. Li, G.-L. Yang, Q. Zhu, X.-H. Zhong, Y.-C. Nie, X.-H. Li, Y. Wang

Department of Hepatology, Third People’s Hospital of Shenzhen City, Shenzhen, China. gl.yang@163.com

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• Gastroenterology

OBJECTIVE: Drug-induced liver injury has become a serious public health problem that cannot be ignored. Although the mechanism of acetaminophen (APAP)-induced liver injury has been investigated for several decades, there are still many deficiencies. However, only a deeper study of its mechanism can provide more effective measures of prevention and treatment for APAP-induced liver injury. The aim of this study was to investigate whether toll-like receptor 4 (TLR4) participates in and regulates APAP-induced liver injury, which may provide a new direction for the prevention and treatment of clinical drug-induced hepatitis.

MATERIALS AND METHODS: WT mice were treated with APAP (300 mg/kg) or equivalent PBS. The livers of mice were taken at 1 h, 3 h, 6 h and 12 h after treatment. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect TLR4 mRNA expression level in the liver. After TLR4 involving in APAP-induced liver injury was confirmed, we investigated the relationship between TLR4 expression and hepatic inflammation. WT and TLR4-/- mice received APAP (3000 mg/kg) intraperitoneal injection after 16 h of fasting; serum was collected after 8 h and 24 h, and serum alanine aminotransferase (ALT) and reduced glutathione (GSH) activity were measured. Rat liver tissue was observed for histological changes by hematoxylin and eosin (H&E) staining. RT-qPCR and enzyme-linked immunosorbent assay (ELISA) assay were performed to analyze proinflammatory cytokines expression (such as TNF-α, IL-1β, MCP-1, IL-6). After isolating mononuclear cells (MNCs) in the liver of mice, flow cytometry was used to detect cell activation level and infiltration of macrophages and neutrophils. Western blotting was used to analyze the activation of phosphorylated JNK and p38 signaling pathways in livers of WT and TLR4-/- mice. In addition, after stimulated with APAP, the silence of TLR4 in RAW264.7 cells could activate phosphorylated JNK and p38 signaling pathways.

RESULTS: After APAP stimulation, WT mice exhibited more severe liver injury than TLR4-/- mice, with higher ALT levels, lower GSH levels, and more necrotic or apoptotic cells. TLR4-/- mice have lower levels of inflammatory cytokines including MCP-1 and IL-6; at the same time, the number of infiltrating macrophages and neutrophils in liver tissue of TLR4-/- mice was significantly lower than that of WT mice. The activation of JNK signaling pathway was strikingly enhanced in WT mice treated with APAP, but no significant difference was observed in the activation of JNK phosphorylation in TLR4-/- mice after the same dose of APAP stimulation. Similarly, in RAW264.7 cells, the activation of phosphorylated JNK and p38 was remarkably inhibited by TLR4-siRNA, but was activated in the control group, which was consistent in vivo.

CONCLUSIONS: APAP-treated TLR4-/- mice showed milder liver injury compared to WT mice. It was confirmed that TLR4 could activate the JNK signaling pathway to induce the secretion of inflammatory factors and the infiltration of macrophages to promote APAP-induced liver injury. This finding might provide a new prevention and treatment idea for clinical drug-induced hepatitis.

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