低氧预处理人牙髓干细胞对缺氧缺血性脑损伤新生大鼠脑神经元自噬及ATG7的影响

doi:10.11852/zgetbjzz2022-1261

中国儿童保健杂志 ›› 2023, Vol. 31 ›› Issue (8): 851-857.DOI: 10.11852/zgetbjzz2022-1261

低氧预处理人牙髓干细胞对缺氧缺血性脑损伤新生大鼠脑神经元自噬及ATG7的影响

高淑君¹, 张浩¹, 武沐洋¹, 张新月², 肖培伦³, 王晓莉², 王凡涛⁴

1.潍坊医学院口腔医学院,山东潍坊 261053;
2.潍坊医学院医学影像学院,山东潍坊 261053;
3.潍坊医学院基础医学院解剖学教研室,山东潍坊 261053;
4.潍坊医学院附属医院口腔科,山东潍坊 261053

收稿日期:2022-11-02 修回日期:2023-02-07 发布日期:2023-08-18
通讯作者: 肖培伦,E-mail:xiaopeilun123@sina.com; 王凡涛,E-mail:wang.fantao@163.com
作者简介:高淑君(1993-),女,山东人,在读硕士研究生,主要研究方向为牙髓干细胞对缺血缺氧性脑病的治疗作用。
基金资助:
山东省自然科学基金(ZR2020MH074);山东省中医药科技发展计划项目(2019-0417,2019-0421);潍坊市科技发展计划(2020YX037);潍坊市科技发展计划(2022YX042)

Effects of hypoxic preconditioning of hDPSCs on autophagy and ATG7 in brain neurons of neonatal HIBD rats

GAO Shujun¹, ZHANG Hao¹, WU Muyang¹, ZHANG Xinyue², XIAO Peilun³, WANG Xiaoli², WANG Fantao⁴

1. School of Stomatology, Weifang Medical University, Weifang, Shangdong 261053, China;
2. School of Medical Imaging, Weifang Medical University, Weifang, Shangdong 261053, China;
3. Department of Anatomy, School of Basic Medical Science, Weifang Medical University, Weifang, Shangdong 261053, China;
4. Department of Stomatology, Affiliated Hospital of Weifang Medical University, Weifang, Shangdong 261053, China

Received:2022-11-02 Revised:2023-02-07 Published:2023-08-18
Contact: XIAO Peilun, E-mail: xiaopeilun123@sina.com; WANG Fantao, E-mail: wang.fantao@163.com

摘要/Abstract

摘要： 目的探讨低氧预处理人牙髓干细胞(hDPSCs)对缺氧缺血性脑损伤(HIBD)新生大鼠脑神经元自噬及ATG7的影响,为hDPSCs临床应用提供理论依据。方法 2021年6月-2022年9月选取7日龄SD大鼠72只随机分为假手术组(Sham组)、HIBD组、常氧预处理hDPSCs组(N-hDPSCs组)及低氧预处理hDPSCs组(H-hDPSCs组)。采用Rice-Vannucci法制备新生大鼠HIBD模型,造模后24h侧脑室移植N-hDPSCs与H-hDPSCs。移植24h后,采用免疫荧光双标染色结合Western Blots检测H-hDPSCs对损伤侧大脑皮层神经元自噬的影响;移植4d,采用苏木素-伊红(hematoxylin and eosin stain,HE)染色法和尼氏染色法观察损伤侧大脑皮层组织病理学变化。结果免疫荧光染色结果示,H-hDPSCs组与N-hDPSCs组大脑皮层神经元LC3-Ⅱ、Beclin-1、ATG7阳性细胞数量显著低于HIBD组,且H-hDPSCs组显著低于N-hDPSCs组(F=326.87、205.87、229.59,P<0.05);H-hDPSCs组与N-hDPSCs组大脑皮层神经元P62阳性细胞数量显著高于HIBD组,且H-hDPSCs组显著高于N-hDPSCs组(F=124.07,P<0.05)。Western Blots结果示,H-hDPSCs组与N-hDPSCs组大脑皮层神经元LC3-Ⅱ、Beclin-1蛋白表达水平显著低于HIBD组,且H-hDPSCs组显著低于N-hDPSCs组(F=46.949、43.330,P<0.05)。HE染色结果示,HIBD组大脑皮层神经细胞形态不规则,排列不整齐;N-hDPSCs组和H-hDPSCs组大脑皮层神经细胞形态较规则,排列较整齐;尼氏染色结果示,N-hDPSCs组和H-hDPSCs组大脑皮层神经细胞数均较HIBD组增加,且H-hDPSCs组显著高于N-hDPSCs组 (F=312.44,P<0.05)。结论 hDPSCs移植可下调HIBD新生大鼠脑神经元自噬相关蛋白Beclin-1、LC3-Ⅱ、ATG7的表达,上调P62的表达,从而抑制自噬,减轻新生大鼠HIBD,促进脑损伤修复,其中,低氧预处理hDPSCs移植的疗效更优。

关键词: 低氧, 人牙髓干细胞, 新生大鼠, 缺氧缺血性脑损伤, 自噬

Abstract: Objective To analyze the effects of hypoxic preconditioning of human dental pulp stem cells (hDPSCs) on autophagy and autophagy related geneATG7 in brain neurons of neonatal rats with hypoxic-ischemic brain injury (HIBD), so as to provide scientific theoretical basis for the clinical application of hDPSCs. Methods From January 2021 to September 2022, totally 72 seven-day-old SD rats were randomly divided into sham operation group (Sham group), HIBD group, normoxia preconditioning hDPSCs group (N-hDPSCs group) and hypoxia preconditioning hDPSCs group (H-hDPSCs group).The HIBD model of neonatal rats was prepared by Rice Vannuis method.N-hDPSCs and H-hDPSCs were transplanted into the lateral ventricle 24 hours after the HIBD model established.Twenty-four hours after transplantation, the effects of hypoxic preconditioning with hDPSCs on autophagy in the injured cerebral cortex neurons of HIBD neonatal rats were detected by immunofluorescence double staining combined with Western Blots.Four days after transplantation, hematoxylin and eosin stain (HE) and Nissl stain were used to observe the pathological changes of cerebral cortex on the injured side of neonatal rats in each group. Results The results of immunofluorescence staining showed that thenumberof LC3-Ⅱ, Beclin-1, and ATG7 positive cells in cerebral cortex neurons of H-hDPSCs group and N-hDPSCs group were significantly lower than those of HIBD group, and these expression of H-hDPSCs group was significantly lower than those of N-hDPSCs group (F=326.87, 205.87, 229.59, P<0.05).The number of P62 positive cells in cerebral cortex neurons of H-hDPSCs group and N-hDPSCs group was significantly higher than that of HIBD group, and P62 expression of H-hDPSCs group was significantly higher than that of N-hDPSCs group (F=124.07, P<0.05).Western blots showed that the expression levels of LC3-Ⅱ and Beclin-1 protein in cerebral cortex neurons of H-hDPSCs group and N-hDPSCs group were significantly lower than those of HIBD group, and H-hDPSCs group was significantly lower than that of N-hDPSCs group (F=46.949, 43.330, P<0.05).HE staining results showed that neurons in cerebral cortex of HIBD group were irregular in shape and arrangement, while the neurons in the cerebral cortex of N-hDPSCs group and H-hDPSCs group had regular morphology and orderly arrangement.The results of Nissl staining showed that the number of neurons in the cerebral cortex of N-hDPSCs group and H-hDPSCs group was more than that of HIBD group, and the number of neurons in H-hDPSCs group was significantly more than that in N-hDPSCs group (F=312.44, P<0.05). Conclusions Transplantation of hDPSCs can downregulate the expression of autophagy related proteins Beclin-1, LC3-Ⅱ, ATG7, and upregulate the expression of P62 in brain neurons of neonatal rats with HIBD, so as to inhibit autophagy, reduce HIBD in neonatal rats and promote the repair of brain damage.Among them, hypoxic preconditioning of hDPSCs is more effective.

Key words: hypoxia, human dental pulp stem cells, neonatal rats, hypoxic-ischemic brain damage, autophagy

中图分类号:

R741

高淑君, 张浩, 武沐洋, 张新月, 肖培伦, 王晓莉, 王凡涛. 低氧预处理人牙髓干细胞对缺氧缺血性脑损伤新生大鼠脑神经元自噬及ATG7的影响[J]. 中国儿童保健杂志, 2023, 31(8): 851-857.

GAO Shujun, ZHANG Hao, WU Muyang, ZHANG Xinyue, XIAO Peilun, WANG Xiaoli, WANG Fantao. Effects of hypoxic preconditioning of hDPSCs on autophagy and ATG7 in brain neurons of neonatal HIBD rats[J]. Chinese Journal of Child Health Care, 2023, 31(8): 851-857.

参考文献

[1] Serrenho I, Rosado M, Dinis A, et al.Stem cell therapy for neonatal hypoxic-ischemic encephalopathy: A systematic review of preclinical studies[J].Int J Mol Sci, 2021,22(6):3142.
[2] Yang X, Wang M, Zhou Q, et al.Macamide B pretreatment attenuates neonatal hypoxic-ischemic brain damage of mice induced apoptosis and regulates autophagy via the PI3K/AKT signaling pathway[J].Mol Neurobiol,2022,59(5):2776-2798.
[3] Fu CH, Lai FF, Chen S, et al.Silencing of long non-coding RNA CRNDE promotes autophagy and alleviates neonatal hypoxic-ischemic brain damage in rats[J].Mol Cell Biochem,2020,472(1-2):1-8.
[4] Chang Y, Kong R.Ganoderic acid A alleviates hypoxia-induced apoptosis, autophagy, and inflammation in rat neural stem cells through the PI3K/AKT/mTOR pathways[J].Phytother Res,2019,33(5):1448-1456.
[5] Huang YG, Tao W, Yang SB, et al.Autophagy: Novel insights into therapeutic target of electroacupuncture against cerebral ischemia/ reperfusion injury[J].Neural Regen Res,2019,14(6):954-961.
[6] Jiang LJ, Xu ZX, Wu MF, et al.Resatorvid protects against hypoxic-ischemic brain damage in neonatal rats[J].Neural Regen Res,2020,15(7):1316-1325.
[7] Tang T, Gao D, Yang X, et al.Exogenous Netrin-1inhibits autophagy of ischemic brain tissues and hypoxic neurons via PI3K/mTOR pathway in ischemic stroke[J].J Stroke Cerebrovasc Dis,2019,28(5):1338-1345.
[8] Zhang DM, Zhang T, Wang MM, et al.TIGAR alleviates ischemia/reperfusion-induced autophagy and ischemic brain injury[J].Free Radic Biol Med,2019,137:13-23.
[9] 李文星,唐军,邹蓉,等.干细胞移植治疗新生儿缺氧缺血性脑病研究现状的可视化分析[J].中国当代儿科杂志,2018,20(12):1002-1007.
Li WX,Tang J,Zou R, et al.Visualization analysis of the research status of stem cell transplantation in the treatment of neonatal hypoxic ischemic encephalopathy[J].Chin J Contemp Pediatr,2018,20(12):1002-1007.(in Chinese)
[10] Kabatas S, Demir CS, Civelek E, et al.Neuronal regeneration in injured rat spinal cord after human dental pulp derived neural crest stem cell transplantation[J].Bratisl Lek Listy, 2018,119(3):143-151.
[11] 王爱,牟青杰,王晓莉,等.牙髓干细胞移植治疗缺氧缺血性脑损伤新生大鼠远期行为学及环磷酸腺苷反应元件结合蛋白的变化[J].中国组织工程研究, 2017, 21(5):701-706.
Wang A,Mu QJ,Wang XL, et al.Long term behavioral changes and cyclic adenosine monophosphate response element binding protein in neonatal rats with hypoxic-ischemic brain injury after dental pulp stem cell transplantation[J].J Clin Rehabil Tis Eng Res,2017,21(5):701-706.(in Chinese)
[12] 杨峥,赵敏,肖琳,等.不同低氧浓度对体外培养人牙髓干细胞生物学特性的影响[J].上海口腔医学,2021,30(3):247-252.
Yang Z,Zhao M,Xiao L, et al.Visualization analysis of the research status of stem cell transplantation in the treatment of neonatal hypoxic-ischemic encephalopathy[J].Shanghai J Stom,2021,30(3):247-252.(in Chinese)
[13] Liu J, He J, Ge L, et al.Hypoxic preconditioning rejuvenates mesenchymal stem cells and enhances neuroprotection following intracerebral hemorrhage via the miR-326-mediated autophagy[J].Stem Cell Res Ther,2021,12(1):413.
[14] 张浩,高淑君,武沐洋,等.低氧浓度促进人牙髓干细胞增殖及神经分化[J].中国组织化学与细胞化学杂志,2022,31(2):133-138.
Zhang H,Gao SJ,Wu MY, et al.Hypoxia promotes the proliferation and neural differentiation of human dental pulp stem cells[J].Chin J Histochem Cytochem,2022,31(2):133-138.(in Chinese)
[15] 陈宁馨,赖光云,汪俊.脱落乳牙牙髓干细胞在口腔颌面部组织再生及免疫调节中应用的研究进展[J].中国实用口腔科杂志,2021,14(2):220-224.
Chen NX,Lai GY,Wang J.Research progress in the application of deciduous teeth pulp stem cells in oral and maxillofacial tissue regeneration and immune regulation[J].Chin J Pract Stomatol,2021,14(2):220-224.(in Chinese)
[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.
[17] 张新月,刘晨萌,马瑜徽等.TXNIP/Trx-1/GPX4通路促进新生大鼠缺氧缺血后海马神经元铁死亡的作用机制[J].中国当代儿科杂志,2022,24(9):1053-1060.
Zhang XY,Liu CM,Ma YH, et al.The mechanism of TXNIP/Trx-1/GPX4 pathway promoting iron death of hippocampal neurons in neonatal rats after hypoxic ischemia[J].Chin J Contemp Pediatr,2022,24(9):1053-1060.(in Chinese)
[18] Tsutsui TW.Dental pulp stem cells: Advances to applications[J].Stem Cells Cloning,2020,13:33-42.
[19] 王鹏,郭铭.探讨低氧预处理骨髓间充质干细胞(MSCs)移植对大鼠缺血性脑损伤后神经功能恢复的作用及机制[J].现代诊断与治疗,2020,31(7):1009-1011.
Wang P,Guo M.To explore the effect and mechanism of transplantation of hypoxic preconditioning bone marrow mesenchymal stem cells (MSCs) on the recovery of neural function after ischemic brain injury in rats[J].Mod Diagn Treat,2020,31(7):1009-1011.(in Chinese)
[20] Jiang LJ, Xu ZX, Wu MF, et al.Resatorvid protects against hypoxic-ischemic brain damage in neonatal rats[J].Neural Regen Res,2020,15(7):1316-1325.
[21] Zhang X, Wei M, Fan J, et al.Ischemia-induced upregulation of autophagy preludes dysfunctional lysosomal storage and associated synaptic impairments in neurons[J].Autophagy,2021,17(6):1519-1542.
[22] Chang E, Kim L, Park SE,et al.Ezetimibe improves hepatic steatosis in relation to autophagy in obese and diabetic rats[J].World J Gastroenterol,2015,21(25):7754-7763.
[23] Wei W, Lu M, Lan X, et al.Neuroprotective effect of verbascoside on hypoxic-ischemic brain damage in neonatal rat[J].Neurosci Lett,2019,711:134415.

低氧预处理人牙髓干细胞对缺氧缺血性脑损伤新生大鼠脑神经元自噬及ATG7的影响

Effects of hypoxic preconditioning of hDPSCs on autophagy and ATG7 in brain neurons of neonatal HIBD rats

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