摘要
目的评价氢对氧糖剥夺-复氧复糖损伤大鼠海马神经保护作用机制与线粒体自噬的关系。方法出生24 h的健康SD大鼠,体外分离并原代培养海马神经元,以7×10^5个/孔的密度将神经元接种于预先用多聚赖氨酸包被过的6孔板中,采用随机数字表法分为5组(n=30):对照组(C组)、氧糖剥夺-复氧复糖组(OGD/R组)、氧糖剥夺-复氧复糖+氢气组(OGD/R+H2组)、氧糖剥夺-复氧复糖+3-甲基腺嘌呤(3-MA)组(OGD/R+3-MA组)和氧糖剥夺-复氧复糖+氢气+3-MA组(OGD/R+H2+3-MA组)。C组正常培养箱(75%N2-20%O2-5%CO2)中培养24 h;OGD/R组、OGD/R+H2组、OGD/R+3-MA组和OGD/R+H2+3-MA组制备神经元氧糖剥夺-复氧复糖损伤模型;OGD/R+H2组造模后,置于富氢培养箱(60%H2-10%O2-5%CO2-25%N2)中培养24 h;OGD/R+3-MA组造模后,加入自噬抑制剂3-MA 10 mmol/L,然后置于正常培养箱中培养24 h;OGD/R+H2+3-MA组造模后,加入3-MA 10 mmol/L,然后置于富氢培养箱中培养24 h。采用MTT法测定神经元存活率,DCFH-DA荧光探针法测定ROS活性,JC-10法测定线粒体膜电位(MPP),流式细胞术测定神经元凋亡率,Western blot法测定微管相关蛋白1轻链3(LC3)、PTEN诱导激酶1(PINK1)和Parkin的表达水平,并计算LC3Ⅱ/LC3Ⅰ比值。结果与C组比较,OGD/R组及OGD/R+H2组神经元存活率和细胞MMP降低,神经元凋亡率和ROS活性升高,PINK1和Parkin的表达水平和LC3Ⅱ/LC3Ⅰ比值升高(P<0.05);与OGD/R组比较,OGD/R+H2组神经元存活率及MMP升高,神经元凋亡率和细胞ROS活性降低,PINK1和Parkin的表达水平和LC3Ⅱ/LC3Ⅰ比值升高,OGD/R+3-MA组神经元存活率及MMP降低,神经元凋亡率及细胞ROS活性升高,PINK1和Parkin的表达水平及LC3Ⅱ/LC3Ⅰ比值降低(P<0.05)。与OGD/R+H2组比较,OGD/R+3-MA组和OGD/R+H2+3-MA组神经元存活率及MMP降低,神经元凋亡率及细胞ROS活性升高,PINK1和Parkin的表达水平及LC3Ⅱ/LC3Ⅰ比值降低(P<0.05)。结论氢对氧糖剥夺-复氧复糖损伤大鼠海马神经保�
Objective To evaluate the relationship between the hippocampal neuron-protective mechanism of hydrogen in a rat model of oxygen-glucose deprivation and restoration(OGD/R)and mitochondrial autophagy.Methods Hippocampal neurons isolated from healthy Sprague-Dawley rats(24 h after birth)were cultured in vitro,seeded in polylysine-coated 6-well plates at a density of 7×10^5 cells/well and then divided into 5 groups(n=30 each)using a random number table method:control group(C group),OGD/R group,OGD/R+H2 group,OGD/R plus 3-methyladenine(3-MA)group(OGD/R+3-MA group),and OGD/R plus H2 plus 3-MA group(OGD/R+H2+3-MA group).The cells were cultured for 24 h in normal culture atmosphere(75%N2-20%O2-5%CO2)in group C,and cells were subjected to oxygen-glucose deprivation for 2 h followed by O2-glucose supply for 24 h to establish the model of OGD/R injury in OGD/R,OGD/R+H2,OGD/R+3-MA and OGD/R+H2+3-MA groups.The cells were cultured for 24 h in a hydrogen-rich incubator(60%H2-10%O2-5%CO2-25%N2)after establishing the model in group OGD/R+H2.Autophagy inhibitor 3-MA 10 mmol/L was added,and then cultured for 24 h in normal culture atmosphere after establishing the model in group OGD/R+3-MA.Autophagy inhibitor 3-MA 10 mmol/L was added,and then cultured for 24 h in hydrogen-rich incubator after establishing the model in group OGD/R+H2+3-MA.The cell survival rate was measured using MTT assay.DCFH-DA fluorescent probe was applied for determination of reactive oxygen species(ROS)activity.The mitochondrial membrane potential was measured using a JC-10 assay kit.The neuronal apoptosis was detected by flow cytometry,and apoptosis rate was calculated.The expression of mitophagy-related protein microtubule-associated protein 1 light chain 3(LC3),PINK1 and Parkin was determined by Western blot,and LC3Ⅱ/LC3Ⅰratio was calculated.Results Compared with group C,the cell survival rate and MMP were significantly decreased,the apoptosis rate and ROS activity were increased,and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰratio were increa
作者
谭永星
董庆华
吴新伟
郝怡萌
储国海
彭洁
谢克亮
于泳浩
Tan Yongxing;Dong Qinghua;Wu Xinwei;Hao Yimeng;Chu Guohai;Peng Jie;Xie Keliang;Yu Yonghao(Department of Anesthesiology,Tianjin Medical University General Hospital Tianjin Research Institute of Anesthesiology,Tianjin 300052,China;Department of Surgical Intensive Care Unit,Affiliated Hospital of Guilin Medical University,Guilin 541001,China)
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2019年第10期1243-1247,共5页
Chinese Journal of Anesthesiology
基金
广西壮族自治区自然科学基金(2015GXNSFAA139130)
广西壮族自治区卫生健康委员会自筹课题(Z2016390,Z20180612)。
关键词
氢
缺氧缺血
脑
再灌注损伤
海马
神经元
线粒体
自噬
Hydrogen
Hypoxia-ischemia
brain
Reperfusion injury
Hippocampus
Neurons
Mitochondria
Autophagy
