Toll样受体4信号通路在心肌微血管内皮细胞缺氧复氧损伤中的变化

目的:探讨Toll样受体4(TLR-4)信号通路在心肌微血管内皮细胞(CMECs)缺氧-复氧(H-R)损伤过程中表达的变化。方法:采用体外培养的CMECs,分为正常组、H-R组,其中H-R组根据复氧时间又分为复氧2 h、12 h及24 h组,用四甲基偶氮唑蓝(MTT)比色法检测内皮细胞增殖的能力。取细胞上清液用ELISA法检测白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的水平;用Western blot法检测各组细胞中TLR-4及核因子-κB(NF-κB)表达的水平。结果:与正常对照组相比,缺氧6 h复氧2 h、12 h、24 h后CMECs增殖分别降低48.6%(P0.01)、49.1%(P0.01)、63.2%(P0.01);TLR-4蛋白水平在复氧2 h以及12 h后明显升高(P0.05),24 h基本恢复正常;NF-κB表达水平在复氧2 h、24 h显著高于对照组(P0.05);而CMECs复氧2 h、12 h、24 h后分泌的IL-6、TNF-α水平均高于对照组(P0.05)。结论:在CMECs H-R损伤中,TLR-4及下游NF-κB的表达增加,炎性因子IL-6和TNF-α的分泌增加,说明H-R损伤可激活CMECs表面的TLR-4信号通路,这一过程可能参与了H-R诱导的CMECs损伤。

Alteration of Toll-like receptor 4 pathway expression in cardiac microvascular endothelial cells during hypoxia-reoxygenation injury

AIM:To detect the alteration of Toll-like receptor 4 (TLR-4) pathway expression in cardiac microvascular endothelial cells (CMECs) during hypoxia-reoxygenation (H-R) injury. METHODS: CMECs isolated from the hearts of adult rats were exposed to hypoxia (94% N2, 5% CO2, 1% O2) for 6 h followed by 2, 12 or 24 h reoxygenation (95% air, 5% CO2). Proliferation of CMECs was assessed by MTT assay, TLR-4 and NF-κB expressions were analyzed by Western blot and levels of IL-6 and TNF-α were detected by ELISA kits. RESULTS: The proliferation ability of CMECs was significantly inhibited by H-R injury (P<0.01) and H-R injury increased the TLR-4 expression 2 and 12 h after reoxygenation (P<0.05). The level of NF-κB increased 2 and 24 h after reoxygenation (P<0.05) and H-R injury enhanced IL-6 and TNF-α secretion compared with those in control groups (P<0.05). CONCLUSION: H-R injury increases the TLR-4 and NF-κB expressions in CMECs and enhances secretion of IL-6 and TNF-α. Activation of TLR-4/NF-κB pathway may participate in H-R injury of CMECs.