TRPA1参与丙泊酚联合布比卡因所致背根神经节细胞损伤

Transient receptor potential channel A1 is involved in propofol combined bupivacaine‑induced cellular injury to dorsal root ganglion neurons

目的:探讨非选择性阳离子通道瞬时受体电位通道A1(transient receptor potential channel A1, TRPA1)是否参与丙泊酚联合布比卡因所致背根神经节(dorsal root ganglion, DRG)细胞损伤。方法:以C57B6/J背景的野生型(wild type, WT)小鼠与TRPA1敲除(TRPA1^(-/-))小鼠为研究对象,分别采集小鼠腰段(L_(3)~L_(5))DRG进行原代细胞培养,培养24 h后进行体外细胞实验。取培养24 h的WT小鼠DRG细胞采用随机数字表法分为4组(每组9孔):对照组(未予以药物处理)、丙泊酚组(丙泊酚10 μmol/L)、布比卡因组(布比卡因2 mmol/L)、丙泊酚+布比卡因组(丙泊酚10 μmol/L、布比卡因2 mmol/L),观察WT小鼠DRG细胞在不同药物处理后的细胞形态变化。取培养24 h的WT小鼠DRG细胞采用随机数字表法分为3组(每组9孔):对照组(未予以药物处理)、丙泊酚+布比卡因组(丙泊酚10 μmol/L、布比卡因2 mmol/L)、HC-030031+丙泊酚+布比卡因组(丙泊酚10 μmol/L、布比卡因2 mmol/L、HC-030031 1 μmol/L),采用细胞计数试剂盒(cell counting kit-8, CCK-8)法检测DRG细胞活力并计算细胞存活率,MitoSOX Red超氧化物指示剂检测线粒体活性氧(reactive oxygen species, ROS)水平,JC-1法检测线粒体膜电位水平。WT小鼠与TRPA1^(-/-)小鼠DRG细胞根据小鼠基因型分为WT组与TRPA1^(-/-)组(每组9孔),比较两组DRG细胞在丙泊酚(10 μmol/L)联合布比卡因(2 mmol/L)处理后线粒体ROS水平及其膜电位的变化。 结果:与对照组、丙泊酚组、布比卡因组比较,布比卡因+丙泊酚组DRG细胞形态改变,甚至细胞破坏。与对照组比较,丙泊酚+布比卡因组细胞存活率降低( P<0.05),ROS水平升高( P<0.05),线粒体膜电位水平降低( P<0.05);与丙泊酚+布比卡因组比较,HC-030031+丙泊酚+布比卡因组细胞存活率升高( P<0.05),ROS水平降低( P<0.05),线粒体膜电位水平升高( P<0.05)。与WT组比较,TRPA1^(-/-)组在丙泊酚联合布比卡因处理后,DRG细胞线粒体ROS水平降低( P<0.05),线粒体膜电位水平增加( P<0.05)。 结论:TRPA1参与丙泊酚联合布比卡因所致体外培养DRG细胞损伤,与线粒体活性氧及膜电位水平有关。

Objective To test whether the combined use of propofol and bupivacaine leads to cellular injury in dorsal root ganglion(DRG)neurons via a non‐selective cation permeable channel,the transient receptor potential channel A1(TRPA1).Meth‑ods Wild type(WT)mice and TRPA1 knockout(TRPA1^(−/−))mice with C57B6/J background were used as research subjects.DRG(L_(3)‒L_(5))from the lumbar segment of mice were collected for primary cell culture,and cultured for 24 h before conducting in vitro experiments.To observe the morphological changes of DRG cells in WT mice treated with different drugs,cells were randomly divided into 4 groups(n=9)using a random number table method:control group(no treatment),propofol group(10μmol/L),bupivacaine group(2 mmol/L),propofol+bupivacaine group(propofol 10μmol/L,bupivacaine 2 mmol/L).Using HC‐030031 to specifically block TRPA1,the effects of HC‐030031 on the cell activity and viability of WT mouse DRG cells induced by propofol combined with bupivacaine[cell counting kit 8(CCK‐8)],mitochondrial reactive oxygen species(ROS)levels(MitoSOX Red superoxide indicator detection),and membrane potential polarization(JC‐1 method)were measured.For this purpose,cells were divide into 3 groups using a random num‐ber table method(n=9):control group(no treatment),and propofol+bupivacaine group(propofol 10μmol/L,bupivacaine 2 mmol/L),HC‐030031+propofol+bupivacaine group(propofol 10μmol/L,bupivacaine 2 mmol/L,HC‐0300311μmol/L).Furthermore,we com‐pared the changes in mitochondrial ROS levels and membrane potential of DRG cells between WT mice(WT group)and TRPA1−/−mice(TRPA1^(−/−) group)(n=9)when using propofol(10μmol/L)combined with bupivacaine(2 mmol/L).Results Compared with the control group,propofol group,and bupivacaine group,the DRG cells in the bupivacaine+propofol group showed morphological changes and even cell destruction.Compared with the control group,the cell survival rate of the propofol+bupivacaine group decreased(P<0.05),the ROS level increased(P<0.05),and the mitochondrial membrane potential level decreased(P<0.05);Compared with the propofol+bupivacaine group,the HC‐030031+propofol+bupivacaine group showed an increase in cell survival rate(P<0.05),a decrease in ROS level(P<0.05),and an increase in mitochondrial membrane potential level(P<0.05).Compared with the WT group,the TRPA1^(−/−) group showed a decrease in mitochondrial ROS levels(P<0.05)and an increase in mitochondrial membrane levels in DRG cells treated with propofol combined with bupivacaine(P<0.05).Conclusions The combination of bupivacaine and propofol can cause injury to the cultured DRG neurons from mice,in which TRPA1 is involved.This effect is related to mitochondrial ROS and membrane potential level.