针刺研究

2021, v.46(01) 45-51

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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

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;

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

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