Sun Wen, Zhao Pengyue, Hu Shidong, Zhao Zhenting, Liu Boyan, Yang Xingpeng, Yang Jiaqi, Fu Ze, Li Songyan, Yu Wenli
The First Central Clinical School, Tianjin Medical University, Tianjin, China; Department of Anesthesiology, the Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China.
Department of General Surgery, First Medical Center of the Chinese PLA General Hospital, Beijing, China.
Neurotoxicology. 2024 May;102:81-95. doi: 10.1016/j.neuro.2024.04.002. Epub 2024 Apr 9.
Propofol can increase neurotoxicity in infants but the precise mechanism is still unknown. Our previous study revealed that nuclear FMR1 interacting protein 1 (NUFIP1), a specific ribophagy receptor, can alleviate T cell apoptosis in sepsis. Yet, the effect of NUFIP1-engineered exosomes elicited from human umbilical cord blood mesenchymal stem cells (hUMSCs) on nerve injury induced by propofol remains unclear. This study intended to investigate the effect of NUFIP1-engineered exosomes on propofol-induced nerve damage in neonatal rats.
Firstly, NUFIP1-engineered exosomes were extracted from hUMSCs serum and their identification was conducted using transmission electron microscopy (TEM), Flow NanoAnalyzer, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot (WB). Subsequently, the optimal exposure duration and concentration of propofol induced apoptosis were determined in SH-SY5Y cell line using WB. Following this, we co-cultured the NUFIP1-engineered exosomes in the knockdown group (NUFIP1-KD) and overexpression group (NUFIP1-OE) with SH-SY5Y cells and assessed their effects on the apoptosis of SH-SY5Y cells using terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay, Hoechst 33258 staining, WB, and flow cytometry, respectively. Finally, NUFIP1-engineered exosomes were intraperitoneally injected into neonatal rats, and their effects on the learning and memory ability of neonatal rats were observed through the righting reflex and Morris water maze (MWM) test. Hippocampi were extracted from different groups for hematoxylin-eosin (HE) staining, immunohistochemistry, immunofluorescence, and WB to observe their effects on apoptosis in neonatal rats.
TEM, Flow NanoAnalyzer, qRT-PCR, and WB analyses confirmed that the exosomes extracted from hUMSCs serum exhibited the expected morphology, diameter, surface markers, and expression of target genes. This confirmed the successful construction of NUFIP1-KD and NUFIP1-OE-engineered exosomes. Optimal exposure duration and concentration of propofol were determined to be 24 hours and 100 µg/ml, respectively. Co-culture of NUFIP1 engineered exosomes and SH-SY5Y cells resulted in significant up-regulation of pro-apoptotic proteins Bax and c-Caspase-3 in the KD group, while anti-apoptotic protein Bcl-2 was significantly decreased. The OE group showed the opposite trend. TUNEL apoptosis assay, Hoechst 33258 staining, and flow cytometry yielded consistent results. Animal experiments demonstrated that intraperitoneal injection of NUFIP1-KD engineered exosomes prolonged the righting reflex recovery time of newborn rats, and MWM tests revealed a significant diminution in the time and number of newborn rats entering the platform. HE staining, immunohistochemistry, immunofluorescence, and WB results also indicated a significant enhancement in apoptosis in this group. Conversely, the experimental results of neonatal rats in the OE group revealed a certain degree of anti-apoptotic effect.
NUFIP1-engineered exosomes from hUMSCs have the potential to regulate nerve cell apoptosis and mitigate neurological injury induced by propofol in neonatal rats. Targeting NUFIP1 may hold great significance in ameliorating propofol-induced nerve injury.
丙泊酚可增加婴儿的神经毒性，但其确切机制仍不清楚。我们之前的研究表明，核FMR1相互作用蛋白1（NUFIP1）是一种特异性的核糖体自噬受体，可减轻脓毒症中的T细胞凋亡。然而，人脐带血间充质干细胞（hUMSCs）来源的NUFIP1工程化外泌体对丙泊酚诱导的神经损伤的影响仍不清楚。本研究旨在探讨NUFIP1工程化外泌体对新生大鼠丙泊酚诱导的神经损伤的影响。
首先，从hUMSCs血清中提取NUFIP1工程化外泌体，并使用透射电子显微镜（TEM）、流式纳米分析仪、定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹（WB）进行鉴定。随后，使用WB在SH-SY5Y细胞系中确定丙泊酚诱导凋亡的最佳暴露时间和浓度。在此之后，我们将敲低组（NUFIP1-KD）和过表达组（NUFIP1-OE）中的NUFIP1工程化外泌体与SH-SY5Y细胞共培养，并分别使用末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）检测、Hoechst 33258染色、WB和流式细胞术评估它们对SH-SY5Y细胞凋亡的影响。最后，将NUFIP1工程化外泌体腹腔注射到新生大鼠体内，并通过翻正反射和莫里斯水迷宫（MWM）试验观察它们对新生大鼠学习和记忆能力的影响。从不同组中提取海马进行苏木精-伊红（HE）染色、免疫组织化学、免疫荧光和WB，以观察它们对新生大鼠凋亡的影响。
TEM、流式纳米分析仪、qRT-PCR和WB分析证实，从hUMSCs血清中提取的外泌体呈现出预期的形态、直径、表面标志物和靶基因表达。这证实了NUFIP1-KD和NUFIP1-OE工程化外泌体的成功构建。丙泊酚的最佳暴露时间和浓度分别确定为２４小时和１００µｇ/ml。NUFIP1工程化外泌体与SH-SY5Y细胞共培养导致KD组中促凋亡蛋白Bax和c-Caspase-3显著上调，而抗凋亡蛋白Bcl-2显著降低。OE组呈现相反的趋势。TUNEL凋亡检测、Hoechst 33258染色和流式细胞术产生了一致的结果。动物实验表明，腹腔注射NUFIP1-KD工程化外泌体延长了新生大鼠的翻正反射恢复时间，MWM试验显示新生大鼠进入平台的时间和次数显著减少。HE染色、免疫组织化学、免疫荧光和WB结果也表明该组凋亡显著增加。相反，OE组新生大鼠的实验结果显示出一定程度的抗凋亡作用。
hUMSCs来源的NUFIP1工程化外泌体有可能调节神经细胞凋亡并减轻新生大鼠丙泊酚诱导的神经损伤。靶向NUFIP1可能对改善丙泊酚诱导的神经损伤具有重要意义。
