蛇毒三肽pENW对LPS诱导人脐静脉内皮细胞中NOS表达的影响

目的:探讨焦谷氨酸-天冬酰胺-色氨酸(蛇毒三肽p ENW)对体外脂多糖(LPS)诱导人脐静脉内皮细胞损伤的保护机制。方法:采用LPS(1 mg·L-1)模拟人脐静脉内皮细胞炎症损伤模型,实验分为空白组、LPS模型组、蛇毒三肽p ENW高、中、低剂量组(10-4,10-5,10-6mol·L-1)、N-甲基-L-精氨酸组(L-NMMA,5×10-4mol·L-1);除空白组外,其余各试验组加入LPS,蛇毒三肽p ENW高、中、低剂量组和L-NMMA组同时给予不同浓度的药物,孵育24 h后,采用噻唑蓝(MTT)比色法检测人脐静脉内皮细胞活力;Griess Reagent法用于检测一氧化氮(NO)的含量;比色法测定一氧化氮合酶(NOS)分型的活力;Western blot分析内皮型一氧化氮合酶(e NOS),诱导型一氧化氮合酶(i NOS)蛋白表达的变化。结果:与空白组比较,模型组LPS显著性损伤人脐静脉内皮细胞并诱导NO大量释放,t NOS,i NOS的活性明显增强(P0.01),i NOS蛋白表达显著上调(P0.01),e NOS的活性和蛋白表达并无差异;与模型组比较,蛇毒三肽p ENW高、中、低剂量能够剂量依赖性的抑制LPS对人脐静脉内皮细胞的损伤,并且能够明显降低NO的释放,同时,蛇毒三肽p ENW高、中、低剂量能够降低LPS诱导人脐静脉内皮细胞中的t NOS,i NOS的活性及i NOS的蛋白表达(P0.05,P0.01),与L-NMMA组作用一致,但蛇毒三肽p ENW对人脐静脉内皮细胞中e NOS的活性及蛋白表达无显著性影响。结论:蛇毒三肽p ENW可降低LPS对人脐静脉内皮细胞的损伤作用,其保护机制可能与逆转LPS诱导的i NOS蛋白表达上调及NO的释放相关。

Effect of Snake Venom Tripeptide pENW on NOS Expression of Human Umbilical Vein Endothelial Cells Induced by LPS

Objective: To investigate the protective effect of snake venom tripeptide pENW on the injury of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS) in vitro and to explore its underlying mechanism. Method: HUVECs were treated with pENW (10^-4, 10^-5, 10^-6 mol · L^-1) and nitric oxide synthase(NOS) inhibitor L-NMMA (5×10^-4 mol · L^-1), then induced with LPS (1 mg · L^-1) for 24 h. The cell viability was evaluated by MTT assay. Nitric oxide (NO) content was measured by Griess Reagent. The nitric-oxide synthase (NOS) activity was assessed by commercially available kits. The NOS expression was measured by Western blot. Result: Compared with normal control group, vascular endothelial cells were obviously damaged and NO release increased by LPS, which could be protected by pENW in a concentration-dependent manner (P<0.05, P<0.01). L-NMMA and pENW could increase the cell viability rates and decrease NO production (P<0.05, P<0.01). Meanwhile, iNOS activity and protein expression were inhibited by pENW(P<0.05, P<0.01). However, there was no significant effect on eNOS activity and protein expression. Conclusion: The results showe that pENW has protective effect on HUVECs induced by LPS. Its mechanism may be related to the suppression of iNOS expression along with decreasing levels of NO and iNOS activity.