RESUMO
BACKGROUND AND OBJECTIVE: Acetaminophen (APAP) is a widely used antipyretic and analgesic. If taken in excess, it can cause severe drug-induced acute liver injury. The purpose of this study was to investigate the effects of anti-TLR4 IgG2 on APAP-induced liver injury and its underlying mechanisms. METHODS: We injected APAP into the abdominal cavity of mice to establish a liver injury model. Mice were divided into the control group, APAP group, and APAP + anti-TLR4 IgG2 group. In order to verify the implication of the toll-like receptor4 and mitogen-activated protein kinases activation (TLR4/MAPKs) signaling pathway, mice were intraperitoneally injected with a TLR4 / MAPKs inhibitor anti-TLR4 IgG2. We evaluated the effects of TLR4 IgG2 on the antioxidant, anti-apoptotic, anti-inflammatory, and liver histopathology of APAP mice. In addition, the expression of the TLR4 / MAPKs signaling pathway was detected by Western blot. RESULTS: Our study showed that APAP mouse models were successfully established; however, pretreatment with anti-TLR4 IgG2 alleviated APAP-induced hepatic injury, as evidenced by the 24-h survival rate. Meanwhile, anti-TLR4 IgG2 prevented the elevation of serum biochemical parameters and lipid profile. Furthermore, compared with the APAP group, hepatic antioxidants, including 3- Nitrotyrosine, high mobility group protein B1, superoxide dismutase, catalase, and glutathione, were increased in APAP + anti-TLR4 IgG2 group. In contrast, a significant decrease was observed in the levels of the malondialdehyde, which is a lipid peroxidation product. Moreover, the western blotting analysis showed that anti-TLR4 IgG2 treatment inhibited the activation of the apoptotic pathway by increasing Bcl-2 and decreasing Bax, P53, and cleaving caspase-3 / caspase-3 protein expression. These results were further validated by TUNEL staining and immunohistochemical. Histopathological observation also revealed that pretreat-ment with anti-TLR4 IgG2 could significantly reverse hepatocyte inflammatory infiltration, congestion, and necrosis in liver tissues by APAP. Importantly, anti-TLR4 IgG2 effectively alleviated APAP-induced liver injury by inhibiting tolllike receptor4 and mitogen-activated protein kinases activation signaling pathways (TLR4/MAPKs). CONCLUSION: The results clearly suggest that the underlying molecular mechanisms in the hepatoprotection of anti-TLR4 IgG2 in APAP-induced hepatotoxicity may be due to its antioxidation, anti-apoptosis, and anti-inflammation effects through inhibition of the TLR4/MAPKs signaling axis.
Assuntos
Acetaminofen , Doença Hepática Crônica Induzida por Substâncias e Drogas , Animais , Camundongos , Acetaminofen/efeitos adversos , Acetaminofen/metabolismo , Receptor 4 Toll-Like/metabolismo , Caspase 3/metabolismo , Doença Hepática Crônica Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Crônica Induzida por Substâncias e Drogas/patologia , Fígado , Transdução de Sinais , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Estresse OxidativoRESUMO
Previous studies have revealed that activation of the Tolllike receptor 4 (TLR4)mediated proinflammatory signaling pathway plays an important role in acute inflammation, sepsis and chronic inflammatory disorders. Moreover, TLR4 significantly contributes to lipopolysaccharide (LPS)induced immune response. Thus, modulation of the TLR4 pathway is an important strategy to specifically target these pathologies. The aim of the present study was to develop a complete human antiTLR4 IgG2 antibody by screening human TLR4 Fab from a phagedisplay library and integrating it with constant regions of the heavy chain of human IgG2 via antibody engineering. ELISA, a BLItz system and fluorescenceactivated cell sorting were used to assess its affinity. Furthermore, mousederived peritoneal macrophages were treated with human antiTLR4 IgG2 and induced with LPS in vitro. Reverse transcriptionquantitative PCR and western blotting were used to determine mRNA expression levels of cytokines and phosphorylation levels of signaling pathways, respectively. It was found that human antiTLR4 IgG2 bound to TLR4 with a high affinity of 8.713x1010 M, and that preincubation with antiTLR4 IgG2 inhibited the LPSinduced production of tumor necrosis factorα, interferonß and interleukin6 mRNA expression levels in mouse peritoneal macrophages. It was also demonstrated that human antiTLR4 IgG2 inhibited LPSinduced TLR4 signaling by reducing the phosphorylation of the NFκB, mitogenactivated protein kinase and interferon regulatory factor 3 signaling pathways. In addition, human antiTLR4 IgG2 protected mice from LPS challenge with a survival rate of 40% and also significantly increased the survival time in the cecal ligation and puncture model. Therefore, it was speculated that human antiTLR4 IgG2 plays a protective role against sepsisassociated injury and is potentially applicable for the treatment of infectionassociated immune dysfunction.