腺苷预处理对脑缺血再灌注损伤大鼠脑组织中微RNA-29b及凋亡蛋白B细胞淋巴瘤-2、磷酸化蛋白激酶B、人髓细胞白血病1表达的影响

Effect of adenosine preconditioning on the expression of microRNA,B-lymphoma-2, phosphorylated protein kinase B and human myeloid leukemia-1 in brain tissue of rats with cerebral ischemia reperfusion injury

目的观察腺苷预处理(AP)对大鼠脑缺血再灌注(IR)后脑组织中微RNA-29b(miR-29b)及凋亡蛋白B细胞淋巴瘤-2(Bcl-2)、磷酸化蛋白激酶B(p-Akt)、人髓细胞白血病1(Mcl-1)表达的影响,探讨AP对大鼠脑缺血再灌注损伤(CIRI)的保护作用及机制。方法将健康雄性清洁级Sprague Dawley大鼠135只随机分为假手术组、IR组、AP组,每组45只。AP组大鼠在手术前3 d腹腔注射2 mL腺苷注射液(1.5 mg·kg^(-1)),每日1次;假手术组和IR组大鼠在手术前3 d每日相同时间点腹腔注射2 mL生理盐水。IR组及AP组大鼠采用改良线栓法行脑中动脉栓塞并于栓塞2 h后拨出栓线恢复血供制备IR模型,假手术组大鼠不做颈动脉结扎,其余手术方法同IR组和AP组。根据5分制神经行为学评分标准对大鼠进行神经功能损伤评分;术后24 h分别取各组造模成功大鼠,断头处死,应用氯化三苯四氮唑染色法观察脑梗死情况,并计算脑梗死体积占比;苏木精-伊红染色观察大鼠海马区神经元细胞存活情况,实时荧光定量聚合酶链反应法检测各组大鼠海马组织内miR-29b表达,Western blot法检测大鼠海马组织中Bcl-2、p-Akt、Mcl-1表达。结果假手术组、IR组和AP组大鼠神经行为学评分和脑梗死体积占比比较差异有统计学意义(F=186.500、191.300,P<0.05),IR组和AP组大鼠神经行为学评分和脑梗死体积占比显著高于假手术组(P<0.05),AP组大鼠神经行为学评分和脑梗死体积占比显著低于IR组(P<0.05)。假手术组大鼠脑皮质神经元形态结构正常;IR组大鼠脑皮质神经元排列紊乱、数目明显减少、结构疏松,细胞体积变小,细胞核溶解、出现空泡,细胞膜完整性破坏,细胞呈三角形或梭形,间质水肿;与IR组比较,AP组大鼠神经元损伤显著减轻。手术后2、6、24、48 h, IR组和AP组大鼠海马组织miR-29b相对表达量显著高于假手术组(P<0.05),AP组大鼠海马组织miR-29b相对表达量显著高于IR组(P<0.05)。假手术组大鼠手术后2、6、24、48 h海马组织miR-29b相对表达量比较差异无统计学意义(P>0.05);IR组大鼠手术后2、6、24 h海马组织miR-29b相对表达量呈增高趋势,手术后48 h开始降低(P<0.05);AP组大鼠手术后2、6、24 h海马组织miR-29b相对表达量呈增高趋势,手术后48 h开始降低(P<0.05)。假手术组大鼠手术后2、6、24、48 h海马组织Bcl-2、Mcl-1和p-Akt相对表达量比较差异无统计学意义(P>0.05);IR组和AP组大鼠手术后2、6、24 h脑组织Bcl-2、Mcl-1和p-Akt相对表达量呈显著增高趋势(P<0.05),手术后48 h开始降低。手术后2、6、24、48 h, IR组和AP组大鼠海马组织Bcl-2相对表达量显著高于假手术组,AP组大鼠海马组织Bcl-2相对表达量显著高于IR组(P<0.05)。IR组大鼠手术后2、6、24 h海马组织Mcl-1相对表达量显著高于假手术组,手术后48 h显著低于假手术组(P<0.05);AP组大鼠手术后2、6、24 h海马组织Mcl-1相对表达量显著高于假手术组(P<0.05);AP组与假手术组大鼠手术后48 h海马组织Mcl-1相对表达量比较差异无统计学意义(P>0.05);AP组大鼠手术后2、6、24、48 h海马组织Mcl-1相对表达量显著高于IR组(P<0.05)。IR组大鼠手术后2、6、24、48 h海马组织p-Akt相对表达量显著高于假手术组(P<0.05);AP组大鼠手术后2、6、24 h海马组织p-Akt相对表达量显著高于假手术组,手术后48 h海马组织p-Akt相对表达量显著低于假手术组(P<0.05);AP组大鼠手术后2、6、24、48 h海马组织p-Akt相对表达量显著低于IR组(P<0.05)。结论 AP可上调大鼠脑IR后miR-29b的表达,miR-29b通过下调海马组织中Akt蛋白的磷酸化水平、促进抗凋亡蛋白Bcl-2和Mcl-1的表达来调控细胞凋亡,从而发挥对CIRI的保护作用。

Objective To observe the adenosine preconditioning(AP) on the expressions of microRNA-29 b(miR-29 b),B-lymphoma-2(Bcl-2),phosphorylated protein kinase B(p-Akt) and human myeloid leukemia-1(Mcl-1) in brain tissue of rats after cerebral ischemia reperfusion(IR),and to investigate the protective effect and mechanism of AP on cerebral ischemia reperfusion injury(CIRI) in rats.Methods One hundred and thirty-five healthy male Sprague Dawley rats were randomly divided into sham operation group, IR group and AP group, with 45 rats in each group.The rats in the AP group were intraperitoneally injected with 2 mL adenosine injection(1.5 mg· kg^(-1)) once a day for 3 days before operation, while the rats in the sham operation group and IR group were intraperitoneally injected with 2 mL saline at the same time point for 3 days before operation.The rats in the IR group and AP group were underwent middle cerebral artery occlusion with improved thread embolization method, 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 ligated, and other operation methods were same as those in the IR group and AP group.The neurobehavioral scoring standard of 5-point system was used to evaluate the neurological injury of rats;24 hours after operation, the rats in each group were decapitated and killed, and the brain tissue was taken, the cerebral infarction was observed by triphenyltetrazolium chloride staining, and the proportion of cerebral infarction volume was calculated.The survival of hippocampal neurons was observed by hematoxylin-eosin staining, the expression of miR-29 b in brain tissue was detected by real-time fluorescence quantitative polymerase chain reaction, and the expression of Bcl-2,p-Akt and Mcl-1 protein in hippocampus were detected by Western blot.Results There were significant differences in the neurobehavioral score and the proportion of cerebral infarction volume of rats among the sham group, IR group and AP group(F=186.500,191.300;P<0.05).The neurobehavioral score and the proportion of cerebral infarction volume of rats in the AP group and IR group were significantly lower than those in the sham operation group(P<0.05),the neurobehavioral score and the proportion of cerebral infarction volume of ratsin the AP group were significantly lower than those in the IR group(P<0.05).The morphology and structure of cerebral cortical neurons of rats in the sham operation group was normal;while in the IR group, the arrangement of cortical neurons was disordered, the number decreased significantly, the structure was loose, the cell volume became smaller, the nucleus dissolved, vacuoles appeared, the integrity of cell membrane was destroyed, the cells were triangular or fusiform, and interstitial edema;compared with the IR group, the neuronal injury of rats in the AP group was significantly lightened.At 2,6,24 and 48 hours after operation, the relative expression of miR-29 b in hippocampus of rats in the IR group and AP group was significantly higher than that in the sham operation group(P<0.05),and the relative expression of miR-29 b in hippocampus of rats in the AP group was significantly higher than that in the IR group(P<0.05).There was no significant difference in the relative expression of miR-29 b in hippocampus of rats at 2,6,24 and 48 hours after operation in the sham operation group(P>0.05);in the IR group, the relative expression of miR-29 b in hippocampus of rats increased at 2,6 and 24 hours after operation, and began to decrease at 48 hours after operation(P<0.05);in the AP group, the relative expression of miR-29 b in hippocampus of rats increased at 2,6 and 24 hours after operation, and began to decrease at 48 hours after operation(P<0.05).There was no significant difference in the relative expression of Bcl-2,Mcl-1 and p-Akt in hippocampus of rats at 2,6,24 and 48 hours after operation in the sham operation group(P>0.05);the relative expressions of Bcl-2,Mcl-1 and p-Akt in hippocampus of rats in the IR group and AP group increased significantly at 2,6 and 24 hours after operation(P<0.05),and began to decrease at 48 hours after operation.At 2,6,24 and 48 hours after operation, the relative expression of Bcl-2 in hippocampus of rats in the IR group and AP group was significantly higher than that in the sham operation group, and the relative expression of Bcl-2 in hippocampus of rats in the AP group was significantly higher than that in the IR group(P<0.05).The relative expression of Mcl-1 in hippocampus of rats in the IR group was significantly higher than that in the sham operation group at 2,6 and 24 hours after operation(P<0.05),and significantly lower than that in the sham operation group at 48 hours after operation(P<0.05);the relative expression of Mcl-1 in hippocampus of rats in the AP group was significantly higher than that in the sham operation group at 2,6 and 24 hours after operation(P<0.05);there was no significant difference in the relative expression of Mcl-1 between the AP group and the sham operation group at 48 hours after operation(P>0.05);the relative expression of Mcl-1 in hippocampus of rats in the AP group was significantly higher than that in the IR group at 2,6,24 and 48 hours after operation(P<0.05).The relative expression of p-Akt in hippocampus of rats in the IR group was significantly higher than that in the sham operation group at 2,6,24 and 48 hours after operation(P<0.05);the relative expression of p-Akt in hippocampus of rats in the AP group at 2,6 and 24 hours after operation was significantly higher than that in the sham operation group, and the expression of p-Akt in hippocampus at 48 after operation hours was significantly lower than that in the sham operation group(P<0.05);the relative expression of p-Akt in hippocampus of rats in the AP group was significantly lower than that in the IR group at 2,6,24 and 48 hours after operation(P<0.05).Conclusion AP can up-regulate the expression of miR-29 b after cerebral IR in rats. MiR-29 b can regulate apoptosis by down-regulating the phosphorylation level of Akt protein in brain tissue and promoting the expression of anti-apoptotic proteins Bcl-2 and Mcl-1, so as to play a protective role for CIRI.