Wang Xiaoqi, Huang Jing, Ou Yanjing, Zhang Shuang, Fan Le, Liang Youde, Wang Yining, Zhou Yi
Department of Stomatology, The First Affiliated Hospital of Soochow University Suzhou, Jiangsu, China.
The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University Wuhan, Hubei, China.
Am J Transl Res. 2023 Jun 15;15(6):4065-4078. eCollection 2023.
Bone marrow-derived mesenchymal stem cells (BMSCs) - based tissue engineering is an important strategy for treatment of bone defects. However, the ischemia environment limits the survival and biological functions of BMSCs. The present study aimed to investigate the effect of leukemia inhibitory factor (LIF) on the apoptosis of BMSCs induced by hypoxia and serum-deprivation (H&SD) as well as the underlying pathway mechanism.
Mitochondrial membrane potential (MMP) was determined by flow cytometry. The apoptotic phenomenon of nuclear morphology was detected by fluorescence microscope. The ratio of apoptotic BMSCs was investigated by Annexin V/propidium iodide (PI) double staining and flow cytometric analysis. The expression of apoptosis-related molecules was detected by quantitative polymerase chain reaction (qPCR) and western blotting.
H&SD treatment induced a series of apoptotic phenotypes, including the downregulation of MMP, the apoptotic phenomenon of nuclear morphology, the increased rate of BMSCs at early and late apoptotic stage, and the reduced B-cell lymphoma-2 (Bcl-2)/Bcl-2-associated X (Bax) ratio. Administration of recombinant LIF alleviated the apoptosis of BMSCs induced by H&SD, which was reflected in recovery of MMP, morphology of nuclei, rate of apoptotic cells and inhibition of cleaved Caspase-3. The results of western blot demonstrated that phosphorylation of janus kinase (JAK) 1 and signal transducer and activator of transcription (STAT) 3 was inhibited by H&SD treatment, which was upregulated by LIF administration. JAK1-specific inhibitor GLPG0634 or STAT3-specific inhibitor S3I-201 eliminated the protective effects of LIF on the apoptosis of BMSCs.
These data indicated that LIF played a protective role in apoptosis of BMSCs induced by ischemia via activating JAK1/STAT3 signaling pathway.
基于骨髓间充质干细胞（BMSCs）的组织工程是治疗骨缺损的重要策略。然而，缺血环境限制了BMSCs的存活和生物学功能。本研究旨在探讨白血病抑制因子（LIF）对缺氧和血清剥夺（H&SD）诱导的BMSCs凋亡的影响及其潜在的信号通路机制。
采用流式细胞术检测线粒体膜电位（MMP）。用荧光显微镜检测细胞核形态的凋亡现象。采用膜联蛋白V/碘化丙啶（PI）双染法和流式细胞术分析检测BMSCs的凋亡率。通过定量聚合酶链反应（qPCR）和蛋白质免疫印迹法检测凋亡相关分子的表达。
H&SD处理诱导了一系列凋亡表型，包括MMP下调、细胞核形态的凋亡现象、早期和晚期凋亡阶段BMSCs比例增加以及B细胞淋巴瘤-2（Bcl-2）/Bcl-2相关X蛋白（Bax）比值降低。给予重组LIF可减轻H&SD诱导的BMSCs凋亡，这体现在MMP的恢复、细胞核形态、凋亡细胞比例以及对裂解的半胱天冬酶-3的抑制上。蛋白质免疫印迹结果表明，H&SD处理抑制了janus激酶（JAK）1和信号转导及转录激活因子（STAT）3的磷酸化，而LIF给药使其上调。JAK1特异性抑制剂GLPG0634或STAT3特异性抑制剂S3I-201消除了LIF对BMSCs凋亡的保护作用。
这些数据表明，LIF通过激活JAK1/STAT3信号通路对缺血诱导的BMSCs凋亡发挥保护作用。
