基于ERK5信号通路探讨薯蓣健脾益智合剂对血管性痴呆大鼠海马神经元凋亡的影响

Effect of Modified Shuyu Pill on Hippocampal Neuron Apoptosis in Vascular Dementia Rats Based on ERK5 Signaling Pathway

目的:探讨薯蓣健脾益智合剂对血管性痴呆(VaD)大鼠海马神经元凋亡的影响以及相关作用机制。方法:采用改良双侧颈总动脉结扎(2-VO)法建立VaD大鼠模型,将120只大鼠随机分为正常组、假手术组、模型组和治疗组,每组30只。治疗组予以10 mL/(kg·d)薯蓣健脾益智合剂灌胃,正常组、假手术组、模型组给予等量生理盐水灌胃,持续8周。观察大鼠一般状态,并于造模后第7天及造模后治疗4、8周分别进行行为学检测,苏木精-伊红(HE)染色观察海马神经元组织学改变,脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)染色观察海马神经元凋亡情况,免疫组织化学法检测丝分裂原活化蛋白激酶K2(MEKK2)、丝分裂原活化蛋白激酶K3(MEKK3)和丝裂原激活蛋白激酶5(MEK5)、细胞外信号调节激酶5(ERK5)表达的变化,蛋白免疫印迹法检测MEKK2、MEKK3和MEK5、ERK5的蛋白表达。结果:与正常组及假手术组比较,模型组行为学表现差异显著,变性神经元增加,神经元凋亡率增加(P<0.01),MEKK2、MEKK3和MEK5、ERK5的表达减少;与模型组比较,治疗组行为学表现改善,变性神经元减少,神经元凋亡率降低(P<0.05或P<0.01),MEKK2、MEKK3和MEK5、ERK5表达增加。结论:薯蓣健脾益智合剂可能通过上调ERK5信号通路,抑制海马神经元凋亡,促进海马神经元的修复进而治疗VaD。

Objective:To investigate the effect of modified Shuyu Pill on hippocampal neuron apoptosis in rats with vascular dementia(VaD).Methods:The modified bilateral common carotid artery ligation(2-VO)was used to establish the VaD rat model.One hundred and twenty rats were randomly divided into normal group,control group,model group,and treatment group,with 30 rats in each group.The treatment group was given 10 mL/(kg·d)modified Shuyu Pill by intragastric administration.The normal group,control group,and model group were given the same volume of normal saline.The intervention lasted for 8 weeks.Behavior detection were performed at the 7th day after modeling,and 4 and 8 weeks,vespectively after treatment.Histological changes of hippocampal neurons were observed by hematoxylin-eosin(HE)staining.The apoptosis of hippocampal neurons was observed by terminal deoxyribonucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL).The expression of mitogen-activated protein kinase K2(MEKK2),mitogen-activated protein kinase K3(MEKK3),mitogen-activated protein kinase 5(MEK5),and extracellular signal-regulated kinase 5(ERK5)were detected by immunohistochemistry.The protein expressions of MEKK2,MEKK3,MEK5,and ERK5 were detected by Western Blot.Results:Compared with the normal group and control group,the behavior of the model group was significantly different,the degeneration of neurons increased,the apoptosis rate of neurons increased(P<0.01),and the expressions of MEKK2,MEKK3,MEK5,and ERK5 decreased.Compared with the model group,the behavioral performance of the treatment group improved,the degeneration of neurons reduced,the apoptosis rate of neurons decreased(P<0.05 or P<0.01),and the expressions of MEKK2,MEKK3,MEK5,and ERK5 significantly increased.Conclusion:Modified Shuyu Pill may inhibit the apoptosis of hippocampal neurons and promote the repair of hippocampal neurons to treat VaD by up-regulating ERK5 signaling pathway.