Objective: To investigate the changes in autophagy of mesenchymal stem cells (MSCs) from patients with ankylosing spondylitis and explore the mechanism for decreased autophagy in ASMSCs.
Methods: MSCs collected from 14 patients with AS (ASMSCs) and from 15 healthy donors (HDMSCs) were cultured in the absence or presence of 25 ng/mL TNF-α for 6 h. Autophagy of the cells was determined by immunofluorescence staining of GFP-LC3B, and the results were confirmed by detecting the protein expressions of autophagy markers LC3 II/LC3 I and P62. The mRNA expressions of the related genes were detected using qRT-PCR, and the protein expressions of the autophagy markers and signaling pathway-related molecules were determined with Western blotting. TG100713 was used to block the PI3K/AKT/mTOR signal pathway, and its effect on autophagy of ASMSCs was evaluated.
Results: ASMSCs showed significantly weaker GFP-LC3B puncta staining and lower protein expression levels of LC3 II/LC3 I but higher levels of P62 protein (P < 0.05), indicating a decreased autophagy capacity as compared with HDMSCs. TNF-α-induced ASMSCs showed significantly higher protein expressions of p-PI3K/ PI3K, p-AKT/AKT and p-mTOR/mTOR than HDMSCs (P < 0.05), suggesting hyperactivation of the PI3K/AKT/mTOR signaling pathway in ASMSCs. Blocking PI3K/AKT/mTOR signaling with TG100713 eliminated the difference in TNF-α-induced autophagy between HDMSCs and ASMSCs.
Conclusion: In patients with AS, hyperactivation of the PI3K/AKT/mTOR signaling pathway results in decreased autophagy of the MSCs and potentially contributes to chronic inflammation.
目的: 比较强直性脊柱炎患者间充质干细胞（ASMSCs）与健康人间充质干细胞（HDMSCs）自噬水平的差异及其机制。
方法: 将ASMSCs和HDMSCs种于6孔板，细胞贴壁后，实验组加入25 ng/mL α-肿瘤坏死因子（TNF-α）及培养液，对照组单纯加入培养液，诱导6 h后进行后续实验。用GFP-LC3B免疫荧光染色检测自噬情况，并通过检测LC3 II/LC3 I及P62的蛋白表达进一步确认。通过qRT-PCR检测基因表达水平，通过Western blot检测蛋白表达水平。使用TG100713特异性阻断PI3K的磷酸化以明确PI3K/AKT/mTOR通路与ASMSCs自噬减弱的关系。
结果: ASMSCs的LC3 II/LC3 I表达水平和GFP-LC3B免疫荧光斑点明显弱于HDMSCs（P < 0.05），而P62表达水平明显高于HDMSCs（P < 0.05）。在自噬过程中，ASMSCs中p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR的表达明显高于HDMSCs（P < 0.05）。阻断PI3K磷酸化后，p-AKT/AKT和p-mTOR/mTOR的表达及ASMSCs的自噬可恢复至HDMSCs的水平。
结论: TNF-α诱导下，PI3K/AKT/mTOR信号通路异常是导致ASMSCs自噬减弱的关键，可能参与AS慢性炎症的发生。
Keywords: ankylosing spondylitis; autophagy; mesenchymal stem cells; tumor necrosis factor-α.