目的 探讨布比卡因和丙泊酚联合应用是否通过激活瞬时受体电位锚蛋白A1（TRPA1）导致神经毒性。方法 选用3-4周雄性WT小鼠，安乐死后取腰骶部（L3-L5）背根神经节（Dorsal root ganglion，DRG）进行原代神经元培养，采用不同浓度布比卡因和/或丙泊酚进行干预，检测神经元活力并观察细胞形态。通过检测线粒体活性氧（Reactive oxygen species，ROS）和膜电位水平，以及Ca2+浓度判断麻醉药物对DRG神经元的影响。使用HC-030031 特异性阻断TRPA1受体后，检测布比卡因和/或丙泊酚对DRG神经元作用有无改变。WT和Trpa1-/-小鼠进行鞘内注射布比卡因（2 mM，6 μl）和腹腔注射丙泊酚（50 mg/kg）1h后进行步态测试。 结果 布比卡因处理DRG神经元2h时50%抑制浓度（Half maximal inhibitory concentration, IC50）为2 mM，1、10和100 μM浓度丙泊酚对神经元活力无显著影响。布比卡因（2 mM）与丙泊酚（1或10 μM）合用使细胞活力显著下降（P＜0.05），DRG神经元形态改变。布比卡因（2 mM）和丙泊酚（10 μM）合用使WT鼠DRG神经元线粒体ROS显著增加与膜电位去极化（P＜0.05）。WT小鼠DRG神经元线粒体Ca2+以布比卡因浓度依赖性方式升高（P＜0.05）。使用HC-030031特异性阻断TRPA1受体后，可明显提高丙泊酚与布比卡因联合处理后的DRG神经元活性（P＜0.05），降低DRG神经元线粒体ROS水平和Ca2+浓度（P＜0.05），减少膜电位去极化（P＜0.05）。鞘内注射布比卡因和腹腔注射丙泊酚1h后，WT小鼠相对于Trpa1-/-小鼠行走时后肢与地面的最大压力接触时间显著缩短（P＜0.05）。 结论 布比卡因与丙泊酚联合使用可通过激活TRPA1造成背根神经节神经元损伤。
Objective To explore whether the combined use of bupivacaine and propofol leads to neurotoxicity by activating transient receptor potential anchor protein 1(TRPA1). Methods Cultivate primary neurons of the lumbar (L3-L5) dorsal root ganglion (DRG) and intervene with different concentrations of bupivacaine and/or propofol to detect neuronal viability and observe cell morphology. The effects of anesthetics on DRG neurons were determined by detecting the levels of mitochondrial superoxide and membrane potential, as well as the concentration of Ca2+. After using HC-030031 to specifically block the TRPA1 receptor, effects of bupivacaine and/or propofol on sensory neurons were measured. WT and Trpa1-/- mice were subjected to intrathecal injection of bupivacaine (2 mM, 6 μl) and intraperitoneal injection of propofol (50 mg/kg), followed by gait testing one hour afterwards. Results The half maximal inhibitory concentration (IC50) of DRG neurons treated with bupivacaine for 2 hours was 2 mM, and propofol at concentrations of 1, 10, and 100 μM had no significant effect on neuronal viability. Bupivacaine (2 mM) and propofol (1 or 10 μM) combined significantly reduced cell viability (P0.05) and altered neuronal morphology. Bupivacaine (2 mM) and propofol (10 μM) combined significantly increased mitochondrial superoxide and membrane potential depolarization in DRG neurons of WT mice (P0.05). The mitochondrial Ca2+ of DRG neurons in WT mice increased in a concentration dependent manner with bupivacaine (P0.05). HC-030031 specific blocking of TRPA1 receptor significantly increased the activity of DRG neurons after combined with propofol and bupivacaine (P0.05), reduced the level of superoxide and concentration of Ca2+ in neuron mitochondria (P0.05), and reduced depolarization (P0.05). One hour after intrathecal injection of bupivacaine and intraperitoneal injection of propofol, the maximum contact time (Max Contact At (%)) between the hind limbs and the ground during walking in WT mice shortened when compared to that from Trpa1-/- mice (P0.05). Conclusions The combination of bupivacaine and propofol can cause damage to dorsal root ganglion neurons by activating TRPA1.
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