瑞马唑仑通过Keap1/Nrf2信号通路减轻大鼠神经元氧糖剥夺–复氧复糖损伤

Remimazolam Alleviates Neuronal Oxygen Glucose Deprivation/Re-Oxygenation Injury in Rats by Keap1/Nrf2 Signaling Pathway

目的:探讨瑞马唑仑是否通过激活Keap1/Nrf2通路减轻海马神经元氧糖剥夺–复氧复糖损伤的功能。方法:取SD大鼠乳鼠海马神经元培养至第8天,采用随机数字表法分为5组:对照组(C组)、模型组(M组)、氧糖剥夺–复氧复糖+瑞马唑仑组(R组)、氧糖剥夺–复氧复糖 + 瑞马唑仑 + 二甲基亚砜组(V组)、氧糖剥夺–复氧复糖 + 瑞马唑仑 + 鸦胆子苦醇组(B组),缺氧6 h后复氧20 h建立缺氧复氧模型,瑞马唑仑于复氧时加入,终浓度100 μM,鸦胆子苦醇及二甲基亚砜在缺氧前4 h加入,鸦胆子苦醇终浓度为500 nM,二甲基亚砜浓度<0.1%。复氧结束后检测CCK8细胞活性,细胞凋亡率,ROS水平,Nrf2、Keap1、Bcl2、Bax蛋白水平,免疫荧光检测Nrf2核转位及线粒体形态。结果:与C组比较,M组细胞活力降低,凋亡率及ROS活性升高,Bax蛋白水平升高,Nrf2、Keap1、Bcl2蛋白水平降低,Nrf2核转位增加,线粒体碎片化程度加重;与M组比较,R组的细胞活力升高,细胞凋亡率及ROS活性降低,Bax蛋白水平降低,Nrf2、Keap1、Bcl2蛋白水平升高,Nrf2核转位增加,线粒体碎片化程度减轻;V组与R组间无统计学差异;与V组比较,B组细胞活力降低,凋亡率及ROS活性升高,Bax蛋白水平升高,Nrf2、Keap1、Bcl2蛋白水平降低,Nrf2核转位减少,线粒体碎片化程度加重。结论:瑞马唑仑可激活Keap1/Nrf2通路,减少ROS蓄积及线粒体损伤,进而减少细胞凋亡,减轻大鼠海马神经元氧糖剥夺–复氧复糖损伤。

Objective: To investigate the activation effect of Remimazolam on Keap1/Nrf2 pathway and its function in reducing Oxygen glucose deprivation/re-oxygenation injury of hippocampal neurons. Methods: Rat hippocampal neurons were cultured to the 8th day, and were divided into 5 groups by random number table method: control group (group C), oxygen glucose deprivation/re-oxy- gena-tion group (group M), oxygen glucose deprivation/re-oxygenation + Remimazolam group (group R), oxygen glucose deprivation/re-oxygenation + Remimazolam + dimethyl sulfoxide group (group V), oxygen glucose deprivation/re-oxygenation + Remimazolam + brusatol group (group B). The oxy-gen glucose deprivation/re-oxygenation model was established by the method of reoxygenation for 20 h after 6 h of oxygen. Remimazolam was added during reoxygenation at a final concentration of 100 μM, bruscoxol and dimethyl sulfoxide were added 4 h before hypoxia, and bruscoxol was added at the end of hypoxia. The concentration was 500 nM, and the dimethyl sulfoxide concentration was <0.1%. After reoxygenation, CCK8 cell activity, apoptosis rate, ROS level, Nrf2, Keap1, Bcl2, Bax protein levels were detected. Mitochondrial morphology and Nrf2 nuclear translocation was de-tected by immunofluorescence. Results: Compared with the C group, the M group had decreased cell viability, increased apoptosis rate and ROS activity, increased Bax protein level, decreased Nrf2, Keap1, Bcl2 protein levels, increased mitochondrial fragmentation and increased Nrf2 nuclear translocation;Compared with the M group, the cell viability in the R group increased, the apoptosis rate and ROS activity decreased, the Bax protein level decreased, the Nrf2, Keap1, Bcl2 protein lev-els increased, and the nuclear translocation of Nrf2 increased and the degree of mitochondrial fragmentation decreased;There was no statistical difference between group V and group R;com-pared with group V, group B had decreased cell viability, increased apoptosis rate and ROS activity, increased Bax protein level, and decreased Nrf2, Keap1, Bcl2 protein levels, reduced Nrf2 nuclear translocation and increased mitochondrial fragmentation. Conclusion: Remimazolam can activate the Keap1/Nrf2 pathway, reduce the accumulation of ROS and mitochondrial damage, reducing apoptosis and reducing the damage of oxygen sugar deprivation-rehy- drosaccharide in rat hippo-campal neurons.