Department of Rheumatology, The Second Hospital of Shanxi Medical University, Taiyuan, Shanxi, China.
Pathology, Joint Program in Transfusion Medicine, Brigham and Women's Hospital/Children's Hospital, Harvard Medical School, Boston, MA, USA.
J Immunol Res. 2018 Feb 18;2018:3619320. doi: 10.1155/2018/3619320. eCollection 2018.
Rheumatoid arthritis (RA) multidrug resistance is associated with P-glycoprotein (P-gp) overexpression. We investigated the effects of methotrexate (MTX) alone and combined with 4-hydroperoxycyclophosphamide (4-HC) on P-gp expression in fibroblast-like synoviocytes (FLSs) from patients with RA and examined the signaling pathway involved.
RA-FLSs were treated with MTX, MTX + 4-HC, AG490 + MTX, or AG490 + MTX + 4-HC for 72 h. Proliferation inhibition rates were determined by MTT assay; P-gp expression was measured by flow cytometry and real-time polymerase chain reaction (RT-PCR); JAK2 and STAT3 were measured by RT-PCR and cell-based ELISA to assess STAT3 signaling.
MTX alone significantly induced P-gp expression and mRNA production in RA-FLSs. P-gp expression and mRNA levels were lower in the MTX + 4-HC group than in the MTX-alone group. In contrast to MTX, MTX + 4-HC reduced the STAT3 phosphorylation and downregulated JAK2 and STAT3 mRNA production. Inhibition of constitutively active STAT3 accompanied by 4-HC suppressed P-gp levels in RA-FLSs. The MTT assays revealed no significant differences in proliferation inhibition rates among groups.
The increased anti-P-gp effect of MTX + 4-HC versus MTX alone in RA-FLSs was mediated via inhibition of the JAK2/STAT3 pathway and may have helped reverse MDR in refractory RA patients with high-P-gp levels.
类风湿关节炎（RA）多药耐药与 P-糖蛋白（P-gp）过表达有关。我们研究了单独使用甲氨蝶呤（MTX）以及联合使用 4-羟环磷酰胺（4-HC）对 RA 患者成纤维样滑膜细胞（FLS）中 P-gp 表达的影响，并探讨了相关的信号通路。
RA-FLS 分别用 MTX、MTX+4-HC、AG490+MTX 和 AG490+MTX+4-HC 处理 72 小时。通过 MTT 法测定增殖抑制率；通过流式细胞术和实时聚合酶链反应（RT-PCR）测定 P-gp 表达；通过 RT-PCR 和基于细胞的 ELISA 测定 JAK2 和 STAT3，以评估 STAT3 信号。
单独的 MTX 可显著诱导 RA-FLS 中 P-gp 的表达和 mRNA 产生。MTX+4-HC 组的 P-gp 表达和 mRNA 水平均低于 MTX 单独组。与 MTX 相反，MTX+4-HC 降低了 STAT3 磷酸化，并下调了 JAK2 和 STAT3 mRNA 的产生。与 4-HC 联合使用抑制组成性激活的 STAT3 可降低 RA-FLS 中的 P-gp 水平。MTT 检测结果显示各组间增殖抑制率无显著差异。
MTX+4-HC 相较于 MTX 单独使用对 RA-FLS 中 P-gp 的抑制作用增强，这是通过抑制 JAK2/STAT3 通路介导的，可能有助于逆转高 P-gp 水平的难治性 RA 患者的 MDR。
