Valipour Behnaz, Fathi Ezzatollah, Farahzadi Raheleh, Naderali Elahe, Behniafar Hamed
Department of Basic Sciences and Health, Sarab Faculty of Medical Sciences, Sarab, East Azerbaijan, Iran.
Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran.
Tissue Cell. 2024 Dec;91:102558. doi: 10.1016/j.tice.2024.102558. Epub 2024 Sep 10.
Stem cell-based therapy has emerged as an attractive approach for regenerative medicine. Poor survival and maintenance of the cells used in regenerative medicine are considered as serious barriers to enhance the efficacy of the cell therapy. Using some antioxidants has been reported to prevent the aging of stem cells, and finding effective factors to reduce the senescence of these cells has impressive potential in cell therapy. The PI3K pathway adversely regulates the transcription factors known as FOXO, which are thought to have an inhibitory influence on cell proliferation. By downregulating FOXO and other targets, PI3K signaling controls the growth of cells. For this reason, the aim of the present study is to investigate the effect of L-carnitine (LC) as antioxidant on the cell proliferation and the protein expression of PI3K and FOXO.
For understanding the in vitro effect of LC on the PI3K and FOXO-1 expression of C-kit hematopoietic progenitor cells, the bone marrow mononuclear cells were isolated, and C-kit cells was enriched by the magnetic-activated cell sorting (MACS). Next, the identification of enriched C-kit cells were done by flowcytometry and immunocytochemistry. Then, C-kit cells were treated with 0.2 mM LC, the cells were collected at the end of the treatment period (48 h), and the proteins were extracted. In the following, the protein expression of PI3K and FOXO-1 was measured by western blotting. In addition, flowcytometry was done to assess the Ki-67 expression as a key marker for cell proliferation investigation.
0.2 mM LC cause to significantly decrease in the protein expression of PI3K and FOXO-1 (*P<0.05 and **P<0.01, respectively). Also, the expression of Ki-67 was significantly increased in the presence of 0.2 mM LC (***P<0.001).
Briefly, LC can be considered an effective factor in increasing the proliferation of C-kit cells via some signaling pathways.
基于干细胞的疗法已成为再生医学中一种有吸引力的方法。再生医学中所用细胞的低存活率和维持率被视为提高细胞疗法疗效的严重障碍。据报道，使用一些抗氧化剂可防止干细胞衰老，而找到有效因素来减少这些细胞的衰老在细胞疗法中具有巨大潜力。PI3K 通路对称为 FOXO 的转录因子产生负向调节作用，而 FOXO 被认为对细胞增殖具有抑制影响。通过下调 FOXO 及其他靶点，PI3K 信号传导控制细胞生长。因此，本研究的目的是探究作为抗氧化剂的左旋肉碱（LC）对细胞增殖以及 PI3K 和 FOXO 蛋白表达的影响。
为了解 LC 对 C-kit 造血祖细胞的 PI3K 和 FOXO-1 表达的体外作用，分离骨髓单个核细胞，并通过磁珠分选法（MACS）富集 C-kit 细胞。接下来，通过流式细胞术和免疫细胞化学对富集的 C-kit 细胞进行鉴定。然后，用 0.2 mM LC 处理 C-kit 细胞，在处理期结束时（48 小时）收集细胞并提取蛋白质。随后，通过蛋白质印迹法测量 PI3K 和 FOXO-1 的蛋白表达。此外，进行流式细胞术以评估 Ki-67 的表达，Ki-67 是细胞增殖研究的关键标志物。
0.2 mM LC 导致 PI3K 和 FOXO-1 的蛋白表达显著降低（分别为*P<0.05 和**P<0.01）。此外，在存在 0.2 mM LC 的情况下，Ki-67 的表达显著增加（***P<0.001）。
简而言之，LC 可被视为通过某些信号通路增加 C-kit 细胞增殖的有效因素。
