吗啡预处理对NGF诱导神经细胞TRPV1通道敏化及ERK蛋白活化的影响

Effects of morphine preconditioning on TRPV1 channel current and ERK phosphorylation sensitized by NGF in neurocytes

目的探讨吗啡预处理(morphine preconditioning,MPC)对神经生长因子(nerve growth factor,NGF)诱导的神经细胞辣椒素受体(transient receptor potential vanilloid 1,TRPV1)敏化,以及细胞外信号调节激酶(extracellular regulated protein kinase,ERK)活化的调控作用。方法原代培养的10日龄SD大鼠T2-T8节段背根神经节(dorsal root ganglia,DRG)神经元及大鼠嗜铬瘤细胞(pheochromocytoma cell,PC12)神经细胞,分别随机分为对照组(C组)、NGF敏化组(NGF组)、吗啡预处理组(MPC 0.3、1.0、3.0μmol·L^(-1)组)。经抗神经元特异性烯醇化酶(neuronal specific enolase,NSE)抗体鉴定DRG神经元后,用吗啡、NGF及辣椒素对两种细胞进行处理,模拟在体MPC对经历心肌缺血/再灌注损伤(ischemia/reperfusion injury,IRI)的T2-T8节段DRG神经元的影响。即刻采用单细胞膜片钳技术检测辣椒素诱发的DRG神经元内向电流;Western blot法检测PC12细胞TRPV1、磷酸化TRPV1(phosphorylated TRPV1,p-TRPV1)及pERK相对表达水平。结果 DRG神经元细胞状态良好,神经元特异性标志物NSE染色阳性;与对照组比较,NGF组的辣椒素诱发内向电流幅度增大(P<0.05),而MPC可以抑制电流幅度增大(P<0.05);NGF孵育可使TRPV1、p-TRPV1及p-ERK蛋白相对表达量上调(P<0.05),而MPC对表达上调的TRPV1、p-TRPV1及p-ERK蛋白具有抑制作用(P<0.05)。结论 MPC可以抑制NGF敏化的神经细胞TRPV1通道电流,其机制可能与降低TRPV1蛋白表达量及TRPV1、ERK蛋白磷酸化水平有关。

Aim To investigate the effects of morphine preconditioning(MPC)on transient receptor potential vanilloid 1(TRPV1)channel current in rat dorsal root ganglia(DRG)neurons and the phosphorylation(p)of TRPV1 and extracellular regulated protein kinases(ERK)in PC12 cells that sensitized by nerve growth factor(NGF).Methods DRG neurons isolated from T2-T8 segments of 10 days old SD rat or pheochromocytoma(PC12)cells were seeded into 24-well plates or 6-well plates,respectively,and randomly divided into 5 groups:control group(group C),NGF sensitization group(group NGF),and morphine preconditioning groups(group MPC 0.3,group MPC 1.0 and group MPC 3.0).DRG neurons were identified by immunofluorescent method with neuronal specific enolase(NSE).Cells were treated by morphine,NGF and capsaicin to simulate the effects of MPC on DRG neurons in T2-T8 segments during myocardial ischemia reperfusion injury(IRI).Afterwards,the inward current of DRG neurons induced by capsaicin in all groups were detected by whole cell recording;the expression and phosphorylation of TRPV1 and ERK in PC12 cells were detected by Western blot.Results DRG neurons survived and grew nicely,and the staining of neuronal specific markers,NSE,was positive.In comparison with group C,the inward current of group NGF was enhanced(P<0.05),while MPC inhibited the enhancement(P<0.05).The relative expression of TRPV1,p-TRPV1 and p-ERK in group NGF was up-regulated when compared with group C(P<0.05).Moreover,the up-regulation was also suppressed by MPC(P<0.05).Conclusions MPC inhibits TRPV1 channel current sensitized by NGF in neurocytes,and the mechanism might be associated with the down-regulation of TRPV1 p-TRPV1 and p-ERK expression.