缺氧前、后处理1-磷酸鞘氨醇对心肌细胞缺氧／复氧损伤拮抗作用的差异及相关机制

目的：探讨1-磷酸鞘氨醇（S1P）缺氧前、后处理对心肌细胞缺氧／复氧（H／R）损伤拮抗作用的差异及相关机制。方法：分离SD乳鼠（1—2d龄）心肌细胞进行体外培养。对培养的心肌细胞进行H／R处理以模拟心脏缺血／再灌注（ischemia／reperfusion，r／R）损伤。将培养的心肌细胞随机分为对照组、H／R组、H／R＋SIP缺氧前处理（H／R’pre—S1P）组及I-I／R＋SIP缺氧后处理（I-I／R＋post—S1P）组，采用四甲基偶氮唑蓝（methylthiazolyltetrazo｜ium。MTT）比色法检测心肌细胞的活力，乳酸脱氢酶（1actatedehydrogenase，LDH）检测试剂盒检测培养基中LDH的水平，caspase-3活性检测试剂盒检测心肌细胞中caspase．3的活性，Westernblot检测ATP激活的蛋白激酶（AMPK）、激活的苏氨酸激酶（Akt）、细胞外信号调节激酶1／2（ERKl／2）蛋白磷酸化水平及SIP裂解酶．1（SIPlyase1，SPLl）蛋白表达的水平。结果：与对照组相比，I-1／R组心肌细胞活力显著降低（P〈0．01），培养基中LDH的浓度和细胞中easpase-3的活性显著增高（P〈0．01）。S1P缺氧前处理可明显增加H／R处理后心肌细胞的活力（P〈0．01），减少LDH释放和caspase-3的活性（P〈0．01）；而I-I／R＋post—S1P组的各项指标与H／R组比较均无明显差异。Westernblot检测发现，S1P缺氧前处理可明显增高心肌细胞中AMPK、Akt和ERkl／2磷酸化的水平（P〈0．05），而SIP缺氧后处理未激活AMPK、Akt和ERKl／2生存信号。Westernblot检测发现，缺氧处理组心肌细胞中SPLl表达的水平较对照组显著升高（P〈0．01）。结论：缺氧前用S1P处理可激活AMPK、Akt、ERKl／2生存信号，显著减少心肌细胞凋亡，增加细胞的存活率，减轻心肌细胞H／R损伤；而S1P缺氧后处理不能发挥以上保护作用，其机制可能是缺氧处理导致心肌细胞SPLl的表达上调，通过降解SIP而减弱了其介导的抗心肌细胞H／R损伤的作用。

Difference and mechanism of SIP-mediated protection against hypoxia/ reoxygenation induced cardiomyocyte injury between pre- and post-hy- poxia treatment

AIM： To investigate the difference of sphingosine-l-phosphate （S1P）-mediated protection against hypoxia/reoxygenation （H/R） induced cardiomyocyte injury between pre- and post-hypoxia treat- ment and the mechanism（s） involved. METHODS： Neonatal rat ventricular myocytes （NRVMs） were isolated from 1- to 2-day-old Sprague Dawley （SD） rats and were subjected to 9 h of hypoxia followed by 3 h of reoxygenation. After pre- or post-hypoxia treatment with 1 μmol/L S1P, cell viability was detected by MTr assay. LDH release and caspase-3 activity were detected by assay kits. AMPK, Akt and ERK1/2 phosphorylation levels and S1P lyase 1 （SPL1） expression levels were analyzed by Western blot. RESULTS：In cultured NRVMs, pre-hypoxia treatment with 1 txmol/L S1P increased AMPK, Akt and ERK1/2 phosphorylation levels （ all P 〈 0. 05 ） increased cell viability and reduced LDH release and caspase-3 activity after H/R （all P 〈 0.01 ）. However, post-hypoxia treatment with 1 txmol/L S1P failed to upreg- ulate AMPK, Akt and ERK1/2 phosphorylation levels and lost its cardioprotective effects compared with those in H/R group. SPL1 expression level in NRVMs increased after hypoxia treatment （P 〈 0.01 ）. CONCLUSION： Pre-hypoxia treatment with S1 P, but not post-hypoxia treatment, exerts strong protec- tive effects against H/R injury in NRVMs by decreasing apoptosis. Pre-hypoxia treatment with SIP acti- vates AMPK, Akt and ERK1/2 survival signaling pathway, whereas post-hypoxia treatment with S1P fails to achieve these effects. The mechanisms may be that hypoxia-induced SPL1 upregulation dismisses the protective role of SIP against H/R induced cardiomyocyte injury.