ERK和CREB磷酸化在硫氢化钠对大鼠脑缺血再灌注神经保护的机制探讨

Roles of ERK and CREB phosphorylation in sodium hydrosulfide-induced neuroprotection after cerebral ischemia-reperfusion in rats

摘要:

目的 探讨磷酸化细胞外调节蛋白激酶(p-ERK)和磷酸化cAMP反应元件结合蛋白(p-CREB)在外源性硫化氢(NaHS)干预下对大鼠脑缺血再灌注的神经保护作用及机制。方法 将180只雄性SD大鼠随机分为3组:假手术组、模型组和NaHS干预组(100 μmol/L),每组60只。线栓法制备大鼠大脑中动脉栓塞模型,2 h后再灌注,灌注前20 min给予生理盐水或NaHS腹腔注射,术后6 h、1 d、2 d、5 d、7 d后行TTC染色观察脑梗死体积,尼氏染色检测神经元表达,Western Blot检测海马p-ERK1/2、p-CREB表达,免疫组化检测Bcl-2表达。结果 干预组脑梗死体积和神经元凋亡相对于模型组显著减少(P<0.05)。与模型组相比,干预组各时间点p-ERK1/2、p-CREB蛋白表达均升高(P<0.05),两者之间呈正相关;干预组Bcl-2相对于模型组升高(P<0.05)。结论 大鼠脑缺血再灌注损伤后NaHS可能通过诱导ERK1/2和CREB蛋白磷酸化,激活下游Bcl-2蛋白表达,起到抗凋亡作用,减少脑梗死体积,发挥神经保护作用。

Abstract:

Objective To investigate the roles and mechanisms of action phosphorylated extracellular signal-regulated kinase1/2 (p-ERK1/2) and phosphorylated cAMP response element binding protein (p-CREB) in exogenous sodium hydrosulfide (NaHS)-induced neuroprotection after cerebral ischemia-reperfusion (I/R) in rats.Methods A total of 180 male Sprague-Dawley rats were randomly and equally divided into sham-operation group, model group, and NaHS (100 μmol/L) intervention group. The rat model of middle cerebral artery occlusion was established by intraluminal suture technique. Reperfusion was performed after 2 hrs. Rats were given saline solution or NaHS by intraperitoneal injection 20 min before reperfusion. At 6 hrs, 1 day, 2 days, 5 days, and 7 days after surgery, triphenyltetrazolium chloride staining was used to observe volume of cerebral infarction; Nissl staining was used to evaluate apoptosis in neurons. Western blot was used to determine the expression of p-ERK1/2 and p-CREB in the hippocampus; immunohistochemical analysis was used to measure the expression of B cell lymphoma/lewkmia-2 (Bcl-2).Results Compared with the model group, the volume of cerebral infarction and apoptosis in neurons were significantly reduced in the NaHS intervention group (P<0.05). At each time point, the protein expression of p-ERK1/2 and p-CREB was significantly higher in the NaHS intervention group than in the model group (P<0.05). The expression of p-ERK1/2 was positively correlated with the expression of p-CREB. Moreover, the NaHS intervention group had significantly higher expression of Bcl-2 than the model group (P<0.05).Conclusions After cerebral I/R injury in rats, NaHS may protect neurons against apoptosis and reduce volume of cerebral infarction by inducing phosphorylation of ERK1/2 and CREB and stimulating expression of downstream Bcl-2.