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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(P<0.01),i NOS蛋白表达显著上调(P<0.01),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(P<0.05,P<0.01),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。
Objective: To investigate the protective mechanism of pyroglutamic acid-asparagine-tryptophan (snake venom tripeptide p ENW) on the injury of human umbilical vein endothelial cells induced by lipopolysaccharide (LPS) in vitro. Methods: The injury model of human umbilical vein endothelial cells was simulated by LPS (1 mg · L-1). The experiment was divided into blank group, LPS model group, snake venom tripeptide p ENW high, medium and low dose groups (10-4,10 -5,10-6 mol·L-1) and N-methyl-L-arginine group (L-NMMA, 5 × 10-4 mol·L-1). Except for the blank group, , Snake venom tripeptide p ENW high, medium and low dose groups and L-NMMA group were given different concentrations of drugs, incubated for 24 h, using MTT colorimetric assay of human umbilical vein endothelial cell viability; Griess Reagent method Was used to detect the content of nitric oxide (NO); the activity of nitric oxide synthase (NOS) typing was determined by colorimetric method; the expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase Changes of enzyme (i NOS) protein expression. Results: Compared with the blank group, LPS significantly damaged human umbilical vein endothelial cells and induced a large number of NO release. The activity of tNOS and iNOS was significantly increased (P <0.01), while the expression of iNOS protein was significantly increased (P <0.01) , eNOS activity and protein expression was no difference; compared with the model group, snake venom tripeptide p ENW high, medium and low dose can inhibit LPS-induced injury of human umbilical vein endothelial cells, and can significantly reduce the NO (P <0.05, P <0.01), and the high, medium and low doses of snake venom tripeptide p ENW could reduce the activity of tNOS and iNOS and the protein expression of iNOS in LPS-induced human umbilical vein endothelial cells L-NMMA group had the same effect, but the snake venom tripeptide p ENW had no significant effect on eNOS activity and protein expression in human umbilical vein endothelial cells. CONCLUSION: The snake venom tripeptide p ENW can reduce the damage of LPS to human umbilical vein endothelial cells. The protective mechanism may be related to up-regulation of iNOS protein expression and NO release induced by LPS.