Zhang Lu, Chen Shan, Zou Renfang, Shu Xin, Zhang Jingxuan, He Xuan, Su Moxi, Wang Luna, Wang Bin, Sha Dujuan
Department of General Practice, Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.
Department of General Practice, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, Jiangsu, China.
Front Neurosci. 2024 Aug 20;18:1424719. doi: 10.3389/fnins.2024.1424719. eCollection 2024.
Apoptosis has been recognized as a critical pathophysiological process during cerebral ischemia. The neuroprotective effect of CART on ischemic brain injury is determined. However, there is little research on the protective effect of CART on neural stem cells (NSCs).
Primary cultured rat NSCs were utilized as the research subject. oxygen glucose deprivation (OGD) treatment was employed, and NSCs were extracted from SD pregnant rats following previous experimental protocols and identified through cell immunofluorescence staining. The appropriate concentration of CART affecting OGD NSCs was initially screened using Cell Counting Kit-8 (CCK-8) and Lactate Dehydrogenase (LDH) assays. EdU staining and Western blotting (WB) techniques were employed to assess the impact of the suitable CART concentration on the proliferation and apoptosis of OGD NSCs. Finally, Western blot analysis was conducted to investigate the cAMP-response element binding protein (CREB) pathway and expression levels of related proteins after KG-501 treatment in order to elucidate the mechanism underlying apoptosis and proliferation regulation in OGD NSCs.
CCK-8 and LDH assays indicated that a concentration of 0.8 nM CART may be the optimal concentration for modulating the proliferation of OGD NSCs. Subsequently, cellular immunofluorescence and EdU detection experiments further confirmed the findings obtained from CCK-8 analysis. Western blot analysis of apoptosis-related protein expression also demonstrated that an appropriate concentration of CART could suppress the apoptosis of OGD NSCs. Finally, Western blotting was conducted to examine the CREB pathway and related protein expression after treatment with KG-501, revealing that an appropriate concentration of CART regulated both apoptosis and proliferation in OGD NSCs through CREB signaling.
The concentration of CART at 0.8 nM may be deemed appropriate for inhibiting apoptosis and promoting proliferation in OGD NSCs . The mechanism maybe through activating the CREB pathway.
细胞凋亡已被公认为是脑缺血过程中的一个关键病理生理过程。CART对缺血性脑损伤的神经保护作用已得到确定。然而，关于CART对神经干细胞（NSCs）的保护作用的研究较少。
以原代培养的大鼠神经干细胞为研究对象。采用氧糖剥夺（OGD）处理，按照先前的实验方案从SD孕鼠中提取神经干细胞，并通过细胞免疫荧光染色进行鉴定。最初使用细胞计数试剂盒-8（CCK-8）和乳酸脱氢酶（LDH）测定法筛选影响OGD神经干细胞的CART的合适浓度。采用EdU染色和蛋白质免疫印迹（WB）技术评估合适浓度的CART对OGD神经干细胞增殖和凋亡的影响。最后，进行蛋白质免疫印迹分析以研究KG-501处理后cAMP反应元件结合蛋白（CREB）途径及相关蛋白的表达水平，以阐明OGD神经干细胞凋亡和增殖调节的潜在机制。
CCK-8和LDH测定表明，0.8 nM的CART浓度可能是调节OGD神经干细胞增殖的最佳浓度。随后，细胞免疫荧光和EdU检测实验进一步证实了CCK-8分析的结果。凋亡相关蛋白表达的蛋白质免疫印迹分析也表明，合适浓度的CART可抑制OGD神经干细胞的凋亡。最后，进行蛋白质免疫印迹以检测KG-501处理后的CREB途径和相关蛋白表达，结果显示合适浓度的CART通过CREB信号调节OGD神经干细胞的凋亡和增殖。
0.8 nM的CART浓度可能被认为适合抑制OGD神经干细胞的凋亡并促进其增殖。其机制可能是通过激活CREB途径。
