电针“夹脊”穴调节细胞自噬及内质网应激对脊髓损伤小鼠的修复作用  

Effects of electroacupuncture at“Jiaji”(EX-B2)on autophagy and endoplasmic reticulum stress in spinal cord injury mice

作　　者：代攀 黄思琴[1] 唐成林[1] 代妮 赵鸿娣 谭燕玲 杨云昊 陶春鹤 DAI Pan;HUANG Si-qin;TANG Cheng-lin;DAI Ni;ZHAO Hong-di;TAN Yan-ling;YANG Yun-hao;TAO Chun-he(College of Traditional Chinese Medicine of Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学中医药学院,重庆400016

出　　处：《针刺研究》2021年第1期45-51,共7页Acupuncture Research

基　　金：国家自然科学基金项目(No.81403466、81273870);重庆市基础研究与前沿探索项目(No.cstc2017jcyjAX0363、cstc2018jcyjAX0036);重庆市卫计委项目(No.ZY201802026);重庆市人社局留创项目(No.cx2018106)。

摘　　要：目的:通过观察电针"夹脊"穴对脊髓损伤(SCI)小鼠细胞自噬及内质网应激水平的影响,探讨电针对SCI小鼠的作用及其相关机制。方法:将雌性C57BL/6小鼠按照随机数字表法分为假手术组、模型组及电针组,每组20只;每组再分成7、14 d两个亚组,每个亚组10只。采用血管夹压迫脊髓法制备SCI模型。电针组于造模后3 h开始采用电针双侧"夹脊"穴,每日1次,分别治疗7、14 d。各组于造模后第7、14天采用Basso mouse scale(BMS)评分评估小鼠后肢运动功能变化;HE染色法观察脊髓组织形态变化;Western blot法测定内质网应激指标葡萄糖调节蛋白-78(GRP78)、半胱氨酸天冬氨酸特异性蛋白酶-12(Caspase-12)及细胞自噬指标微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)、P62的表达变化;免疫荧光染色法检测SCI区CCAAT增强子结合蛋白同源蛋白(CHOP)和P62的蛋白表达。结果:造模后第7、14天,与假手术组比较,模型组小鼠的BMS评分显著下降(P<0.05);脊髓组织中细胞核固缩、肿胀,神经元坏死数目相对较多;LC3Ⅱ蛋白表达水平下降(P<0.05),P62、GRP78、Caspase-12蛋白表达水平明显升高(P<0.05);CHOP和P62阳性表达增高(P<0.05)。与模型组比较,电针组小鼠的BMS评分显著升高(P<0.05);脊髓组织中细胞核固缩、肿胀减轻,神经元坏死数目相对减少;LC3Ⅱ蛋白表达水平升高(P<0.05),P62、GRP78、Caspase-12蛋白表达水平明显下降(P<0.05);CHOP和P62的阳性表达显著减少(P<0.05)。结论:电针"夹脊"可以通过抑制小鼠SCI后内质网应激和促进细胞自噬,从而促进神经功能恢复,其机制可能与电针调节自噬及内质网应激的相关蛋白表达有关。Objective To observe the effect of electroacupuncture(EA)at"Jiaji"(EX-B2)on the levels of autophagy and endoplasmic reticulum stress in mice with spinal cord injury(SCI),so as to explore its mechanism underlying improvement of SCI.Methods A total of 60 female C57BL/6 mice were randomly divided into sham operation,model and EA groups,which were further divided into 7 d and 14 d subgroups(10 mice in each subgroup).The SCI model was established by pressing the exposed spinal cord(L1)with a vascular clamp for 15 s.EA was applied to bilateral EX-B23 h after modeling,once a day for 7 and 14 d,respectively.Basso Mouse Scale(BMS)for locomotion was used to evaluate hindlimb motor function on day 7 and 14 after SCI.H.E.staining was used to observe histopathologic changes of the injured spinal cord tissue,and Western blot employed to detect the expression of glucose regulatory protein-78(GRP78),Caspase-12,microtubule-associated protein light chain 3 Ⅱ(LC-Ⅱ)and P62(also known as sqstm1/Sequestome1)proteins.Immunofluorescence staining was used to detect the immunoacti-vities of spinal CCAAT/enhancer-binding protein(C/EBP)homologous protein(CHOP,an endoplasmic reticulum stress-inducible protein)and P62.Results On the 7^(th) and 14^(th) day after SCI,the BMS scores and expression levels of LC3Ⅱ protein were significantly down-regulated(P<0.05),and the expression levels of P62,GRP78 and Caspase-12 proteins,the immunoactivities of CHOP and P62 were all significantly up-regulated on both day 7 and 14 in the model group than in the sham operation group(P<0.05).Compared with the model group,the BMS scores and the expression levels of LC3Ⅱ protein were significantly increased on both day 7 and 14(P<0.05),while the expression levels of P62,GRP78 and Caspase-12 proteins,and the immunoactivities of CHOP and P62 were obviously decreased on day 7 and 14 in the EA group(P<0.05).Outcomes of H.E.stain showed that the cells with nuclei pyknosis and swelling and the necrotic cells appeared in the model group,which was relatively fewer

关 键 词：电针 脊髓损伤 内质网应激 细胞自噬 

分 类 号：R245.97[医药卫生—针灸推拿学]