摘要
目的探讨硫氧还蛋白相互作用蛋白(TXNIP)在布比卡因介导氧化应激引起脊髓神经毒性中的机制。方法无特定病原体(SPF)级健康雄性SD大鼠48只,体重110~130 g,采用随机数字表法分为4组(每组12只):对照组(C组)、布比卡因组(B组)、布比卡因+腺相关病毒(AAV)‑TXNIP短发夹RNA(shRNA)(BT组)、布比卡因+shRNA组(BS组)。C组、B组、BT组、BS组大鼠均行L_(5)‑L_(6)节段鞘内置管。C组和B组均鞘内注射5μl生理盐水;BT组和BS组分别鞘内注射5μl AAV‑TXNIP shRNA(病毒滴度为1.01×10^(12)VG/ml)和5μl无义对照序列shRNA。随后拔除鞘管,待所有大鼠饲养至250~300 g时,再次行L_(5)‑L_(6)节段鞘内置管,B组、BT组、BS组鞘内注射3次5%布比卡因0.12μl/g,每次间隔90 min,C组在相应时间内鞘内注射等量生理盐水。各组于相应处理后采用大鼠甩尾潜伏期(TFL)换算而来的最大抗伤害效应百分比(%MPE)、巴索‑比蒂‑布雷斯纳汉(BBB)评分法于鞘内注射布比卡因或等量生理盐水后当天(T_(0))、鞘内注射后第1天(T_(1))、鞘内注射后第2天(T_(2))、鞘内注射后第3天(T_(3))测定大鼠的尾部感觉和下肢运动功能;苏木精‑伊红染色(H‑E染色)观察脊髓组织损伤情况,尼氏染色观察存活神经元;生化检测丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽(GSH)含量,免疫荧光多重染色法检测活性氧(ROS)水平,实时荧光定量聚合酶链反应(FQ‑PCR)检测TXNIP信使RNA(mRNA)水平,免疫印迹法(Western blot)检测脊髓神经组织中TXNIP、谷胱甘肽过氧化物酶4(GPX4)的蛋白水平。结果与C组比较:B组、BT组、BS组T_(0)、T_(1)、T_(2)、T_(3)时%MPE均较高,BBB评分均较低(均P<0.05);B组和BS组脊髓组织少突胶质细胞和小胶质细胞增多,神经元细胞水肿或萎缩,存活神经元较少(均P<0.05);B组、BT组、BS组的MDA含量较高(均P<0.05),SOD、GSH、CAT含量较低(均P<0.05);B组和BS组ROS水平较高(均P<0.05),TXNIP mRNA水平较高(均P<0.05);BT组TXNIP mRNA水平较低(P<0.05);B组和BS组TXNIP蛋白水平较高(均P<0.05),GPX4蛋白水平较低(P<0.05)。与B组比较,BT组T_(1)、T_(2)、T_(3)时%MPE较低,BBB评分较高(均P<0.05),灰质及白质水肿空泡减少,受损的神经元减少,存活神经元较多(P<0.05),MDA含量较低(P<0.05),SOD、GSH、CAT含量较高(均P<0.05),ROS水平较低(P<0.05),TXNIP mRNA水平、TXNIP蛋白水平较低(均P<0.05),GPX4蛋白水平较高(P<0.05)。结论布比卡因通过激活TXNIP介导氧化应激产生脊髓神经毒性,敲低TXNIP可通过抑制氧化应激缓解布比卡因导致的脊髓神经毒性。
Objective To investigate the mechanism by which thioredoxin‑interacting protein(TXNIP)mediates oxidative stress in bupivacaine‑induced spinal neurotoxicity.Methods A total of 48 specific pathogen‑free(SPF)healthy male SD rats,weighing 110−130 g,were selected.According to the random number table method,they were divided into four groups(n=12):a control group(group C),a bupivacaine group(group B),a bupivacaine+adeno‑associated virus(AAV)‑TXNIP short hairpin RNA(shRNA)group(group BT),and a bupivacaine+shRNA group(group BS).Intrathecal catheterization was conducted at the L_(5)‑L_(6)segments of rats in groups C,B,BT and BS.Rats in group C and group B were intrathecally injected with 5μl normal saline,while those in group BT and group BS were intrathecally injected with 5μl of AAV‑TXNIP shRNA(virus titer:1.01×10^(12)VG/ml)and 5μl of the nonsense control sequence shRNA,respectively.Subsequently,the sheath tube was removed.When all the rats were raised to 250−300 g,the L_(5)‑L_(6)segment was intrathecally catheterized again.Groups B,BT,and BS were intrathecally injected with 5%bupivacaine at 0.12μl/g for three times,with an interval of 90 min between each session.Group C was injected with the same amount of normal saline at the corresponding time points.After the corresponding treatments,the tail sensation and lower limb motor function of rats were measured by the percent of maximum possible effect(%MPE)converted from tail‑flick latency(TFL)in rats,and the Basso Beatty Bresnahan(BBB)scoring method on the day of intrathecal injection of bupivacaine or an equal amount of normal saline(T_(0)),day 1 after intrathecal injection(T_(1)),day 2 after intrathecal injection(T_(2)),and day 3 after intrathecal injection(T_(3)).The injury of spinal cord tissue was observed by hematoxylin eosin staining(H‑E staining),while Nissl staining was used to observe survival neurons.The contents of malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT),and glutathione(GSH)were detected by biochemical methods.The levels of reactive oxygen species(ROS)were detected by immunofluorescence staining.The levels of TXNIP messenger RNA(mRNA)were examined by real‑time fluorescence quantitative polymerase chain reaction(FQ‑PCR).The levels of TXNIP and glutathione peroxidase 4(GPX4)in spinal cord nerve tissue was detected by Western blot.Results Compared with group C,groups B,BT,and BS showed increases in%MPE and decreases in BBB scores at T_(0),T_(1),T_(2),and T_(3)(all P<0.05);group B and group BS had an increased number of oligodendrocytes and microglia,edema or atrophy in neuronal cells,and a decreased number of survival neurons(all P<0.05);groups B,BT,and BS showed increases in the contents of MDA(all P<0.05),and decreases in the contents of SOD,GSH,and CAT(all P<0.05);groups B and BS presented increases in the levels of ROS(all P<0.05),and TXNIP mRNA levels(P<0.05);group BT showed decreases in TXNIP mRNA levels(P<0.05);and groups B and BS presented increases in TXNIP protein expression(all P<0.05),and decreases in GPX4 protein expression(all P<0.05).Compared with group B,group BT showed decreases in%MPE,but increases in BBB score at T_(1),T_(2),and T_(3)(all P<0.05),as well as reduced edema and vacuoles in the gray and white matter,with a reduced number of damaged neurons,and an elevated number of survival neurons(P<0.05),decreases in MDA contents(P<0.05),increases in the contents of SOD,GSH and CAT(all P<0.05),decreases in the level of ROS(P<0.05),reductions in TXNIP mRNA and protein levels(all P<0.05),and increases in GPX4 protein expression(P<0.05).Conclusions Bupivacaine induces spinal neurotoxicity by activating TXNIP‑mediated oxidative stress.Knockdown of TXNIP can alleviate bupivacaine‑induced spinal neurotoxicity by inhibiting oxidative stress.
作者
陈园园
赵漾
罗云鹏
赖坚
刘子儒
罗茜
刘敬臣
Chen Yuanyuan;Zhao Yang;Luo Yunpeng;Lai Jian;Liu Ziru;Luo Xi;Liu Jingchen(Department of Anesthesiology,the First Afiliated Hospital of Guangxi Medical University,Nanning 530021,China)
出处
《国际麻醉学与复苏杂志》
CAS
2024年第5期449-456,共8页
International Journal of Anesthesiology and Resuscitation
基金
国家自然科学基金(81660623)
广西研究生创新项目(YCBZ2022091)。
