Piao Jiyuan, Hong Hyun Sook, Son Youngsook
Department of Genetic Engineering, College of Life Science and Graduate School of Biotechnology, Kyung Hee University, Yong In, Korea.
East-West Medical Research Institute/Department of Biomedical Science and Technology, Graduate School, Kyung Hee University, Seoul, Korea.
Microcirculation. 2018 Apr;25(3):e12443. doi: 10.1111/micc.12443.
The aim of this study was to explore the beneficial effects of SP on NO production and inflammation-induced vascular endothelium cell death.
To mimic the inflammatory environment, TNF-α was treated with HUVECs, and SP was added prior to TNF-α to determine its protective effect. WST-1 assay was performed to detect cell viability. NO level in conditioned medium was measured by Griess Reagent System. The protein level of cleaved caspase-3, eNOS, and phosphorylated Akt was detected by Western blot analysis.
TNF-α declined endothelial cell viability by downregulating Akt and NO production. TNF-α-induced cell death was reliably restored by NO, confirming the requirement of NO for cell survival. By contrast, pretreatment of SP attenuated TNF-α-induced cellular apoptosis, accompanied by an increase in the phosphorylation of Akt, eNOS expression, and NO production. Blockage of NK-1R, phosphorylated Akt or eNOS by CP-96345, A6730, or L-NAME entirely eliminated the effect of SP.
SP can protect the vascular endothelium against inflammation-induced damage through modulation of the Akt/eNOS/NO signaling pathway.
本研究旨在探讨速激肽（SP）对一氧化氮（NO）生成及炎症诱导的血管内皮细胞死亡的有益作用。
为模拟炎症环境，用肿瘤坏死因子-α（TNF-α）处理人脐静脉内皮细胞（HUVECs），并在加入TNF-α之前添加SP以确定其保护作用。采用WST-1法检测细胞活力。用格里斯试剂系统测定条件培养基中的NO水平。通过蛋白质印迹分析检测裂解的半胱天冬酶-3、内皮型一氧化氮合酶（eNOS）和磷酸化Akt的蛋白质水平。
TNF-α通过下调Akt和NO生成降低内皮细胞活力。NO可可靠地恢复TNF-α诱导的细胞死亡，证实NO对细胞存活的必要性。相比之下，SP预处理可减轻TNF-α诱导的细胞凋亡，同时伴有Akt磷酸化增加、eNOS表达增加和NO生成增加。CP-96345、A6730或L-NAME阻断神经激肽-1受体（NK-1R）、磷酸化Akt或eNOS可完全消除SP的作用。
SP可通过调节Akt/eNOS/NO信号通路保护血管内皮免受炎症诱导的损伤。
