Nitric oxide mediates the lipopolysaccharide dependent upregulation of the heme oxygenase-1 gene expression in cultured rat Kupffer cells
Nitric oxide mediates the lipopolysaccharide dependent upregulation of the heme oxygenase-1 gene expression in cultured rat Kupffer cells
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DOI：
10.1016/s0168-8278(99)80008-7
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发表时间：
1999-01-01
影响因子：
25.7
通讯作者：
Ramadori, G
中科院分区：
文献类型：
--
作者：
Immenschuh, S;Tan, M;Ramadori, G
背景/目的：血红素加氧酶催化血红素降解的限速酶步骤。血红素加氧酶的诱导异构体血红素加氧酶-1在大多数组织中表达水平较低，受底物血红素和各种应激刺激的上调。库普弗细胞代表了人体组织巨噬细胞中最大的群体，在防御各种病原体（如脂多糖）方面具有生理功能。本研究的目的是研究脂多糖处理大鼠肝脏库普弗细胞和离体库普弗细胞中血红素加氧酶-1基因的表达。方法：采用大鼠血红素加氧酶-1和ED2特异性抗体免疫荧光双染色法对正常大鼠肝脏冷冻切片进行研究。采用标准方案对大鼠肝脏的Kupffer细胞和原代肝细胞进行分离和细胞培养，并进行Northern和Western blot分析。结果：血红素加氧酶-1蛋白在正常大鼠肝大窦细胞中高表达，巨噬细胞表面标记物ED2染色鉴定为Kupffer细胞。血红素加氧酶-1在肝实质细胞中未见表达。免疫细胞化学染色以及Western和Northern blot分析也发现，培养的离体Kupffer细胞中血红素加氧酶-1的高表达。脂多糖处理Kupffer细胞后，血红素加氧酶-1在蛋白质和mRNA水平上呈时间和剂量依赖性上调，这种由脂多糖引起的血红素加氧酶-1表达的增加被一氧化氮抑制剂n - g -单甲基- l-精氨酸阻止，并被过量的l-精氨酸逆转。不同一氧化氮供体上调Kupffer细胞血红素加氧酶-1 mRNA表达。结论：Kupffer细胞中高表达的血红素加氧酶-1基因的脂多糖依赖性上调是通过一氧化氮依赖性机制介导的。
Background/Aims: Heme oxygenase catalyzes the rate-limiting enzymatic step of heme degradation. The inducible isoform of heme oxygenase, heme oxygenase-1, is expressed at a low level in most tissues and is upregulated by its substrate heme and various stress stimuli. Kupffer cells which represent the largest population of the body's tissue macrophages serve physiological functions in the defense against various pathogens such as lipopolysaccharide. The goal of the present study was to investigate the heme oxygenase-1 gene expression in Kupffer cells of rat liver and in isolated Kupffer cell cultures during treatment with lipopolysaccharide.Methods: Cryostat sections of normal rat liver were investigated by immunofluorescence double-staining using specific antibodies for rat heme oxygenase-1 and ED2. Isolation and cell culture of Kupffer cells and primary hepatocytes from rat liver, as well as Northern and Western blot analysis, were performed with standard protocols.Results: Heme oxygenase-1 protein was highly expressed in large sinusoidal cells of normal rat liver, which were identified as Kupffer cells by staining with the macrophage surface marker ED2. By contrast, no expression of heme oxygenase-1 was detected in liver parenchymal cells. High expression of heme oxygenase-1 was also found in isolated Kupffer cells in culture by immunocytochemical staining as well as by Western and Northern blot analysis. After treatment of Kupffer cells cultures with lipopolysaccharide, heme oxygenase-1 was upregulated on the protein and mRNA level in a time- and dose-dependent manner, This increase in heme oxygenase-1 expression by lipopolysaccharide was prevented by the nitric oxide inhibitor N-G-monomethyl-L-arginine which was reversed by an excess of L-arginine. Various nitric oxide donors up-regulated heme oxygenase-1 mRNA expression in Kupffer cells.Conclusions: The lipopolysaccharide-dependent upregulation of the heme oxygenase-1 gene which is highly expressed in Kupffer cells is mediated by a nitric oxide-dependent mechanism.