文章摘要
陈双,江伟,陈洁,肖农.视黄酸通过RARα对缺氧缺血性脑损伤后神经元凋亡的影响[J].中华物理医学与康复杂志,2018,40(4):247-252
视黄酸通过RARα对缺氧缺血性脑损伤后神经元凋亡的影响
Retinoic acid affects apoptosis of neurons injured by hypoxic-ischemic brain damage via retinoic acid receptor alpha
  
DOI:
中文关键词: 视黄酸核受体α  神经元凋亡  腺病毒  神经营养因子  半胱氨酸天冬氨酸蛋白酶-8
英文关键词: Retinoic acid receptor alpha  Neuron apoptosis  Adenovirus  Glial cell-derived neurotrophic factor  Caspase-8
基金项目:国家自然科学基金(81571091)
作者单位
陈双,江伟,陈洁,肖农 400014 重庆重庆医科大学附属儿童医院康复科儿童发育疾病研究教育部重点实验室儿科学重庆市重点实验室(陈双江伟肖农)重庆医科大学附属儿童医院营养研究中心(江伟陈洁) 
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中文摘要:
      目的 探讨视黄酸通过视黄酸核受体α(RARα)对缺氧缺血性脑损伤(HIBD)后神经元凋亡的影响。 方法 采用维生素A缺乏饲料和维生素A正常的普通饲料分别构建视黄酸缺乏(RAD)和视黄酸正常(RAN)新生SD模型大鼠,随机选取24只RAD新生模型鼠作为RAD组,选取48只RAN新生模型鼠随机分为RAN组和假手术组,每组24只。RAN组和RAD组采用经典Rice法进行分离结扎左侧颈总动脉建立HIBD模型,假手术组只做颈部皮肤切开缝合。分别于术后第3天和第7天,采用TUNEL法检测脑皮质神经元凋亡情况,于术后第7、14、21和28天进行神经功能缺损评估。体外分离培养原代神经元,建立缺氧缺糖损伤(OGD)模型,将原代神经元细胞分为对照组、OGD组、视黄酸OGD组,采用实时荧光定量聚合酶链反应(Real-time PCR)和Western blot法检测各组RARα、胶质细胞源性神经营养因子(GDNF)及半胱氨酸天冬氨酸蛋白酶-8(Caspase-8)的mRNA及蛋白表达水平。采用RARα基因小干扰RNA重组腺病毒(Ad-si RARα)感染原代神经元,将原代神经元分为沉默组和阴性对照组,检测RARα、GDNF、Caspase-8的mRNA和蛋白表达水平。 结果 ①RAD组和RAN组大鼠在缺氧缺血后第3天到第7天,皮质凋亡神经元数量逐渐增加,且RAD组神经元凋亡数量明显多于RAN组;RAD组大鼠各时间点的神经功能损伤评分均高于RAN组(P<0.05)。②视黄酸OGD组神经元的RARα、GDNF mRNA表达水平[(1.49±0.05)、(0.61±0.02)]均高于OGD组[(0.51±0.04)、(0.21±0.02)],Caspase-8的mRNA表达水平显著低于OGD组[(1.13±0.09) vs (1.79±0.11)],且组间差异均有统计学意义(P<0.01);各组神经元的蛋白表达水平与mRNA表达水平基本一致。③沉默组神经元的RARα、GDNF mRNA表达水平均低于阴性对照组[RARα(1.07±0.12) vs (2.33±0.08),P<0.01];GDNF[(0.11±0.01) vs (0.13±0.01),P<0.05],沉默组神经元的Caspase-8 mRNA表达水平较阴性对照组高[(2.92±0.20) vs (2.40±0.18),P<0.01];各组蛋白表达水平与mRNA表达水平基本一致。 结论 视黄酸可通过RARα上调GDNF表达,下调Caspase-8的表达,进而抑制HIBD后神经元的凋亡。
英文摘要:
      Objective To explore the effect of the retinoic acid (RA) on the apoptosis of neurons caused by hypoxic ischemic brain damage (HIBD). Methods Seventy-two newborn Sprague-Dawley rats were randomly divided into an RA deficiency (RAD) group, an RA normal (RAN) group and a control group, each of 24. The HIBD model was established in the RAD and RAN groups using Rice′s method. The left common carotid artery was exposed, ligated and cut, inducing hypoxia. In the control group the left common carotid artery was exposed without any other treatment. Three and 7 days after the operation, neuron apoptosis in the brain tissue was evaluated using TUNEL staining. The degree of HIBD was quantified using modified neurological severity scores (mNSS) 7, 14, 21 and 28 days after the operation. Primary neurons were cultured in vitro, and oxygen glucose deprivation (OGD) was induced, then control, OGD and RA+OGD groups were formed. The gene transcription and the protein activity of retinoic acid receptor alpha (RARα), GDNF (glial cell line-derived neurotrophic factor) and Caspase-8 were examined with polymerase chain reactions (PCR) and Western blotting. The RA+OGD group was exposed to RA and SiRNA adenovirus, and divided into a silenced group and a negative transfection group according to the infection. Results The average mNSS of the RAD group was significantly higher than that of the RAN group. TUNEL staining showed that the apoptotic cells in the cortex increased from day 3 to 7 after the operation, but significantly more in the RAD group than in the RAN group. The gene transcription and protein activity of RARα and GDNF in the RA+OGD group were significantly higher than in the OGD group, and those of Caspase-8 were significantly lower. The gene transcription and protein activity of RARα and GDNF in the silenced group were significantly lower than in the negative transfection group, while those of Caspase-8 were just the opposite. Conclusion RA can inhibit the apoptosis of primary neurons after HIBD by up-regulating the expression of GDNF and down-regulating that of Caspase-8 via RARα.
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