Triptolide Inhibits Expression of Inflammatory Cytokines and Proliferation of Fibroblast-like Synoviocytes Induced by IL-6/sIL-6R-Mediated JAK2/STAT3 Signaling Pathway

Abstract

Triptolide, a component of the Chinese herb Tripterygium wilfordii Hook F, has been proved to be effective in the treatment of rheumatoid arthritis (RA). However, its underlying mechanisms on RA have not yet been well established. We observed the inhibitory effect of triptolide on the expression of inflammatory cytokines and proliferation of fibroblast-like synoviocytes (FLS) induced by the complex of interleukin-6 (IL-6) and the soluble form of the IL-6 receptor (sIL-6R). Furthermore, to clarify the underlying mechanisms, we treated FLS with the Janus-activated kinase 2 (JAK2) inhibitor/signal transducer and activator of transcription 3 (STAT3) activation blocker AZD1480. In this study, immunohistochemical staining was used to identify vimentin (+) and CD68 (−) in FLS. The FLS proliferation was measured by cell proliferation assay, and the cell cycles were analyzed by flow cytometry. Furthermore, ELISA was used to detect the expression of the inflammatory factors in culture solution. The expression levels of p-JAK2, JAK2, p-STAT3 and STAT3 were investigated through Western blotting analysis. The results showed that IL-6/sIL-6R significantly increased the cell proliferation and expression of inflammatory cytokines, including IL-6, interleukin-1β (IL-1β) and vascular endothelial growth factor (VEGF). Triptolide or AZD1480 inhibited the cell proliferation and inflammatory cytokine expression in IL-6/sIL-6R-stimulated FLS by suppressing JAK2/STAT3. The study suggested that the physiological effects of triptolide on RA were due to its contribution to the inhibition of the inflammatory cytokine expression and FLS proliferation by suppressing the JAK2/STAT3 signaling pathway. It may provide an innovative insight into the effect of triptolide in preventing RA pathogenesis.

Keywords

triptolide / inflammatory cytokines / proliferation / fibroblast-like synoviocytes / JAK2/STAT3

References

[1] NiuHQ, ZhaoWP, ZhaoXC, et al.. Combination of 4-hydroperoxy cyclophosphamide and methotrexate inhibits IL-6/sIL-6R-induced RANKL expression in fibroblast-like synoviocytes via suppression of the JAK2/STAT3 and p38MAPK signaling pathway. Int Immunopharmacol, 2018, 61: 45-53

[2] WuJ, QuY, ZhangYP, et al.. RHAMM induces progression of rheumatoid arthritis by enhancing the functions of fibroblast-like synoviocytes. BMC Musculoskelet Disord, 2018, 19(1): 455

[3] BartokB, FiresteinGS. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev, 2010, 233(1): 233-255

[4] ChangSK, GuZ, BrennerMB. Fibroblast-like synoviocytes in inflammatory arthritis pathology: the emerging role of cadherin-11. Immunol Rev, 2010, 233(1): 256-266

[5] MgaB, CejW, WiegertjesR, et al.. A three-dimensional model to study human synovial pathology. ALTEX, 2019, 36(1): 18-28

[6] SchellerJ, KovalevaM, RabeB, et al.. Development of a monoclonal antibody-based enzyme-linked immunoabsorbent assay for the binding of gp130 to the IL-6/IL-6R complex and its competitive inhibition. J Immunol Methods, 2004, 291(1–2): 93-100

[7] SchellerJ, OhnesorgeN, Rose-JohnS. Interleukin-6 trans-signalling in chronic inflammation and cancer. Scand J Immunol, 2006, 63(5): 321-329

[8] MigitaK, KogaT, KomoriA, et al.. Influence of Janus kinase inhibition on interleukin 6-mediated induction of acute-phase serum amyloid A in rheumatoid synovium. J Rheumatol, 2011, 38(11): 2309-2317

[9] YangY, YeY, QiuQ, et al.. Triptolide inhibits the migration and invasion of rheumatoid fibroblast-like synoviocytes by blocking the activation of the JNK MAPK pathway. Int Immunopharmacol, 2016, 41: 8-16

[10] TaoX, FanF, HoffmannV, et al.. Therapeutic impact of the ethyl acetate extract of Tripterygium wilfordii Hook F on nephritis in NZB/W F1 mice. Arthritis Res Ther, 2006, 8(1): R24

[11] ZhangL, ChangJ, ZhaoY, et al.. Fabrication of a triptolide-loaded and poly-γ-glutamic acid-based amphiphilic nanoparticle for the treatment of rheumatoid arthritis. Int J Nanomedicine, 2018, 13: 2051-2064

[12] QianK, ZhangL, ShiK. Triptolide prevents osteoarthritis via inhibiting hsa-miR-20b. Inflammopharmacology, 2019, 27(1): 109-119

[13] RitterJM. Drugs and the skin: psoriasis. Br J Clin Pharmacol, 2012, 74(3): 393-395

[14] HuberLC, DistlerO, TarnerI, et al.. Synovial fibroblasts: key players in rheumatoid arthritis. Rheumatology (Oxford), 2006, 45(6): 669-675

[15] WenHL, YangG, DongQR. Ellipticine inhibits the proliferation and induces apoptosis in rheumatoid arthritis fibroblast-like synoviocytes via the STAT3 pathway. Immunopharmacol Immunotoxicol, 2017, 39(4): 219-224

[16] KusunokiN, YamazakiR, KitasatoH, et al.. Triptolide, an active compound identified in a traditional Chinese herb, induces apoptosis of rheumatoid synovial fibroblasts. BMC Pharmacol, 2004, 4: 2

[17] VernalR, VelásquezE, GamonalJ, et al.. Expression of proinflammatory cytokines in osteoarthritis of the temporomandibular joint. Arch Oral Biol, 2008, 53(10): 910-915

[18] YouS, YooSA, ChoiS, et al.. Identification of key regulators for the migration and invasion of rheumatoid synoviocytes through a systems approach. Proc Natl Acad Sci USA, 2014, 111(1): 550-555

[19] Li Y, Xu JZ, Gu CX, et al. Carvacrol suppresses inflammatory responses in rheumatoid arthritis fibroblast-like synoviocytes. J Cell Biochem, 2018

[20] BiggioggeroM, CrottiC, BeccioliniA, et al.. Tocilizumab in the treatment of rheumatoid arthritis: an evidence-based review and patient selection. Drug Des Devel Ther, 2018, 13: 57-70

[21] KayJ, CalabreseL. The role of interleukin-1 in the pathogenesis of rheumatoid arthritis. Rheumatology (Oxford), 2004, 43(Suppl3): iii2-2iii9

[22] KondoY, SuzukiK, InoueY, et al.. Significant association between joint ultrasonographic parameters and synovial inflammatory factors in rheumatoid arthritis. Arthritis Res Ther, 2019, 21(1): 14

[23] GaoW, SweeneyC, WalshC, et al.. Notch signalling pathways mediate synovial angiogenesis in response to vascular endothelial growth factor and angiopoietin 2. Ann Rheum Dis, 2013, 72(6): 1080-1088

[24] ChoeJY, ParkKY, ParkSH, et al.. Regulatory effect of calcineurin inhibitor, tacrolimus, on IL-6/sIL-6R-mediated RANKL expression through JAK2-STAT3-SOCS3 signaling pathway in fibroblast-like synoviocytes. Arthritis Res Ther, 2013, 15(1): R26

[25] KimSK, ParkKY, YoonWC, et al.. Melittin enhances apoptosis through suppression of IL-6/sIL-6R complex-induced NF-κB and STAT3 activation and Bcl-2 expression for human fibroblast-like synoviocytes in rheumatoid arthritis. Joint Bone Spine, 2011, 78(5): 471-477