RAI | 雷帕霉素抑制类风湿关节炎滑膜细胞增殖并诱导凋亡的新机制

山西医科大学第二医院李小峰教授团队在Rheumatology & Autoimmunity 在线发表最新研究，深入探讨了雷帕霉素对类风湿关节炎（RA）患者滑膜细胞（FLSs）的增殖抑制和凋亡诱导作用，为RA的临床治疗提供了新的分子机制和治疗策略。

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研 究 背 景 

类风湿关节炎（RA）是一种慢性炎症性自身免疫性疾病，其特征是关节滑膜的过度增生和炎症，导致关节损伤和功能障碍。RA的病理过程中，滑膜细胞（FLSs）的异常增殖和抗凋亡特性起着关键作用。李小峰教授团队采用细胞计数试剂盒-8检测雷帕霉素对RA-FLSs增殖的影响，并通过实时聚合酶链反应和蛋白质印迹分析，探讨了雷帕霉素通过AKT/mTORC1信号通路调控RA-FLSs的分子机制。

研 究 结 果  

研究发现，雷帕霉素能够显著抑制RA-FLSs的增殖，并以剂量依赖性方式诱导细胞凋亡。在分子水平上，雷帕霉素显著上调了促凋亡蛋白Bax和caspase家族成员的表达，同时降低了抗凋亡蛋白Bcl-2的表达。此外，雷帕霉素还抑制了AKT/mTORC1信号通路的关键分子，包括p-AKT、p-mTOR、p-S6K和p-4EBP1的磷酸化水平，从而抑制了RA-FLSs的增殖并促进了细胞凋亡。

Figure 1 Identification of RA-FLSs. Flow cytometry to check the expression of cell surface markers CD68, CD90, FAP-α on RA-FLSs. (A) Blank. (B) RA-FLSs. APC, allophycocyanin; FAP, fibroblast activation protein; PE, phycoerythrin; RA-FLS, rheumatoid arthritis fibroblast-like synoviocytes.

Figure 2 Cell inhibition rate of rheumatoid arthritis fibroblast-like synoviocytes after intervention with different concentrations of rapamycin. (A) Cell inhibition rate after 24 h intervention. (B) Cell inhibition rate after 48 h intervention. **p < 0.01, rapamycin group (1, 5, 10, 15, 20, 25 nmol/L) versus the control group.

Figure 3 Cell apoptosis determined by flow cytometry. (A) Flow rate of apoptosis in control group. (B) Flow rate of apoptosis in rapamycin group (10 nmol/L). (C) Histogram of apoptosis rate of RA-FLSs. ***p < 0.001. ADC, apparent diffusion coefficient; FITC, fluorescein isothiocyanate; FL1, plotting green fluorescence; FL4, red fluorescence intensity; PI, propidium iodide; RA-FLS, rheumatoid arthritis fibroblast-like synoviocytes.

Figure 4 RA-FLSs were treated with rapamycin (10 nmol/L) for 48 h. (A) Relative expression of Bax and Bcl-2 mRNA. (B) Western blotting analysis was performed to detect the protein levels of Bax and Bcl-2. (C) Rapamycin increases Bax, caspase 3, and caspase 9 expression levels and decreases Bcl-2 expression level in RA-FLSs. *p < 0.05; **p < 0.01. Bax, Bcl-2-associated X protein; Bcl-2, B cell lymphoma 2; RA--FLS, rheumatoid arthritis-fibroblast-like synoviocytes.

Figure 5 RA-FLSs were treated with rapamycin (10 nmol/L) for 48 h. (A) Expression level of CD1, CDK2, and β-actin in RA-FLSs by Western blotting analysis. (B) Quantitative analysis of CDK2, CD1 expression in RA-FLSs by Western blotting analysis. *p < 0.05, **p < 0.01. CDK2, cyclin-dependent kinase 2; RA-FLS, rheumatoid arthritis fibroblast-like synoviocytes.

Figure 6 RA-FLSs were treated with rapamycin (10nmol/L) for 48 h. (A) Expression level of AKT and mTOR mRNA. (B) Relative expression of p-AKT/AKT, p-mTOR/mTOR, p-S6K/S6K, p-4EBP1/4EBP1; (C) Quantitative analysis of AKT/mTORC1 pathway expression in RA-FLSs by Western blotting analysis, **p < 0.01, *p < 0.05. 4EBP1, 4E-binding protein 1; AKT, protein kinase B; Bax, Bcl-2-associated X protein; Bcl-2, B cell lymphoma 2; mTOR, mammalian target of rapamycin; RA-FLSs, rheumatoid arthritis-fibroblast-like synoviocytes; S6K, S6 kinase.

结 论 

该研究系统性地揭示了雷帕霉素通过靶向AKT/mTORC1信号通路，对RA-FLSs的增殖和凋亡具有显著的调控作用。这一发现为RA的临床治疗提供了新的分子靶点和治疗策略。李小峰教授指出，雷帕霉素作为一种新型的免疫调节剂，不仅能通过调控Treg细胞的增殖和稳态诱导免疫平衡，亦可以直接作用于RA的滑膜细胞发挥治疗作用，其在RA等诸多自身免疫性疾病的治疗中的潜力值得关注。