过表达circ_ITCH通过调节星形胶质细胞活化对大鼠脊髓损伤修复的促进作用

doi:10.13481/j.1671-587X.20260306

摘要/Abstract

摘要：

目的探讨过表达环状RNA（circRNA）_ITCH对大鼠脊髓损伤（SCI）的促进作用，并阐明其可能的作用机制。方法采用改良Allen’s打击法建立SCI大鼠模型，将造模成功大鼠随机分为假手术组、SCI模型组（SCI组）、空载腺相关病毒组（AAV-NC组）和circ_ITCH过表达腺相关病毒组（AAV-circ_ITCH组），每组15只。使用微量注射器向大鼠损伤脊髓内注射6 μL AVV-circ_ITCH和AAV-NC进行干预，假手术组和SCI组大鼠注射等量生理盐水。采用斜板实验和BBB运动评定量表评估各组大鼠后肢运动功能，实时荧光定量PCR（RT-qPCR）法检测各组大鼠脊髓损伤组织中circ_ITCH表达水平，HE染色观察各组大鼠脊髓损伤组织病理形态表现，酶联免疫吸附试验（ELISA）法检测各组大鼠脊髓损伤组织中白细胞介素（IL）-1β、IL-6和肿瘤坏死因子α（TNF-α）水平，Western blotting法检测各组大鼠脊髓损伤组织中胶质纤维酸性蛋白（GFAP）、神经蛋白聚糖（Neurocan）和波形蛋白（Vimentin）表达水平，免疫荧光法检测各组大鼠脊髓损伤组织中星形胶质细胞活化标志物GFAP蛋白表达情况。结果与假手术组比较，SCI组大鼠脊髓组织损伤明显，斜板角度和BBB评分明显降低（P<0.05），脊髓损伤组织中circ_ITCH表达水平明显降低（P<0.05），IL-1β、IL-6和TNF-α水平明显升高（P<0.05），GFAP、Neurocan和Vimentin蛋白表达水平及GFAP蛋白荧光强度明显升高（P<0.05）。与SCI组比较，AAV- circ_ITCH组大鼠脊髓组织损伤明显改善，斜板角度和BBB评分明显升高（P<0.05），脊髓损伤组织中circ_ITCH表达水平明显升高（P<0.05），IL-1β、IL-6和TNF-α水平明显降低（P<0.05），GFAP、Neurocan和Vimentin蛋白表达水平及GFAP蛋白荧光强度明显降低（P<0.05）；AAV-NC组以上指标差异均无统计学意义（P>0.05）。结论过表达circ_ITCH可减轻脊髓损伤大鼠的脊髓组织损伤，促进大鼠运动功能恢复，其机制可能与抑制神经炎症和星形胶质细胞活化有关。

关键词: 脊髓损伤, 环状RNA_ITCH, 星形胶质细胞, 神经功能, 运动功能

Abstract:

Objective To discuss the promotion effect of overexpression of circular RNA （circRNA）_ITCH on spinal cord injury （SCI） in the rats， and to clarify its possible mechanism. Methods The rat model of SCI was established by modified Allen’s method. The successfully modeled rats were randomly divided into sham operation group， SCI model group （SCI group）， empty adeno-associated virus group （AAV-NC group） and circ_ITCH overexpression adeno-associated virus group （AAV-circ_ITCH group）， with 15 rats in each group. Microsyringe was used to inject 6 μL of AAV-circ_ITCH and AAV-NC into the injured spinal cord of rats for intervention， and the rats in sham group and SCI group were injected with the same volume of normal saline. Inclined plane test and BBB locomotor rating scale were used to evaluate the hind limb motor function of rats in various groups； real-time fluorescence quantitative PCR was used to detect the expression levels of circ_ITCH in the spinal cord injury tissue of rats in various groups； HE staining was used to observe the pathomorphological manifestations of the spinal cord injury tissue of the rats in various groups； enzyme-linked immunosorbent assay was used to detect the levels of interleukin-1β （IL-1β）， interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in spinal cord injury tissue of the rats in various groups； Western blotting method was used to detect the expression levels of glial fibrillary acidic protein （GFAP）， neurocan and vimentin proteins in spinal cord injury tissue of the rats in various groups； immunofluorescence method was used to detect the expression of GFAP protein， a marker of astrocyte activation， in the spinal cord injury tissue of rats in various groups. Results Compared with sham operation group， the rats in SCI group showed obvious spinal cord tissue injury， the inclined plane angle and BBB score were significantly decreased （P<0.05）， the expression level of circ_ITCH in the spinal cord injury tissue was significantly decreased （P<0.05）， the levels of IL-1β， IL-6 and TNF-α were significantly increased （P<0.05）， and the expression levels of GFAP， neurocan and vimentin proteins as well as the fluorescence intensity of GFAP protein were significantly increased （P<0.05）. Compared with SCI group， the rats in AAV-circ_ITCH group showed significantly ameliorated spinal cord tissue injury， the inclined plane angle and BBB score were significantly increased （P<0.05）， the expression level of circ_ITCH in the spinal cord injury tissue was significantly increased （P<0.05）， the levels of IL-1β， IL-6 and TNF-α were significantly decreased （P<0.05）， and the expression levels of GFAP， neurocan and vimentin proteins as well as the fluorescence intensity of GFAP protein were significantly decreased （P<0.05）； in AAV-NC group， there were no statistically significant differences in the above indicators （P>0.05）. Conclusion Overexpression of circ_ITCH can alleviate spinal cord tissue injury in the rats with SCI and promote the recovery of motor function in the rats； its mechanism may be related to inhibiting neuroinflammation and astrocyte activation.

Key words: Spinal cord injury, Circular RNA_ITCH, Astrocytes, Neurological function, Motor function

中图分类号:

R744.9

赵焱,吴华伟. 过表达circ_ITCH通过调节星形胶质细胞活化对大鼠脊髓损伤修复的促进作用[J]. 吉林大学学报(医学版), 2026, 52(3): 633-640.

Yan ZHAO,Huawei WU. Promotion effect of overexpression of circ_ITCH on repairment of spinal cord injury by regulating astrocyte activation in rats[J]. Journal of Jilin University(Medicine Edition), 2026, 52(3): 633-640.

图/表 6

图1

图2

表1

图3

表2

图4

参考文献 24

[1]	IZZY S. Traumatic spinal cord injury［J］. Continuum （Minneap Minn）， 2024， 30（1）： 53-72.
[2]	QUADRI S A， FAROOQUI M， IKRAM A， et al. Recent update on basic mechanisms of spinal cord injury［J］. Neurosurg Rev， 2020， 43（2）： 425-441.
[3]	REYES C， MOKALLED M H. Astrocyte-neuron interactions in spinal cord injury［J］. Adv Neurobiol， 2024， 39： 213-231.
[4]	XU J H， FANG S， DENG S X， et al. Generation of neural organoids for spinal-cord regeneration via the direct reprogramming of human astrocytes［J］. Nat Biomed Eng， 2023， 7（3）： 253-269.
[5]	LONG C M， LI Z， SONG W， et al. The roles of non-coding RNA targeting astrocytes in cerebral ischemia［J］. Mol Neurobiol， 2024， 61（8）： 5814-5825.
[6]	WANG D， GAO Q， HUANG S J， et al. Circular E3 ubiquitin-protein ligase improves renal function and alleviates inflammation and renal injury in chronic glomerulonephritis rats via the microRNA-146a-5p/tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma axis［J］. J Physiol Pharmacol， 2024， 75（3）： 327-337.
[7]	YANG F， GUO Z F， WU J W， et al. Circ-ITCH inhibits bladder cancer progression through miR-184/FOXO3 axis［J］. Am J Transl Res， 2024， 16（12）： 7911-7923.
[8]	LI S L， ZHOU J B， ZHANG J J， et al. Construction of rat spinal cord injury model based on Allen’s animal model［J］. Saudi J Biol Sci， 2019， 26（8）： 2122-2126.
[9]	QIN C， LIU Y， XU P P， et al. Inhibition by rno-circRNA-013017 of the apoptosis of motor neurons in anterior horn and descending axonal degeneration in rats after traumatic spinal cord injury［J］. Front Neurosci， 2022， 16： 1065897.
[10]	RIVLIN A S， TATOR C H. Objective clinical assessment of motor function after experimental spinal cord injury in the rat［J］. J Neurosurg， 1977， 47（4）： 577-581.
[11]	BASSO D M， BEATTIE M S， BRESNAHAN J C. A sensitive and reliable locomotor rating scale for open field testing in rats［J］. J Neurotrauma， 1995， 12（1）： 1-21.
[12]	ANJUM A， YAZID M D， FAUZI DAUD M， et al. Spinal cord injury： pathophysiology， multimolecular interactions， and underlying recovery mechanisms［J］. Int J Mol Sci， 2020， 21（20）： 7533.
[13]	方露，谢财忠，王红星，等. 脊髓损伤后痉挛的机制及其治疗研究进展［J］. 中国康复医学杂志， 2020， 35（1）： 112-118.
[14]	FRANTSUZOV R， MONDAL S， WALSH C M， et al. A finite element model of contusion spinal cord injury in rodents［J］. J Mech Behav Biomed Mater， 2023， 142： 105856.
[15]	ZHANG F， LIN F L， XU Z W， et al. Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration［J］. Aging， 2021， 13（10）： 14185-14197.
[16]	刘京松，史旭，万然，等. 外泌体及其在继发性脊髓损伤中作用的研究进展［J］. 中国脊柱脊髓杂志， 2020， 30（1）： 71-75.
[17]	唐润妍，惠首智，张明，等. PD1基因敲除加重小鼠脊髓损伤后继发性损伤的研究［J］. 神经解剖学杂志， 2021， 37（5）： 517-524.
[18]	刘丹丹，秦合伟，高洋，等. 干细胞调控巨噬细胞极化缓解脊髓损伤的研究进展［J］. 解放军医学杂志， 2025， 50（9）： 1195-1208.
[19]	CLIFFORD T， FINKEL Z， RODRIGUEZ B， et al. Current advancements in spinal cord injury research-glial scar formation and neural regeneration［J］. Cells， 2023， 12（6）： 853.
[20]	刘曌，王丽君，万东君. 抗髓鞘少突胶质细胞糖蛋白IgG抗体相关疾病的临床特征分析［J］. 中风与神经疾病杂志， 2025， 42（3）： 233-238.
[21]	CAI J J， KONG J D， MA S， et al. Upregulation of TRPC6 inhibits astrocyte activation and proliferation after spinal cord injury in rats by suppressing AQP4 expression［J］. Brain Res Bull， 2022， 190： 12-21.
[22]	石美，汤云萍，刘绛巍，等. HIF-1α通过调控TRPV通道对细胞功能的影响［J］. 中国兽医学报， 2025， 45（8）： 1784-1791， 1800.
[23]	CHEN K Z， LIU S X， LI Y W， et al. Vimentin as a potential target for diverse nervous system diseases［J］. Neural Regen Res， 2023， 18（5）： 969-975.
[24]	KAN S L， LIU C J， ZHAO X Y， et al. Resveratrol improves the prognosis of rats after spinal cord injury by inhibiting mitogen-activated protein kinases signaling pathway［J］. Sci Rep， 2023， 13（1）： 19723.

Group	IL-1β	IL-6	TNF-α
Sham operation	86.43±8.04	42.55±5.17	34.56±4.62
SCI	402.80±27.22^*	274.61±24.28^*	313.88±19.01^*
AAV-NC	389.54±20.37	288.52±19.80	297.45±22.87
AAV-circ_ITCH	184.36±12.30^△	135.01±14.92^△	108.13±9.25^△

Group	GFAP	Neurocan	Vimentin
Sham operation	0.09±0.02	0.17±0.03	0.06±0.01
SCI	0.54±0.04^*	0.87±0.08^*	0.36±0.03^*
AAV-NC	0.53±0.03	0.83±0.06	0.35±0.04
AAV-circ_ITCH	0.18±0.02^△	0.29±0.02^△	0.20±0.02^△