南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (2): 272-277.doi: 10.12122/j.issn.1673-4254.2022.02.15

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PI3K/AKT/mTOR信号通路异常激活导致强直性脊柱炎患者的间充质干细胞自噬减弱

刘振华，闵少雄，卢秀仪，岑水忠，陈志鹏，王 涛，李建君，曾炜波，邱素均   

  1. 南方医科大学珠江医院脊柱外科，广东 广州 510282；北京大学深圳医院脊柱外科，广东 深圳 518034；广州医科大学附属第四医院皮肤科，广东 广州 510316；中山大学孙逸仙纪念医院骨外科，广东 广州 510120
  • 出版日期:2022-02-20 发布日期:2022-03-16

Hyperactivation of PI3K/AKT/mTOR signal pathway impairs TNF-α-induced autophagy in mesenchymal stem cells from patients with ankylosing spondylitis

LIU Zhenhua, MIN Shaoxiong, LU Xiuyi, CEN Shuizhong, CHEN Zhipeng, WANG Tao, LI Jianjun, ZENG Weibo, QIU Sujun   

  1. Department of Orthopedics, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China; Department of Orthopedics, Shenzhen Hospital, Peking University, Shenzhen 518034, China; Department of Dermatology, Fourth Affiliated Hospital of Guangzhou Medical University, Guangzhou 510316, China; Department of Orthopedics, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
  • Online:2022-02-20 Published:2022-03-16

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摘要： 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、pAKT/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。

关键词: 强直性脊柱炎；间充质干细胞；肿瘤坏死因子-α；自噬

Abstract: 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.

Key words: ankylosing spondylitis; mesenchymal stem cells; tumor necrosis factor-α; autophagy