Wang Chenxu, Zhou Yan, Xie Keliang, Yuan Yuan, Wang Guolin, Yu Yonghao
Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin Research Institute of Anesthesiology, Tianjin 300052, China. Corresponding author: Yu Yonghao, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2023 Feb;35(2):189-194. doi: 10.3760/cma.j.cn121430-20210926-01402.
To investigate whether propofol can cause injury to hippocampal mitochondria in neonatal rats and the regulation of excitatory amino acid receptor AMPA receptor.
Forty-eight Sprague-Dawley (SD) rats aged 7 days were randomly divided into control group, propofol group, propofol+AMPA receptor agonist AMPA group (propofol+AMPA group) and propofol+AMPA receptor inhibitor CNQX group (propofol+CNQX group), with 12 rats in each group. The rats in the propofol groups were intraperitoneally injected with 30 mg/kg propofol, while in control group with 3 mg/kg normal saline. Each group was given 1/2 of the first dose every 20 minutes after the first administration, three times a day, for three consecutive days. The rats in the propofol+AMPA group and the propofol+CNQX group were injected with 1 g/L AMPA or CNQX 5 μL through left ventricle after the first administration. Three days after administration, the rats were sacrificed to obtain brain tissue. Western blotting was used to determine the expression of AMPA receptor glutamate receptors (GluR1, GluR2) subunit totally (T) and on membrane (M) in hippocampus. The expression of dynamin-related protein-1 (DRP-1) and phosphorylated-DRP-1 (p-DRP-1) and mitofusin 2 (Mfn2) related to mitochondrial fission and fusion were determined. The adenosine triphosphate (ATP) content and ATPase activity were determined.
Compared with the control group, GluR1 expression and its M/T ratio were significantly increased after treatment of propofol, GluR2 expression and its M/T ratio were significantly decreased, the ATP content and ATP-related enzyme activity were decreased significantly, while the expression of DRP-1 and its phosphorylation was significantly increased, and the expression of Mfn2 was significantly decreased. The changes indicated that repeated intraperitoneal injection of 30 mg/kg propofol leading to the injury of mitochondria in neural cells. Compared with the propofol group, the GluR1 expression and its M/T ratio further increased after AMPA agonist administration [T-GluR1 protein (T-GluR1/β-actin): 2.41±0.29 vs. 1.72±0.11, M-GluR1 protein (M-GluR1/β-actin): 1.18±0.15 vs. 0.79±0.09, M/T ratio: 0.78±0.12 vs. 0.46±0.08, all P < 0.01], GluR2 expression was significantly increased [T-GluR2 protein (T-GluR2/β-actin): 0.65±0.13 vs. 0.30±0.14, P < 0.01; M-GluR2 protein (M-GluR2/β-actin): 0.17±0.05 vs. 0.13±0.07, P > 0.05], but its M/T ratio was further decreased (0.27±0.10 vs. 0.41±0.08, P < 0.05). The ATP-related enzyme activity was further decreased, and the ATP content was further decreased (μmol/g: 0.32±0.07 vs. 0.70±0.10, P < 0.01). Mitochondria DRP-1 expression and its phosphorylation were further increased [DRP-1 protein (DRP-1/GAPDH): 2.75±0.36 vs. 1.70±0.19, p-DRP-1 protein (p-DRP-1/GAPDH): 0.99±0.14 vs. 0.76±0.15, both P < 0.05], and Mfn2 expression was further decreased (Mfn2/GAPDH: 0.23±0.12 vs. 0.54±0.12, P < 0.05). This indicated that the AMPA agonist increased the expression of the AMPA receptor GluR1 subunit on the cell membrane and shifted the GluR2 into the cell, thus increasing the mitochondrial injury caused by propofol. Compared with the propofol group, the GluR1 expression and its M/T ratio decreased significantly after AMPA inhibitor administration [T-GluR1 protein (T-GluR1/β-actin): 0.99±0.14 vs. 1.72±0.11, M-GluR1 protein (M-GluR1/β-actin): 0.21±0.07 vs. 0.79±0.09, M/T ratio: 0.21±0.07 vs. 0.46±0.08, all P < 0.01], the change of GluR2 expression was not significant, but its M/T ratio was significantly increased (0.59±0.09 vs. 0.41±0.08, P < 0.05). The ATP-related enzyme activity was increased significantly, and the ATP content was increased significantly (μmol/g: 0.87±0.12 vs. 0.70±0.10, P < 0.05). Mitochondria DRP-1 expression and its phosphorylation were significantly decreased [DRP-1 protein (DRP-1/GAPDH): 1.18±0.17 vs. 1.70±0.19, p-DRP-1 protein (p-DRP-1/GAPDH): 0.37±0.10 vs. 0.76±0.10, both P < 0.05], and Mfn2 expression was significantly increased (Mfn2/GAPDH: 0.78±0.10 vs. 0.54±0.12, P < 0.05). This indicated that AMPA inhibitor promoted the movement to the cell membrane of GluR2 subunits meanwhile inhibited the expression of GluR1 subunits, thus alleviating the injury of mitochondrial caused by propofol in the brain.
Repeated intraperitoneal injection of 30 mg/kg propofol for 3 days can increase the expression of GluR1 subunits of AMPA receptor in 7-day neonatal rats hippocampus mainly distributing in the cell membrane, decrease the expression of GluR2 subunits moving into the cell, thus causing injury of mitochondrial function and dynamics, which can be aggravated by AMPA receptor agonist and alleviated by AMPA receptor inhibitors.
探讨丙泊酚是否会导致新生大鼠海马线粒体损伤以及对兴奋性氨基酸受体AMPA受体的调节作用。
将48只7日龄的Sprague-Dawley（SD）大鼠随机分为对照组、丙泊酚组、丙泊酚+AMPA受体激动剂AMPA组（丙泊酚+AMPA组）和丙泊酚+AMPA受体抑制剂CNQX组（丙泊酚+CNQX组），每组12只。丙泊酚组大鼠腹腔注射30mg/kg丙泊酚，对照组注射3mg/kg生理盐水。首次给药后每隔20分钟给予首剂量的1/2，每日3次，连续3天。丙泊酚+AMPA组和丙泊酚+CNQX组大鼠在首次给药后经左心室注射1g/L AMPA或5μL CNQX。给药3天后，处死大鼠获取脑组织。采用蛋白质免疫印迹法检测海马中AMPA受体谷氨酸受体（GluR1、GluR2）亚基总表达量（T）和膜表达量（M）。检测与线粒体分裂融合相关的动力相关蛋白-1（DRP-1）、磷酸化-DRP-1（p-DRP-1）和线粒体融合蛋白2（Mfn2）的表达。测定三磷酸腺苷（ATP）含量和ATP酶活性。
与对照组相比，丙泊酚处理后GluR1表达及其M/T比值显著升高，GluR2表达及其M/T比值显著降低，ATP含量和ATP相关酶活性显著降低，而DRP-1表达及其磷酸化显著增加，Mfn2表达显著降低。这些变化表明，腹腔重复注射30mg/kg丙泊酚可导致神经细胞线粒体损伤。与丙泊酚组相比，给予AMPA激动剂后GluR1表达及其M/T比值进一步升高[T-GluR1蛋白（T-GluR1/β-肌动蛋白）：2.41±0.29 vs. 1.72±0.11，M-GluR1蛋白（M-GluR1/β-肌动蛋白）：1.18±0.15 vs. 0.79±0.09，M/T比值：0.78±0.12 vs. 0.46±0.08，均P<0.01]，GluR2表达显著增加[T-GluR2蛋白（T-GluR2/β-肌动蛋白）：0.65±0.13 vs. 0.30±0.14，P<0.01；M-GluR2蛋白（M-GluR2/β-肌动蛋白）：0.17±0.05 vs. 0.13±0.07，P>0.05]，但其M/T比值进一步降低（0.27±0.10 vs. 0.41±0.08，P<0.05）。ATP相关酶活性进一步降低，ATP含量进一步降低（μmol/g：0.3
