尼莫地平对蛛网膜下腔出血大鼠海马CA1区自噬的影响

Effect of nimodipine on autophagy in hippocampal CA1 area of rats with subarachnoid hemorrhage

目的探讨尼莫地平对蛛网膜下腔出血(SAH)大鼠海马CA1区自噬的影响。方法雄性Sprague-Dawley大鼠48只,按随机数字表法分为Sham组(假手术组,12只)、SAH组(12只)、NMDP组(SAH+尼莫地平,12只)、3-MA组(SAH+尼莫地平+3-甲基腺嘌呤,12只)。颈内动脉刺破法制备SAH动物模型。NMDP组造模成功后30 min腹腔注射尼莫地平。3-MA组于造模前1 h腹腔注射3-MA,且造模后30 min腹腔注射尼莫地平。干预24 h后给予穿梭箱实验观察大鼠神经功能,随后取海马CA1区脑组织进行苏木素-伊红染色观察细胞形态,免疫组化及Western blotting观察自噬相关蛋白[Beclin-1及微管相关蛋白1轻链3Ⅱ(LC3-Ⅱ)]的表达。结果与Sham组比较,SAH组大鼠躲避反应次数减少[(17. 9±2. 6)次比(26. 3±2. 7)次],躲避反应时间延长[(10. 0±3. 3) s比(3. 5±1. 0) s],海马CA1区正常形态神经细胞数目减少[(72±28)个比(118±27)个,P <0. 05],Beclin-1及LC3-Ⅱ阳性细胞增多[分别为(39±9)个比(22±11)个、(39±12)个比(12±5)个],Beclin-1蛋白表达水平升高(0. 59±0. 12比0. 36±0. 03),LC3-Ⅱ/LC3-Ⅰ比率上升(0. 51±0. 13比0. 20±0. 05),差异均有统计学意义(均P <0. 05);与SAH组比较,NMDP组大鼠躲避反应次数[(22. 3±2. 3)次]增多,躲避反应时间[(5. 0±1. 5) s]减少,海马CA1区正常神经细胞数目[(86±20)个]增多,Beclin-1及LC3-Ⅱ阳性细胞[分别为(50±11)、(48±11)个]增多,Beclin-1蛋白表达水平(0. 97±0. 24)增高,LC3-Ⅱ/LC3-Ⅰ比率(0. 87±0. 12)上升,差异均有统计学意义(均P <0. 05),而3-MA组大鼠各项差异均无统计学意义(均P>0. 05)。结论尼莫地平可增强自噬的激活,减少海马CA1区神经细胞损伤,改善大鼠的神经功能。

Objective To investigate the effect of nimodipine on autophagy in hippocampal CA1 area of rats with subarachnoid hemorrhage(SAH).Methods Forty-eight male Sprague-Dawley rats were randomly divided into sham operation group(sham group),SAH group,SAH+nimodipine group(NMDP group),and SAH+nimodipine+3-methyladenine group(3-MA group,n=12 in each group).A SAH animal model was induced by internal carotid artery puncture method.Nimodipine was intraperitoneally injected 30 min after successful modeling in the NMDP group.The 3-MA group was intraperitoneally injected with 3-MA 1 h before modeling,and nimodipine was intraperitoneally injected 30 min after modeling.After 24 h of intervention,a shuttle box test was performed to observe the neurological function of the rats,then the brain tissue of hippocampal CA1 area was taken for hematoxylin-eosin staining in order observe the cell morphology.Immunohistochemistry and Western blotting were used to observe the expression of autophagy-related protein(Beclin-1 and microtubule-associated protein 1 light chain 3Ⅱ[LC3-Ⅱ]).Results Compared with the sham group,the times of rat avoidance reaction were decreased in the SAH group(17.9±2.6 times vs.26.3±2.7 times),the avoidance reaction time was prolonged(10.0±3.3 s vs.3.5±1.0 s),the numbers of normal neurons in hippocampus CA1 area were reduced(72±28 vs.118±27,P<0.05),the positive cells of Beclin-1 and LC3-Ⅱwere increased(39±9 vs.22±11 and 39±12 vs.12±5 respectively),the expression level of Beclin-1 protein was increased(0.59±0.12 vs.0.36±0.03),and the ratio of LC3-Ⅱ/LC3-Ⅰwas increased(0.51±0.13 vs.0.20±0.05).The differences were statistically significant(P<0.05).Compared with the SAH group,the times of avoidance reaction(22.3±2.3 times)were increased,the time of avoidance reaction(5.0±1.5 s)was decreased,the numbers of normal neurons(86±20)in hippocampal CA1 area were increased,the numbers of Beclin-1 and LC3-Ⅱpositive cells(50±11 and 48±11 respectively)were increased,the expression level of Beclin-1 protein(0.97±0.24)was increased,and the ratio of LC3-Ⅱ/LC3-Ⅰ(0.87±0.12)was increased in the NMDP group.The differences were statistically significant(all P<0.05),and there were no significant differences in each difference in the 3-MA group(all P >0.05).Conclusion Nimodipine may enhance the activation of autophagy,reduce neuronal damage in hippocampus CA1 areas,and improve neurological function of rats.