Hippocampal neuron-protective mechanism of hydrogen in a rat model of oxygen-glucose deprivation and restoration: promoting mitochondrial autophagy
10.3760/cma.j.issn.0254-1416.2019.10.024
- VernacularTitle: 氢对氧糖剥夺-复氧复糖损伤大鼠海马神经元保护作用机制：促进线粒体自噬
- Author:
Yongxing TAN
1
;
Qinghua DONG
2
;
Xinwei WU
2
;
Yimeng HAO
2
;
Guohai CHU
2
;
Jie PENG
2
;
Keliang XIE
3
;
Yonghao YU
3
Author Information
1. Department of Anesthesiology, Tianjin Medical University General Hospital Tianjin Research Institute of Anesthesiology, Tianjin 300052, China (is working on the Department of Surgical Intensive Care Unit, Affiliated Hospital of Guilin Medical University, Guilin 541001, China)
2. Department of Surgical Intensive Care Unit, Affiliated Hospital of Guilin Medical University, Guilin 541001, China
3. Department of Anesthesiology, Tianjin Medical University General Hospital Tianjin Research Institute of Anesthesiology, Tianjin 300052, China
- Publication Type:Journal Article
- Keywords:
Hydrogen;
Hypoxia-ischemia, brain;
Reperfusion injury;
Hippocampus;
Neurons;
Mitochondria;
Autophagy
- From:
Chinese Journal of Anesthesiology
2019;39(10):1243-1247
- CountryChina
- Language:Chinese
-
Abstract:
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 7×105 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/LC3Ⅰ ratio 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/LC3Ⅰ ratio were increased in OGD/R and OGD/R+ H2 groups (P<0.05). Compared with group OGD/R, the cell survival rate and MMP were significantly increased, the apoptosis rate and ROS activity were decreased, and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were increased in group OGD/R+ H2(P<0.05), and 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/LC3Ⅰ ratio were decreased in group OGD/R+ 3-MA (P<0.05). Compared with group OGD/R+ H2, 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/LC3Ⅰ ratio were decreased in OGD/R+ 3-MA and OGD/R+ H2+ 3-MA groups (P<0.05).
Conclusion:Hippocampal neuron-protective mechanism of hydrogen against OGDR injury is related to promoting mitochondrial autophagy in rats.
