Hseu You Cheng, Wang Shi Ying, Chen Hwei Yan, Lu Fung Jou, Gau Rung Jiun, Chang Weng Cheng, Liu Tsan Zon, Yang Hsin Ling
Department of Medical Technology, Fooyin Institute of Technology, Kaohsiung, Taiwan.
Free Radic Biol Med. 2002 Apr 1;32(7):619-29. doi: 10.1016/s0891-5849(02)00752-9.
Humic acid (HA) has been implicated as an etiological factor in the peripheral vasculopathy of blackfoot disease (BFD). In this study, we examined the effects of HA upon the generation of nitric oxide (NO) during the process of lethal cell injury in cultured human umbilical vein endothelial cells (HUVECs). NO production was measured by the formation of nitrite (NO(2)(-)), the stable end-metabolite of NO. Cell death was assessed by measuring the release of intracellular lactate dehydrogenase (LDH). Treatment HUVECs with HA at a concentration of 50, 100, and 200 microg/ml concentration-dependently increased nitrite levels, reaching a peak at 12 h subsequent to HA treatment, with a maximal response of approximately 400 pmole nitrite (from 1 x 10(4) cells). HA-induced nitrite formation was blocked completely by N(G)-nitro-L-arginine methyl ester (L-NAME) and also by N(G)-methyl-L-arginine (L-NMA), both being specific inhibitors of NO synthase. The LDH released from endothelial cells was evoked at from 24 h after the addition of HA (50, 100, 200 microg/ml) in a concentration- and time-dependent manner. The HA-induced LDH release was also reduced by the presence of both L-NAME and L-NMA. The addition of Ca(2+) chelator (BAPTA) inhibited both nitrite formation and LDH release by HA. Moreover, the antioxidants (superoxide dismutase, vitamin C, vitamin E) and protein kinase inhibitor (H7) effectively suppressed HA-induced nitrite formation. These results suggest that HA treatment of endothelial cells stimulates NO production, which can elicit cell injury via the stimulation of Ca(2+)-dependent NO synthase activity by increasing cytosolic Ca(2+) levels. Because the destruction of endothelial cells has been implicated in triggering the onset of BFD, the induction of excessive levels of NO and consequent endothelial-cell injury may be important to the etiology of HA-induced vascular disorders associated with BFD for humans.
腐殖酸（HA）被认为是黑脚病（BFD）外周血管病变的一个病因。在本研究中，我们检测了HA对培养的人脐静脉内皮细胞（HUVECs）致死性细胞损伤过程中一氧化氮（NO）生成的影响。通过测量亚硝酸盐（NO₂⁻）的形成来检测NO的产生，亚硝酸盐是NO的稳定终代谢产物。通过测量细胞内乳酸脱氢酶（LDH）的释放来评估细胞死亡。用浓度为50、100和200μg/ml的HA处理HUVECs，亚硝酸盐水平呈浓度依赖性增加，在HA处理后12小时达到峰值，最大反应约为400皮摩尔亚硝酸盐（来自1×10⁴个细胞）。HA诱导的亚硝酸盐形成被N⁰-硝基-L-精氨酸甲酯（L-NAME）以及N⁰-甲基-L-精氨酸（L-NMA）完全阻断，这两种都是NO合酶的特异性抑制剂。内皮细胞释放的LDH在添加HA（50、100、200μg/ml）后24小时开始以浓度和时间依赖性方式诱发。L-NAME和L-NMA的存在也降低了HA诱导的LDH释放。添加Ca²⁺螯合剂（BAPTA）抑制了HA诱导的亚硝酸盐形成和LDH释放。此外，抗氧化剂（超氧化物歧化酶、维生素C、维生素E）和蛋白激酶抑制剂（H7）有效抑制了HA诱导的亚硝酸盐形成。这些结果表明，HA处理内皮细胞会刺激NO生成，这可通过增加胞质Ca²⁺水平刺激Ca²⁺依赖性NO合酶活性来引发细胞损伤。由于内皮细胞的破坏与BFD的发病有关，过量NO的诱导以及随之而来的内皮细胞损伤可能对人类HA诱导的与BFD相关的血管疾病的病因学很重要。
