Department of Pharmacology, Faculty of Pharmacy, Mersin University, 33169 Mersin, Turkey.
Nitric Oxide. 2013 Sep 1;33:18-41. doi: 10.1016/j.niox.2013.05.001. Epub 2013 May 14.
We have previously demonstrated that a stable synthetic analog of 20-hydroxyeicosatetraenoic acid (20-HETE), N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE), prevents vascular hyporeactivity, hypotension, tachycardia, and inflammation in rats treated with lipopolysaccharide (LPS) and mortality in endotoxemic mice. These changes were attributed to decreased production of inducible nitric oxide (NO) synthase (iNOS)-derived NO, cyclooxygenase (COX)-2-derived vasodilator prostanoids, and proinflammatory mediators associated with increased cyctochrome P450 (CYP) 4A1-derived 20-HETE and CYP2C23-dependent antiinflammatory mediator formation. The aim of this study was to determine whether decreased expression and activity of iNOS, soluble guanylyl cyclase (sGC), protein kinase G (PKG), COX-2, gp91(phox) (NOX2; a superoxide generating NOX enzyme), and peroxynitrite production associated with increased expression of COX-1 and CYP4A1 and 20-HETE formation in renal and cardiovascular tissues of rats contributes to the effect of 5,14-HEDGE to prevent vasodilation, hypotension, tachycardia, and inflammation in response to systemic administration of LPS. Mean arterial pressure fell by 28mmHg and heart rate rose by 47beats/min in LPS (10mg/kg, i.p.)-treated rats. Administration of LPS also increased mRNA and protein expression of iNOS and COX-2 associated with a decrease in COX-1 and CYP4A1 mRNA and protein expression. Increased NOS activity, iNOS-heat shock protein 90 complex formation (an index for iNOS activity), protein expression of phosphorylated vasodilator stimulated phosphoprotein (an index for PKG activity), gp91(phox), p47(phox) (NOXO2; organizer subunit of gp91(phox)), and nitrotyrosine (an index for peroxynitrite production) as well as cGMP (an index for sGC activity), 6-keto-PGF1α (a stable metabolite PGI2) and PGE2 levels (indexes for COX activity), and nitrotyrosine levels by LPS were also associated with decreased CYP hydroxylase activity as measured by 20-HETE formation from arachidonic acid in renal microsomes of LPS-treated rats. These effects of LPS, except iNOS mRNA and COX-1 protein expression, were prevented by 5,14-HEDGE (30mg/kg, s.c.; 1h after LPS). A competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (30mg/kg, s.c.; 1h after LPS) reversed the effects of 5,14-HEDGE, except iNOS and COX-1 mRNA and protein expression as well as expression of CYP4A1 mRNA. These results suggest that increased CYP4A1 expression and 20-HETE formation associated with suppression of iNOS/sGC/PKG pathway, COX-2, and gp91(phox) participate in the protective effect of 5,14-HEDGE against vasodilation, hypotension, tachycardia, and inflammation in the rat model of septic shock.
我们之前的研究表明，20-羟二十碳四烯酸（20-HETE）的稳定合成类似物 N-[20-羟二十碳五烯酰基]甘氨酸（5,14-HEDGE）可预防脂多糖（LPS）处理的大鼠血管低反应性、低血压、心动过速和炎症以及内毒素血症小鼠的死亡率。这些变化归因于诱导型一氧化氮合酶（iNOS）衍生的 NO、环氧化酶（COX）-2 衍生的血管扩张性前列腺素和与细胞色素 P450（CYP）4A1 衍生的 20-HETE 增加和 CYP2C23 依赖性抗炎介质形成相关的促炎介质产生减少。本研究的目的是确定肾和心血管组织中大鼠 LPS 治疗后 iNOS、可溶性鸟苷酸环化酶（sGC）、蛋白激酶 G（PKG）、COX-2、gp91（phox）（产生超氧化物的 NOX 酶）和过氧亚硝酸盐产生的减少与 COX-1 和 CYP4A1 表达增加和 20-HETE 形成相关，这是否有助于 5,14-HEDGE 预防 LPS 全身给药引起的血管扩张、低血压、心动过速和炎症。LPS（10mg/kg，腹腔注射）处理的大鼠平均动脉压下降 28mmHg，心率升高 47 次/分钟。LPS 还增加了 iNOS 和 COX-2 的 mRNA 和蛋白表达，同时降低了 COX-1 和 CYP4A1 的 mRNA 和蛋白表达。NOS 活性增加，iNOS-热休克蛋白 90 复合物形成（NOS 活性指标），磷酸化血管扩张刺激磷酸蛋白（PKG 活性指标）、gp91（phox）、p47（phox）（gp91（phox）的组织者亚基）和硝基酪氨酸（过氧亚硝酸盐产生的指标）以及 cGMP（sGC 活性指标）、6-酮-PGF1α（PGI2 的稳定代谢物）和 PGE2 水平（COX 活性指标）在 LPS 处理的大鼠肾微粒体中由花生四烯酸形成的 20-HETE 测定的 CYP 羟化酶活性也增加。除 iNOS mRNA 和 COX-1 蛋白表达外，LPS 的这些作用（除 iNOS mRNA 和 COX-1 蛋白表达外）均被 5,14-HEDGE（30mg/kg，皮下注射；LPS 后 1 小时）预防。20-HETE 血管收缩作用的竞争性拮抗剂 20-羟二十碳六烯酸（30mg/kg，皮下注射；LPS 后 1 小时）逆转了 5,14-HEDGE 的作用，除了 iNOS 和 COX-1 mRNA 和蛋白表达以及 CYP4A1 mRNA 表达。这些结果表明，与抑制 iNOS/sGC/PKG 途径、COX-2 和 gp91（phox）相关的 CYP4A1 表达和 20-HETE 形成参与了 5,14-HEDGE 对败血症休克大鼠血管扩张、低血压、心动过速和炎症的保护作用。
