Ebisawa Yoshiaki, Kono Toru, Yoneda Masashi, Asama Toshiyuki, Chisato Naoyuki, Sugawara Mutsubu, Ishikawa Kazushi, Iwamoto Jun, Ayabe Tokiyoshi, Kohgo Yutaka, Kasai Shinichi
Department of Surgery II, Asahikawa Medical College, Asahikawa, Hokkaido, Japan.
J Surg Res. 2004 May 15;118(2):183-9. doi: 10.1016/S0022-4804(03)00348-2.
The role of nitric oxide (NO) in lipopolysaccharide (LPS) tolerance in the liver has been investigated in a number of previous studies, but it is still not clear whether NO is cytotoxic or cytoprotective. The aims of this study were to investigate whether low-dose LPS (LLPS)-induced hepatic production of NO is beneficial and to clarify the origins of cytoprotective NO-producing cells in the liver during LPS tolerance.
Male Wistar rats received saline or LLPS intraperitoneally (i.p.; 0.01-1000 microg/kg) followed by a high dose of LPS (HLPS, 5 mg/kg) at various time intervals (4-16 h). NG-nitro-L-arginine methyl ester (L-NAME) was used to investigate the effects of inhibition of NOS. 4,5-Diaminofluorescein (DAF-2) was used to identify NO-producing cells in isolated liver cells in vitro. At various time points (4-16 h) after saline or LLPS (1 microg/kg, i.p.) injection, hepatocytes and Kupffer cells were isolated, incubated in 7 microm DAF-2 diacetate, and perfused with Krebs solution. Illumination at 495 nm revealed DAF-fluorescence (515 nm) in isolated cells under confocal laser fluorescence microscopy. The NO production in hepatocytes and Kupffer cells was assessed by the number of labeled cells per 1000 cells or per 100 cells, respectively.
Pretreatment with LLPS (0.1-100 microg/kg) resulted in a significant reduction (maximal at 8 h) of the HLPS-induced liver damage. L-NAME abolished the LLPS-induced protection. The NO production in hepatocytes was significantly increased and reached a maximum of 84% of all cells 8 h after LLPS administration. By contrast, the NO production in Kupffer cells remained constant at 95%, even following preinjection of LLPS.
LLPS-induced NO in hepatocytes, but not in Kupffer cells, exhibits cytoprotective effects on HLPS-induced liver damage, suggesting that NO has a beneficial role in the induction of the early phase of LPS tolerance.
一氧化氮（NO）在肝脏脂多糖（LPS）耐受中的作用已在多项先前研究中得到探讨，但NO究竟是具有细胞毒性还是细胞保护作用仍不清楚。本研究的目的是调查低剂量LPS（LLPS）诱导的肝脏NO生成是否有益，并阐明LPS耐受期间肝脏中产生细胞保护性NO的细胞来源。
雄性Wistar大鼠腹腔注射（i.p.）生理盐水或LLPS（0.01 - 1000微克/千克），随后在不同时间间隔（4 - 16小时）给予高剂量LPS（HLPS，5毫克/千克）。使用NG-硝基-L-精氨酸甲酯（L-NAME）研究抑制一氧化氮合酶（NOS）的作用。4,5-二氨基荧光素（DAF-2）用于在体外鉴定分离的肝细胞中产生NO的细胞。在腹腔注射生理盐水或LLPS（1微克/千克）后的不同时间点（4 - 16小时），分离肝细胞和库普弗细胞，在7微摩尔DAF-2二乙酸酯中孵育，并用克雷布斯溶液灌注。在共聚焦激光荧光显微镜下，495纳米的光照显示分离细胞中的DAF荧光（515纳米）。分别通过每1000个细胞或每100个细胞中标记细胞的数量来评估肝细胞和库普弗细胞中的NO生成。
用LLPS（0.1 - 100微克/千克）预处理可显著降低（8小时时最大）HLPS诱导的肝损伤。L-NAME消除了LLPS诱导的保护作用。LLPS给药8小时后，肝细胞中的NO生成显著增加，达到所有细胞的84%的最大值。相比之下，即使预先注射LLPS，库普弗细胞中的NO生成仍保持在95%不变。
LLPS诱导肝细胞而非库普弗细胞产生NO，对HLPS诱导的肝损伤具有细胞保护作用，这表明NO在LPS耐受早期诱导中具有有益作用。
