治疗性低温减轻大鼠脑缺血再灌注损伤时GSTM1与ASK1-JNK-p38MAPK信号通路的关系

Relationship between GSTM1 and ASK1-JNK-p38 MAPK signaling pathway during therapeutic hypothermia-induced reduction of cerebral ischemia-reperfusion injury in rats

目的评价治疗性低温减轻大鼠脑缺血再灌注损伤(CIRI)时谷胱甘肽S转移酶μ1(GSTM1)与细胞凋亡信号调节激酶1(ASK1)-c-Jun氨基末端激酶(JNK)/p38丝裂原活化蛋白激酶(p38 MAPK)信号通路的关系。方法清洁级健康雄性SD大鼠100只,8周龄,体质量260~280 g,采用随机数字表法分为5组(n=20):假手术组(S组)、脑缺血再灌注组(I/R组)、治疗性低温组(H组)、GSTM1抑制剂+治疗性低温组(IH组)和GSTM1抑制剂+ASK1抑制剂+治疗性低温组(IAH组)。采用线栓法建立CIRI模型,阻断大鼠左侧大脑中动脉2 h后拔出线栓恢复血流灌注,手术期间维持大鼠脑温36~37℃。H组于再灌注即刻用75%酒精擦拭大鼠头部及颈部,维持脑温32~33℃3 h,其余同I/R组;IH组分别于造模前24和1 h时腹腔注射GSTM1抑制剂依他尼酸8.6 mg/kg,其余同H组;IAH组于造模前4 d开始口服ASK1抑制剂Selonsertib 10 mg/kg,1次/d,连续4 d,其余同IH组。于再灌注24 h行改良神经功能缺损评分(mNSS),随后处死大鼠后取脑,采用TTC染色法观察脑梗死情况并计算梗死体积百分比,Western blot法检测GSTM1、ASK1、磷酸化ASK1(p-ASK1)、JNK、磷酸化JNK(p-JNK)、p-38 MAPK、磷酸化p-38 MAPK(p-p38 MAPK)的表达水平;HE染色观察神经细胞形态改变,TUNEL法检测神经细胞凋亡率。结果与S组比较,I/R组mNSS、神经细胞凋亡率、脑梗死体积百分比、p-ASK1/ASK1比值、p-JNK/JNK比值及p-p38 MAPK/p38MAPK比值升高,GSTM1表达下调(P<0.05);与I/R组和IH组比较,H组mNSS、神经细胞凋亡率、脑梗死体积百分比、p-ASK1/ASK1比值、p-JNK/JNK比值及p-p38 MAPK/p38 MAPK比值降低,GSTM1表达上调(P<0.05),神经细胞损伤减轻;与IH组比较,IAH组mNSS、神经细胞凋亡率、脑梗死体积百分比、p-ASK1/ASK1比值、p-JNK/JNK比值及p-p38 MAPK/p38 MAPK比值降低(P<0.05),GSTM1表达差异无统计学意义(P>0.05),神经细胞损伤减轻。结论治疗性低温减轻大鼠CIRI的机制与上调GSTM1表达,抑制ASK1-JNK-p38 MAPK信号通路激活有关。

Objective To evaluate the relationship between glutathione S-transferaseμ1(GSTM1)and the apoptosis signal-regulating kinase 1(ASK1)-c-Jun N-terminal kinase(JNK)/p38 mitogen-activated protein kinase(MAPK)signaling pathway during therapeutic hypothermia-induced reduction of cerebral ischemia-reperfusion injury(CIRI)in rats.Methods One hundred clean-grade healthy male Sprague-Dawley rats,aged 8 weeks,weighing 260-280 g,were divided into 5 groups(n=20 each)using a random number table method:sham operation group(S group),cerebral ischemia-reperfusion group(I/R group),therapeutic hypothermia group(H group),GSTM1 inhibitor+therapeutic hypothermia group(IH group),and GSTM1 inhibitor+ASK1 inhibitor+therapeutic hypothermia group(IAH group).CIRI model was developed by occlusion of the left middle cerebral artery for 2 h,followed by restoration of the blood flow.A nylon thread was inserted into the internal carotid artery and advanced cephalad until resistance was met.The brain temperature was maintained at 36-37℃during surgery.In H group,the head and neck were wiped with 75%alcohol immediately after reperfusion,and the brain temperature was maintained at 32-33℃for 3 h,and the rest procedures were the same as those previously described in I/R group.In IH group,GSTM1 inhibitor itaconic acid 8.6 mg/kg was intraperitoneally injected at 24 and 1 h before developing the model,and the rest procedures were the same as those previously described in H group.In IAH group,ASK1 inhibitor selonsertib 10 mg/kg was given orally once a day for 4 consecutive days starting from 4 days before developing the model,and the rest procedures were the same as those previously described in IH group.Modified Neurological Severity Score(mNSS)was assessed at 24 h of reperfusion,then the rats were sacrificed and brains were harvested for microscopic examination of brain infarction,neuronal morphology(using HE staining)and for determination of the expression of GSTM1,ASK1,phosphorylated ASK1(p-ASK1),JNK,phosphorylated JNK(p-JNK),p-38 MAPK and phosphorylated p-38 MAPK(p-p38 MAPK)(by Western blot)and neuronal apoptosis(by TUNEL assay).The percentage of the infarct size was calculated using TTC staining.The apoptosis rate was calculated.Results Compared with S group,the mNSS,apoptosis rate of neurons,percentage of the cerebral infarct size,p-ASK1/ASK1 ratio,p-JNK/JNK ratio and p-p38 MAPK/p38 MAPK ratio were significantly increased,and the expression of GSTM1 was down-regulated in I/R group(P<0.05).Compared with I/R group and IH group,the mNSS,apoptosis rate of neurons,percentage of the cerebral infarct size,p-ASK1/ASK1 ratio,p-JNK/JNK ratio and p-p38 MAPK/p38 MAPK ratio were significantly decreased,the expression of GSTM1 was up-regulated(P<0.05),and the neuronal injury was significantly attenuated in H group.Compared with IH group,the mNSS,apoptosis rate of neurons,percentage of the cerebral infarct size,p-ASK1/ASK1 ratio,p-JNK/JNK ratio and p-p38 MAPK/p38 MAPK ratio were significantly decreased(P<0.05),no significant change was found in GSTM1 expression(P>0.05),and the neuronal damage was significantly attenuated in IAH group.Conclusions The mechanism by which therapeutic hypothermia alleviates CIRI is related to up-regulating the expression of GSTM1 and inhibiting the activation of the ASK1-JNK-p38 MAPK signaling pathway in rats.