内毒素耐受导致的GSK-3抑制对急性内毒素性肝损伤的保护机制

LPS tolerance-induced GSK-3 inhibition protects endotoxemia rats from acute liver injury

目的探讨内毒素(lipopolysaccharide,LPS)耐受导致的糖原合成酶激酶-3(glycogen synthase kinase-3,GSK-3)活性抑制对急性内毒素性肝损伤产生保护效应的潜在机制。方法建立LPS和GSK-3抑制剂氯化锂(LiCl)预处理的SD大鼠模型,采用半定量RT-PCR检测肝组织肿瘤坏死因子-α(TNF-α)和白细胞介素-10(IL-10)mRNA表达水平,以专用试剂盒检测代表肝组织中性粒细胞浸润程度的髓过氧化物酶(MPO)活性;用密度梯度离心法分离Kupffer细胞,建立LPS耐受和GSK-3抑制剂(LiCl和胰岛素)预处理的Kupffer细胞模型,用中性红法测定细胞吞噬活性;采用激光共聚焦显微镜和Western blot实验观察Kupffer细胞核因子-κB(NF-κB)p65的亚细胞定位和核转位情况。结果 LPS耐受和GSK-3抑制剂LiCl预处理均可减少大剂量LPS攻击导致的肝组织TNF-αmRNA上调表达(P<0.05)、促进IL-10 mRNA上调表达(P<0.05),减少LPS攻击导致的MPO活性升高(P<0.05);LPS耐受和GSK-3抑制剂(LiCl和胰岛素)均能增强Kupffer细胞吞噬活性(P<0.05);LPS耐受和GSK-3抑制剂LiCl预处理均可减少大剂量LPS导致的NF-κB p65核转位(P<0.05)。结论 LPS耐受导致的GSK-3功能活性抑制,可能通过对NF-kappaB p65核转位、炎症细胞因子分泌、中性粒细胞浸润和Kupffer细胞吞噬活性等下游事件产生影响,从而保护急性内毒素性肝脏损伤。

Objective To probe into the protective mechanism of LPS tolerance-related GSK-3 inhibition on endotoxin-induced acute liver injury in rats with endotoxemia. Methods After the SD rat models of LPS tolerance with or without GSK-3 inhibitor LiC1 pretreatment were established, and the mRNA levels of TNF-α and IL-10 in hepatic tissue were measured by semi-quantitative RT-PCR in the normal rats, and above model groups. The activity of myeloperoxidase （MPO） in the liver tissue was detected by using special kit to evaluate the neutrophils infiltration in the liver tissue. Kupffer cells were isolated by Ficoll density gradient centrifugation, and the cell models of endotoxin tolerance and GSK-3 inhibition （ LiC1 and insulin） were established. The phagocytosis activity of Kupffer cells was measured with Neutral Red assy. Confocal analysis and Western blotting were used to observe the subcellular localization and nuclear translocation of NF-KB p65. Results LPS tolerance as well as GSK-3 inhibitor LiC1 pretreatment decreased the up-regulated TNF-ot mRNA （P 〈 0.05） and promoted IL-10 mRNA （P 〈 0. 05 ） induced by high-dosed endotoxin attack, and the enhanced activity of MPO was also decreased during this process （ P 〈 0. 05 ）. On the other hand, both endotoxin tolerance and GSK-3 inhibition （LiC1 and insulin） enhanced the phagocytic activity of Kupffer cells （P 〈 0. 05 ）. Both LPS tolerance and GSK-3 inhibition LiC1 decreased the nuclear translocation of NF-KB p65 induced by large-dosed LPS （P 〈 0. 05）. Conclusion The inhibition of GSK-3 functional activity induced by LPS tolerance may be one of the protective mechanisms against endotoxin induced acute liver injury, probably via affecting nuclear translocation of NF-KB p65, secretion of inflammation related cytokines, infiltration of neutrophils, phagocytic activity of Kupffer cells and other downstream events.