摘要
目的探讨抗氧化剂MitoQ对异氟醚诱导的新生大鼠海马神经元细胞损伤的影响及潜在机制。方法 SPF级健康SD大鼠15只,7日龄,体重15~20g。采用随机数字表法分为三组:对照组(C组)、异氟醚组(I组)和异氟醚+MitoQ组(IM组),每组5只。C组吸入空-氧混合气体。Ⅰ组于出生后7、14和21d吸入1.5%异氟醚2h,IM组在每次吸入异氟醚前腹腔注射MitoQ0.4ml/kg。于出生后28d采用HE染色观察各组大鼠海马CA1区海马神经元细胞形态。分离培养新生大鼠原代海马神经元细胞培养并分组处理,采用MTT法和TUNEL原位荧光染色法检测细胞存活率和凋亡率;采用硫代巴比妥酸法和黄嘌呤氧化酶法检测细胞中丙二醛(MDA)浓度和超氧化物歧化酶(SOD)活性;采用Rhodamine 123染色荧光显微镜照相法检测线粒体膜电位(MMP),DCFH-DA染色荧光显微镜照相法检测细胞内活性氧簇(ROS)生成量,采用Western blot法检测海马神经元细胞中Bax、Bcl-2和caspase-3蛋白含量。结果与C组比较,Ⅰ组大鼠海马组织神经细胞受损明显,细胞数目减少,Ⅰ组细胞存活率明显降低,细胞凋亡率明显升高,MDA浓度明显升高,SOD活性明显降低,ROS生成量明显增加,MMP水平明显降低,Bax和caspase-3蛋白含量明显升高,Bcl-2蛋白含量明显降低(P<0.05);与Ⅰ组比较,IM组大鼠海马组织神经细胞损伤减少,细胞存活率明显升高,细胞凋亡率明显降低,MDA浓度明显降低,SOD活性明显升高,ROS生成量明显减少,MMP水平明显升高,Bax和caspase-3蛋白含量明显降低,Bcl-2蛋白含量明显升高(P<0.05)。结论抗氧化剂MitoQ可明显抑制异氟醚诱导的海马神经元细胞损伤,这与其拮抗细胞氧化应激和维持线粒体功能作用密切相关。
Objective To explore the impacts and potential mechanisms of MitoQ on isoflurane-induced injury of primary cultured hippocampal neurons in newborn rats.Methods Fifteen healthy SPF Sprague-Dawley rats of both sex were randomly divided into three groups（n=5 each）using a random number table：control group（group C）,multiple exposures to isoflurane anesthesia group（group Ⅰ）and multiple exposures to isoflurane anesthesia＋MitoQ group（group IM）.On postnatal days 7,14 and 21,1.5% isoflurane was inhaled for 2 hin group Ⅰ.MitoQ was intraperitoneally administered in a volume of 0.4 ml/kg before isoflurane anesthesia in group IM,while a mixture of oxygen and air was inhaled instead of isoflurane in group C.HE staining was carried out on postnatal day 28 to observe the morphological changes in hippocampal CA1 region of rat neural cell structures.Hippocampal neuron cells were dissected from clean Sprague-Dawley rats born in 24 h.After primary culture for seven days,MTT assay and TUNEL assay was respectively performed to measure the cell viability and apoptosis of hippocampal neurons.The malondialdehyde（MDA）content and superoxide dismutase（SOD）activity were detected respectively using the thiobarbituric method and xanthinoxidase method.Mitochondrial membrane potential（MMP）was measured by rhodamine 123 staining,intracellular levels of reactive oxygen species（ROS）were tested by DCFH-DA staining.Western blot was used to analyze the protein levels of Bax,Bcl-2 and caspase-3.Results Compared with group C,group Ⅰ decreased the number of neural cells and the cell survival rate;the apoptotic rate was significantly increased;MDA contents and ROS production were significantly increased;SOD activity and MMP level were significantly decreased;the expression of Bax and caspase-3 were significantly increased,while the expression of Bcl-2 was significantly decreased（P 〈0.05）.Compared with the group Ⅰ,the damaged neural cells were decreased,the cell survival rate was significantly increased,the apoptotic rate was significantly decreased in group IM;MDA contents and ROS production were significantly decreased;SOD activity and MMP level were significantly increased;the expression of Bax and caspase-3 were significantly decreased,while the expression of Bcl-2 was significantly increased（P 〈0.05）.Conclusion Antioxidant MitoQ attenuates isoflurane-induced neuron damage,which may be associated with the inhibition on oxidative stress and mitochondrial dysfunction.
作者
明少鹏
周凤坤
MING Shaopeng ZHOU Fengkun(Department of Anesthesiology, Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine, Nanning 530011, China)
出处
《临床麻醉学杂志》
CAS
CSCD
北大核心
2017年第10期1001-1005,共5页
Journal of Clinical Anesthesiology
