儿茶素清除超氧阴离子对Ang II诱导的内皮祖细胞NO与eNOS表达及凋亡影响

Effects of clearance of superoxide anion by catechin on the expression of NO and eNOS and apoptosis in endothelial progenitor cells induced by angiotensin II

摘要:

目的：探讨儿茶素通过清除超氧阴离子(O2—?)对血管紧张素Ⅱ(Ang II)诱导的内皮祖细胞(EPCs)、一氧化氮(NO)与内皮型一氧化氮合酶(eNOS)表达及凋亡的影响。方法：分离Sprague-Dawley大鼠骨髓内皮祖细胞，将其分为对照组、Ang II组与Ang II+儿茶素组，培养48 h。实验末，利用NBT还原法测定培养上清O2—?浓度、硝酸还原酶法测定培养上清NO浓度；TUNEL法测定EPCs凋亡率；RT-PCR测定eNOS mRNA表达；Western blot测定eNOS蛋白表达。结果：MTT实验确定10-6 mol/L Ang II为实验细胞诱导浓度，400 mg/L儿茶素为实验干预剂量。实验末，对照组、Ang II组及Ang II+儿茶素组的凋亡率别为(24.8±1.2)‰，(541.8±7.7)‰，(168.7±3.5)‰，三组间差异有非常显著性(P<0.01)；对照组、Ang II组及Ang II+儿茶素组细胞培养上清O2—?分别为(33.7±2.8)、(81.7±3.6)、(62.3±2.2)U/L，NO分别为(105.8±9. 8)、(189.8±9.0)、(276.4±10.1)μmol/L，AngⅡ组与Ang II+儿茶素组细胞培养上清中O2—?及NO浓度较对照组均有非常显著增高(P<0.01)。Ang II+儿茶素组浓度与AngⅡ组相比则有明显降低(P<0.05) ，但NO浓度则明显增高(P<0.05)。与对照组相比，AngⅡ组与Ang II+儿茶素组eNOS mRNA（P<0.05）和蛋白表达（P<0.01）均明显增高。Ang II+儿茶素组eNOS蛋白表达较AngⅡ组显著增加 (P<0.05 )。结论：Ang II可诱导EPCs产生O2—?、灭活NO、刺激eNOS基因与蛋白表达增高，儿茶素可能是通过有效清除O2—?、减少NO的失活、降低eNOS蛋白的解偶联以减少EPCs的凋亡。［中国当代儿科杂志，2009，11（6）：476-480］

Abstract:

OBJECTIVE: To evaluate the effect of clearance of superoxide anion (O2—?) by catechin on the expression of nitrogen monoxidum (NO) and endothelial nitricoxide synthase (eNOS) and apoptosis in endothelial progenitor cells (EPCs) induced by angiotensin II (Ang II). METHODS: The marrow endothelial progenitor cells of Sprague-Dawley rats were isolated and assigned to control (no treatment), Ang II treatment and Ang II + catechin treatment groups. After 48 hrs of culture, the concentration of O2—?in the supernate was measured by the NBT method, and NO concentration in the supernate was measured by the nitrate reductase method; the apoptosis rate of EPCs was detected by the TUNEL method; the mRNA expression of eNOS was detected by RT-PCR; the protein expression of eNOS was detected by Western blot analysis. RESULTS: Ang II of 10-6 mol/L was determined as the suitable concentration for cell induction by the MTT test. Catechin of 400 mg/L was determined as an advisable intervention dosage. The apoptosis rate of EPCs in the control, the Ang II and the Ang II+catechin treatment groups were 24.8±1.2‰, 541.8±7.7‰ and 168.7±3.5‰, respectively, and there were significant differences among the three groups (P<0.01). The O2—? concentration in the Ang II and the Ang II+catechin treatment groups (81.7±3. 6 and 62.3±2. 2 U/L respectively) was significantly higher than that in the control group (33.7±2.8 U/L) (P<0.01). An increased NO concentration was also found in the Ang II (189. 8±9.0 μmol/L) and the Ang II+catechin treatment groups (276.4±10.1 μmol/L) compared with that in the control group (105.8±9.8 μmol/L) (P<0.01). There were significant differences in the concentrations of O2—? and NO between the Ang II and the Ang II+catechin treatment groups (P<0.05). The mRNA (P<0.05) and protein expression (P<0.01) of eNOS in the Ang II and the Ang II+catechin treatment groups increased significantly compared with those in the control group. The Ang II+catechin treatment group showed increased eNOS protein expression compared with the Ang II group (P<0.05). CONCLUSIONS: Ang II may induce the generation of O2—?, inactivate NO and increase gene and protein expression of eNOS in EPCs. Catechin might decrease the apoptosis of EPCs through the effective clearance of O2—? and the reduction of NO inactivation and of eNOS protein uncoupling.［Chin J Contemp Pediatr, 2009, 11 (6):476-480］