Macrophage-induced inhibition of nitric oxide production in primary rat hepatocyte cultures via prostaglandin E2 release
Macrophage-induced inhibition of nitric oxide production in primary rat hepatocyte cultures via prostaglandin E2 release
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DOI：
10.1002/hep.510280519
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发表时间：
1998-11-01
期刊：
影响因子：
13.5
通讯作者：
Sergent, O
中科院分区：
文献类型：
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作者：
Griffon, B;Cillard, J;Sergent, O
枯否细胞和其它巨噬细胞在肝脏毒物的发病机制中起重要作用。本研究的目的是评估巨噬细胞对肝细胞产生一氧化氮（NO）的影响，这是以前报道的对原代大鼠肝细胞中诱导的氧化应激具有保护作用。为此，将RAW 264.7巨噬细胞以不同的巨噬细胞和肝细胞之间的比例添加到原代大鼠肝细胞中。这些共培养物补充有脂多糖（LPS）和干扰素γ（IFN-γ）23小时以诱导NO合酶并触发NO产生。NO的产生随后通过定量培养基中的亚硝酸盐和从巨噬细胞分离后的完整肝细胞中的二亚硝酰铁复合物（DNIC）。在与LPS和IFN-γ孵育的共培养肝细胞中，DNIC和亚硝酸盐水平与在相同条件下无巨噬细胞培养的肝细胞中观察到的水平相比降低。此外，在肝细胞共培养物中NO产生的抑制是巨噬细胞数量依赖性的。巨噬细胞条件培养液也抑制肝细胞NO的产生，表明巨噬细胞的作用是由可溶性因子介导的。在已知的降低NO水平的可溶性因子中，有一些细胞因子、生长因子、活性氧和胰高血糖素。通过500-d膜超滤巨噬细胞条件培养基以排除较高分子量的分子，如抗炎细胞因子和生长因子，未能恢复NO的产生。以同样的方式，使用超氧化物歧化酶（SOD）和过氧化氢酶（CAT），以消除活性氧产生的大相没有导致肝细胞中的NO水平的恢复。然而，当用N-G-单甲基-L-精氨酸（L-NMMA）抑制巨噬细胞NO合成时，肝细胞恢复产生NO的能力，同时观察到巨噬细胞释放前列腺素E-2（PGE（2））的净减少，并且消炎痛抑制巨噬细胞产生PGE（2）导致NO水平恢复。综上所述，我们的观察结果表明，由巨噬细胞合成的NO可以通过释放PGE（2）减少肝细胞中NO的产生。由于NO对许多肝损伤的保护作用，可以假设巨噬细胞通过这种机制促进肝损伤。
Kupffer cells and other macrophages play an important role in pathogenesis of toxicants in the liver. The aim of this study was to evaluate the effect of macrophages on hepatocyte production of nitric oxide (NO), which has been previously reported to be protective toward oxidative stress induced in primary rat hepatocytes, For this purpose, RAW 264.7 macrophages were added to primary rat hepatocytes at various ratios between macrophages and hepatocytes. These cocultures were supplemented with lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) for 23 hours to induce NO synthase and trigger NO production. NO production was followed by quantification of nitrites in culture medium and dinitrosyl iron complexes (DNIC) in intact hepatocytes after separation from macrophages, In cocultured hepatocytes incubated with LPS and IFN-gamma, DNIC and nitrite levels decreased compared with those observed in hepatocytes cultured without macrophages in the same conditions. Moreover, inhibition of NO production in hepatocyte cocultures was macrophage-number-dependent. Macrophage-conditioned medium also inhibited NO production in hepatocytes, suggesting that the effect of macrophages was mediated by soluble factors. Among the soluble factors known to decrease NO levels are some cytokines, growth factors, reactive oxygen species, and prostaglandins, Ultrafiltration of macrophage-conditioned medium through a 500-d membrane to rule out higher-molecular-weight molecules, such as anti-inflammatory cytokines and growth factors, failed to restore NO production. In the same way, the use of superoxide dismutase (SOD) and catalase (CAT) to eliminate reactive oxygen species produced by macrophases did not lead to recovery of NO levels in hepatocytes. However, when NO synthesis was inhibited in macrophages by N-G-monomethyl-L-arginine (L-NMMA), hepatocytes recovered the capacity to produce NO. A net decrease of prostaglandin E-2 (PGE(2)) release by macrophages was concomitantly observed, Moreover, inhibition of PGE(2) production in macrophages by indomethacin led to restoration of NO levels. Taken together, our observations suggest that NO synthesized by macrophages can decrease NO production in hepatocytes via PGE(2) release. Because of the protective role of NO toward many liver injuries, it may be postulated that macrophages contribute through this mechanism to liver damage.