Hippocampal neuron-protective mechanism of hydrogen in a rat model of oxygen-glucose deprivation and restoration: promoting mitochondrial autophagy / 中华麻醉学杂志

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.