Okayama Naotsuka, Omi Hitoshi, Okouchi Masahiro, Imaeda Kenro, Kato Takashi, Akao Masaya, Imai Seiji, Shimizu Manabu, Fukutomi Tatsuya, Itoh Makoto
First Department of Internal Medicine, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.
J Diabetes Complications. 2002 Sep-Oct;16(5):321-6. doi: 10.1016/s1056-8727(02)00178-2.
We have previously reported that endothelial cells cultured in the presence of high concentrations of glucose (27.8 and 55.5 mM) exhibited enhanced neutrophil adhesion through increased expression of endothelial adhesion molecules via the activation of a protein kinase C (PKC)-dependent pathway. We also found that the aldose reductase inhibitor, epalrestat, inhibited these events, but the mechanisms for this inhibition remained unclear. In this study, we further investigated the inhibitory mechanisms of epalrestat with reference to PKC activation and nitric oxide (NO) production.
Human umbilical vein endothelial cells (HUVECs) were cultured for 48 h in glucose-rich medium and neutrophils from healthy volunteers were then added and allowed to adhere for 30 min. Adhered neutrophils were quantified by measuring myeloperoxidase (MPO) activity and surface expression of endothelial adhesion molecules was determined by enzyme immunoassay.
Culture in the presence of a high concentration of glucose (27.8 mM for 48 h) increased neutrophil-endothelial cell adhesion and surface expression of intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin on endothelial cells. These phenomena were significantly inhibited by epalrestat (10 microM), while NO synthase (NOS) inhibitors reduced the inhibitory effects of this compound. In contrast, 10 nM phorbol 12-myristate 13-acetate (PMA), a PKC activator, showed similar effects as high glucose, and these effects were also inhibited by epalrestat.
Our data suggested that epalrestat inhibited high glucose-mediated neutrophil-endothelial cell adhesion and expression of endothelial adhesion molecules not only through inhibition of a PKC-dependent pathway, but also through increased endothelial NO production.
我们之前报道过，在高浓度葡萄糖（27.8和55.5 mM）存在的情况下培养的内皮细胞，通过蛋白激酶C（PKC）依赖性途径的激活，增加内皮黏附分子的表达，从而增强中性粒细胞黏附。我们还发现醛糖还原酶抑制剂依帕司他可抑制这些事件，但这种抑制的机制仍不清楚。在本研究中，我们参照PKC激活和一氧化氮（NO）生成情况，进一步研究了依帕司他的抑制机制。
人脐静脉内皮细胞（HUVECs）在富含葡萄糖的培养基中培养48小时，然后加入健康志愿者的中性粒细胞并使其黏附30分钟。通过测量髓过氧化物酶（MPO）活性对黏附的中性粒细胞进行定量，并通过酶免疫测定法测定内皮黏附分子的表面表达。
在高浓度葡萄糖（27.8 mM，培养48小时）存在的情况下进行培养，可增加中性粒细胞与内皮细胞的黏附以及内皮细胞上细胞间黏附分子-1（ICAM-1）、P-选择素和E-选择素的表面表达。这些现象被依帕司他（10 microM）显著抑制，而一氧化氮合酶（NOS）抑制剂降低了该化合物的抑制作用。相比之下，10 nM佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA），一种PKC激活剂，显示出与高糖相似的作用，并且这些作用也被依帕司他抑制。
我们的数据表明，依帕司他抑制高糖介导的中性粒细胞与内皮细胞的黏附以及内皮黏附分子的表达，不仅是通过抑制PKC依赖性途径，还通过增加内皮NO的生成。
