Rockey D C, Chung J J, McKee C M, Noble P W
Department of Medicine, San Francisco General Hospital, University of California, USA.
Hepatology. 1998 Jan;27(1):86-92. doi: 10.1002/hep.510270115.
Hepatic injury and chronic wounding are characterized by increased synthesis of extracellular matrix proteins including hyaluronan (HA). Recently, it has been recognized that low-molecular-weight fragments of HA, but not native HA (e.g., high-molecular-weight HA), induce inflammatory gene expression, and activate the transcriptional regulator, nuclear factor kappaB (NF-kappaB). The inducible isoform of nitric oxide synthase (iNOS) is induced by cytokines and/or lipopolysaccharide (LPS) through the NF-kappaB signal transduction pathway. Because of this association, we hypothesized that HA fragments might also stimulate iNOS gene transcription. The aims of this study were therefore to determine whether HA or HA fragments induced iNOS in hepatic cells, and to characterize the signaling pathway. HA fragments (100 microg/mL) markedly stimulated iNOS messenger RNA (mRNA) in endothelial and Kupffer cells, but minimally induced this mRNA in hepatocytes and stellate cells. High-molecular-weight HA (200 microg/mL) had no effect on iNOS mRNA in any cell type. The addition of interferon gamma (IFN-gamma) to HA fragments resulted in stimulation of iNOS mRNA 2-, 3-, 4-, and 10-fold above that for HA fragments alone in hepatocytes, endothelial, Kupffer, and stellate cells, respectively. The combination of HA fragments and LPS did not result in an incremental increase in iNOS mRNA induction. iNOS protein and nitrite levels (used as a measure of NO production and NOS enzymatic activity) paralleled closely iNOS mRNA expression and increased proportionally to HA fragment concentration in a dose-dependent fashion. At 1 hour following stimulation, NF-kappaB DNA binding activity was detected in extracts from Kupffer cells stimulated with HA fragments, but not in those exposed to media alone or to high-molecular-weight HA. Finally, inhibitors of NF-kappaB blocked HA fragment-dependent iNOS mRNA induction in Kupffer and sinusoidal endothelial cells. The data indicate that HA fragments, but not high-molecular-weight HA, induce iNOS in liver, having the greatest effects on endothelial and Kupffer cells. We speculate that HA fragments may be an important stimulus for NO production in various forms of liver disease, particularly as a cofactor with inflammatory cytokines.
肝损伤和慢性创伤的特征是细胞外基质蛋白（包括透明质酸，HA）合成增加。最近人们认识到，低分子量的HA片段而非天然HA（如高分子量HA）可诱导炎症基因表达，并激活转录调节因子核因子κB（NF-κB）。诱导型一氧化氮合酶（iNOS）的同工型由细胞因子和/或脂多糖（LPS）通过NF-κB信号转导途径诱导产生。鉴于这种关联，我们推测HA片段可能也会刺激iNOS基因转录。因此，本研究的目的是确定HA或HA片段是否能在肝细胞中诱导iNOS，并对其信号通路进行表征。HA片段（100μg/mL）可显著刺激内皮细胞和库普弗细胞中的iNOS信使核糖核酸（mRNA），但对肝细胞和星状细胞中该mRNA的诱导作用极小。高分子量HA（200μg/mL）对任何细胞类型中的iNOS mRNA均无影响。在肝细胞、内皮细胞、库普弗细胞和星状细胞中，向HA片段中添加干扰素γ（IFN-γ）分别导致iNOS mRNA的刺激程度比单独使用HA片段时高出2倍、3倍、4倍和10倍。HA片段与LPS的组合并未导致iNOS mRNA诱导的增量增加。iNOS蛋白和亚硝酸盐水平（用作NO产生和NOS酶活性的指标）与iNOS mRNA表达密切平行，并以剂量依赖方式与HA片段浓度成比例增加。刺激后1小时，在用HA片段刺激的库普弗细胞提取物中检测到NF-κB DNA结合活性，但在单独暴露于培养基或高分子量HA的提取物中未检测到。最后，NF-κB抑制剂可阻断库普弗细胞和窦状内皮细胞中HA片段依赖性iNOS mRNA的诱导。数据表明，HA片段而非高分子量HA可在肝脏中诱导iNOS，对内皮细胞和库普弗细胞的影响最大。我们推测，HA片段可能是各种肝病中NO产生的重要刺激因素，尤其是作为炎症细胞因子的辅助因子。
