缺氧条件下CXCL13通过MAPK信号通路促进人骨髓间充质干细胞增殖和自噬

CXCL13 promotes proliferation and autophagy of human mesenchymal stem cells through MAPK signaling pathway under hypoxic condition

目的探索CXC趋化因子配体13(CXCL13)对人骨髓间充质干细胞(hBMSC)自噬和增殖的影响及机制。方法 hBMSC分为对照组、缺氧组、 CXCL13处理组、 CXCL13和SB203580的联合处理组。除对照组外,其余各组均在920 mL/L N_2、 30 mL/L O_2和50 mL/L CO_2建立的细胞缺氧模型下进行培养。缺氧组不进行任何处理, CXCL13处理组给予50 ng/mL CXCL13重组蛋白刺激,联合处理组则预先添加20μmol/L丝裂原激活蛋白激酶(MAPK)抑制剂SB203580,培养30 min后,再以50 ng/mL CXCL13刺激。分别采用噻唑蓝(MTT)法检测hBMSC增殖活性, annexinⅤ-FITC/PI双标记结合流式细胞术检测hBMSC的凋亡, Western blot法检测hBMSC的核因子κB(NF-κB)、 beclin-1蛋白水平, ELISA检测细胞裂解液自噬相关蛋白7(ATG7)的含量。结果与缺氧组及联合处理组相比, CXCL13处理组hBMSC的细胞增殖活性、 beclin-1及NF-κB蛋白水平均升高、凋亡率明显降低;细胞裂解液ATG7水平升高。结论缺氧环境下, CXCL13通过MAPK/NF-κB信号通路促进hBMSC自噬和增殖。

Objective To explore the possible effect of C-X-C motif chemokine ligands13(CXCL13) on the autophagy and proliferation of human mesenchymal stem cells(hBMSCs) and the underlying mechanisms. Methods The hBMSCs were divided into control group, hypoxia group, CXCL13 treatment group, and CXCL13 combined with SB203580 group. Except the control group, the rest of cells were cultured under the established hypoxia condition of 920 mL/L N2, 30 mL/L O2 and 50 mL/L CO2. The cells of CXCL13 treatment group were stimulated with 50 ng/mL recombinant CXCL13. In the combination group, the cells were preincubated with 20 μmol/L of MAPK inhibitor SB203580 for 30 minutes and subsequently treated with 50 ng/mL recombinant CXCL13. MTT assay was used to determine the proliferation of hBMSCs. The apoptosis rate of hBMSCs was analyzed by annexin Ⅴ-FITC/PI double labeling and flow cytometry. The protein levels of nuclear factor-κB(NF-κB) and beclin-1 were measured by Western blot analysis. ELISA was used to measure the content of autophagy-related protein 7(ATG7) in cell lysis buffer. Results Compared with the hypoxia group and the combination group. The cell proliferation, beclin-1 and NF-κB protein levels increased significantly in the CXCL13 treatment group however, the apoptosis rate decreased, and the level of ATG7 increased significantly in cell lysate. Conclusion Under hypoxic condition, CXCL13 promotes autophagy and proliferation of hBMSCs through the MAPK/NF-κB signaling pathway.