AMPA受体对丙泊酚诱导新生大鼠海马线粒体损伤的调控作用

Regulation of AMPA receptor on propofol induced hippocampal mitochondrial injury in neonatal rats

目的探讨丙泊酚是否对新生大鼠海马线粒体造成损伤,以及兴奋性氨基酸受体AMPA受体的调控作用。方法将48只7日龄SD大鼠按随机数字表法分为对照组、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。丙泊酚各组大鼠腹腔注射30 mg/kg丙泊酚,对照组注射生理盐水3 mg/kg;各组均于首次给药后每隔20 min给予首剂量的1/2,每日3次,连续注射3 d。丙泊酚+AMPA组和丙泊酚+CNQX组分别于首次给药后经左侧脑室注射1 g/L的AMPA或CNQX 5μL。各组给药3 d后处死大鼠取脑组织,采用蛋白质免疫印迹试验(Western blotting)检测海马AMPA受体谷氨酸受体(GluR1、GluR2)亚单位的总蛋白(T)及膜蛋白(M)表达量,并计算二者比值(M/T);采用Western blotting检测线粒体动力相关蛋白-1(DRP-1)、磷酸化DRP-1(p-DRP-1)和线粒体融合蛋白2(Mfn2)表达;同时测定海马三磷酸腺苷(ATP)含量及ATP相关酶活性。结果与对照组相比,丙泊酚处理后大鼠海马GluR1的表达及其M/T比值均明显升高,而GluR2的表达及其M/T比值则明显降低,ATP含量及ATP相关酶活性明显下降,同时DRP-1表达及其磷酸化水平明显升高,而Mfn2表达明显下降,说明反复多次腹腔注射30 mg/kg丙泊酚可导致神经细胞线粒体损伤。与丙泊酚组比较,加入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-actin):2.41±0.29比1.72±0.11,M-GluR1蛋白(M-GluR1/β-actin):1.18±0.15比0.79±0.09,M/T比值:0.78±0.12比0.46±0.08,均P<0.01〕;GluR2表达明显升高〔T-GluR2蛋白(T-GluR2/β-actin):0.65±0.13比0.30±0.14,P<0.01;M-GluR2蛋白(M-GluR2/β-actin):0.17±0.05比0.13±0.07,P>0.05〕,但其M/T比值进一步降低(0.27±0.10比0.41±0.08,P<0.05);ATP相关酶活性进一步降低,ATP含量进一步下降(μmol/g:0.32±0.07比0.70±0.10,P<0.01);线粒体DRP-1表达及其磷酸化水平进一步升高〔DRP-1蛋白(DRP-1/GAPDH):2.75±0.36比1.70±0.19,p-DRP-1蛋白(p-DRP-1/GAPDH):0.99±0.14比0.76±0.15,均P<0.05〕,Mfn2表达进一步下降(Mfn2/GAPDH:0.23±0.12比0.54±0.12,P<0.05),说明AMPA激动剂增加了大鼠神经细胞膜上AMPA受体GluR1亚单位的表达,同时使GluR2向细胞内移动,从而加剧了丙泊酚所致的线粒体损伤。而加入AMPA抑制剂后,与丙泊酚组比较,GluR1表达及其M/T比值均明显降低〔T-GluR1蛋白(T-GluR1/β-actin):0.99±0.14比1.72±0.11,M-GluR1蛋白(M-GluR1/β-actin):0.21±0.07比0.79±0.09,M/T比值:0.21±0.07比0.46±0.08,均P<0.01〕;GluR2表达变化则不明显,但其M/T比值明显升高(0.59±0.09比0.41±0.08,P<0.05);ATP酶活性明显升高,且ATP含量明显上升(μmol/g:0.87±0.12比0.70±0.10,P<0.05);线粒体DRP-1表达及其磷酸化水平明显下降〔DRP-1蛋白(DRP-1/GAPDH):1.18±0.17比1.70±0.19,p-DRP-1蛋白(p-DRP-1/GAPDH):0.37±0.10比0.76±0.15,均P<0.05〕,而线粒体Mfn2表达则明显升高(Mfn2/GAPDH:0.78±0.10比0.54±0.12,P<0.05),说明AMPA抑制剂通过抑制GluR1亚单位的表达,促使GluR2亚单位向细胞膜移动,从而缓解了丙泊酚导致的大鼠脑组织线粒体产能及动力学损伤。结论连续3 d多次腹腔注射丙泊酚30 mg/kg,可引起7日龄新生大鼠海马AMPA受体GluR1亚单位表达升高且主要分布于细胞膜,GluR2亚单位表达下降且向细胞内移动,从而导致线粒体功能及动力学损伤;AMPA受体激动剂可加重上述损伤,而AMPA受体抑制剂可减轻这一损伤。

Objective To investigate whether propofol can cause injury to hippocampal mitochondria in neonatal rats and the regulation of excitatory amino acid receptor AMPA receptor.Methods 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 5μL 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.Results 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.Conclusions 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.