氙气后处理对脊髓缺血再灌注损伤的效果：Akt信号通路和自噬机制

doi:10.3969/j.issn.1006-9771.2023.02.006

摘要/Abstract

摘要：

目的探讨氙气后处理对大鼠脊髓缺血再灌注损伤(SCIRI)后自噬的影响及其与苏氨酸蛋白激酶(Akt)信号通路的关系。方法 30只健康雄性Sprague-Dawley大鼠随机分为假手术组(Sham组)、脊髓缺血再灌注组(I/R组)和氙气后处理组(Xe组)，每组10只。后两组通过夹闭腹主动脉85 min，再灌注4 h建立大鼠SCIRI模型。于再灌注1 h时，Xe组经呼吸机吸入50%氙气+50%氧气混合气1 h。其余两组吸入50%氮气+50%氧气混合气1 h。再灌注4 h时，对大鼠行BBB评分和斜板试验后，收集L_3-5脊髓，Nissl染色计数正常神经元数量，Western blotting检测脊髓组织中Akt、磷酸化Akt(p-Akt)、自噬蛋白〔p62、Beclin 1、微管相关蛋白1轻链3 (LC3)Ⅰ和LC3 Ⅱ〕的表达，逆转录实时定量聚合酶链反应检测脊髓组织中p62、Beclin 1和LC3 Ⅱ mRNA的表达。结果与Sham组相比，I/R组后肢BBB评分明显降低(P < 0.01)，斜板试验最大倾斜度明显减小(P < 0.01)，正常神经元数量明显减少(P < 0.01)；p-Akt/Akt比值明显降低(P < 0.01)；Beclin 1蛋白表达明显上调(P < 0.01)，LC3 Ⅱ/LC3 Ⅰ比值明显升高(P < 0.01)，p62蛋白表达下调，且Beclin 1 mRNA和LC3 Ⅱ mRNA的含量增加，p62 mRNA含量明显减少(P < 0.01)。与I/R组相比，Xe组后肢BBB评分明显升高(P < 0.01)，斜板试验最大倾斜度明显增大(P < 0.01)，正常神经元数量明显增加(P < 0.01)；p-Akt蛋白表达上调，p-Akt/Akt比值明显升高(P < 0.01)；Beclin 1蛋白的表达明显下调(P < 0.01)，LC3 Ⅱ/ LC3 Ⅰ比值明显降低(P < 0.01)，p62蛋白的表达明显上调(P < 0.01)，p62 mRNA的含量明显增加(P < 0.01)，LC3 Ⅱ mRNA 的含量减少(P < 0.05)。结论氙气后处理可以减轻SCIRI，可能与激活Akt通路，抑制自噬水平有关。

关键词: 脊髓, 缺血再灌注, 氙气后处理, 自噬, 苏氨酸蛋白激酶, 大鼠

Abstract:

Objective To investigate the effect of xenon post-conditioning on autophagy after spinal cord ischemia/reperfusion injury (SCIRI) in rats and its relationship with protein kinase B (Akt) signaling pathway. Methods A total of 30 male rats were randomized into sham-operated group (sham group), spinal cord ischemia/reperfusion injury group (I/R group) and I/R + xenon post-conditioning group (Xe group), with ten rats in each group. In the latter two groups, SCIRI was induced by clamping the abdominal aorta for 85 minutes followed by reperfusion for four hours. Xe group inhaled xenon and oxygen (1∶1) for one hour at one hour after initiation of reperfusion, while the other groups inhaled nitrogen and oxygen (1∶1) for one hour. After the reperfusion, they were assessed with Basso-Beattie-Bresnahan (BBB) scale and slanting board test. And then, their spinal cords of L_3-5 were obtained. Nissl staining was used to count the number of normal neurons. Western blotting was used to detect the protein expression of Akt, p-Akt, p62, Beclin 1, microtubule-associated protein 1 light chain 3 (LC3) Ⅰ, LC3 Ⅱ. The mRNA expression of Beclin 1, p62 and LC3 Ⅱ in the spinal cord was measured with reverse transcription real-time quantitative polymerase chain reaction. Results Compared with the sham group, the BBB score and the maximum inclination of the slanting board test decreased, the count of normal neurons decreased, the protein expression of p62 and the p-Akt/Akt ratio decreased (P< 0.01), the protein and mRNA expression of Beclin 1 and LC3 Ⅱ, and the LC3 Ⅱ/LC3 Ⅰ ratio increased, the p62 mRNA expression decreased (P< 0.01) in the I/R group. Compared with the I/R group, the BBB score and the maximum inclination of the slanting board test increased, the count of normal neurons increased, the protein expression of p-Akt and p62 increased, the p-Akt/Akt ratio increased, the protein and mRNA expression of Beclin 1, LC3 Ⅱ and LC3 Ⅱ/LC3 Ⅰ ratio decreased, and the mRNA expression of p62 increased (P< 0.01) in Xe group.

Conclusion Xenon post-conditioning may relieve SCIRI in rats, which is related to activating Akt signaling pathway to inhibit autophagy.

Keywords: spinal cord; ischemia/reperfusion; xenon post-conditioning; autophagy; protein kinase B; rats

中图分类号:

R651.2

罗兰, 李璐, 金沐. 氙气后处理对脊髓缺血再灌注损伤的效果：Akt信号通路和自噬机制[J]. 《中国康复理论与实践》, 2023, 29(2): 174-181.

LUO Lan, LI Lu, JIN Mu. Effect of xenon post-conditioning on spinal cord ischemia/reperfusion injury in rats: regulating Akt signaling pathway and autophagy[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2023, 29(2): 174-181.

图/表 7

表1

图1

表2

图2

图3

表3

表4

参考文献 43

[1]	GAUDINO M, KHAN F M, RAHOUMA M, et al. Spinal cord injury after open and endovascular repair of descending thoracic and horacoabdominal aortic aneurysms: a meta-analysis[J]. J Thorac Cardiovasc Surg, 2022, 163(2): 552-564. doi: 10.1016/j.jtcvs.2020.04.126
[2]	KHACHATRYAN Z, HAUNSCHILD J, VON ASPERN K, et al. Ischemic spinal cord injury-experimental evidence and evolution of protective measures[J]. Ann Thorac Surg, 2022, 113(5): 1692-1702. doi: 10.1016/j.athoracsur.2020.12.028
[3]	MAZE M, LAITIO T. Neuroprotective properties of xenon[J]. Mol Neurobiol, 2020, 57(1): 118-124. doi: 10.1007/s12035-019-01761-z pmid: 31758401
[4]	LIU F, LIU S L, PATTERSON T A, et al. Effects of xenon-based anesthetic exposure on the expression levels of polysialic acid neural cell adhesion molecule (PSA-NCAM) on human neural stem cell-derived neurons[J]. Mol Neurobiol, 2020, 57(1): 217-225. doi: 10.1007/s12035-019-01771-x pmid: 31522383
[5]	罗兰, 佟家祺, 李璐, 等. 氙气后处理对大鼠脊髓缺血再灌注损伤起保护作用:基于下调mTOR通路和抑制内质网应激介导的神经元凋亡[J]. 南方医科大学学报, 2022, 42(8): 1256-1262.
	LUO L, TONG J Q, LI L, et al. Xenon post-conditioning protects against spinal cord ischemia-reperfusion injury in rats by downregulating mTOR pathway and inhibiting endoplasmic reticulum stress-induced neuronal apoptosis[J]. J South Med Univ, 2022, 42(8): 1256-1262.
[6]	侯思雨, 杨彦伟, 金沐, 等. 大鼠脊髓缺血再灌注损伤后ERK、Akt的表达与细胞凋亡关系的研究[J]. 心肺血管病杂志, 2015, 34(1): 62-64.
	HOU S Y, YANG Y W, JIN M, et al. Neuronal apoptosis and expressions of ERK, Akt in rats with spinal cord ischemia reperfusion injury[J]. J Cardiov Pulm Dis, 2015, 34(1): 62-64.
[7]	YANG Y W, LU J K, QING E M, et al. Post-conditioning by xenon reduces ischaemia-reperfusion injury of the spinal cord in rats[J]. Acta Anaesthesiol Scand, 2012, 56(10): 1325-1331. doi: 10.1111/j.1399-6576.2012.02718.x pmid: 22621442
[8]	YANG Y W, CHENG W P, LU J K, et al. Timing of xenon-induced delayed postconditioning to protect against spinal cord ischemia-reperfusion injury in rats[J]. Br J Anaesth, 2014, 113(1): 168-176. doi: 10.1093/bja/aet352
[9]	YANG Y W, WANG Y L, LU J K, et al. Delayed xenon post-conditioning mitigates spinal cord ischemia/reperfusion injury in rabbits by regulating microglial activation and inflammatory factors[J]. Neural Regen Res, 2018, 13(3): 510-517. doi: 10.4103/1673-5374.228757
[10]	LIU S Y, YANG Y W, JIN M, et al. Xenon-delayed postconditioning attenuates spinal cord ischemia/reperfusion injury through activation Akt and ERK signaling pathways in rats[J]. J Neurol Sci, 2016, 368: 277-284. doi: 10.1016/j.jns.2016.07.009 pmid: 27538649
[11]	LIU G Y, SABATINI D M. mTOR at the nexus of nutrition, growth, ageing and disease[J]. Nat Rev Mol Cell Biol, 2020, 21(4): 183-203.
[12]	BERTACCHINI J, HEIDARI N, MEDIANI L, et al. Targeting PI3K/AKT/mTOR network for treatment of leukemia[J]. Cell Mol Life Sci, 2015, 72(12): 2337-2347. doi: 10.1007/s00018-015-1867-5 pmid: 25712020
[13]	REZQ S, HASSAN R, MAHMOUD M F. Rimonabant ameliorates hepatic ischemia/reperfusion injury in rats: involvement of autophagy via modulating ERK- and PI3K/AKT-mTOR pathways[J]. Int Immunopharmacol, 2021, 100: 108140. doi: 10.1016/j.intimp.2021.108140
[14]	SHI B H, MA M Q, ZHENG Y T, et al. mTOR and Beclin1: two key autophagy-related molecules and their roles in myocardial ischemia/reperfusion injury[J]. J Cell Physiol, 2019, 234(8): 12562-12568. doi: 10.1002/jcp.28125 pmid: 30618070
[15]	ZHOU K L, ZHENG Z L, LI Y, et al. TFE3, a potential therapeutic target for spinal cord injury via augmenting autophagy flux and alleviating ER stress[J]. Theranostics, 2020, 10(20): 9280-9302. doi: 10.7150/thno.46566 pmid: 32802192
[16]	MUÑOZ-GALDEANO T, REIGADA D, et al. Cell specific changes of autophagy in a mouse model of contusive spinal cord injury[J]. Front Cell Neurosci, 2018, 12: 164.
[17]	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. doi: 10.1089/neu.1995.12.1
[18]	GUO Y, MA Y, PAN Y L, et al. Jisuikang, a Chinese herbal formula, increases neurotrophic factor expression and promotes the recovery of neurological function after spinal cord injury[J]. Neural Regen Res, 2017, 12(9): 1519-1528. doi: 10.4103/1673-5374.215264 pmid: 29089999
[19]	MORAIS R, ANDRADE L, LOURENÇO A, et al. How xenon works: neuro and cardioprotection mechanisms[J]. Acta Med Port, 2014, 27(2): 259-265. doi: 10.20344/amp.4782
[20]	LIANG M, AHMAD F, DICKINSON R. Neuroprotection by the noble gases argon and xenon as treatments for acquired brain injury: a preclinical systematic review and meta-analysis[J]. Br J Anaesth, 2022, 129(2): 200-218. doi: 10.1016/j.bja.2022.04.016 pmid: 35688658
[21]	ZHANG Y R, ZHANG M D, LIU S H, et al. Xenon exerts anti-seizure and neuroprotective effects in kainic acid-induced status epilepticus and neonatal hypoxia-induced seizure[J]. Exp Neurol, 2019, 322: 113054. doi: 10.1016/j.expneurol.2019.113054
[22]	DANDEKAR M P, YIN X, PENG T, et al. Repetitive xenon treatment improves post-stroke sensorimotor and neuropsychiatric dysfunction[J]. J Affect Disord, 2022, 301: 315-330. doi: 10.1016/j.jad.2022.01.025
[23]	DE DEKEN J, REX S, LERUT E, et al. Postconditioning effects of argon or xenon on early graft function in a porcine model of kidney autotransplantation[J]. Br J Surg, 2018, 105(8): 1051-1060. doi: 10.1002/bjs.10796 pmid: 29603122
[24]	PENG T, BRITTON G L, KIM H, et al. Therapeutic time window and dose dependence of xenon delivered via echogenic liposomes for neuroprotection in stroke[J]. CNS Neurosci Ther, 2013, 19(10): 773-784. doi: 10.1111/cns.12159 pmid: 23981565
[25]	ZHAO H L, HUANG H, OLOGUNDE R, et al. Xenon treatment protects against remote lung injury after kidney transplantation in rats[J]. Anesthesiology, 2015, 122(6): 1312-1326. doi: 10.1097/ALN.0000000000000664 pmid: 25856291
[26]	GU C J, LI L W, HANG Y F, et al. Salidroside ameliorates mitochondria-dependent neuronal apoptosis after spinal cord ischemia-reperfusion injury partially through inhibiting oxidative stress and promoting mitophagy[J]. Oxid Med Cell Longev, 2020, 2020: 3549704.
[27]	ZHENG W J, LIU B, SHI E Y. Perillaldehyde alleviates spinal cord ischemia-reperfusion injury via activating the Nrf2 pathway[J]. J Surg Res, 2021, 268: 308-317. doi: 10.1016/j.jss.2021.06.055 pmid: 34399353
[28]	LUO C L, TAO L Y. The function and mechanisms of autophagy in spinal cord injury[J]. Adv Exp Med Biol, 2020, 1207: 649-654. doi: 10.1007/978-981-15-4272-5_47 pmid: 32671782
[29]	LIAO H Y, WANG Z Q, RAN R, et al. Biological functions and therapeutic potential of autophagy in spinal cord injury[J]. Front Cell Dev Biol, 2021, 9: 761273. doi: 10.3389/fcell.2021.761273
[30]	ZHANG D, WANG F, ZHAI X, et al. Lithium promotes recovery of neurological function after spinal cord injury by inducing autophagy[J]. Neural Regen Res, 2018, 13(12): 2191-2199. doi: 10.4103/1673-5374.241473 pmid: 30323152
[31]	LI W C, YAO S P, LI H R, et al. Curcumin promotes functional recovery and inhibits neuronal apoptosis after spinal cord injury through the modulation of autophagy[J]. J Spinal Cord Med, 2021, 44(1): 37-45. doi: 10.1080/10790268.2019.1616147
[32]	VARGOVA I, MACHOVA URDZIKOVA L, KAROVA K, et al. Involvement of mTOR pathways in recovery from spinal cord injury by modulation of autophagy and immune response[J]. Biomedicines, 2021, 9(6): 593. doi: 10.3390/biomedicines9060593
[33]	ZHOU K L, SANSUR C A, XU H Z, et al. The temporal pattern, flux, and function of autophagy in spinal cord injury[J]. Int J Mol Sci, 2017, 18(2): 466. doi: 10.3390/ijms18020466
[34]	DUPONT N, JIANG S Y, PILLI M, et al. Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1β[J]. EMBO J, 2011, 30(23): 4701-4711. doi: 10.1038/emboj.2011.398 pmid: 22068051
[35]	CHEN H C, FONG T H, LEE A W, et al. Autophagy is activated in injured neurons and inhibited by methylprednisolone after experimental spinal cord injury[J]. Spine, 2012, 37(6): 470-475. doi: 10.1097/BRS.0b013e318221e859
[36]	MARQUEZ R T, XU L. Bcl-2: Beclin 1 complex: multiple, mechanisms regulating autophagy/apoptosis toggle switch[J]. Am J Cancer Res, 2012, 2(2): 214-221.
[37]	RONG Y L, FAN J, JI C Y, et al. USP11 regulates autophagy-dependent ferroptosis after spinal cord ischemia-reperfusion injury by deubiquitinating Beclin 1[J]. Cell Death Differ, 2022, 29(6): 1164-1175. doi: 10.1038/s41418-021-00907-8
[38]	KANNO H, OZAWA H, SEKIGUCHI A, et al. The role of autophagy in spinal cord injury[J]. Autophagy, 2009, 5(3): 390-392. pmid: 19158496
[39]	HOU H P, ZHANG L H, ZHANG L C, et al. Acute spinal cord injury in rats induces autophagy activation[J]. Turk Neurosurg, 2014, 24(3): 369-373. doi: 10.5137/1019-5149.JTN.8623-13.0 pmid: 24848176
[40]	ZHANG D, YUAN Y, ZHU J C, et al. Insulin-like growth factor 1 promotes neurological functional recovery after spinal cord injury through inhibition of autophagy via the PI3K/Akt/mTOR signaling pathway[J]. Exp Ther Med, 2021, 22(5): 1265. doi: 10.3892/etm.2021.10700 pmid: 34594402
[41]	CHEN Z, FU Q G, SHEN B L, et al. Enhanced p62 expression triggers concomitant autophagy and apoptosis in a rat chronic spinal cord compression model[J]. Mol Med Rep, 2014, 9(6): 2091-2096. doi: 10.3892/mmr.2014.2124 pmid: 24715058
[42]	JEONG S J, ZHANG X, RODRIGUEZ-VELEZ A, et al. p62/SQSTM1 and selective autophagy in cardiometabolic diseases[J]. Antioxid Redox Signal, 2019, 31(6): 458-471. doi: 10.1089/ars.2018.7649
[43]	LAMARK T, SVENNING S, JOHANSEN T. Regulation of selective autophagy: the p62/SQSTM1 paradigm[J]. Essays Biochem, 2017, 61(6): 609-624. doi: 10.1042/EBC20170035 pmid: 29233872

组别	n	BBB评分	斜板试验最大倾斜度/°
Sham组	10	21.000±0.000	62.300±2.908
I/R组	10	5.243±3.236^a	41.800±4.492^a
Xe组	10	12.850±1.462^a,b	50.000±6.236^a,b
F值		144.172	47.301
P值		< 0.001	< 0.001

组别	n	正常神经元计数	p-Akt/Akt
Sham组	5	11.920±0.867	1.156±0.103
I/R组	5	5.560±0.573^a	0.781±0.84^a
Xe组	5	8.840±0.385^a,b	0.963±0.75^a,b
F值		123.564	27.218
P值		< 0.001	< 0.001

组别	n	p62/actin	Beclin 1/actin	LC3 Ⅱ/LC3 Ⅰ
Sham组	5	1.148±0.091	0.797±0.033	0.268±0.023
I/R组	5	0.710±0.640^a	1.214±0.426^a	0.521±0.011^a
Xe组	5	0.883±0.059^a,b	0.963±0.344^a,b	0.441±0.014^a,b
F值		55.396	161.836	292.112
P值		< 0.001	< 0.001	< 0.001

组别	n	p62	Beclin 1	LC3 Ⅱ
Sham组	5	1.003±0.901	1.003±0.094	1.013±0.174
I/R组	5	0.546±0.148^a	1.303±0.141^a	1.469±0.167^a
Xe组	5	0.886±0.681^b	1.131±0.169	1.181±0.245^c
F值		24.352	5.857	6.716
P值		< 0.001	0.017	0.011