p53-PUMA通路在富氢液减轻大鼠全脑缺血再灌注损伤中的作用

Role of p53-PUMA pathway in mitigation of global cerebral ischemia-reperfusion injury by hydrogen-rich saline in rats

目的 评价肿瘤抑制因子p53-p53上调凋亡调控因子（PUMA）通路在富氢液减轻大鼠全脑缺血再灌注损伤中的作用.方法 雄性SD大鼠60只,体重280-320 g,3月龄,采用随机数字表法分为6组（n=10）：假手术组（S组）、脑缺血再灌注组（I/R组）、富氢液组（H组）、PBS溶剂对照组（PBS组）、p53抑制剂pifithrin-α组（P组）和pifithrin-α＋富氢液组（PH组）.I/R组、H组、P组和PH组采用经食管心脏起搏诱发心脏骤停后心肺复苏建立大鼠全脑缺血再灌注损伤模型.H组和PH组于再灌注即刻和6h时腹腔注射富氢液5 ml/kg,I/R组腹腔注射等容量生理盐水;缺血前15 min时P组和PH组于右侧股静脉注射pifithrin-α 4 mg/kg,PBS组右侧股静脉注射等容量PBS.再灌注24h时行神经功能评分,然后处死大鼠,取海马组织,采用TUNEL进行凋亡神经元计数,采用Western blot测定p53和PUMA的蛋白表达.结果 与S组比较,I/R组和PBS组神经功能评分降低,海马CA1区凋亡神经元计数增加,海马组织p53和PUMA蛋白表达上调（P＜0.05）;与I/R组比较,H组、P组和PH组神经功能评分升高,海马CA1区凋亡神经元计数减少,海马组织p53和PUMA蛋白表达下调（P＜0.05）;与H组比较,PH组神经功能评分升高,海马CA1区凋亡神经元计数减少,海马组织p53和PUMA蛋白表达下调（P＜0.05）.结论 富氢液通过抑制p53-PUMA通路激活减轻大鼠全脑缺血再灌注损伤.

Objective To evaluate the role of p53-PUMA pathway in mitigation of global cerebral ischemia-reperfusion （I/R） injury by hydrogen-rich saline in rats.Methods Sixty male Sprague-Dawley rats,aged 3 months,weighing 280-320 g,were randomly divided into 6 groups （n =10 each）：sham operation group（group S）,I/R group,hydrogen-rich saline group （group H）,PBS vehicle group （group PBS）,p53 inhibitor pifithrin-α group （group P）,and pifithrin-α ＋ hydrogen-rich saline group （group PH）.Cardiac arrest （CA） was induced by transoesophageal cardiac pacing followed by cardiopulmonary resuscitation to establish the global cerebral I/R model.In H and PH groups,hydrogen-rich saline 5 ml/kg was injected intraperitoneally immediately after onset of reperfusion and at 6 h of reperfusion,while the equal volume of normal saline was given in I/R group.At 15 min before ischemia,pifithrin-α 4 mg/kg was injected via the right femoral vein in P and PH groups,and the equal volume of PBS was given in PBS group.Neurodeficit Score （NDS） was evaluated at 24 h of reperfusion.The rats were then sacrificed at 24 h of reperfusion,and then the bilateral hippocampi were removed for detection of cell apoptosis （using TUNEL） and the protein expression of p53 and PUMA （using Western blot）.Results Compared with group S,NDS was significantly decreased,the number of apoptotic neurons was increased,and the protein expression of p53 and PUMA was up-regulated in I/R and PBS groups.Compared with group I/R,NDS was significantly increased,the number of apoptotic neurons was decreased,and the protein expression of p53 and PUMA was down-regulated in H,P and PH groups.Compared with group H,NDS was significantly increased,the number of apoptotic neurons was decreased,and the protein ex.pression of p53 and PUMA was down-regulated in group PH.Conclusion Hydrogen-rich saline mitigates global cerebral I/R injurythrough inhibiting the activation of p53-PUMA pathway in rats.