目的 观察人脐带间充质干细胞（human umbilical cord mesenchymal stem cell, hUC-MSC）移植对新生大鼠脑白质损伤（white matter injury, WMI）的修复作用，并通过小胶质细胞介导的核因子κB（nuclear factor kappa B, NF-κB）信号通路探讨其机制。 方法 将2日龄Sprague-Dawley新生大鼠随机分为假手术组、WMI组和hUC-MSC组，每组18只。建模后14 d，通过苏木精-伊红染色法观察脑白质病理变化，免疫荧光染色法检测离子钙结合适配器分子1（ionized calcium-binding adapter molecule 1, Iba1）表达，免疫印迹法检测核因子κB抑制蛋白α（inhibitory subunit of nuclear factor-kappa B alpha, IκBα）、磷酸化IκBα（p-IκBα）、磷酸化NF-κB p65（p-NF-κB p65）、髓鞘碱性蛋白（myelin basic protein, MBP）、神经元核蛋白（neuron-specific nuclear protein, NeuN）蛋白表达，实时荧光定量聚合酶链反应法检测肿瘤坏死因子（tumor necrosis factor-α, TNF-α）、白介素-1β（interleukin-1β, IL-1β）、MBP、NeuN mRNA表达，免疫组化法检测MBP、NeuN蛋白表达水平；建模后28 d，利用Morris水迷宫实验评估大鼠空间认知能力。 结果 建模后14 d，假手术组脑白质区域组织结构完整，细胞形态正常、神经纤维排列规则；WMI组大量细胞变性坏死、神经纤维排列紊乱；hUC-MSC组细胞形态相对正常，神经纤维排列较整齐。WMI组Iba1阳性细胞占比、p-IκBα和p-NF-κB p65蛋白表达，以及TNF-α和IL-1β mRNA表达较假手术组增加，IκBα蛋白表达及MBP和NeuN阳性表达、蛋白和mRNA表达较假手术组减少（P<0.05）；hUC-MSC组Iba1阳性细胞占比、p-IκBα和p-NF-κB p65蛋白表达，以及TNF-α、IL-1β mRNA表达较WMI组减少，IκBα蛋白表达及MBP和NeuN阳性表达、蛋白和mRNA表达较WMI组增加（P<0.05）。建模后28 d，水迷宫结果显示与假手术组比较，WMI组逃避潜伏期时间延长、穿越平台次数较减少（P<0.05）；与WMI组比较，hUC-MSC组逃避潜伏期时间缩短、穿越平台次数增加（P<0.05）。 结论 hUC-MSC可修复新生大鼠WMI，促进少突胶质细胞成熟和神经元存活，其机制可能与抑制小胶质细胞介导的NF-κB信号通路激活相关。
Abstract
Objective To observe the reparative effects of human umbilical cord mesenchymal stem cell (hUC-MSC) transplantation on white matter injury (WMI) in neonatal rats and explore its mechanism through the nuclear factor-kappa B (NF-κB) signaling pathway mediated by microglial cells. Methods Sprague-Dawley rats, aged 2 days, were randomly divided into three groups: sham-operation,WMI, and hUC-MSC (n=18 each). Fourteen days after modeling, hematoxylin-eosin staining was used to observe pathological changes in the white matter, and immunofluorescence staining was used to measure the expression level of ionized calcium-binding adapter molecule 1 (Iba1). Western blotting was used to measure the protein expression levels of inhibitory subunit of nuclear factor-kappa B alpha (IκBα), phosphorylated IκBα (p-IκBα), phosphorylated NF-κB p65 (p-NF-κB p65), myelin basic protein (MBP), and neuron-specific nuclear protein (NeuN). Quantitative real-time PCR was used to assess the mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), MBP, and NeuN. Immunohistochemistry was used to measure the protein expression levels of MBP and NeuN. On day 28, the Morris water maze test was used to evaluate spatial cognitive ability. Results Fourteen days after modeling, the sham-operation group exhibited intact white matter structure with normal cell morphology and orderly nerve fiber arrangement. In the WMI group, large-scale cell degeneration and necrosis were observed, and nerve fiber arrangement was disordered. The hUC-MSC group showed relatively normal cell morphology and more orderly nerve fibers. Compared with the sham-operation group, the WMI group had significantly higher proportions of Iba1-positive cells, increased protein levels of p-IκBα and p-NF-κB p65, and higher mRNA levels of TNF-α and IL-1β. The protein expression of IκBα and the positive expression of MBP and NeuN, as well as their protein and mRNA levels, were significantly reduced in the WMI group (P<0.05). Compared with the WMI group, the hUC-MSC group showed reduced proportions of Iba1-positive cells, decreased protein levels of p-IκBα and p-NF-κB p65, and lower mRNA levels of TNF-α and IL-1β. Furthermore, IκBα protein expression and MBP and NeuN expression (both at the protein and mRNA levels) were significantly increased in the hUC-MSC group (P<0.05). On day 28, the Morris water maze results showed that compared with the sham-operation group, the WMI group had significantly longer escape latency and fewer platform crossings (P<0.05). In contrast, the hUC-MSC group had significantly shorter escape latency and more platform crossings than the WMI group (P<0.05). Conclusions hUC-MSC transplantation can repair WMI in neonatal rats, promote the maturation of oligodendrocytes, and support neuronal survival, likely by inhibiting activation of the NF-κB signaling pathway mediated by microglial cells.
关键词
人脐带间充质干细胞 /
脑白质损伤 /
NF-κB通路 /
新生大鼠
Key words
Human umbilical cord mesenchymal stem cell /
White matter injury /
Nuclear factor-kappa B pathway /
Neonatal rat
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
1 Volpe JJ. Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances[J]. Lancet Neurol, 2009, 8(1): 110-124. PMID: 19081519. PMCID: PMC2707149. DOI: 10.1016/S1474-4422(08)70294-1.
2 Gall AR, Amoah S, Kitase Y, et al. Placental mediated mechanisms of perinatal brain injury: evolving inflammation and exosomes[J]. Exp Neurol, 2022, 347: 113914. PMID: 34752783. PMCID: PMC8712107. DOI: 10.1016/j.expneurol.2021.113914.
3 Motavaf M, Piao X. Oligodendrocyte development and implication in perinatal white matter injury[J]. Front Cell Neurosci, 2021, 15: 764486. PMID: 34803612. PMCID: PMC8599582. DOI: 10.3389/fncel.2021.764486.
4 Schang AL, Van Steenwinckel J, Ioannidou ZS, et al. Epigenetic priming of immune/inflammatory pathways activation and abnormal activity of cell cycle pathway in a perinatal model of white matter injury[J]. Cell Death Dis, 2022, 13(12): 1038. PMID: 36513635. PMCID: PMC9748018. DOI: 10.1038/s41419-022-05483-4.
5 De Vlaminck K, Van Hove H, Kancheva D, et al. Differential plasticity and fate of brain-resident and recruited macrophages during the onset and resolution of neuroinflammation[J]. Immunity, 2022, 55(11): 2085-2102.e9. PMID: 36228615. DOI: 10.1016/j.immuni.2022.09.005.
6 Lee J, Hamanaka G, Lo EH, et al. Heterogeneity of microglia and their differential roles in white matter pathology[J]. CNS Neurosci Ther, 2019, 25(12): 1290-1298. PMID: 31733036. PMCID: PMC6887901. DOI: 10.1111/cns.13266.
7 Shao R, Sun D, Hu Y, et al. White matter injury in the neonatal hypoxic-ischemic brain and potential therapies targeting microglia[J]. J Neurosci Res, 2021, 99(4): 991-1008. PMID: 33416205. DOI: 10.1002/jnr.24761.
8 Tang H, Jiang Y, Zhang JH. Stem cell therapy for brain injury[J]. Stem Cells Dev, 2020, 29(4): 177. PMID: 32013774. DOI: 10.1089/scd.2020.29005.tan.
9 Ding DC, Chang YH, Shyu WC, et al. Human umbilical cord mesenchymal stem cells: a new era for stem cell therapy[J]. Cell Transplant, 2015, 24(3): 339-347. PMID: 25622293. DOI: 10.3727/096368915X686841.
10 Tscherrig V, Cottagnoud S, Haesler V, et al. MicroRNA cargo in Wharton's jelly MSC small extracellular vesicles: key functionality to in vitro prevention and treatment of premature white matter injury[J]. Stem Cell Rev Rep, 2023, 19(7): 2447-2464. PMID: 37523115. PMCID: PMC10579138. DOI: 10.1007/s12015-023-10595-1.
11 Robertson NJ, Meehan C, Martinello KA, et al. Human umbilical cord mesenchymal stromal cells as an adjunct therapy with therapeutic hypothermia in a piglet model of perinatal asphyxia[J]. Cytotherapy, 2021, 23(6): 521-535. PMID: 33262073. PMCID: PMC8139415. DOI: 10.1016/j.jcyt.2020.10.005.
12 Liu G, Wang D, Jia J, et al. Neuroprotection of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in alleviating ischemic stroke-induced brain injury by regulating inflammation and oxidative stress[J]. Neurochem Res, 2024, 49(10): 2871-2887. PMID: 39026086. DOI: 10.1007/s11064-024-04212-x.
13 Jensen A. Cerebral palsy: brain repair with stem cells[J]. J Perinat Med, 2022, 51(6): 737-751. PMID: 36503655. DOI: 10.1515/jpm-2022-0505.
14 Thomi G, Surbek D, Haesler V, et al. Exosomes derived from umbilical cord mesenchymal stem cells reduce microglia-mediated neuroinflammation in perinatal brain injury[J]. Stem Cell Res Ther, 2019, 10(1): 105. PMID: 30898154. PMCID: PMC6429800. DOI: 10.1186/s13287-019-1207-z.
15 Che J, Wang H, Dong J, et al. Human umbilical cord mesenchymal stem cell-derived exosomes attenuate neuroinflammation and oxidative stress through the NRF2/NF-κB/NLRP3 pathway[J]. CNS Neurosci Ther, 2024, 30(3): e14454. PMID: 37697971. PMCID: PMC10916441. DOI: 10.1111/cns.14454.
16 Rice JE, Vannucci RC, Brierley JB. The influence of immaturity on hypoxic-ischemic brain damage in the rat[J]. Ann Neurol, 1981, 9(2): 131-141. PMID: 7235629. DOI: 10.1002/ana.410090206.
17 张军, 李明霞, 王超, 等. 不同剂量人脐带间充质干细胞移植对新生大鼠脑白质损伤的修复作用[J]. 中国当代儿科杂志, 2024, 26(4): 394-402. PMID: 38660904. PMCID: PMC11057307. DOI: 10.7499/j.issn.1008-8830.2310081.
18 PaxinosG, WatsonC. 大鼠脑立体定位图谱[M]. 诸葛启钏, 译. 北京: 人民卫生出版社, 2005: 1-63.
19 Learish RD, Brüstle O, Zhang SC, et al. Intraventricular transplantation of oligodendrocyte progenitors into a fetal myelin mutant results in widespread formation of myelin[J]. Ann Neurol, 1999, 46(5): 716-722. PMID: 10553988.
20 Sun C, Zou N, Chen H, et al. The effect of magnetic guiding BMSCs on hypoxic-ischemic brain damage via magnetic resonance imaging evaluation[J]. Magn Reson Imaging, 2021, 79: 59-65. PMID: 33727146. DOI: 10.1016/j.mri.2021.03.008.
21 Lear BA, Lear CA, Dhillon SK, et al. Is late prevention of cerebral palsy in extremely preterm infants plausible?[J]. Dev Neurosci, 2022, 44(4-5): 177-185. PMID: 34937030. DOI: 10.1159/000521618.
22 Back SA. White matter injury in the preterm infant: pathology and mechanisms[J]. Acta Neuropathol, 2017, 134(3): 331-349. PMID: 28534077. PMCID: PMC5973818. DOI: 10.1007/s00401-017-1718-6.
23 Hagberg H, Peebles D, Mallard C. Models of white matter injury: comparison of infectious, hypoxic-ischemic, and excitotoxic insults[J]. Ment Retard Dev Disabil Res Rev, 2002, 8(1): 30-38. PMID: 11921384. DOI: 10.1002/mrdd.10007.
24 Dean JM, Moravec MD, Grafe M, et al. Strain-specific differences in perinatal rodent oligodendrocyte lineage progression and its correlation with human[J]. Dev Neurosci, 2011, 33(3-4): 251-260. PMID: 21865655. PMCID: PMC3225247. DOI: 10.1159/000327242.
25 Semple BD, Blomgren K, Gimlin K, et al. Brain development in rodents and humans: identifying benchmarks of maturation and vulnerability to injury across species[J]. Prog Neurobiol, 2013, 106-107: 1-16. PMID: 23583307. PMCID: PMC3737272. DOI: 10.1016/j.pneurobio.2013.04.001.
26 巴依尔才次克, 王彦梅, 朱艳萍. 间充质干细胞移植改善脑室周围白质软化损伤作用的研究[J]. 实验动物科学, 2021, 38(2): 22-29. DOI: 10.3969/j.issn.1006-6179.2021.02.004.
27 Gao C, Jiang J, Tan Y, et al. Microglia in neurodegenerative diseases: mechanism and potential therapeutic targets[J]. Signal Transduct Target Ther, 2023, 8(1): 359. PMID: 37735487. PMCID: PMC10514343. DOI: 10.1038/s41392-023-01588-0.
28 Kent SA, Miron VE. Microglia regulation of central nervous system myelin health and regeneration[J]. Nat Rev Immunol, 2024, 24(1): 49-63. PMID: 37452201. DOI: 10.1038/s41577-023-00907-4.
29 Liu X, Zhang M, Liu H, et al. Bone marrow mesenchymal stem cell-derived exosomes attenuate cerebral ischemia-reperfusion injury-induced neuroinflammation and pyroptosis by modulating microglia M1/M2 phenotypes[J]. Exp Neurol, 2021, 341: 113700. PMID: 33741350. DOI: 10.1016/j.expneurol.2021.113700.
30 Anilkumar S, Wright-Jin E. NF-κB as an inducible regulator of inflammation in the central nervous system[J]. Cells, 2024, 13(6): 485. PMID: 38534329. PMCID: PMC10968931. DOI: 10.3390/cells13060485.
31 Lawrence T. The nuclear factor NF-kappaB pathway in inflammation[J]. Cold Spring Harb Perspect Biol, 2009, 1(6): a001651. PMID: 20457564. PMCID: PMC2882124. DOI: 10.1101/cshperspect.a001651.
32 You MJ, Bang M, Park HS, et al. Human umbilical cord-derived mesenchymal stem cells alleviate schizophrenia-relevant behaviors in amphetamine-sensitized mice by inhibiting neuroinflammation[J]. Transl Psychiatry, 2020, 10(1): 123. PMID: 32341334. PMCID: PMC7186225. DOI: 10.1038/s41398-020-0802-1.
33 Wei P, Jia M, Kong X, et al. Human umbilical cord-derived mesenchymal stem cells ameliorate perioperative neurocognitive disorder by inhibiting inflammatory responses and activating BDNF/TrkB/CREB signaling pathway in aged mice[J]. Stem Cell Res Ther, 2023, 14(1): 263. PMID: 37735415. PMCID: PMC10512658. DOI: 10.1186/s13287-023-03499-x.
34 Liu Y, Shen X, Zhang Y, et al. Interactions of glial cells with neuronal synapses, from astrocytes to microglia and oligodendrocyte lineage cells[J]. Glia, 2023, 71(6): 1383-1401. PMID: 36799296. DOI: 10.1002/glia.24343.
35 Di Fiore JM, Raffay TM. The relationship between intermittent hypoxemia events and neural outcomes in neonates[J]. Exp Neurol, 2021, 342: 113753. PMID: 33984336. PMCID: PMC8192474. DOI: 10.1016/j.expneurol.2021.113753.
36 Zhu LH, Bai X, Zhang N, et al. Improvement of human umbilical cord mesenchymal stem cell transplantation on glial cell and behavioral function in a neonatal model of periventricular white matter damage[J]. Brain Res, 2014, 1563: 13-21. PMID: 24680746. DOI: 10.1016/j.brainres.2014.03.030.
37 Liu S, Fan M, Xu JX, et al. Exosomes derived from bone-marrow mesenchymal stem cells alleviate cognitive decline in AD-like mice by improving BDNF-related neuropathology[J]. J Neuroinflammation, 2022, 19(1): 35. PMID: 35130907. PMCID: PMC8822863. DOI: 10.1186/s12974-022-02393-2.
38 Fujiwara S, Nakano-Doi A, Sawano T, et al. Administration of human-derived mesenchymal stem cells activates locally stimulated endogenous neural progenitors and reduces neurological dysfunction in mice after ischemic stroke[J]. Cells, 2024, 13(11): 939. PMID: 38891071. PMCID: PMC11171641. DOI: 10.3390/cells13110939.
基金
国家自然科学基金地区科学基金项目（82060288）。
PDF(2623 KB)
