Department of Thoracic and Cardiovascular Surgery, Jiangsu Province People's Hospital and the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, People's Republic of China.
Mol Cell Biochem. 2013 Aug;380(1-2):267-75. doi: 10.1007/s11010-013-1683-x. Epub 2013 May 28.
This study was designed to investigate the effect and mechanism of lipopolysaccharide (LPS) preconditioning on survival and connexin 43 (CX43) expression in rat bone marrow mesenchymal stem cells (bMSCs) under hypoxia and serum deprivation (Hypoxia/SD) conditions. Whole marrow cells were obtained from the femora and tibiae of SD rats, and bMSCs were isolated by density gradient centrifugation and attachment culture. Surface antigens were determined by FACS before the experiment using antibodies conjugated directly against anti-rat CD34, anti-CD45, anti-CD29, and anti-CD44. Passage 3 bMSCs were used for all experiments. The effect of LPS preconditioning on bMSCs apoptosis in response to Hypoxia/SD was investigated by an Annexin V-FITC/PI binding assay and a mitochondrial membrane potential (△Ψm) assay. Cyc-c released into the cytosol from mitochondria and CX43 in bMSCs was determined by Western blot before and after LPS preconditioning. Subsequently, extracellular signal-regulated kinase (ERK) was inhibited with PD98059 to analyze the role of ERK in modulating CX43 expression after LPS preconditioning. The bMSCs surface antigen profiles obtained by flow cytometry were positive for CD29 and CD44 and negative for CD34 and CD45. The Hypoxia/SD conditions induced significant apoptosis of bMSCs. Compared with the Hypoxia/SD group, cells treated with LPS prevented △Ψm from falling significantly. LPS inhibited Hypoxia/SD-induced Cyc-c release. These results were consistent with the total analysis of apoptosis of MSCs. Compared with the control group, the level of CX43 expression in the Hypoxia/SD group and LPS + Hypoxia/SD group decreased significantly at each time point. The level of CX43 expression in the Hypoxia/SD group was lower than that in the LPS + Hypoxia/SD group, while the difference was not significant between the PD98059 + LPS + Hypoxia/SD group and the PD98059 + Hypoxia/SD group (P > 0.05). Compared with the LPS + Hypoxia/SD group, CX43 level in the PD98059 + LPS + Hypoxia/SD group and PD98059 + Hypoxia/SD group decreased significantly (P < 0.05). These results demonstrated that Hypoxia/SD conditions could induce apoptosis of bMSCs markedly. Low-dose LPS preconditioning may preserve the mitochondrial function by maintaining the mitochondrial transmembrane potential and inhibiting Cyc-c release in Hypoxia/SD-induced bMSCs apoptosis. LPS preconditioning also had a stabilizing effect on the cell membrane by inhibiting the decrease of CX43, and this modulating mechanism may be related to the ERK signaling pathway.
本研究旨在探讨脂多糖（LPS）预处理对缺氧和血清剥夺（Hypoxia/SD）条件下大鼠骨髓间充质干细胞（bMSCs）存活和连接蛋白 43（CX43）表达的影响及机制。SD 大鼠股骨和胫骨获得全骨髓细胞，通过密度梯度离心和贴壁培养分离 bMSCs。实验前用直接偶联抗大鼠 CD34、抗 CD45、抗 CD29 和抗 CD44 的抗体通过流式细胞术测定表面抗原。所有实验均使用传代 3 的 bMSCs。通过 Annexin V-FITC/PI 结合测定和线粒体膜电位（△Ψm）测定研究 LPS 预处理对 bMSCs 在 Hypoxia/SD 下凋亡的影响。用 Western blot 法测定 LPS 预处理前后 bMSCs 中从线粒体释放到胞质中的 Cyc-c 和 CX43。随后，用 PD98059 抑制细胞外信号调节激酶（ERK），以分析 LPS 预处理后 ERK 调节 CX43 表达的作用。流式细胞术获得的 bMSCs 表面抗原谱 CD29 和 CD44 阳性，CD34 和 CD45 阴性。Hypoxia/SD 条件诱导 bMSCs 明显凋亡。与 Hypoxia/SD 组相比，用 LPS 处理的细胞明显阻止了 △Ψm 的下降。LPS 抑制 Hypoxia/SD 诱导的 Cyc-c 释放。这些结果与 MSCs 总凋亡分析一致。与对照组相比，Hypoxia/SD 组和 LPS + Hypoxia/SD 组在各个时间点的 CX43 表达水平均显著降低。Hypoxia/SD 组的 CX43 表达水平低于 LPS + Hypoxia/SD 组，但 PD98059 + LPS + Hypoxia/SD 组和 PD98059 + Hypoxia/SD 组之间差异无统计学意义（P>0.05）。与 LPS + Hypoxia/SD 组相比，PD98059 + LPS + Hypoxia/SD 组和 PD98059 + Hypoxia/SD 组的 CX43 水平明显降低（P<0.05）。这些结果表明，Hypoxia/SD 条件可明显诱导 bMSCs 凋亡。低剂量 LPS 预处理可能通过维持 Hypoxia/SD 诱导的 bMSCs 凋亡中线粒体跨膜电位和抑制 Cyc-c 释放来维持线粒体功能。LPS 预处理还通过抑制 CX43 减少对细胞膜具有稳定作用，这种调节机制可能与 ERK 信号通路有关。
