Acteoside inhibits inflammatory response via JAK/STAT signaling pathway in osteoarthritic rats

Qiao et al. BMC Complementary and Alternative Medicine https://doi.org/10.1186/s12906-019-2673-7 (2019) 19:264 RESEARCH ARTICLE Open Access Acteoside inhibits inflammatory response via JAK/STAT signaling pathway in osteoarthritic rats Zhiguang Qiao†, Jiaxin Tang†, Wen Wu, Jian Tang and Ming Liu* Abstract Background: Osteoarthritis (OA) is a common degenerative disease of synovial joints caused by inflammation. Acteoside (ACT), a major component and lipase inhibitor from the Chinese tea Ligustrum purpurascens kudingcha, has been reported to regulate the inflammation and immune response. The study aims to investigate the effects of ACT on inflammatory responses and joint protection in OA rats. Methods: Cell proliferation was examined by MTT and colony formation assay. Apoptosis was analyzed using flow cytometry with Annexin V/PI staining. ELISA was employed to examine the concentration of inflammatory cytokines. OA rat model was established by surgery stimulation. Results: ACT treatment significantly inhibited the upregulation of inflammatory cytokines induced by IL-1β in primary chondrocytes, including IL-6, IL-12, TNF-α and IFN-γ. ACT stimulation also enhanced the cell proliferation, while inhibited cell apoptosis in IL-1β-treated chondrocytes. Consistently, ACT treatment led to downregulation of cleaved-caspase-3 and apoptosis regulator Bax, and upregulation of Bcl-2. Furthermore, ACT treatment inhibited IL1β-induced activation of JAK/STAT pathway. The results were confirmed in surgery-induced OA rat model. Moreover, ACT treatment significantly inhibited synovial inflammation and articular chondrocyte apoptosis in OA rats. Conclusion: Our findings indicate that ACT has the potential therapeutic effect on OA through inhibiting the inflammatory responses via inactivating JAK/STAT signaling pathway. Keywords: Acteoside, Apoptosis, Inflammation, Osteoarthritis, JAK/STAT Background Osteoarthritis (OA) is a common chronic arthritis that might lead to disability worldwide, especially for the older people in the developing country [1]. The clinical pathological characters of OA include progressive loss of articular cartilage, integrity destruction, increased joint friction, bone hyperplasia and persistent pain [2, 3]. Though much progress has been achieved, the pathogenesis and mechanism of OA, remains illusive and the definitive cure is still not available [4]. OA could be characterized as an inflammatory disease as various inflammatory cytokines are involved in OA [5, 6]. * Correspondence: † Zhiguang Qiao and Jiaxin Tang contributed equally to this work. Department of Orthopedic, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiaotong University, No.639 Zhizao Ju Road, Shanghai City 200011, People’s Republic of China Enhanced expression of IL-1β was observed in OA patients’ cartilage, synovial fluid and membrane [7]. Chondrocytes stimulated by IL-1β had elevated a variety of inflammatory cytokines such as IL-6, IL-8, and TNF-α [8]. Multiple reports suggested that inhibition of inflammatory response by targeting IL-1β could be a first-line therapeutic treatment for OA patients [9, 10]. Numerous studies suggest that various signaling pathways participate in the pathogenesis of OA, including TGF-β pathways, NF-κB pathways and AMPK/SIRT-1/PGC-1α pathways [11–14]. Lim et.al reported that p38 MAPK/c-Fos/AP-1 signaling cascade and JAK/STAT pathways had also been activated in IL-1β stimulated chondrocytes [15]. Traditional herbal medicines have been commonly used to treat OA [16, 17]. Xue et al. reported that herbal formula Xianlinggubao could improve the pain and © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Qiao et al. BMC Complementary and Alternative Medicine (2019) 19:264 knee/hand OA [18]. Morin, which was isolated from Moraceae family, showed anti-inflammatory function on IL-1β stimulated chondrocytes [19]. Acteoside (ACT), a major component and lipase inhibitor from the Chinese tea Ligustrum purpurascens kudingcha, has been reported to regulate the inflammation and immune response [20, 21]. In dextran sulphate sodiuminduced colitis model, Hausmann et al. demonstrated that ACT treatment ameliorated intestinal inflammation [22]. ACT also showed anti-inflammatory effects via blocking TLR4 dimerization in mouse model of xylene-induced ear oedema, LPS-induced endotoxic shock and LPS-induced acute kidney injury [21, 23]. However, whether ACT exhibits therapeutic function on OA and the anti-inflammatory mechanism in OA remains unclear. Here, we found that ACT inhibited the upregulation of inflammatory cytokines (such as IL-6, IL-12, TNF-α and IFN-γ) induced by IL-1β in primary chondrocytes. In addition, ACT enhanced the cell proliferation, while inhibited cell apoptosis in IL-1β-treated chondrocytes. Mechanistically, ACT treatment inhibited the activation of JAK/STAT signaling induced by IL-1β stimulation. Thus, our data indicates that ACT might be used to as an allopathic molecule to treat the OA. Methods Chondrocyte isolation, culture and treatment ACT (purity ≥98%), and dimethylsulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). ACT was dissolved in DMSO as a 100 mg/ml stock solution and stored at 4 °C. Further dilution was done in cell culture medium. Sprague-Dawley rats (male, 1–2 weeks old) were purchased from Shanghai SLAC Animal Co. (Shanghai, China). Articular cartilage was isolated and cut into small pieces, followed by digestion with 0.2% Collagenase II at 37 °C for 6 h. Chondrocytes was pelleted by centrifuge after digestion. Chondrocytes were maintained in DMEM/ F-12 medium (Gibco, Carlsbad, CA, USA) supplemented with 20% FBS plus 1% antibiotic mixture of Penicillin and Streptomycin) in a 5% CO2 incubator at 37 °C. Cells were seeded in a 6-well plate (2 × 105 cells/mL) and cultured for 24 h, and then stimulated with 10 ng/ml IL-1β (Peprotech, USA) to establish cellular OA, then different concentrations of ACT (0, 10, 50, 100 μM) or aceclofenac (positive control, ACE 30 μM) were added to the medium and further incubated for another 24 h. Immunocytochemistry staining Primary chondrocytes cells were seeded in a 6-well plate (2 × 105 cells/mL) covered with coverslips. The coverslips were removed after cell adhesion. The cultured cells were rinsed using PBS followed by toluidine blue Page 2 of 8 staining. Briefly, cells were fixed with formaldehyde for 2 h and t (...truncated)