钙调蛋白抑制剂对N-甲基-D-天冬氨酸诱导的大鼠皮质神经元损伤的保护作用  

Protective effect of calmodulin antagonist on N-methyl-D-aspartic acid-induced cortical neuronal injury in rats

作　　者：李洪凤[1,2] 刘学文[1] 

机构地区：[1]辽宁医学院神经内科,锦州121001 [2]淮安市淮阴医院神经内科,223300

出　　处：《中国脑血管病杂志》2010年第5期263-267,共5页Chinese Journal of Cerebrovascular Diseases

基　　金：辽宁省科技厅博士启动基金资助项目(20091049)

摘　　要：目的观察钙调蛋白抑制剂W-7对N-甲基-D-天冬氨酸(NMDA)诱导的皮质神经元损伤的影响,并探讨其可能的机制。方法原代培养SD大鼠(120只)皮质神经元,神经元特异性稀醇化酶(NSE)染色方法确定神经元的比例;添加不同浓度的NMDA,制备细胞损伤模型,MTT法评估细胞生存力;原代大鼠皮质神经元培养后,随机分为对照组、损伤组(NMDA)、不同剂量W-7保护组(25、50、100μmol/L),MTT法评价细胞生存力;对上述各组进行吖啶橙荧光染色,观察细胞凋亡形态;采用Western Blot方法 ,测定上述各组神经元的p38MAPK和NF-κBp65表达情况。结果①以NSE抗体标记培养的神经元,阳性神经元的比例为90.86%。②不同浓度的NMDA对培养大鼠皮质神经细胞具有损伤作用,实验结果表明50μmol/L的NMDA对细胞的损伤以凋亡为主,以此剂量做后续实验。③与对照组比较,损伤组的吸光度(A)值降低,差异有统计学意义,P<0.01;与损伤组比较,W-7各保护组A值均升高(P<0.01或P<0.05),差异有统计学意义。④吖啶橙荧光染色可观察到凋亡细胞,保护组随着W-7剂量的增加凋亡细胞数量减少。⑤Western Blot结果显示,W-7可以使p38MAPK和NF-κB p65表达下降,差异有统计学意义(P<0.05或P<0.01)。结论 W-7可能通过降低p38MAPK和NF-κBp65的途径,对NMDA诱导的神经元损伤起保护作用。Objective To observe the impact of ealmodulin inhibitor W-7 on N-methyl-D-aspartic acid （NMDA）-induced conical neuron injury and its possible mechanisms. Methods After primary rat cortical neurons were cultured, neuron-specific enolase （NSE） staining was used to determine the propor- tion of neurons, adding different concentrations of NMDA, and preparing a cell injury model ; after primary rat cortical neurons were cultured, the rats were randomly divided into control, injury （NMDA 50 μmol/L） and W-7 protection groups （25,50, and 100 μ mol/L）. MTT assay was used to evaluate cell survival activity and to determine the protective effects of W-7. Acridine orange （AO） staining was performed in the above-mentioned groups and the apoptotic morphology was observed; the Western blot method was used to determine the expression of neuronal p38 MAPK and NF-KBp65 in the above-mentioned groups. Results （1）Neurons were labeled by NSE antibody, and the proportion of positive neurons was 90.86%. （2）The different concentrations of NMDA had damage effect on the cultured rat cerebral cortical neurons. The experimental results showed that 50 p, mol/L NMDA caused apoptosis of neurons; therefore, this dose was used in the follow-up experiments. （3）Compared to the control group, the absorbance （A） value in the injury group was decreased; the difference was statistically significant （P 〈 0.01 ） ; compared to the injury group, the A values in each protection group were increased; those differences were statistically significant （ P 〈 0. 01 or P 〈 O. 05）. （4）The apoptotic cells were observed by using AO staining. The number of apoptotic cells was decreased with the increased W-7 dosage. （5）Western Blot results showed that W-7 decreased the expression of p38 MAPK and NF-KBp65, and there were significant differences as compared to the injury group （P 〈 0.05 or P 〈 0.01 ）. Conclusion W-7 has protective effect on the NMDA-induced neuronal injury, which may be attributable to

关 键 词：钙调蛋白 脑损伤 N一甲基天冬氨酸 凋亡诱导因子 

分 类 号：R741[医药卫生—神经病学与精神病学]