代攀;黄思琴;唐成林;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;

• 1:重庆医科大学中医药学院

摘要(Abstract)：

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 C57 BL/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-B2 3 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 LC3 II 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 LC3 II 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 in the EA group.Conclusion EA of EX-B2 can improve the locomotor function in SCI mice, which may be related to its effects in up-regulating the expression of LC3 II(to promote cell autophagy), and down-regulating the expression of P62, GRP78, Caspase-12 and CHOP proteins(to inhibit endoplasmic reticulum stress) in the spinal cord tissue.

关键词(KeyWords)： 电针;脊髓损伤;内质网应激;细胞自噬
Electroacupuncture;Spinal cord injury;Endoplasmic reticulum stress;Cell autophagy

Abstract:

Keywords:

基金项目(Foundation): 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)

作者(Author): 代攀;黄思琴;唐成林;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;

Email:

DOI: 10.13702/j.1000-0607.200229

参考文献(References)：

• [1] ALIZADEH A,DYCK S M,KARIMI-ABDOLREZAEE S.Traumatic spinal cord injury:an overview of pathophysiology,models and acute injury mechanisms[J].Front Neurol,2019,10:282.
• [2] 黄柏南,孙善全,汪克建,等.大鼠脊髓缺血再灌注损伤后Caspase-12 表达与细胞凋亡[J].解剖学杂志,2008,31(2):222-225.
• [3] 黄柏南,孙善全,汪克建,等.内质网分子伴侣GRP78 在大鼠脊髓缺血再灌注损伤中的表达变化[J].基础医学与临床,2007,27(11):1198-1202.
• [4] KUROIWA M,WATANABE M,KATOH H,et al.Effect of amiloride on endoplasmic Reticulum stress response in the injured spinal cord of rats[J].Eur J Neurosci,2014,40(7):3120-3127.
• [5] ZHANG X Y,ZHANG T T,SONG D D,et al.Endoplasmic reticulum chaperone GRP78 is involved in autophagy activation induced by ischemic preconditioning in neural cells[J].Mol Brain,2015,8:20.
• [6] LIN C W,CHEN B,HUANG K L,et al.Inhibition of autophagy by estradiol promotes locomotor recovery after spinal cord injury in rats[J].Neurosci Bull,2016,32(2):137-144.
• [7] HUANG S Q,TANG C L,SUN S Q,et al.Demyelination initiated by oligodendrocyte apoptosis through enhancing endoplasmic reticulum-mitochondria interactions and Id2 expression after compressed spinal cord injury in rats[J].CNS Neurosci Ther,2014,20(1):20-31.
• [8] 代妮,黄思琴,唐成林,等.电针对脊髓损伤小鼠运动功能及炎性和氧化应激反应的影响[J].针刺研究,2019,44(11):781-786.
• [9] JESSEN K R,MIRSKY R.The origin and development of glial cells in peripheral nerves[J].Nat Rev Neurosci,2005,6(9):671-682.
• [10] 霍则军,牒军,徐基民.电针对兔脊髓缺血再灌注损伤组织肿瘤坏死因子-α和白细胞介素-6的影响[J].针刺研究,2012,37(4):308-311.
• [11] YOSHIDA H,MATSUI T,YAMAMOTO A,et al.XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor[J].Cell,2001,107(7):881-891.
• [12] 吕威,李冰,景泉凯,等.电针“大椎”“命门”对脊髓损伤大鼠神经元细胞凋亡及JNK信号通路相关蛋白表达的影响[J].针刺研究,2017,42(1):14-19.
• [13] 李晓宁,吴磊,迟蕾,等.不同治疗周期夹脊电针对急性脊髓损伤大鼠运动功能及细胞凋亡的影响[J].针刺研究,2016,41(6):492-496.
• [14] HUANG S Q,TANG C L,SUN S Q,et al.Protective effect of electroacupuncture on neural myelin sheaths is mediated via promotion of oligodendrocyte proliferation and inhibition of oligodendrocyte death after compressed spinal cord injury[J].Mol Neurobiol,2015,52(3):1870-1881.
• [15] 谭程方,黄思琴,唐成林,等.电针联合雪旺氏细胞移植对脊髓压迫性损伤后脊髓内CD4、CD8表达及髓鞘修复的影响[J].针刺研究,2019,44(6):391-398.
• [16] 唐祎周,孙忠人,张翀.夹脊电针对脊髓损伤大鼠内质网应激相关因IRE1影响的实验研究[J].针灸临床杂志,2013,29(8):55-58.
• [17] 尹洪娜,孙忠人,李全.电针夹脊穴对脊髓损伤大鼠内质网应激相关因子Caspase-12影响的实验研究[J].中医药学报,2016,44(3):33-36.
• [18] 尹洪娜,孙忠人,李全.电针夹脊穴对脊髓损伤大鼠内质网应激相关因子CHOP影响的实验研究[J].中医药信息,2016,33(4):35-38.
• [19] 冯德琳.夹脊电针调节细胞自噬促进脊髓损伤修复的实验研究[D].哈尔滨:黑龙江中医药大学,2018.
• [20] PATERNITI I,ESPOSITO E,CUZZOCREA S.An in vivo compression model of spinal cord injury[J].Methods Mol Biol,2018,1727:379-384.
• [21] 李辞蓉,宋大鲁,华兴邦,等.小鼠常用针灸穴位[J].实验动物与动物实验,1992,2:85-87.
• [22] BASSO D M,FISHER L C,ANDERSON A J,et al.Basso mouse scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains[J].J Neurotrauma,2006,23(5):635-659.
• [23] 胡华辉,黄小龙,刘飞,等.电针夹脊穴治疗脊髓损伤机制的实验研究进展[J].上海针灸杂志,2016,35(12):1480-1483.
• [24] 张立峰,张慧,刘妍妍,等.夹脊脉冲电刺激在脊髓损伤患者康复中的应用[J].中国中医药信息杂志,2013,20(3):84-85.
• [25] 李晓宁,迟蕾.夹脊配合督脉电针治疗脊髓损伤后功能障碍临床观察[J].上海针灸杂志,2015,34(10):972-975.
• [26] FANG B,QIN M M,LI Y,et al.Electroacupuncture preconditioning and postconditioning inhibit apoptosis and neuroinflammation induced by spinal cord ischemia reperfusion injury through enhancing autophagy in rats[J].Neurosci Lett,2017,642:136-141.
• [27] WANG H L,WU Y Q,HAN W,et al.Hydrogen sulfide ameliorates blood-spinal cord barrier disruption and improves functional recovery by inhibiting endoplasmic Reticulum stress-dependent autophagy[J].Front Pharmacol,2018,9:858.
• [28] DONG D Z,NI M,LI J Z,et al.Critical role of the stress chaperone GRP78/BiP in tumor proliferation,survival,and tumor angiogenesis in transgene-induced mammary tumor development[J].Cancer Res,2008,68(2):498-505.
• [29] 朱永林.PI3K/Akt信号通路和内质网应激在继发性脊髓损伤中的作用[D].济南:山东大学,2019.
• [30] OHRI S S,MADDIE M A,ZHANG Y P,et al.Deletion of the pro-apoptotic endoplasmic reticulum stress response effector CHOP does not result in improved locomotor function after severe contusive spinal cord injury[J].J Neurotrauma,2012,29(3):579-588.
• [31] 郭卫东,李刚,范仲凯.二甲双胍对大鼠脊髓损伤后内质网应激和细胞凋亡的影响[J].解剖学杂志(社会科学版),2019,42(2):161-166.
• [32] YANG Z F,KLIONSKY D J.Eaten alive:a history of macroautophagy[J].Nat Cell Biol,2010,12(9):814-822.
• [33] YU H,LI L S,LIU R,et al.Autophagy in long propriospinal neurons is activated after spinal cord injury in adult rats[J].Neurosci Lett,2016,634:138-145.
• [34] NIKOLETOPOULOU V,PAPANDREOU M E,TAVERNARAKIS N.Autophagy in the physiology and pathology of the central nervous system[J].Cell Death Differ,2015,22(3):398-407.
• [35] GALLUZZI L,BRAVO-SAN PEDRO J M,BLOMGREN K,et al.Autophagy in acute brain injury[J].Nat Rev Neurosci,2016,17(8):467-484.
• [36] LIPINSKI M M,WU J F,FADEN A I,et al.Function and mechanisms of autophagy in brain and spinal cord trauma[J].Antioxid Redox Signal,2015,23(6):565-577.
• [37] KABEYA Y,MIZUSHIMA N,UENO T,et al.LC3,a mammalian homologue of yeast Apg8p,is localized in autophagosome membranes after processing[J].EMBO J,2000,19(21):5720-5728.
• [38] TANIDA I,UENO T,KOMINAMI E.LC3 conjugation system in mammalian autophagy[J].Int J Biochem Cell Biol,2004,36(12):2503-2518.
• [39] SUN Y M,LIU D,SU P,et al.Changes in autophagy in rats after spinal cord injury and the effect of hyperbaric oxygen on autophagy[J].Neurosci Lett,2016,618:139-145.
• [40] TANABE F,YONE K,KAWABATA N,et al.Accumulation of p62 in degenerated spinal cord under chronic mechanical compression:functional analysis of p62 and autophagy in hypoxic neuronal cells[J].Autophagy,2011,7(12):1462-1471.
• [41] BJ?RK?Y G,LAMARK T,BRECH A,et al.p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death[J].J Cell Biol,2005,171(4):603-614.
• [42] ZHOU K L,ZHOU Y F,WU K,et al.Stimulation of autophagy promotes functional recovery in diabetic rats with spinal cord injury[J].Sci Rep,2015,5:17130.
• [43] ZHANG D,XUAN J,ZHENG B B,et al.Metformin improves functional recovery after spinal cord injury via auto-phagy flux stimulation[J].Mol Neurobiol,2017,54(5):3327-3341.
• [44] ZHOU Y L,ZHENG B B,YE L B,et al.Retinoic acid prevents disruption of blood-spinal cord barrier by inducing autophagic flux after spinal cord injury[J].Neurochem Res,2016,41(4):813-825.
• [45] ZHOU K L,CHEN D H,JIN H M,et al.Effects of calcitriol on experimental spinal cord injury in rats[J].Spinal Cord,2016,54(7):510-516.
• [46] GOLDSHMIT Y,KANNER S,ZACS M,et al.Rapamycin increases neuronal survival,reduces inflammation and astrocyte proliferation after spinal cord injury[J].Mol Cell Neurosci,2015,68:82-91.
• [47] OGATA M,HINO S,SAITO A,et al.Autophagy is activated for cell survival after endoplasmic reticulum stress[J].Mol Cell Biol,2006,26(24):9220-9231.
• [48] KOUROKU Y,FUJITA E,TANIDA I,et al.ER stress (PERK/eIF2α phosphorylation) mediates the polyglutamine-induced LC3 conversion,an essential step for autophagy formation[J].Cell Death Differ,2007,14(2):230-239.
• [49] B’CHIR W,MAURIN A C,CARRARO V,et al.The eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression[J].Nucleic Acids Res,2013,41(16):7683-7699.
• [50] YIN J J,GU L P,WANG Y F,et al.Rapamycin improves palmitate-induced ER stress/NFκB pathways associated with stimulating autophagy in adipocytes[J].Mediators Inflamm,2015,2015:272313.
• [51] SHENG R,LIU X Q,ZHANG L S,et al.Autophagy regulates endoplasmic Reticulum stress in ischemic preconditio-ning[J].Autophagy,2012,8(3):310-325.
• [52] GUO Y,WANG F,LI H P,et al.Metformin protects against spinal cord injury by regulating autophagy via the mTOR signaling pathway[J].Neurochem Res,2018,43(5):1111-1117.
• [53] LIU S,SARKAR C,DINIZO M,et al.Disrupted autophagy after spinal cord injury is associated with ER stress and neuronal cell death[J].Cell Death Dis,2015,6:e1582.