Orexin-A通过调节促离子型谷氨酸受体促进脊髓损伤大鼠运动功能恢复

doi:10.12122/j.issn.1673-4254.2025.05.15

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (5): 1023-1030.doi: 10.12122/j.issn.1673-4254.2025.05.15

Orexin-A通过调节促离子型谷氨酸受体促进脊髓损伤大鼠运动功能恢复

何光侣¹^,³(), 储婉玉¹^,³, 李妍¹^,³, 盛鑫¹^,³, 罗浩³, 徐爱萍³, 卞命杰³, 张环环², 汪萌芽³(), 郑超¹()

^1.皖南医学院生理科学研究所，神经生物学研究室，安徽芜湖 241002
^2.皖南医学院生理科学研究所，心理生理学研究室，安徽芜湖 241002
^3.皖南医学院生理科学研究所，细胞电生理研究室，安徽芜湖 241002

收稿日期:2024-07-20 出版日期:2025-05-20 发布日期:2025-05-23
通讯作者: 汪萌芽,郑超 E-mail:1596104580@qq.com;wangmy@wnmc.edu.cn;chaozheng10@fudan.edu.cn
作者简介:何光侣，在读硕士研究生，E-mail: 1596104580@qq.com
基金资助:
国家自然科学基金(31200828);安徽省自然科学基金(2108085MC91);安徽省高校学科拔尖人才学术资助项目(gxbjZD2021061);安徽省教育厅自然科学研究项目(2024AH051954)

Orexin-A promotes motor function recovery of rats with spinal cord injury by regulating ionotropic glutamate receptors

Guanglü HE¹^,³(), Wanyu CHU¹^,³, Yan LI¹^,³, Xin SHENG¹^,³, Hao LUO³, Aiping XU³, Mingjie BIAN³, Huanhuan ZHANG², Mengya WANG³(), Chao ZHENG¹()

^11.Neurobiology Laboratory, 2Psychophysiology Laboratory, 3Cell Electrophysiology Laboratory, Institute of Physiological Sciences, Wannan Medical College, Wuhu 241002, China

Received:2024-07-20 Online:2025-05-20 Published:2025-05-23
Contact: Mengya WANG, Chao ZHENG E-mail:1596104580@qq.com;wangmy@wnmc.edu.cn;chaozheng10@fudan.edu.cn
Supported by:
National Natural Science Foundation of China(31200828)

摘要/Abstract

摘要：

目的探讨Orexin-A对促离子型谷氨酸受体的调节在大鼠脊髓损伤（SCI）运动功能恢复中的作用。方法 36只7~14 d新生SD大鼠随机平均分为6组：对照组、假手术组、SCI+生理盐水组、SCI+DNQX组、SCI+Orexin组和SCI+Orexin+DNQX组。除了对照组和假手术组外，其他各组施以SCI手术，术后连续3 d，每天分别向SCI各组鞘内注射对应的药物1次。分别在术前1 d，术后1、3和7 d，应用BBB评分和斜板试验评估各组大鼠运动功能；在膜片钳实验中，将对照组、假手术组、SCI组和SCI+Orexin组大鼠麻醉后，制作脊髓切片，通过酶消化和机械吹打分离出腹角神经元，通过灌流谷氨酸，比较各组大鼠脊髓腹角神经元谷氨酸受体介导电流的翻转电位、动力学指标的差异。结果在术后3 d、7 d，SCI+Orexin组的BBB评分和抓握倾斜角度比SCI+生理盐水组、SCI+DNQX组和SCI+Orexin+DNQX组皆升高（P<0.01）。在术后第7天，SCI+Orexin+DNQX组高于SCI+DNQX组（P<0.01）。SCI组脊髓腹角神经元谷氨酸电流的翻转电位高于SCI+Orexin组和假手术组（P<0.01）。在谷氨酸电流动力学指标中，SCI组的上升斜率比SCI+Orexin组和假手术组皆增大（P<0.05）。在衰减时间项，SCI组比SCI+Orexin组、假手术组皆减小（P<0.05）。结论 Orexin-A可能通过调节（改善）促离子型谷氨酸受体的功能促进脊髓损伤后运动功能的恢复。

关键词: Orexin-A, 促离子型谷氨酸受体, 脊髓损伤, 脊髓腹角神经元, 膜片钳记录

Abstract:

Objective To investigate the effect of orexin-A-mediated regulation of ionotropic glutamate receptors for promoting motor function recovery in rats with spinal cord injury (SCI). Methods Thirty-six newborn SD rats (aged 7-14 days) were randomized into 6 groups (n=6), including a normal control group, a sham-operated group, and 4 SCI groups with daily intrathecal injection of saline, DNQX, orexin-A, or orexin-A+DNQX for 3 consecutive days after PCI. Motor function of the rats were evaluated using blood-brain barrier (BBB) score and inclined plane test 1 day before and at 1, 3, and 7 days after SCI. For patch-clamp experiment, spinal cord slices from newborn rats in the control, sham-operated, SCI, and SCI+orexin groups were prepared, and ventral horn neurons were acutely isolated to determine the reversal potential and dynamic indicators of glutamate receptor-mediated currents under glutamate perfusion. Results At 3 and 7 days after SCI, the orexin-A-treated rats showed significantly higher BBB scores and grip tilt angles than those with other interventions. Compared with those treated with DNQX alone, the rats receiving the combined treatment with orexin and DNQX had significantly higher BBB scores and grip tilt angles on day 7 after PCI. In the patch-clamp experiment, the ventral horn neurons from SCI rat models exhibited obviously higher reversal potential and greater rise slope of glutamate current with shorter decay time than those from sham-operated and orexin-treated rats. Conclusion Orexin-A promotes motor function recovery in rats after SCI possibly by improving the function of the ionotropic glutamate receptors.

Key words: orexin-A, ionotropic glutamate receptor, spinal cord injury, spinal cord ventral horn neurons, patch-clamp recording

何光侣, 储婉玉, 李妍, 盛鑫, 罗浩, 徐爱萍, 卞命杰, 张环环, 汪萌芽, 郑超. Orexin-A通过调节促离子型谷氨酸受体促进脊髓损伤大鼠运动功能恢复[J]. 南方医科大学学报, 2025, 45(5): 1023-1030.

Guanglü HE, Wanyu CHU, Yan LI, Xin SHENG, Hao LUO, Aiping XU, Mingjie BIAN, Huanhuan ZHANG, Mengya WANG, Chao ZHENG. Orexin-A promotes motor function recovery of rats with spinal cord injury by regulating ionotropic glutamate receptors[J]. Journal of Southern Medical University, 2025, 45(5): 1023-1030.

图/表 5

图1 动物行为学实验

Fig.1 Results of behavioral test of the rats in different groups. A, B: BBB scores in the 6 groups. C: Results of inclined plane test in the 6 groups. D: Comparisons of inclined plane test among SCI+Orexin group, SCI+DNQX group and SCI+Orexin+DNQX group。 aP<0.01, bP<0.01 vs Sham group; cP<0.01 vs SCI+Saline group; dP<0.01, fP<0.01 vs SCI+DNQX group; eP<0.01 vs SCI+Orexin+DNQX group.

图2 高倍镜下拍摄的一例脊髓腹角分离出的神经元

Fig.2 A neuron isolated from the ventral horn of the spinal cord captured under high magnification showing clear protrusions from the cell body.

图3 Orexin对脊髓损伤后腹角神经元谷氨酸电流翻转电位的影响

Fig.3 Effect of orexin on reversal potential of glutamate-induced currents in spinal cord ventral horn neurons of rats after spinal cord injury (SCI). A, B: Changes in glutamate-induced currents in normal group under different holding potentials (-70 to +40 mV) and linear fit of the current-voltage relationship curve (the intersection point on the X-axis represents reversal potential of glutamate-induced currents). C, D: Changes in glutamate-induced currents in Sham group under different holding potentials and linear fit of the current-voltage relationship curve. E, F: Changes in glutamate-induced currents in SCI group under different holding potentials and linear fit of the current-voltage relationship curve. G, H: Changes in glutamate-induced currents in SCI+Orexin group under different holding potentials and linear fit of the current-voltage relationship curve.

图4 不同组脊髓腹角神经元谷氨酸电流的翻转电位的比较

Fig.4 Comparisons of the reversal potentials of glutamate currents in the spinal cord ventral horn neurons among different groups. **P<0.01.

图5 不同组脊髓腹角神经元谷氨酸电流动力学指标的比较

Fig.5 Comparisons of the dynamic index of glutamate currents in spinal cord ventral horn neurons among the groups. A: Comparison of the rise time of glutamate current. B: Comparison of the rise slope of glutamate current. C: Comparisons of decay time of glutamate current. D: Comparison of decay slope of glutamate current. *P<0.05, **P<0.01.

参考文献 36

1	Wagner FB, Mignardot JB, Le Goff-Mignardot CG, et al. Targeted neurotechnology restores walking in humans with spinal cord injury[J]. Nature, 2018, 563(7729): 65-71.
2	Angeli CA, Boakye M, Morton RA, et al. Recovery of over-ground walking after chronic motor complete spinal cord injury[J]. N Engl J Med, 2018, 379(13): 1244-50.
3	Sakurai T, Amemiya A, Ishii M, et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior[J]. Cell, 1998, 92(4): 573-85.
4	Chieffi S, Carotenuto M, Monda V, et al. Orexin system: the key for a healthy life[J]. Front Physiol, 2017, 8: 357.
5	Yamuy J, Fung SJ, Xi MC, et al. State-dependent control of lumbar motoneurons by the hypocretinergic system[J]. Exp Neurol, 2010, 221(2): 335-45.
6	Carpi M, Mercuri NB, Liguori C. Orexin receptor antagonists for the prevention and treatment of Alzheimer's disease and associated sleep disorders[J]. Drugs, 2024, 84(11): 1365-78.
7	Becquet L, Abad C, Leclercq M, et al. Systemic administration of orexin A ameliorates established experimental autoimmune encephalomyelitis by diminishing neuroinflammation[J]. J Neuroinflammation, 2019, 16(1): 64.
8	Hwang YT, Piguet O, Hodges JR, et al. Sleep and orexin: a new paradigm for understanding behavioural-variant frontotemporal dementia[J]? Sleep Med Rev, 2020, 54: 101361.
9	Lee MG, Hassani OK, Jones BE. Discharge of identified orexin/hypocretin neurons across the sleep-waking cycle[J]. J Neurosci, 2005, 25(28): 6716-20.
10	Mileykovskiy BY, Kiyashchenko LI, Siegel JM. Behavioral correlates of activity in identified hypocretin/orexin neurons[J]. Neuron, 2005, 46(5): 787-98.
11	Takakusaki K, Takahashi K, Saitoh K, et al. Orexinergic projections to the cat midbrain mediate alternation of emotional behavioural states from locomotion to cataplexy[J]. J Physiol, 2005, 568(Pt 3): 1003-20.
12	Zhang J, Li B, Yu L, et al. A role for orexin in central vestibular motor control[J]. Neuron, 2011, 69(4): 793-804.
13	Yamuy J, Fung SJ, Xi MC, et al. Hypocretinergic control of spinal cord motoneurons[J]. J Neurosci, 2004, 24(23): 5336-45.
14	Kang JW, Ren BK, Huang LH, et al. Orexin-a alleviates ferroptosis by activating the Nrf2/HO-1 signaling pathway in traumatic brain injury[J]. Aging, 2024, 16(4): 3404-19.
15	Russell Huie J, Stuck ED, Lee KH, et al. AMPA receptor phosphorylation and synaptic colocalization on motor neurons drive maladaptive plasticity below complete spinal cord injury[J]. eNeuro, 2015, 2(5): ENEURO.0091-15.2015.
16	Follwell MJ, Ferguson AV. Cellular mechanisms of orexin actions on paraventricular nucleus neurones in rat hypothalamus[J]. J Physiol, 2002, 545(3): 855-67.
17	Borgland SL, Storm E, Bonci A. Orexin B/hypocretin 2 increases glutamatergic transmission to ventral tegmental area neurons[J]. Eur J Neurosci, 2008, 28(8): 1545-56.
18	Lambe EK, Aghajanian GK. Hypocretin (orexin) induces calcium transients in single spines postsynaptic to identified thalamocortical boutons in prefrontal slice[J]. Neuron, 2003, 40(1): 139-50.
19	Niknia S, Kaeidi A, Hajizadeh MR, et al. Neuroprotective and antihyperalgesic effects of orexin-a in rats with painful diabetic neuropathy[J]. Neuropeptides, 2019, 73: 34-40.
20	Jin N, Zhu SY, Yang XY, et al. Orexin-a potentiates Glycine currents by activating OX₁R and IP₃/Ca²⁺/PKC signaling pathways in spinal cord ventral horn neurons[J]. Brain Res Bull, 2021, 169: 196-204.
21	Thomas A, Miller A, Roughan J, et al. Efficacy of intrathecal morphine in a model of surgical pain in rats[J]. PLoS One, 2016, 11(10): e0163909.
22	Rana S, Sieck GC, Mantilla CB. Diaphragm electromyographic activity following unilateral midcervical contusion injury in rats[J]. J Neurophysiol, 2017, 117(2): 545-55.
23	Fan L, Li XB, Liu T. Asiaticoside inhibits neuronal apoptosis and promotes functional recovery after spinal cord injury in rats[J]. J Mol Neurosci, 2020, 70(12): 1988-96.
24	Anjum A, Yazid MD, Fauzi Daud M, et al. Spinal cord injury: pathophysiology, multimolecular interactions, and underlying recovery mechanisms[J]. Int J Mol Sci, 2020, 21(20): 7533.
25	Reshamwala R, Eindorf T, Shah M, et al. Induction of complete transection-type spinal cord injury in mice[J]. J Vis Exp, 2020(159): (159).
26	Pan LL, Qi RR, Wang JQ, et al. Evidence for a role of orexin/hypocretin system in vestibular lesion-induced locomotor abnormalities in rats[J]. Front Neurosci, 2016, 10: 355.
27	Yan GL, Li FG, Tao ZW, et al. Effects of vestibular damage on the sleep and expression level of orexin in the hypothalamus of rats and its correlation with autophagy and Akt tumor signal pathway[J]. J Oncol, 2022, 2022: 2514555.
28	Iacobucci GJ, Popescu GK. Ca²⁺-dependent inactivation of GluN2A and GluN2B NMDA receptors occurs by a common kinetic mechanism[J]. Biophys J, 2020, 118(4): 798-812.
29	Fani G, Mannini B, Vecchi G, et al. Aβ oligomers dysregulate calcium homeostasis by mechanosensitive activation of AMPA and NMDA receptors[J]. ACS Chem Neurosci, 2021, 12(4): 766-81.
30	Yasuda R, Hayashi Y, Hell JW. CaMKII: a central molecular organizer of synaptic plasticity, learning and memory[J]. Nat Rev Neurosci, 2022, 23(11): 666-82.
31	Kosenkov AM, Gaidin SG, Sergeev AI, et al. Fast changes of NMDA and AMPA receptor activity under acute hyperammonemia in vitro [J]. Neurosci Lett, 2018, 686: 80-6.
32	Plaza-Zabala A, Li X, Milovanovic M, et al. An investigation of interactions between hypocretin/orexin signaling and glutamate receptor surface expression in the rat nucleus accumbens under basal conditions and after cocaine exposure[J]. Neurosci Lett, 2013, 557 Pt B(0 0): 101-6.
33	Dong YJ, Jiang NH, Zhan LH, et al. Soporific effect of modified Suanzaoren Decoction on mice models of insomnia by regulating Orexin-A and HPA axis homeostasis[J]. Biomed Pharmacother, 2021, 143: 112141.
34	Palomba L, Motta A, Imperatore R, et al. Role of 2-arachidonoyl-glycerol and CB1 receptors in orexin-A-mediated prevention of oxygen-glucose deprivation-induced neuronal injury[J]. Cells, 2020, 9(6): 1507.
35	Zhang DX, Cui Y, Zhao MM, et al. Orexin-a exerts neuroprotective effect in experimental intracerebral hemorrhage by suppressing autophagy via OXR1-mediated ERK/mTOR signaling pathway[J]. Front Cell Neurosci, 2022, 16: 1045034.
36	Nepovimova E, Janockova J, Misik J, et al. Orexin supplementation in narcolepsy treatment: a review[J]. Med Res Rev, 2019, 39(3): 961-75.

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Orexin-A通过调节促离子型谷氨酸受体促进脊髓损伤大鼠运动功能恢复

Orexin-A promotes motor function recovery of rats with spinal cord injury by regulating ionotropic glutamate receptors

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