摘要
目的探讨微小RNA-199a(miR-199a)对脑梗死大鼠神经元凋亡的影响及对沉默信息调节因子1(SIRT1)的调控作用。方法构建脑梗死大鼠实验模型,将60只大鼠随机分为假手术组、脑梗死组、miR-199a抑制组、miR-199a阴性对照组、miR-199a抑制+SIRT1抑制组,每组12只。通过神经行为学评估判断大鼠神经功能变化,采用苏木精-伊红(HE)染色法观察各组大鼠脑组织神经元病理变化,采用流式细胞术检测脑皮质神经元凋亡情况,采用荧光定量聚合酶链反应(PCR)检测各组脑组织中miR-199a、SIRT1 mRNA表达水平,并通过蛋白质印迹法(Western blot)检测各组脑组织SIRT1、活化型半胱氨酸天冬氨酸蛋白酶-3(Cleaved caspase-3)蛋白表达量。采用双荧光素酶报告基因系统验证miR-199a和SIRT1的靶向调控关系。结果与假手术组相比,脑梗死组大鼠脑组织出现变性、神经元坏死情况,神经元凋亡率、Cleaved caspase-3蛋白表达量、神经学评分和miR-199a表达量升高,SIRT1 mRNA和SIRT1蛋白表达量降低,差异有统计学意义(P<0.05);与miR-199a阴性对照组相比,miR-199a抑制组神经元坏死程度减弱,脑组织结构逐渐恢复,神经元凋亡率、Cleaved caspase-3蛋白表达量、神经学评分和miR-199a表达量降低,SIRT1 mRNA和SIRT1蛋白表达量升高,差异有统计学意义(P<0.05);与miR-199a抑制组相比,miR-199a抑制+SIRT1抑制组神经元坏死及脑组织变性程度加剧,神经元凋亡率、Cleaved caspase-3蛋白表达量和神经学评分升高,SIRT1 mRNA和SIRT1蛋白表达量降低,差异有统计学意义(P<0.05);miR-199a阴性对照组以上指标与脑梗死组比较,差异无统计学意义(P>0.05)。双荧光素酶报告基因实验结果显示,miR-199a可与野生型SIRT13′-UTR特异性结合,两者具有靶向调控关系。结论抑制miR-199a表达可靶向上调SIRT1表达水平,缓解脑梗死大鼠神经元凋亡,改善神经功能。
Objective To explore the effect of microRNA-199a(miR-199a)on neuronal apoptosis in rats with cerebral infarction and its role in regulating silent information regulator 1(SIRT1).Methods The experimental model of cerebral infarction rats was constructed,and 60 rats were randomly divided into sham operation group,cerebral infarction group,miR-199a inhibition group,miR-199a negative control group,and miR-199a inhibition+SIRT1 inhibition group,with 12 rats in each group.Neurobehavioral tests were used to evaluate and judge the changes of nerve function in rat,the hematoxylin-eosin(HE)staining method was used to observe the pathological changes of neurons in the brain tissue of rats in each group,flow cytometry was used to detect the apoptosis of cerebral cortex neurons,fluorescence quantitative polymerase chain reaction(PCR)was used to detect the expression levels of miR-199a and SIRT1 mRNA in the brain tissues of each group,and Western blot method was used to detect the expression levels of SIRT1 and Cleaved caspase-3 proteins in the brain tissues of each group.Dual luciferase reporter system was used to verify the targeted regulation relationship between miR-199a and SIRT1.Results Compared with the sham operation group,rats in the cerebral infarction group had degeneration and neuronal necrosis in the brain tissues,which were accompanied by the neuronal apoptosis rate,protein expression of Cleaved caspase-3,neurological score and miR-199a expression significantly increased,and the SIRT1 mRNA and protein expression of SIRT1 significantly reduced(P<0.05);compared with the miR-199a negative control group,the degree of neuronal necrosis in the miR-199a inhibition group was weakened and the structure of brain tissue gradually recovered,which were accompanied by the neuronal apoptosis rate,protein expression of Cleaved caspase-3,neurological score and miR-199a expression significantly reduced,and the SIRT1 mRNA and protein expression of SIRT1 significantly increased(P<0.05);compared with miR-199a inhibition group,the degrees of neuron necrosis and brain tissue degeneration in the miR-199a inhibition+SIRT1 inhibition group became severer,which were accompanied by the neuronal apoptosis rate,protein expression of Cleaved caspase-3 and neurological score significantly increased,and the SIRT1 mRNA and protein expression of SIRT1 significantly reduced(P<0.05);there were no significant differences in the indicators mentioned above between the miR-199a negative control group and the cerebral infarction group(P>0.05);the results of double luciferase reporter gene experiment showed that miR-199a could specifically bind to wild-type SIRT13′-UTR,and there was a targeted regulation relationship between two indexes.Conclusion Inhibiting the expression of miR-199a can targeted increase the expression level of SIRT1,relieve neuronal apoptosis in rats with cerebral infarction,and improve neurological function.
作者
林影
李建红
卢宏全
陈俊玲
LIN Ying;LI Jianhong;LU Hongquan;CHEN Junling(Department of Neurology,Central Hospital of Western Hainan,Danzhou,Hainan,571700;Department of Neurology,the Second Affiliated Hospital of Hainan Medical University,Haikou,Hainan,570100;Department of Oncology,Central Hospital of Western Hainan,Danzhou,Hainan,571700;Department of Neurology,Haikou City People′s Hospital of Hainan Province,Haikou,Hainan,570100)
出处
《实用临床医药杂志》
CAS
2022年第6期26-32,共7页
Journal of Clinical Medicine in Practice
