RhoA/ROCK2信号通路在三叶苷减轻大鼠脑缺血再灌注损伤中的作用

Role of RhoA/ROCK2 signaling pathway in trilobatin-induced reduction of cerebral ischemia-reperfusion injury in rats

目的:评价Rho亚家族蛋白A(RhoA)/Rho相关的卷曲连接蛋白激酶2(ROCK2)信号通路在三叶苷减轻大鼠脑缺血再灌注损伤中的作用。方法:清洁级健康雄性SD大鼠80只,6~8周龄,体重230~280 g,采用随机数字表法分为4组(n=20):假手术组(S组)、脑缺血再灌注组(IR组)、脑缺血再灌注+三叶苷组(T组)和脑缺血再灌注+三叶苷+RhoA/ROCK2信号通路激动剂AA组(A组)。采用大脑中动脉栓塞法制备大鼠局灶性脑缺血再灌注损伤模型。T组和A组大鼠于缺血前3 d给予三叶苷15 mg/kg灌胃,2次/d,连续3 d,A组大鼠于每次灌胃前腹腔注射RhoA/ROCK2信号通路激动剂AA 10 mg/kg。于再灌注24 h时行神经行为学评分,随后处死大鼠取海马组织,采用流式细胞术检测海马神经元凋亡率,TTC染色确定脑梗死体积,采用Western blot法检测磷酸化RhoA(p-RhoA)、ROCK2和裂解的caspase-3(cleaved caspase-3)的表达,透射电镜下观察海马神经元超微结构。结果:与S组比较,IR组大鼠神经行为学评分和海马神经元凋亡率升高,脑梗死体积增加,p-RhoA、ROCK2和cleaved caspase-3表达上调(P<0.05),海马神经元病理学损伤加重;与IR组比较,T组大鼠神经行为学评分和海马神经元凋亡率降低,脑梗死体积减少,p-RhoA、ROCK2和cleaved caspase-3表达下调(P<0.05),海马神经元病理学损伤减轻;与T组比较,A组大鼠神经行为学评分和海马神经元凋亡率升高,脑梗死体积增加,p-RhoA、ROCK2和cleaved caspase-3表达上调(P<0.05),海马神经元病理学损伤加重。结论:RhoA/ROCK2信号通路参与了三叶苷减轻大鼠脑缺血再灌注损伤的过程,可能与抑制海马神经元凋亡有关。

Objective To evaluate role of Ras homolog gene family member A(RhoA)/Rho-associated coiled-coil protein kinase 2(ROCK2)signaling pathway in trilobatin-induced reduction of cerebral ischemia-reperfusion(I/R)injury in rats.Methods Eighty clean-grade healthy male Sprague-Dawley rats,aged 6-8 weeks,weighing 230-280 g,were divided into 4 groups(n=20 each)using a random number table method:sham operation group(group S),cerebral I/R group(group IR),cerebral I/R plus trilobatin group(group T)and cerebral I/R plus trilobatin plus RhoA/ROCK2 signaling pathway agonist AA group(group A).The model of focal cerebral I/R injury was developed by middle cerebral artery occlusion in anesthetized animals.Trilobatin 15 mg/kg was given by gavage twice a day for 3 consecutive days in T and A groups.RhoA/ROCK2 signaling pathway agonist AA 10 mg/kg was intraperitoneally injected before each administration by gavage in group A.Neurobehavioral score was assessed at 24 h of reperfusion,then the rats were sacrificed,and the hippocampal tissues were isolated for determination of the apoptosis rate of hippocampal neurons(by flow cytometry),cerebral infarction volume(by TTC staining),and expression of phosphorylated RhoA(p-RhoA),ROCK2 and cleaved caspase-3(by Western blot)and for microscopic examination of ultrastructure of hippocampal neurons(with a transmission electron microscope).Results Compared with group S,the neurobehavioral score,apoptosis rate of hippocampal neurons,and cerebral infarction volume were significantly increased,the expression of p-RhoA,ROCK2 and cleaved caspase-3 was up-regulated(P<0.05),and the pathological damage to hippocampal neurons was aggravated in group IR.Compared with group IR,the neurobehavioral score,apoptosis rate of hippocampal neurons and cerebral infarction volume were significantly decreased,the expression of p-RhoA,ROCK2 and cleaved caspase-3 was down-regulated(P<0.05),and the pathological damage to hippocampal neurons was alleviated in group T.Compared with group T,the neurobehavioral score,apoptosis rate of hippocampal neurons,and cerebral infarction volume were significantly increased,the expression of p-RhoA,ROCK2 and cleaved caspase-3 was up-regulated(P<0.05),and the pathological damage to hippocampal neurons was aggravated in group A.Conclusions RhoA/ROCK2 signaling pathway is involved in trilobatin-induced reduction of cerebral I/R injury,which may be related to inhibition of apoptosis in hippocampal neurons of rats.