miR-143-3p对脑梗死大鼠神经细胞凋亡及Nrg-1/ErbB4信号通路的影响

Effects of miR-143-3p on neuronal apoptosis and Nrg-1/ErbB4 signaling pathway in rats with cerebral infarction

目的探讨微小核糖核酸(miR)-143-3p对神经调节蛋白(Nrg)-1/erb-b2受体酪氨酸激酶(ErbB)4信号通路的调节作用,及对脑梗死大鼠神经细胞凋亡的影响.方法将2018年3月-2019年3月在南充精神卫生中心医院神经内科就诊的30例脑梗死患者,分为脑梗死轻度组(脑梗死体积<5cm^(3))、中度组(脑梗死体积在5~15cm^(3))、重度组(脑梗死体积>15cm^(3)),每组10例,另取同期在本院接受体检的健康志愿者10例,作为正常组,用RT-qPCR法测血清中miR-143-3p表达水平.取SD大鼠,随机分为假手术组、模型组、miR-143-3p沉默(si-miR-143-3p)组(尾静脉注射含miR-143-3p低表达序列的腺病毒载体溶液)、空载体质粒(si-eGFP)组(尾静脉注射腺病毒空载体溶液)、Nrg-1基因沉默(Si-Nrg-1)组(尾静脉注射含Nrg-1低表达序列的腺病毒载体溶液)、si-miR-143-3p+Si-Nrg-1组(同时注射si-miR-143-3p及Si-Nrg-1载体溶液),每组15只;中动脉闭塞栓塞术制备脑梗死模型,Longa法对神经功能缺损进行评分;TTC法测脑梗死面积;尼氏及TUNEL法测海马神经元损伤及凋亡;RT-qPCR法测脑海马组织miR-143-3p表达;免疫组化法测Nrg-1阳性表达;Western blot法测Nrg-1、ErbB4、X连锁凋亡抑制蛋白(XIAP)、半胱氨酸天冬氨酸蛋白水解酶(caspase)-3、caspase-9蛋白表达.结果与正常组相比,脑梗死患者血清中miR-143-3p表达升高(P<0.05),且脑梗死越严重miR-143-3p表达越高(P<0.05).与假手术组相比,模型组大鼠死亡、脑梗死面积、神经功能缺损评分升高,海马神经元损伤及凋亡加重,miR-143-3p及Nrg-1/ErbB4通路蛋白表达均升高(P<0.05).沉默miR-143-3p后,大鼠Nrg-1/ErbB4通路蛋白表达升高,海马神经元损伤及凋亡缓解,脑梗死面积及神经功能缺损症评分降低(P<0.05).Si-Nrg-1可逆转沉默miR-143-3p介导的Nrg-1/ErbB4神经保护途径激活,进而加重脑梗死大鼠神经元损伤及凋亡进程(P<0.05).结论miR-143-3p在脑梗死患者血清中表达上调,沉默miR-143-3p可通过上调Nrg-1表达,促进Nrg-1/ErbB4介导的神经元保护途径激活,缓解脑梗死大鼠海马神经元损伤及凋亡.

Objective To investigate the regulation of microribonucleic acid(miR)-143-3p on neuregulin(Nrg)-1/erb-b2 receptor tyrosine kinase(ErbB) 4 signaling pathway,and its effect on neuronal apoptosis in rats with cerebral infarction.Methods Thirty patients with cerebral infarction who were treated in our hospital from March 2018 to March 2019 were divided into mild cerebral infarction group(cerebral infarction volume <5 cm^(3)) and moderate cerebral infarction group(cerebral infarction volume between 5-15 cm^(3)),severe cerebral infarction group(cerebral infarction volume>15 cm^(3)),with 10 cases in each group,in addition,ten healthy volunteers who received physical examination in this hospital during the same period were selected as the normal group,the expression level of miR-143-3p in serum was measured by RT-qPCR method.SD rats were taken and randomly divided into sham operation group,model group,miR-143-3p silencing(si-miR-143-3p) group(tail vein injection of adenovirus vector solution containing miR-143-3p low expression sequence),empty vector plasmid(si-eGFP) group(tail vein injection of adenovirus empty vector solution),Nrg-1 gene silencing(Si-Nrg-1) group(tail vein injection of adenovirus vector solution containing Nrg-1 low expression sequence),si-miR-143-3p+Si-Nrg-1 group(simultaneous injection of si-miR-143-3p and Si-Nrg-1 vector solution),with 15 mice in each group;middle artery occlusion embolization was used to prepare cerebral infarction model,Longa method was used to score the neurological deficits;TTC method was used to measure the area of cerebral infarction;Nissl and TUNEL methods were used to measure hippocampal neuron damage and apoptosis;RT-qPCR method was used to measure the expression of miR-143-3p in hippocampus;immunohistochemical method was used to detect the positive expression of Nrg-1;Western blot was used to detect the protein expression of Nrg-1,ErbB4,X-linked inhibitor of apoptosis protein(XIAP),caspase-3 and caspase-9.Results Compared with the normal group,the expression of miR-143-3p in the serum of patients with cerebral infarction increased(P<0.05),and the more severe the cerebral infarction,the higher the expression of miR-143-3p(P<0.05).Compared with the sham operation group,rats in the model group had increased death,cerebral infarction area,and neurological deficit scores,aggravated hippocampal neuron damage and apoptosis,and increased expression of miR-143-3p and Nrg-1/ErbB4 pathway proteins(P<0.05).After silencing miR-143-3p,the expression of Nrg-1/ErbB4 pathway protein in rats increased,the hippocampal neuron damage and apoptosis were relieved,the cerebral infarction area and neurological deficit score decreased(P<0.05).Si-Nrg-1 could reverse the activation of the Nrg-1/ErbB4 neuroprotective pathway mediated by silencing miR-143-3p,and then aggravate the neuronal damage and apoptosis in rats with cerebral infarction(P<0.05).Conclusion The expression of miR-143-3p is up-regulated in the serum of patients with cerebral infarction.Silencing miR-143-3p can increase the expression of Nrg-1,promote the activation of Nrg-1/ErbB4-mediated neuron protection pathways,and alleviate the damage and apoptosis of hippocampal neurons in rats with cerebral infarction.