基于HIF-1α/ERK通路探讨异丙酚对缺氧大鼠海马神经元线粒体损伤的影响

Effect of propofol on mitochondrial damage of hippocampal neurons in hypoxic rats based on HIF-1α/ERK pathway

摘要:

目的 观察异丙酚通过缺氧诱导因子-1α（HIF-1α）/细胞外调节激酶（ERK）通路对缺氧大鼠海马神经元线粒体损伤的影响。方法 新生SD大鼠离体培养原代海马神经元细胞，随机分为6组。对照组（高糖DMEM培养液）、缺氧组（低糖DMEM培养液）、LP组（3 mg/L异丙酚的低糖DMEM培养液）、MP组（6 mg/L异丙酚的低糖DMEM培养液）、HP组（12 mg/L异丙酚的低糖DMEM培养液）及U0126组（终浓度12 mg/L异丙酚+50 μmol/L U0126的低糖DMEM培养液）。缺氧组、LP组、MP组、HP组及U0126组于缺氧条件培养24 h；对照组于有氧条件培养24 h。透射电镜观察各组海马神经元细胞形态；MTT法检测细胞存活率；激光共聚焦显微镜检测Ca²⁺、活性氧（ROS）的荧光强度；酶联免疫吸附试验检测细胞钙调神经磷酸酶（GaN）活性；Western blotting检测HIF-1α、ERK1/2、p-ERK1/2、天冬氨酸特异性半胱氨酸蛋白酶3（Caspase-3）蛋白表达。结果 缺氧组线粒体损伤严重，LP、MP和HP组线粒体损伤均有所减轻，HP组减轻最为明显，U0126组线粒体损伤较HP组严重。与缺氧组海马神经元细胞存活率（42.30±3.64）%比较，MP组（59.54±5.19）%、HP组（70.81±6.47）%及U0126组（53.08±5.61）%均升高，且HP组高于MP组和U0126组（P <0.05）。与缺氧组Ca²⁺、ROS荧光强度［（0.107±0.004）、（0.087±0.003）］比较，LP组ROS荧光强度（0.078±0.003）及MP组、HP组及U0126组Ca²⁺荧光强度［（0.085±0.003）、（0.067±0.002）、（0.090±0.003）］、ROS荧光强度［（0.060±0.002）、（0.051±0.005）、（0.076±0.003）］均降低（P <0.05），HP组低于MP组和U0126组（P <0.05）。与缺氧组GaN活性（0.61±0.05）u/mg比较，MP组、HP组及U0126组［（0.50±0.05）u/mg、（0.37±0.04）u/mg、（0.54±0.04）u/mg］降低，且HP组低于MP组和U0126组（P <0.05）。与缺氧组比较，LP组、MP组、HP组及U0126组HIF-1α、p-ERK1/2蛋白相对表达量升高，且HP组高于LP组、MP组和U0126组（P <0.05）；与缺氧组比较，LP组、MP组、HP组及U0126组Caspase-3蛋白相对表达量降低，且HP组低于LP组、MP组和U0126组（P <0.05）。结论 异丙酚可减轻缺氧大鼠海马神经元线粒体损伤，其调控机制可能与HIF-1α/ERK通路有关。

Abstract:

Objective To observe the effect of propofol on the mitochondrial damage of hippocampal neurons in hypoxic rats through the hypoxia-inducible factor-1α/extracellular regulatory kinase (HIF-1α/ERK) pathway.Method Newborn SD rats were cultured in vitro with primary hippocampal neurons and randomly divided into 6 groups, hypoxia group (low glucose DMEM medium), LP group, MP group, HP group (final concentrations of 3, 6, 12 mg/L, respectively) propofol low-sugar DMEM culture medium), and U0126 group (final concentration of 12 mg/L propofol + 50 μmol/L U0126 low-sugar DMEM culture medium), cultured under hypoxia for 24 hours. The control group (high-sugar DMEM medium) was cultured for 24 hours under aerobic conditions. The morphology of hippocampal neurons in each group was observed by transmission electron microscope. Cell survival rate was detected by MTT method. Ca²⁺ and reactive oxygen species (ROS) content were detected by laser confocal microscope. The activity of calcineurin (GaN) in cells was detected by ELISA. The protein expressions of HIF-1α, ERK1/2, p-ERK1/2, and aspartate specific cysteine protease 3 (Caspase-3) were detected by Western blotting.Results The mitochondrial damage in the hypoxia group was severe, and the mitochondrial damage in the LP, MP, and HP groups were all reduced. The HP group had the most significant reduction. The U0126 group had more severe mitochondrial damage than the HP group. Compared with the hippocampal neuron survival rate (42.30 ±3.64)% in the hypoxia group, the MP group (59.54 ± 5.19) %, the HP group (70.81 ± 6.47) %, and the U0126 group (53.08 ± 5.61) % all increased, and the HP group Higher than MP group and U0126 group (P < 0.05). Compared with the fluorescence intensity of Ca²⁺ and ROS [ (0.107 ± 0.004), (0.087 ± 0.003) ] in the hypoxia group, the fluorescence intensity of ROS in the LP group (0.078 ± 0.003) and the Ca²⁺ fluorescence intensity in the MP, HP, and U0126 groups [ (0.085 ± 0.003), (0.067 ± 0.002), (0.090 ± 0.003) ], ROS fluorescence intensity [ (0.060 ± 0.002), (0.051 ± 0.005), (0.076 ± 0.003) ] all decreased (P < 0.05), and the HP group was lower than MP group and U0126 group (P < 0.05). Compared with the GaN activity (0.61 ± 0.05) U/mg in the hypoxia group, the MP group, HP group, and U0126 group [ (0.50 ± 0.05) U/mg, (0.37 ± 0.04) U/mg, (0.54 ± 0.04) U/mg] decreased, and HP group was lower than MP group and U0126 group (P < 0.05). Compared with the hypoxia group, the relative expression of HIF-1α and p-ERK1/2 protein in the LP group, MP group, HP group, and U0126 group increased, and the HP group was higher than the LP group, MP group, and U0126 group (P < 0.05); Compared with the hypoxia group, the relative expression of Caspase-3 protein in the LP, MP, HP, and U0126 groups decreased, and the HP group was lower than the LP, MP, and U0126 groups (P < 0.05).Conclusion Propofol can reduce mitochondrial damage of hippocampal neurons in hypoxic rats, and its regulatory mechanism may be related to the HIF-1α/ERK pathway.