纳洛酮对蛛网膜下腔出血大鼠海马区神经细胞自噬的影响

Effect of naloxone on autophagy in hippocampus of rats with subarachnoid hemorrhage

目的探讨纳洛酮对蛛网膜下腔出血(SAH)大鼠海马区神经细胞自噬的影响。方法将108只健康雄性清洁级SD大鼠按随机数字表法分为假手术组、SAH组、纳洛酮组,每组36只。假手术组只进行造模手术不刺破血管,SAH组利用颈内动脉穿刺法制成大鼠SAH动物模型,纳洛酮组在造模成功后立即给予纳洛酮(1 mg∕kg体质量)腹腔注射,每12小时注射1次,用药至处死的各时相点。假手术组和SAH组采用同样方法注射同等体积的0.9%氯化钠溶液。每组又按6 h、24 h、72 h时间点分为3个亚组(每个亚组12只SD大鼠)。穿梭箱实验观察三组大鼠的行为学变化;苏木素-伊红(HE)染色观察海马区神经细胞形态变化;免疫组织化学检测自噬特异性标志物Beclin-1和微管相关蛋白1轻链3(LC3-Ⅱ)在大鼠海马区神经细胞的表达。结果与假手术组相比,SAH组6 h、24 h、72 h各时间点逃避反应次数减少[(24.83±3.24)次比(14.41±1.98)次,(25.33±2.64)次比(16.25±2.18)次,(25.16±2.82)次比(18.33±2.57)次](P<0.05),逃避反应时间增加[(3.54±0.99)s比(9.09±1.38)s,(3.55±0.84)s比(8.73±1.13)s,(3.54±0.83)s比(8.00±1.04)s](P<0.05),海马区正常神经细胞数量减少[(122.67±5.55)个比(84.25±12.63)个,(122.25±5.50)个比(75.58±11.11)个,(121.58±5.68)个比(67.75±10.55)个](P<0.05),表达Beclin-1和LC3-Ⅱ蛋白的阳性细胞数量增高[(20.50±3.45)个比(31.41±4.96)个,(20.33±2.77)个比(47.67±7.35)个,(20.50±3.59)个比(43.17±6.46)个;(17.08±3.00)个比(27.42±3.40)个,(17.50±2.31)个比(39.83±4.67)个,(17.41±2.57)个比(31.50±4.06)个](P<0.05)。与SAH组比较,纳洛酮组各时间点逃避反应次数增多[(14.41±1.98)次比(16.50±1.93)次,(16.25±2.18)次比(19.25±2.30)次,(18.33±2.57)次比(22.50±2.20)次](P<0.05),逃避反应时间减少[(9.09±1.38)s比(8.05±1.24)s,(8.73±1.13)s比(6.56±1.09)s,(8.00±1.04)s比(5.36±0.97)s](P<0.05),海马区正常神经细胞数量增多[(84.25±12.63)个比(94.75±11.06)个,(75.58±11.11)个比(84.50±9.21)个,(67.75±10.55)个比(77.33±9.88)个](P<0.05),表达Beclin-1和LC3-Ⅱ蛋白的阳性细胞数量增高[(31.41±4.96)个比(36.17±4.06)个,(47.67±7.35)个比(57.58±6.68)个,(43.17±6.46)个比(52.75±7.35)个;(27.42±3.40)个比(30.92±4.19)个,(39.83±4.67)个比(48.33±5.66)个,(31.50±4.06)个比(38.58±5.40)个](P<0.05)。结论纳洛酮的干预可适度激活SAH大鼠海马区神经细胞自噬的表达,并具有脑保护作用。

Objective To investigate the effect of naloxone on autophagy in hippocampus of rats with subarachnoid hemorrhage(SAH).Methods 108 healthy male clean grade SD rats were randomly divided into sham operation group,SAH group,SAH com-bined with naloxone treatment group(naloxone group),36 rats in each group.The sham operation group only performed the model-ing operation without puncturing the blood vessels.The SAH group was made into the rat SAH animal model by internal carotid ar-tery puncture.The naloxone group was given naloxone(1 mg∕kg Body mass)intraperitoneally after the successful modeling,Injectonce every 12 hours,from medication to the point of death.The same method was used to inject the same volume of normal salinein the sham operation group and the SAH group.Each group was further divided into 3 subgroups(12 SD rats in each subgroup)at6 h,24 h,and 72 h.The behavioral changes of the three groups were observed in the shuttle box.HE staining was used to observethe morphological changes of neurons in the hippocampus.Immunohistochemistry was used to detect the expression of autophagy-specific markers Beclin-1(Autophagy related protein) and LC3-II(Microtubule associated protein 1 light chain 3)in the hippo-campus of rats.Results Compared with the sham operation group,the number of escape response decreased[(24.83±3.24)vs.(14.41±1.98),(25.33±2.64)vs.(16.25±2.18),(25.16±2.82)vs.(18.33±2.57)](P<0.05),the time of escape response increased[(3.54±0.99)s vs.(9.09±1.38)s,(3.55±0.84)s vs.(8.73±1.13)s,(3.54±0.83)s vs.(8.00±1.04)s](P<0.05),the number of nor-mal nerve cells in the hippocampus decreased[(122.67±5.55)vs(.84.25±12.63),(122.25±5.50)vs.(75.58±11.11),(121.58±5.68)vs.(67.75±10.55)](P<0.05),and the expressions of Beclin-1 and LC3-II increased[(20.50±3.45)vs.(31.41±4.96),(20.33±2.77)vs.(47.67±7.35),(20.50±3.59)vs.(43.17±6.46);(17.08±3.00)vs.(27.42±3.40),(17.50±2.31)vs.(39.83±4.67),(17.41±2.57)vs.(31.50±4.06)](P<0.05)at each time point in the SAH group.Compared with the SAH group,the number of es-cape response increased[(14.41±1.98)vs.(16.50±1.93),(16.25±2.18)vs.(19.25±2.30),(18.33±2.57)vs.(22.50±2.20)](P<0.05),the time of escape response decreased[(9.09±1.38)s vs.(8.05±1.24)s,(8.73±1.13)s vs.(6.56±1.09)s,(8.00±1.04)s vs.(5.36±0.97)s](P<0.05),the number of normal nerve cells in the hippocampus increased[(84.25±12.63)vs.(94.75±11.06),(75.58±11.11)vs.(84.50±9.21),(67.75±10.55)vs.(77.33±9.88)](P<0.05),and the expressions of Beclin-1 and LC3-II in-creased[(31.41±4.96)vs.(36.17±4.06),(47.67±7.35)vs.(57.58±6.68),(43.17±6.46)vs.(52.75±7.35);(27.42±3.40)vs.(30.92±4.19),(39.83±4.67)vs.(48.33±5.66),(31.50±4.06)vs.(38.58±5.40)](P<0.05)at each time point in the naloxonegroup.Conclusion The intervention of naloxone can moderately activate the expression of autophagy in the hippocampus of SAH ratsand have a protective effect on the brain.