Xu Fa Liang, You Hai Bo, Li Xu Hong, Chen Xian Feng, Liu Zuo Jin, Gong Jian Ping
Chongqing Medical University, Chongqing, PRC.
Am J Surg. 2008 Jul;196(1):139-48. doi: 10.1016/j.amjsurg.2007.09.045.
Several experimental studies have observed better outcomes after glycine treatment in patients with endotoxin-induced liver injuries, but its molecular mechanism is not yet fully understood. The purpose of this study was to evaluate the hypothesis that glycine attenuates endotoxin-induced liver injury by affecting endotoxin signal transduction in liver macrophages.
An animal model of endotoxin-induced liver injury was established by intraperitoneally injecting mice with 10 mg/kg body weight endotoxin fed a pretreatment diet with or without 5% (w/w) glycine. Blood and liver samples were obtained for analysis of liver morphology and to determine concentrations of alanine aminotransferase, endotoxin receptor Toll-like receptor 4 (TLR4), tumor necrosis factor-alpha (TNF-alpha), and interleukin (IL)-10 at various time points after injection. To investigate the effect of glycine on liver macrophages, Kupffer cells (KCs) were isolated and challenged by LPS (100 ng/mL), with or without glycine (4 mmol/l) pretreatment, and the expressions of TLR4, IL-10, and TNF-alpha were assayed at mRNA and protein levels. DNA-binding activity of nuclear factor-kappa B (NF-kappaB) was also analyzed using enzyme-linked immunosorbent assay.
Dietary glycine significantly improved the survival rate of endotoxemic mice (P < .05), whereas serum alanine aminotransferase and TNF-alpha levels were significantly decreased at different time points (P < .05); IL-10 levels were increased (P < .05). Concurrently, LPS-induced hepatic tissue injury was attenuated as indicated by morphologic analysis; secretion of IL-10 in liver tissue (P < .05) was enhanced; and expression of TLR4 and TNF-alpha in liver tissue was downregulated (P < .05). Consistent with these in vivo experiments, enhanced secretion of IL-10 and inhibited expression of TLR4 and TNF-alpha caused by glycine pretreatment were also observed in LPS-stimulated KCs. NF-kappaB DNA-binding activity was also significantly inhibited by glycine (P < .05, respectively).
Dietary glycine improved survival rates and liver function in endotoxemic mice by regulating the production of proinflammatory or anti-inflammatory cytokines in liver. It attenuated liver injury by deactivating KCs through inhibiting TNF-alpha secretion and increasing IL-10 production. The downregulative effect of glycine on the endotoxin signaling pathway and TLR4/NF-kappaB/TNF-alpha may be a novel potential mechanism by which glycine inhibits KC activity.
多项实验研究观察到，甘氨酸治疗内毒素诱导的肝损伤患者后效果更佳，但其分子机制尚未完全明确。本研究旨在评估甘氨酸通过影响肝巨噬细胞内毒素信号转导来减轻内毒素诱导的肝损伤这一假说。
通过给小鼠腹腔注射10mg/kg体重的内毒素建立内毒素诱导的肝损伤动物模型，小鼠预先喂食含或不含5%（w/w）甘氨酸的饮食。在注射后不同时间点采集血液和肝脏样本，用于分析肝脏形态，并测定丙氨酸转氨酶、内毒素受体Toll样受体4（TLR4）、肿瘤坏死因子-α（TNF-α）和白细胞介素（IL）-10的浓度。为研究甘氨酸对肝巨噬细胞的影响，分离库普弗细胞（KCs），用脂多糖（LPS，100ng/mL）刺激，有无甘氨酸（4mmol/L）预处理，检测TLR4、IL-10和TNF-α在mRNA和蛋白水平的表达。还采用酶联免疫吸附测定法分析核因子-κB（NF-κB）的DNA结合活性。
饮食中的甘氨酸显著提高了内毒素血症小鼠的存活率（P<.05），而血清丙氨酸转氨酶和TNF-α水平在不同时间点显著降低（P<.05）；IL-10水平升高（P<.05）。同时，形态学分析表明LPS诱导的肝组织损伤减轻；肝组织中IL-10的分泌增强（P<.05）；肝组织中TLR4和TNF-α的表达下调（P<.05）。与这些体内实验一致，在LPS刺激的KCs中也观察到甘氨酸预处理导致IL-10分泌增加以及TLR4和TNF-α表达受到抑制。甘氨酸也显著抑制了NF-κB的DNA结合活性（P分别<.05）。
饮食中的甘氨酸通过调节肝脏中促炎或抗炎细胞因子的产生，提高了内毒素血症小鼠的存活率和肝功能。它通过抑制TNF-α分泌和增加IL-10产生使KCs失活，从而减轻肝损伤。甘氨酸对内毒素信号通路以及TLR4/NF-κB/TNF-α的下调作用可能是其抑制KCs活性的一种新的潜在机制。
