Lycium ruthenicum Murr. anthocyanins inhibit hyperproliferation of synovial fibroblasts from rheumatoid patients and the mechanism study powered by network pharmacology

Abstract

Background: Rheumatoid arthritis (RA), is a typical autoimmune disease affecting nearly 1% of the world's population. The dysfunctional hyperproliferation of synovial fibroblast (SF) in articular cartilage of RA patients is considered as the essential etiology. Traditional chemotherapeutic agents for RA treatment are imperfect for their high cost and unpredictable side-effects. L. ruthenicum anthocyanins (LRAC) is a natural product that of potential for therapeutic application against RA.

Methods: LRAC was characterized by UPLC-MS/MS. Bioinformatics analyses based on network pharmacology were applied to predict the potential targets of LRAC, and to select DEGs (differentially expressed genes) caused by RA pathogenesis from GSE77298. Interactions between LRAC and the predicted targets were evaluated by molecular docking. Effects of LRAC on SFs from RA patients were examined by in vitro assays, which were analyzed by flow cytometry and western blotting (WB).

Results: LRAC was able to inhibit the abnormal proliferation and aggressive invasion of SFs from RA patients. LRAC was mainly constituted by petunidin (82.7%), with small amount of delphinidin (12.9%) and malvidin (4.4%) in terms of anthocyanidin. Bioinformatics analyses showed that in 3738 RA-related DEGs, 58 of them were collectively targeted by delphinidin, malvidin and delphinidin. AR, CDK2, CHEK1, HIF1A, CXCR4, MMP2 and MMP9, the seven hub genes constructed a central network mediating the signal transduction. Molecular docking confirmed the high affinities between the LRAC ligands and the protein receptors encoded by the hub genes. The in vitro assays validated that LRAC repressed the growth of RASF by cell cycle arresting and cell invasion paralyzing (c-Myc/p21/CDK2), initiating cell apoptosis (HIF-1α/CXCR4/Bax/Bcl-2), and inducing pyroptosis via ROS-dependent pathway (NOX4/ROS/NLRP3/IL-1β/Caspase-1).

Conclusion: LRAC can selectively inhibit the proliferation of RASFs, without side-effecting immunosuppression that usually occurred for RA treatment using MTX (methotrexate). These findings demonstrate the potential application of LRAC as a phytomedicine for RA treatment, and provide a valid approach for exploring natural remedies against autoimmune diseases.

Keywords: Anthocyanins; HIF-1α; Lycium ruthenicum Murr.; Rheumatoid arthritis; Synovial fibroblast.