Virdis Agostino, Colucci Rocchina, Fornai Matteo, Blandizzi Corrado, Duranti Emiliano, Pinto Stefania, Bernardini Nunzia, Segnani Cristina, Antonioli Luca, Taddei Stefano, Salvetti Antonio, Del Tacca Mario
Department of Internal Medicine, University of Pisa, Via Roma, 67, 56100 Pisa, Italy.
J Pharmacol Exp Ther. 2005 Mar;312(3):945-53. doi: 10.1124/jpet.104.077644. Epub 2004 Nov 16.
We investigated whether cyclooxygenase (COX) isoforms (COX-1 and COX-2) and decreased NO availability contribute to endothelial dysfunction in endotoxemic rats. The involvement of reactive oxygen species (ROS) was also evaluated. Rats were injected with Salmonella-derived lipopolysaccharide or saline. After 6 h, endothelial function of mesenteric resistance arteries was evaluated. In controls, acetylcholine (ACh)-induced relaxation was inhibited by the nitric-oxide synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA) and unaffected by 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)-phenyl-2(5H)-furanone (DFU) (COX-2 inhibitor). In lipopolysaccharide (LPS)-treated rats, the response to ACh was blunted compared with controls, less sensitive to l-NMMA, and enhanced by DFU. COX-2 blockade also improved the inhibitory effect of l-NMMA on cholinergic relaxation. SC-560 [5-(4-clorophenyl)-1-(4-metoxyphenyl)-3-trifluoromethylpirazole] (COX-1 inhibitor) did not modify the response to ACh in both groups. LPS-induced endothelial dysfunction was unaffected by the thromboxane A(2) (TxA(2)) receptor antagonist SQ-29548 (7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1] hept-2-yl]-[1S(1alpha,2alpha(Z),3alpha,4alpha)]-5-heptenoic acid). In vivo inducible nitric-oxide synthase (iNOS) inhibition by S-methylisothiourea partly attenuated LPS-induced endothelial dysfunction. The antioxidants ascorbic acid and superoxide dismutase normalized endothelium-dependent relaxation and restored the inhibitory action of l-NMMA on ACh. Responses to sodium nitroprusside were similar in both groups. In LPS-treated rats, reverse transcription-polymerase chain reaction showed a marked increase in mesenteric iNOS and COX-2 expressions, whereas endothelial nitric-oxide synthase and COX-1 were unchanged. LPS-induced COX-2 overexpression was reduced but not abrogated by S-methylisothiourea. LPS-induced COX-2 up-regulation was also documented by immunohistochemistry. In conclusion, mesenteric resistance vessels from endotoxemic rats show impaired endothelial function due to reduced NO availability, a condition that is partly ascribable to an iNOS-dependent enhanced COX-2 expression, whereas TxA(2) does not seem to be involved. Oxidative stress is the main mechanism responsible for reduced NO availability, and COX-2 might act as a source of ROS.
我们研究了环氧化酶（COX）同工型（COX-1和COX-2）以及一氧化氮（NO）可用性降低是否会导致内毒素血症大鼠的内皮功能障碍。还评估了活性氧（ROS）的参与情况。给大鼠注射沙门氏菌衍生的脂多糖或生理盐水。6小时后，评估肠系膜阻力动脉的内皮功能。在对照组中，一氧化氮合酶抑制剂N(G)-单甲基-L-精氨酸（L-NMMA）可抑制乙酰胆碱（ACh）诱导的舒张，而5,5-二甲基-3-(3-氟苯基)-4-(4-甲磺酰基)苯基-2(5H)-呋喃酮（DFU，COX-2抑制剂）对此无影响。在脂多糖（LPS）处理的大鼠中，与对照组相比，对ACh的反应减弱，对L-NMMA不敏感，而DFU可增强该反应。COX-2阻断也改善了L-NMMA对胆碱能舒张的抑制作用。SC-560 [5-(4-氯苯基)-1-(4-甲氧基苯基)-3-三氟甲基吡唑]（COX-1抑制剂）在两组中均未改变对ACh的反应。血栓素A2（TxA2）受体拮抗剂SQ-29548（7-[3-[[2-[(苯基氨基)羰基]肼基]甲基]-7-氧杂双环[2.2.1]庚-2-基]-[1S(1α,2α(Z),3α,4α)]-5-庚烯酸）对LPS诱导的内皮功能障碍无影响。S-甲基异硫脲体内抑制诱导型一氧化氮合酶（iNOS）可部分减轻LPS诱导的内皮功能障碍。抗氧化剂抗坏血酸和超氧化物歧化酶使内皮依赖性舒张恢复正常，并恢复了L-NMMA对ACh的抑制作用。两组对硝普钠的反应相似。在LPS处理的大鼠中，逆转录-聚合酶链反应显示肠系膜iNOS和COX-2表达显著增加，而内皮型一氧化氮合酶和COX-1未改变。S-甲基异硫脲可降低但不能消除LPS诱导的COX-2过表达。免疫组织化学也证实了LPS诱导的COX-2上调。总之，内毒素血症大鼠的肠系膜阻力血管因NO可用性降低而出现内皮功能受损，这种情况部分归因于iNOS依赖性增强的COX-2表达，而TxA2似乎未参与其中。氧化应激是导致NO可用性降低的主要机制，COX-2可能是ROS的来源。
