汉黄芩苷对大鼠脊髓损伤的修复效果

doi:10.16098/j.issn.0529-1356.2022.02.006

摘要/Abstract

摘要：

目的通过观察不同剂量汉黄芩苷对大鼠脊髓损伤后介导的炎症反应的干预情况，探讨汉黄芩苷对脊髓损伤的影响。方法建立大鼠脊髓横断损伤模型（n=95），随机分成5组：正常组（N）、生理盐水组（NS）、汉黄芩苷低剂量组（WG12.5）、汉黄芩苷中剂量组（WG25）及汉黄芩苷高剂量组（WG50）。采用ELISA检测炎性因子肿瘤坏死因子α（TNF-α）及白细胞介素1（IL-1）的表达水平；静态脚印分析脚趾扩张（TS）和中间脚趾扩张（ITS）的变化；HE染色观察脊髓损伤大鼠组织学情况；尼氏染色观察脊髓损伤大鼠神经元数目变化；透射电子显微镜观察轴突形态及再生现象；劳克坚牢蓝（LFB）髓鞘染色观察脱髓鞘现象；免疫组织化学观察胶质纤维酸性蛋白（GFAP）和神经生长相关蛋白43（GAP43）的表达变化。结果 NS组与N组相比,TNF-α及IL-1表达明显升高（P＜0.01），TS和ITS明显减少（P＜0.01），脊髓形态显著异常，神经元严重丢失，髓鞘形态异常，髓鞘严重脱色（P＜0.01），GFAP表达明显增多，GAP43表达稍有增加；WG组与NS组相比，TNF-α和IL-1表达明显降低（P＜0.01），TS和ITS增加（P＜0.01），脊髓形态有所恢复，神经元丢失减少，髓鞘形态趋于正常且有新生轴突出现，髓鞘脱色减少（P＜0.01），GFAP表达明显减少，GAP43表达明显增多，且呈现剂量依赖性。结论汉黄芩苷能改善脊髓损伤局部微环境，减少胶质瘢痕形成，促进轴突再生，减少局部神经元丢失、空洞形成等，进而促进脊髓损伤的修复。

关键词: 汉黄芩苷, 脊髓损伤, 炎性反应, 胶质纤维酸性蛋白, 神经生长相关蛋白43, 透射电子显微术, 免疫组织化学, 大鼠

Abstract:

Objective To assess the effect of wogonoside on the inflammation after rat spinal cord injury. Methods Rats (n=95) were subjected to dorsal spinal cord transection at T9-10 vertebra. The rats were randomly divided into 5 groups: normal group (N), normal saline group (NS), low-dose wogonoside group (WG12.5), medium-dose wogonoside group (WG25) and high-dose wogonoside group (WG50). The expression levels of inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1) were detected by ELISA. The levels of toe spreading (TS) and intermediate toe spreading (ITS) were evaluated with static footprint analysis. HE staining was performed to observe the histology of spinal cord injury rats. The number of neurons in rats with spinal cord injury was observed by Nissl staining, the morphology and regeneration of axons by transmission electron microscope, the demyelination by myelin sheath staining, and the expression of glial fibrillary acidic protein (GFAP) and nerve growth associated protein 43 (GAP43) by immunohistochemistry. Results Compared with the N group, NS group showed higher expression of TNF-α and IL-1（P＜0.01）, higher levels of TS and ITS（P＜0.01）, abnormal morphology of spinal cord, severer loss of neurons, abnormal myelin sheath morphology, more serious decolorization of myelin sheath（P＜0.01）, significantly increased GFAP expression, slightly increased GAP43 expression. The WG group showed decreased expression of TNF-α and IL-1（P＜0.01）, lower levels of TS and ITS（P＜0.01）, restored morphology of spinal cord, inhibited neuron loss, restored myelin sheath morphology and new neural axis, reduced decolorization of myelin sheath（P＜0.01）, decreased expression of GFAP, and increased expression of GAP43. All these reversed results were dose-dependent. Conclusion Wogonoside can improve the local microenvironment of spinal cord injury, reduce the formation of glial scar, promote axon regeneration, inhibit local neuron loss and cavity formation, all contributing to the repair of spinal cord injury.

Key words: Wogonoside, Spine cord injury, Inflammatory reaction, Glial fibrillary acidic protein, Nerve growth associated protein 43, Transmission electron microscopy, Immunohistochemistry, Rat

中图分类号:

Q189

王笑刘卿谷成旭李希凯张璐萍. 汉黄芩苷对大鼠脊髓损伤的修复效果[J]. 解剖学报, 2022, 53(2): 173-182.

WANG Xiao LIU Qing GU Cheng-xu LI Xi-kai ZHANG Lu-ping. Repair effect of wogonoside on rat spinal cord injury[J]. Acta Anatomica Sinica, 2022, 53(2): 173-182.

参考文献

［1］ Duan YY, Chai YQ, Chai Y, et al. Role and mechanism of stabilizing microtubules of endothelial cells and pericytes in improving the microvasculature dysfunction after spinal cord injury［J］. Acta Anatomica Sinica, 2021,52(1):21-29.(in Chinese)

段洋洋,张雅群,柴勇,等.稳定内皮和周细胞微管改善脊髓损伤中微循环障碍的作用及其机制［J］.解剖学报,2021,52(1):21-29.

［2］ Hong JY, Lee SH, Lee SC, et al. Therapeutic potential of induced neural stem cells for spinal cord injury［J］.J Biol Chem, 2014, 289(47):32512-32525

［3］ Zhang Q, Yang H, An J, et al. Therapeutic effects of traditional Chinese medicine on spinal cord injury: a promising supplementary treatment in future［J］. Evid Based Complement Alternat Med, 2016,2016:8958721.

［4］ Shi Z, Yuan S, Shi L, et al. Programmed cell death in spinal cord injury pathogenesis and therapy［J］. Cell Prolif, 2021, 54(3):e12992.

［5］ Piri SM, Ghodsi Z, Shool S, et al. Macrophage migration inhibitory factor as a therapeutic target after traumatic spinal cord injury: a systematic review ［J］. Eur Spine J, 2021，30（6）：1474-1494.

［6］ Yu H, Zhang P, Zhou W, et al. Alkaline-phosphatase triggered self-assemblies enhances the anti-inflammatory property of methylprednisolone in spinal cord injury ［J］. J Appl Biomater Funct Mater, 2020, 18:588110665.

［7］ Zhang P, Ma X. Therapeutic potential of flavonoids in spinal cord injury ［J］.Rev Neurosci, 2017, 28(1):87-101.

［8］ Shen YF, Fan XQ. Progress on pharmacological research of wogonoside ［J］.Journal of Shanghai University of Traditional Chinese Medicine, 2016, 30(4):98-101.(in China)

申云富, 范小青.汉黄芩苷的药理活性研究进展［J］.上海中医药大学学报, 2016, 30(4):98-101.

［9］ Huang S, Fu Y, Xu B, et al. Wogonoside alleviates colitis by improving intestinal epithelial barrier function via the MLCK/pMLC2 pathway［J］. Phytomedicine, 2020,68:153179.

［10］Tang Q, Zheng C, Feng Z, et al, Wogonoside inhibits IL-1β induced catabolism and hypertrophy in mouse chondrocyte and ameliorates murine osteoarthritis ［J］.Oncotarget, 2017, 8(37): 61440-61456

［11］ Arevalo-Martin A, Garcia-Ovejero D, Molina-Holgado E.The endocannabinoid 2-arachidonoylglycerol reduces lesion expansion and white matter damage after spinal cord injury.［J］.Neurobiol Dis, 2010, 38(2):304-312.

［12］ Dinarello CA. Overview of the IL-1 family in innate inflammation and acquired immunity［J］. Immunol Rev, 2018, 281(1):8-27.

［13］ Bessis A, Bernard D, Triller A. Tumor necrosis factor-alpha and neuronal development.［J］.Neuroscientist, 2005, 11(4):277-281.

［14］ Jiang Z, Zhang J. Mesenchymal stem cell-derived exosomes containing miR-145-5p reduce inflammation in spinal cord injury by regulating the TLR4/NF-kappa B signaling pathway ［J］. Cell Cycle, 2021,21(10):993-1009.

［15］ Wen J, Sun D, Tan J, et al. A consistent, quantifiable, and graded rat lumbosacral spinal cord injury model［J］. J Neurotrauma, 2015, 32(12):875-892.

［16］ Afshary K, Chamanara M, Talari B,et al. Therapeutic effects of minocycline pretreatment in the locomotor and sensory complications of spinal cord injury in an animal model［J］.J Mol Neurosci, 2020,70(7):1064-1072.

［17］ Liu NK, Xu XM. Neuroprotection and its molecular mechanism following spinal cord injury［J］. Neural Regen Res, 2012,7(26):2051-2062.

［18］ Liu J, Li R, Huang Z, et al. Rapamycin preserves neural tissue, promotes schwann cell myelination and reduces glial scar formation after hemi-contusion spinal cord injury in mice［J］. Front Mol Neurosci, 2021,13:574041.

［19］ Sun X, Zhang C, Xu J, et al.Neurotrophin-3-loaded multichannel nanofibrous scaffolds promoted anti-inflammation, neuronal differentiation, and functional recovery after spinal cord injury［J］.ACS Biomater Sci Eng, 2020, 6(2):1228-1238.

［20］ Hara M, Kobayakawa K, Ohkawa Y, et al. Interaction of reactive astrocytes with type Ⅰ collagen induces astrocytic scar formation through the integrin-N-cadherin pathway after spinal cord injury［J］.Nat Med, 2017, 23(7):818-828.

［21］ Sobani ZA, Quadri SA, Enam SA. Stem cells for spinal cord regeneration: current status ［J］. Surg Neurol Int, 2010, 1:93.

［22］ Karimi-Abdolrezaee S, Billakanti R. Reactive astrogliosis after spinal cord injury-beneficial and detrimental effects.［J］.Mol Neurobiol, 2012, 46(2):251-264.

［23］ Leal-Filho MB. Spinal cord injury: from inflammation to glial scar ［J］. Surg Neurol Int, 2011,2:112.

［24］ Fehlings MG, Hawryluk GW. Scarring after spinal cord injury［J］.J Neurosurg Spine, 2010, 13(2):165-168.

［25］ Wang GY, Cheng ZhJ, Yang BH, et al.The expression of chondroitin sulfate proteoglycan and GFAP after spinal cord injury［J］.Biological Orthopedic Materials and Clinical Research,2020,17(1):43-46.(in Chinese)

王国毓,程志坚,杨保辉,等.脊髓损伤后硫酸软骨素蛋白多糖及GFAP表达的变化［J］.生物骨科材料与临床研究,2020,17(1):43-46.

［26］ Zhang QL, Li JC, Ding PP, et al. Study on the time phases of GAP-43 expression following spinal cord injury［J］.Sichuan Journal of Anatomy, 2010, 18（2):1-3.(in Chinesr)

张奇兰, 李俊岑, 丁培培, 等.神经生长相关蛋白-在脊髓损伤后不同时段的表达［J］.四川解剖学杂志, 2010, 18(2):1-3.

[1]	谢惠兰方芳林毅. Chir99021调控Wnt/β-catenin 信号通路抑制大鼠牙髓干细胞成骨诱导分化的机制[J]. 解剖学报, 2024, 55(1): 67-72.
[2]	魏茜茜李超红赵晨露唐家萍刘玉珍. 大鼠颈上神经节中Ⅰ组代谢型谷氨酸受体表达及慢性间歇性低氧对其的影响 [J]. 解剖学报, 2024, 55(1): 3-9.
[3]	袁蕾杨智伟杨惠刘政海何洁万炜. 三七总皂苷抑制小鼠星形胶质细胞活化缓解完全弗氏佐剂所致炎性痛 [J]. 解剖学报, 2024, 55(1): 25-31.
[4]	郑丽平刘霞杜晓华吴亚娟刘珊珊 . 脑源性神经营养因子在牦牛与黄牛端脑不同区域的表达与分布 [J]. 解剖学报, 2024, 55(1): 10-16.
[5]	苏芃王兴仪梁景岩熊天庆. 一种低密度及高纯度胎鼠原代海马神经元培养方法的优化及鉴定[J]. 解剖学报, 2024, 55(1): 113-119.
[6]	郝永明郭磊周程辉裴汉军李莉赵哲 . 改良腹主动脉缩窄法建立心肌肥厚模型[J]. 解剖学报, 2024, 55(1): 120-124.
[7]	许亚平余悦欣陈金福王柯柯郭志坤 . 高龄大鼠心室肌间质弹性纤维和胶原纤维的结构分布特征及其机制 [J]. 解剖学报, 2023, 54(6): 716-721.
[8]	刘丽平朱梦妮徐慧 . 白细胞介素6对脂多糖诱导所致类子痫前期大鼠有核红细胞的影响 [J]. 解剖学报, 2023, 54(6): 722-729.
[9]	赵卫明李晓余国营王改平常翠芳 . 丝氨酸肽酶抑制因子Kazal型1对肝癌细胞增殖的影响及其机制 [J]. 解剖学报, 2023, 54(6): 695-702.
[10]	王文娟丁雨桐苏昊任捷孙艳荣王涵菲陆嘉莉张林倩白钰秦丽华. 低雌激素状态下大鼠海马及纹状体Nogo-A的表达变化[J]. 解剖学报, 2023, 54(6): 620-627.
[11]	梁盼盼禹爱梅杜静寇文辉王欢欢宋爱霞. 基于c-Jun氨基末端激酶/p38丝裂原活化蛋白激酶信号通路探讨阿魏酸钠对偏头痛大鼠炎症反应的抑制作用[J]. 解剖学报, 2023, 54(6): 652-659.
[12]	姚素华鄢骏谢芳林春华黄争春 . 奥氮平介导环磷酸腺苷反应元件结合蛋白/脑源性神经营养因子/酪氨酸蛋白激酶受体B通路对精神分裂模型大鼠的神经修复作用 [J]. 解剖学报, 2023, 54(6): 660-667.
[13]	徐颖刘斌郑兴何宗钊. 补体C3a受体在盲肠结扎穿孔致脓毒症大鼠认知障碍中的作用及机制 [J]. 解剖学报, 2023, 54(6): 668-675.
[14]	李霞朱世杰唐中生罗亚非范瑞娟谢高宇寇云芳陆莹 . 电针智三针调节酪氨酸激酶受体的配体通路改善血管性痴呆的作用机制[J]. 解剖学报, 2023, 54(6): 689-694.
[15]	刘叶楚亚楠徐岑璐何佳澄苏炳银太颢然. Roscovitine挽救帕金森病小鼠相关脑区神经元丢失和神经炎症[J]. 解剖学报, 2023, 54(6): 635-643.