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

doi:10.11852/zgetbjzz2022-1261

Abstract

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

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

CLC Number:

R741

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.

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.

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