Abstract: Objective To investigate the effects of hypoxia-preconditioned human dental pulp stem cells (H-hDPSCs) transplantation on neuronal ferroptosis in neonatal rats with hypoxic-ischemic brain damage (HIBD), in order to provide novel therapeutic insights for neonatal HIBD. Methods A total of 64 7-day-old Sprague Dawley rats were randomly assigned to four groups: sham-operated (Sham), HIBD, normoxic-cultured hDPSCs (N-hDPSCs), and hypoxia-preconditioned hDPSCs (H-hDPSCs), with 16 rats in each group.The HIBD model was established using the Rice-Vannucci method.N-hDPSCs or H-hDPSCs were transplanted into the lateral ventricle 24 hours post-modeling.At 72 hours post-transplantation, histopathological changes were assessed by hematoxylin-eosin (HE) and Nissl staining, serum and cortical iron levels were measured by ELISA.Expression levels of ACSL4, GPX4, and SLC7A11 mRNA in the cerebral cortex detected by real-time quantitative fluorescence polymerase chain reaction (qRT-PCR). Results The results of HE and Nissl staining showed that compared with the Sham group, HIBD group showed neuronal disarray, pyknotic nuclei, and reduced Nissl bodies, while both hDPSCs groups exhibited attenuated damage.Compared with the Sham group, serum iron and iron content of the cortical tissue on the injured side were as the followings in the remaining three groups: HIBD>N-hDPSCs>Sham (F=79.565, 121.065, P<0.001).In terms of ferroptosis markers: GPX4+/SLC7A11+ neurons were as the followings: Sham>H-hDPSCs>HIBD (F=30.896, 75.089, P<0.001), ACSL4+ neurons were as the followings: HIBD>N-hDPSCs>Sham (F=87.724, P<0.001).H-hDPSCs showed higher GPX4 or SLC7A11 and lower ACSL4 expression than N-hDPSCs at both mRNA (qRT-PCR) and protein (Western blot) levels (P<0.001). Conclusions Transplantation of hDPSCs mitigates HIBD by suppressing neuronal ferroptosis, with hypoxia-preconditioned cells demonstrating superior therapeutic effects through regulation of ferroptosis-related pathways (GPX4/SLC7A11 upregulation and ACSL4 downregulation).

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

摘要： 目的 探究低氧预处理人牙髓干细胞(H-hDPSCs)移植对缺氧缺血性脑损伤(HIBD)新生大鼠脑神经元铁死亡的影响,为新生儿HIBD的治疗提供新思路。方法 选取健康7d Sprague Dawley大鼠64只,随机分为对照(Sham)组、HIBD组、常氧培养hDPSCs(N-hDPSCs)组和低氧预处理hDPSCs(H-hDPSCs)组,每组16只。HIBD 模型采用经典Rice-Vannucci法建立, N-hDPSCs或H-hDPSCs建模后24h移植至侧脑室。移植后72h,采用苏木素伊红(HE)与尼氏染色法观察损伤侧大脑皮层组织病理学变化;ELISA法检测血清铁和损伤侧大脑皮层组织铁含量的变化;NeuN/ACSL4、NeuN/GPX4、NeuN/SLC7A11免疫荧光双标染色与Western blot检测ACSL4、GPX4、SLC7A11蛋白在损伤侧大脑皮层神经元的表达水平;实时荧光定量聚合酶链式反应法(qRT-PCR)检测大脑皮层ACSL4、GPX4、SLC7A11 mRNA的表达水平。 结果 HE及尼氏染色结果显示,与Sham组相比,HIBD组神经细胞排列紊乱,细胞核深染固缩,尼氏小体数量较少,N-hDPSCs组和H-hDPSCs组与HIBD组相比损伤减轻。血清铁和大脑皮层组织铁含量HIBD组最高,N-hDPSCs组次之,Sham组最低(F=79.565、121.065,P<0.001)。免疫荧光染色示Sham组NeuN⁺GPX4⁺和NeuN⁺SLC7A11⁺细胞数最高,H-hDPSCs组次之,HIBD组最低(F=30.896、75.089,P<0.001);NeuN⁺ACSL4⁺细胞数HIBD组最高,N-hDPSCs组次之,Sham组最低(F=87.724,P<0.001)。qRT-PCR和Western blot结果示,H-hDPSCs组和N-hDPSCs组GPX4,SLC7A11的mRNA及蛋白表达水平显著高于HIBD组,且H-hDPSCs组显著高于N-hDPSCs组(F=1 008.209、41.765、48.178、43.303,P<0.001);H-hDPSCs组和N-hDPSCs组ACSL4的mRNA及蛋白表达水平显著低于HIBD组,且H-hDPSCs组显著低于N-hDPSCs组(F=234.513、164.942,P<0.001)。结论 hDPSCs移植可通过抑制大脑皮层铁死亡相关基因的表达,抑制神经元铁死亡发生,新生大鼠HIBD得以减轻,且H-hDPSCs效果优于N-hDPSCs。

关键词: 低氧, 人牙髓干细胞, 新生大鼠, 缺氧缺血性脑损伤, 铁死亡