腺苷预处理对脑缺血再灌注损伤大鼠脑组织中CCAAT/增强子结合蛋白同源蛋白表达的影响

目的　观察腺苷预处理（AP）对脑缺血再灌注损伤大鼠脑组织中CCAAT/增强子结合蛋白同源蛋白（CHOP）表达的影响，探讨AP对脑缺血再灌注损伤大鼠的神经保护作用机制。方法　将36只健康雄性Sprague Dawley大鼠按随机数字表法分为假手术组、缺血再灌注（IR）组和AP组，每组12只。AP组大鼠术前3 d腹腔注射1.5 mg·kg^-1腺苷注射液，每日1次；假手术组和IR组大鼠术前3 d腹腔注射等量生理盐水，每日1次。IR组及AP组大鼠采用线栓法制备大脑中动脉栓塞（MCAO）模型，并于栓塞2 h后拔出栓线恢复血供制备IR模型；假手术组大鼠不插入栓线，余处理同IR组及AP组。于再灌注24 h时，根据5分制神经行为学评分标准对3组大鼠进行神经功能缺损评分；然后，断头处死3组大鼠，取脑组织；应用苏木精-伊红染色法观察3组大鼠脑组织病理学改变，实时荧光定量聚合酶链式反应法检测3组大鼠脑组织中CHOP mRNA相对表达量，免疫组织化学法检测3组大鼠脑组织中CHOP蛋白阳性表达情况，脱氧核糖核苷酸末端转移酶介导的缺口末端标记法检测3组大鼠脑组织细胞凋亡情况。结果　3组大鼠神经功能缺损评分比较差异有统计学意义（F=157.923，P<0.05）；IR 组和AP组大鼠神经功能缺损评分显著高于假手术组(t=17.663、7.133，P<0.05)，AP组大鼠神经功能缺损评分显著低于IR 组(t=-10.530，P<0.05)。假手术组大鼠脑组织神经元形态结构正常，细胞核完整，间质无水肿；IR组大鼠脑组织神经元数目减少、细胞核溶解、出现空泡，间质水肿，组织疏松；AP组大鼠神经元损伤程度显著低于IR组。3组大鼠脑组织中CHOP mRNA相对表达量比较差异有统计学意义（F=2 989.751，P<0.05）；IR组和AP组大鼠脑组织中CHOP mRNA相对表达量显著高于假手术组 (t=75.514、23.502，P<0.05)，AP组大鼠脑组织中CHOP mRNA相对表达量显著低于IR 组(t=-52.171，P<0.05)。3组大鼠脑组织中CHOP 蛋白阳性细胞表达率比较差异有统计学意义（F=2 257.096，P<0.05）；IR组和AP组大鼠脑组织中CHOP 蛋白阳性细胞表达率显著高于假手术组(t=65.927、21.744，P<0.05)，AP组大鼠脑组织中CHOP 蛋白阳性细胞表达率显著低于IR组(t=-44.183，P<0.05)。3组大鼠脑组织细胞凋亡率比较差异有统计学意义（F=1 519.372，P<0.05）；IR组和AP组大鼠脑组织细胞凋亡率显著高于假手术组(t=5.512、2.693，P<0.05)，AP组大鼠脑组织细胞凋亡率显著低于IR组(t=-2.818，P<0.05)。结论　AP可通过抑制CHOP的表达减轻脑缺血再灌注损伤。

Effect of adenosine pretreatment on expression of CCAAT/enhancer binding protein homologous protein in brain tissue of rats with cerebral ischemia-reperfusion injury

Objective　To observe the effect of adenosine pretreatment (AP) on the expression of CCAAT/enhancer binding protein homologous protein (CHOP) in brain tissue of rats with cerebral ischemia-reperfusion injury,and to explore the neuroprotective mechanism of AP on cerebral ischemia-reperfusion injury in rats.
Methods　Thirty-six healthy male Sprague Dawley rats were divided into sham operation group,ischemia reperfusion(IR) group and AP group according to random number table method,with 12 rats in each group.The rats in the AP group were intraperitoneally injected with 1.5 mg·kg^-1 adenosine injection on three days before surgery,once a day;the rats in the sham operation group and IR group were intraperitoneally injected with the same amount of normal saline on three days before surgery,once a day.The middle cerebral artery occlusion(MCAO) models were established by wire embolization in the IR group and AP group,and the thread was pulled out at 2 hours after embolization to prepare the IR models.The rats in the sham operation group were not inserted with wire embolization,and the residual treatments were same as those in the IR group and AP group.At 24 hours of reperfusion,the rats in the three groups were performed with the neurological impairment score according to the 5-point neurobehavioral scale.Then,the rats in the three groups were sacrificed,and the brain tissues of the rats were taken;the histopathological changes in brain tissues of rats in the three groups were observed by hematoxylin-eosin,the relative expression of CHOP mRNA in the brain tissues of rats in the three groups was detected by quantitative real-time polymerase chain reaction,the positive expression of CHOP protein in the brain tissues of rats in the three groups was detected by immunohistochemistry,and the cell apoptosis in the brain tissues of rats in the three groups was detected by TdT mediated dUTP nick end labeling method.
Results　There was significant difference in neurological impairment score of rats among the three groups (F=157.923,P<0.05).The neurological impairment score of rats in the IR group and AP group was significantly higher than that in the sham operation group (t=17.663,7.133;P<0.05),and the neurological impairment score of rats in the AP group was significantly lower than that in the IR group (t=-10.530,P<0.05).In the sham operation group,the morphology and structure of brain neurons of rats were normal,the nuclei were intact,and the interstitial edema was not observed.In the IR group,the number of neurons decreased,the nuclei dissolved,the vacuoles appeared,the interstitial edema was observed and the tissue was loose.The injury degree of neurons of rats in the AP group was significantly lower than that in the IR group.There was significant difference in the relative expression level of CHOP mRNA in brain tissues of rats among the three groups (F=2 989.751,P<0.05).The relative expression level of CHOP mRNA in brain tissues of rats in the IR group and AP group was significantly higher than that in the sham operation group (t=75.514,23.502;P<0.05),and the relative expression level of CHOP mRNA in brain tissues of rats in the AP group was significantly lower than that in the IR group (t=-52.171,P<0.05).There was significant difference in the rate of CHOP protein positive cells in brain tissues of rats among the three groups(F=2 257.096,P<0.05).The rate of CHOP protein positive cells in brain tissue of rats in the IR group and AP group was significantly higher than that in the sham operation group (t=65.927,21.744;P<0.05),and the rate in CHOP protein positive cells in brain tissue of rats in the AP group was significantly lower than that in the IR group (t=-44.183,P<0.05).There was significant difference in the cell apoptosis rate in brain tissue of rats among the three groups (F=1 519.372,P<0.05).The cell apoptosis rate in brain tissue of rats in the IR group and AP group was significantly higher than that in the sham operation group (t=5.512,2.693;P<0.05),and the cell apoptosis rate in brain tissue of rats in the AP group was significantly lower than that in the IR group (t=-2.818,P<0.05).
Conclusion　AP can reduce cerebral ischemia-reperfusion injury by inhibiting the expression of CHOP.