Functional inhibition of TIM-4 in Kupffer cells alleviates hepatic ischemia-reperfusion injury in mice

Abstract

Objective To investigate the effects of functional inhibition of TIM-4 in Kupffer cells(KCs) in protecting against hepatic ischemia-reperfusion injury in mice and explore the mechanism. Methods We determined the expression of TIM-4 in KCs and dendritic cells(DCs) in a mouse model of hepatic ischemia-reperfusion injury using Western blotting and immunohistochemistry. The mouse models were treated with PBS(sham group), a monoclonal antibody of TIM-4(TIM-4 mAb group) or a control antibody(Ctr Ig group), and the liver functions, inflammatory factors and products of oxidative stress were assayed. HE staining was used to observe the pathological changes in the liver tissue, and TUNEL and flow cytometry were used to detect apoptosis of the hepatocytes. Western blotting was used to detect the expression of nuclear factor-κB(NF-κB) pathway-related proteins. Results In the mouse model of hepatic ischemia-reperfusion injury, the expression of TIM-4 protein in KCs was increased progressively as the reperfusion time extended, while TIM-4 expression in the DCs showed no significant variations with time. Immunohistochemical staining identified TIM-4 expression mainly in the hepatic sinusoidal macrophages; The serum levels of hepatic function-related factors, pro-inflammatory cytokines in the hepatic homogenate, and oxidative stress products were significantly reduced, while anti-inflammatory cytokine(IL-10) and anti-oxidative stress products(CAT and SOD) were increased significantly in TIM-4 mAb group as compared with Ctr Ig group(P < 0.05). Histopathological examination of the liver tissues revealed inflammatory cell infiltration and mild congestion in the hepatic sinusoids in Ctr Ig group, but no obvious pathological changes in TIM-4 mAb group. The apoptotic index was significantly lower in TIM-4 mAb group than in Ctr Ig group; the early apoptosis rates in the 2 groups were(5.35±0.33)% and(25.56±1.26)%, respectively, showing a significant difference between them(P < 0.05). Functional blocking of TIM-4 in the KCs lowered the expression levels of TLR4 and the downstream p-IKKα, p-IκBα, p-p65 proteins. Pre-treatment of the mice with TPCA-1, an IKK-2 blocker, further validated that blocking TIM-4 in the KCs inhibited NF-κB signaling pathway and improved hepatic ischemia-reperfusion injury. Conclusion Functional inhibition of TIM-4 in the KCs can inhibit the activation of NF-κB inflammatory pathway and reduce the inflammatory response and hepatocyte apoptosis to protect against hepatic ischemia-reperfusion injury in mice.

Keywords

• t cell immunoglobulin and mucin-domain-containing molecule 4
• kupffer cells
• hepatic ischemia-reperfusion injury
• nuclear factor-κb