线粒体钙单向转运体在布比卡因加重糖尿病大鼠脊髓神经损伤中的作用

Role of MCU in bupivacaine-induced spinal cord neurotoxic injury in diabetic rats

目的:探讨线粒体钙单向转运体(MCU)在布比卡因(B)致糖尿病(D)大鼠脊髓神经毒性损伤中表达的变化及其作用。方法:健康雄性SD大鼠,体重180～220 g,分为正常组与造模组,腹腔注射链脲佐菌素制备糖尿病大鼠模型后,随机选取48只,分为6组,每组8只,即鞘内注射生理盐水正常大鼠(C)组、鞘内注射生理盐水糖尿病大鼠(D)组、鞘内注射布比卡因正常大鼠(C+B)组、鞘内注射布比卡因糖尿病大鼠(D+B)组、鞘内注射10μmol/L Ru360+布比卡因糖尿病大鼠(D+R_1+B)组和鞘内注射50μmol/L Ru360+布比卡因糖尿病大鼠(D+R_2+B)组;各组在造模前、造模成功后、鞘内注射药物后12 h、鞘内注射药物后24 h和鞘内注射药物后48 h测定机械刺激缩足阈值(PWMT)与热刺激缩足潜伏期(PWTL)改变;测定完毕后处死大鼠,取脊髓腰膨大行RT-qPCR与Western blot测定MCU表达水平,ELISA法测定氧化损伤产物丙二醛(MDA)与8-羟基脱氧鸟苷(8-OHdG)的含量,TUNEL法测定脊髓神经细胞凋亡率。结果:与D组比较,D+B组MCU表达显著上调(P <0. 05),PWMT、PWTL及MDA与8-OHdG含量显著升高(P <0. 05),脊髓神经细胞凋亡率显著提高(P <0. 05);与D+B组比较,D+R_2+B组MCU表达显著下调(P <0. 05),PWMT、PWTL及MDA与8-OHdG表达均显著降低(P <0. 05),脊髓神经细胞凋亡率显著下降(P <0. 05)。结论:布比卡因通过上调MCU表达活性,增强氧化应激,加重糖尿病大鼠脊髓神经损伤与提高其机械痛与热痛阈值。

AIM:To investigate the role of mitochondrial calcium uniporter(MCU)in bupivacaine(B)-induced spinal cord injury in diabetic(D)rats.METHODS:Healthy male Sprague-Dawley rats,weighing 180~220 g,were divided into normal rats and diabetic rats.After intraperitoneal injection of streptozotocin for building diabetic rat mo-del,48 male rats were randomly divided into 6 groups with 8 rats in each group as following:control(C)group(normal rats by intrathecal injection of normal saline),D group(diabetic rats by intrathecal injection of normal saline),C+B group(normal rats by intrathecal injection of bupivacaine),D+B group(diabetic rats by intrathecal injection of bupivacaine),D+R 1+B group(diabetic rats by intrathecal injection of 10μmol/L Ru360 and bupivacaine)and D+R 2+B group(diabetic rats by intrathecal injection of 50μmol/L Ru360 and bupivacaine).The changes of paw withdrawal mechanical threshold(PWMT)and paw withdrawal thermal latency(PWTL)were measured before modeling,after modeling,and 12 h,24 h and 48 h after intrathecal injection.Lumbar enlargement was removed from spinal cord after rats were killed.MCU expression was tested by RT-qPCR and Western bolt.The levels of malondialdehyde(MDA)and 8-hydroxydeoxyguanosine(8-OHdG)were determined by ELISA.Spinal neuronal apoptosis was measured using TUNEL assay.RESULTS:Compared with D group,the expression of MCU,the values of PWMT and PWTL,the levels of MDA and 8-OHdG,and the apoptotic rate of spinal cord neurons were significantly increased in D+B group(P<0.05).Compared with D+B group,the expression of MCU,the values of PWMT and PWTL,the levels of MDA and 8-OHdG,and the apoptotic rate of spinal cord neurons were significantly decreased in D+R 2+B group(P<0.05).CONCLUSION:Bupivacaine enhances oxidative stress and aggravates spinal cord injury via up-regulating MCU activity in diabetic rats.