Institute of Clinical Pharmacology, Anhui Medical University, Key Laboratory of Anti-inflammatory and Immune Medicine, Ministry of Education, Anhui Collaborative Innovation Center of Anti-inflammatory and Immune Medicine, Meishan Road 81, Hefei, 230032, China.
Institute of Clinical Pharmacology, Anhui Medical University, Key Laboratory of Anti-inflammatory and Immune Medicine, Ministry of Education, Anhui Collaborative Innovation Center of Anti-inflammatory and Immune Medicine, Meishan Road 81, Hefei, 230032, China.
Biochem Biophys Res Commun. 2020 Jun 4;526(3):805-812. doi: 10.1016/j.bbrc.2020.03.164. Epub 2020 Apr 5.
This study aims to explore the effect of paeoniflorin-6'-O-benzene sulfonate (CP-25) on the migration of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) and the mechanism focused on CXCR4-Gβγ-PI3K/AKT signaling.
Human synovial tissues were collected from RA and osteoarthritis (OA) patients. Immunohistochemistry (IHC) and Western blot were used to detect the protein expression of CXCR4, GRK2, Gβγ, PI3K, p-PI3K, AKT and p-AKT. Transwell was adopted to analyse the migration of fibroblast-like synoviocytes (FLS). Co-immunoprecipitation (Co-IP) and laser scanning confocal microscopy (LSCM) were used to detect the combination of GRK2 and Gβγ, the combination of PI3K and Gβγ.
The expression level of CXCR4, GRK2, Gβγ, p-p85 and p-AKT were increased in RA synovial tissue according to the results of IHC and Western blot. In vitro, the migration of FLS was increased after stimulation of CXCL12, inhibition of Gβγ suppressed the migration and phosphorylation of p85 and AKT induced by CXCL12 in FLS, and CP-25 had the same effect as inhibition of Gβγ. The membrane expression of GRK2, Gβγ, PI3K and the combination of GRK2 and Gβγ, the combination of PI3K and Gβγ in FLS were increased after the stimulation of CXCL12, and CP-25 had an ability in reducing the membrane expression and the combination of these proteins.
Excessive migration of FLS in RA was associated with over-activation of PI3K/AKT signaling, and the activity of Gβγ was involved in the over-activation of PI3K/AKT. CP-25 down-regulated CXCR4-Gβγ-PI3K/AKT signals by inhibiting GRK2-Gβγ complex membrane translocation.
本研究旨在探讨苯磺酸丹皮酚（CP-25）对类风湿关节炎（RA）成纤维样滑膜细胞（FLS）迁移的影响，并重点探讨 CXCR4-Gβγ-PI3K/AKT 信号通路。
收集 RA 和骨关节炎（OA）患者的滑膜组织，采用免疫组织化学（IHC）和 Western blot 检测 CXCR4、GRK2、Gβγ、PI3K、p-PI3K、AKT 和 p-AKT 的蛋白表达。采用 Transwell 分析成纤维样滑膜细胞（FLS）的迁移。采用免疫共沉淀（Co-IP）和激光共聚焦显微镜（LSCM）检测 GRK2 和 Gβγ 的结合、PI3K 和 Gβγ 的结合。
根据 IHC 和 Western blot 的结果，RA 滑膜组织中 CXCR4、GRK2、Gβγ、p-p85 和 p-AKT 的表达水平升高。体外实验中，CXCL12 刺激后 FLS 迁移增加，Gβγ 抑制后抑制 CXCL12 诱导的 FLS 迁移和 p85 和 AKT 的磷酸化，CP-25 具有与 Gβγ 抑制相同的作用。CXCL12 刺激后，FLS 中 GRK2、Gβγ、PI3K 的膜表达以及 GRK2 和 Gβγ 的结合、PI3K 和 Gβγ 的结合增加，CP-25 具有降低这些蛋白膜表达和结合的能力。
RA 中 FLS 的过度迁移与 PI3K/AKT 信号过度激活有关，Gβγ 的活性参与了 PI3K/AKT 的过度激活。CP-25 通过抑制 GRK2-Gβγ 复合物的膜转位，下调 CXCR4-Gβγ-PI3K/AKT 信号。
