Rheumatoid arthritis (RA) is a common autoimmune disease characterized by immune dysfunction, chronic inflammation, synovial tissue hyperplasia, cartilage/bone damaging, and accompanied with progressive loss of joint function (Scott et al., 2010), which significantly affects the life quality of the patients and brings huge economic burden as well as the serious social problems (Scott et al., 2010). Compared with the current modern drugs (e.g. the non-steroidal anti-inflammatory agents, NSAIDs; corticosteroids; disease modifying anti-rheumatic drugs, DMARDs; and biologics, etc.) in clinical application for RA, traditional herbal medicines with the advantages of low-price, good safety, and wide cultural recognition still occupy a huge market. Investigations into the multi-biological mechanisms and quality control of herb medicines could promote the precise applications in clinic and be beneficial to such a group of people.

Sigesbeckia herba (SH) widely distributed throughout the world (Pradhan et al., 2018) is a traditional herb medicine with a long history for management of various inflammation-related diseases (especially for rheumatism) in China (Zhong et al., 2019) and some other Asian countries (Jang et al., 2018). In United States, SH was approved to be used for promoting healthy joints in dietary supplements. In 2015, a preparation with SH extract (Phynova Joint and Muscle Relief Tablets™) was licensed by Medicines and Healthcare Products Regulatory Agency (MHRA) in United Kingdom as a traditional herbal medicine for joint and muscle pain. With the increasing concerns on this herb medicine for its anti-inflammation and joint protection, it is critical to demonstrate the anti-arthritic effects and mechanisms of SH with modern research approaches.

In China, the officially authorized plant origins for SH include S. pubescens Makino (SP), Sigesbeckia orientalis L. (SO), and S. glabrescens Makino (SG). Huh et al. had reported that the therapeutic effect of SP on cartilage protection in a rabbit collagen type II (C II)-induced model of osteoarthritis (Huh et al., 2008). In terms of SG and SO, there is not any direct and scientific demonstration related to the RA. Hong et al. reported that SO attenuated λ-carrageenan–induced paw edema and LPS-induced systemic inflammation in mice (Hong et al., 2014), which indicated the potential of SO on RA and valuable to be further investigated. Our previous works have made a systemic analysis on the chemistry for SH species (Tao et al., 2018; Tao et al., 2020) and identified some potential quality control markers for SO (Linghu et al., 2020b). Growing in vitro researches showing the anti-inflammatory effects of SO on macrophages (Hong et al., 2014; Guo et al., 2018; Zhang et al., 2019; Linghu et al., 2020b), which indicates that anti-inflammation could be one of the anti-arthritic mechanisms of SO. An additional therapeutic and mechanical demonstration of SO on RA animal model and other potential target cells are essential to elucidate the anti-arthritic effects and mechanisms of SO comprehensively.

The pathophysiological mechanism of RA is very complicated. Generally, systemic immune imbalance accompanying with local inflammatory infiltration to synovium and joints go through the whole process of RA (Smolen et al., 2016). Various cells such as macrophages, lymphocytes, and fibroblasts have been identified to be participated in the development and deterioration of RA (Branimir Anić, 2014). To be an important part in synovial tissue, the synovial fibroblasts (also known as fibroblast-like synovial cells, FLS) has been demonstrated to play a key role in the destruction of joints by secreting various cytokines, proteases, and arachidonic acid metabolites (Huber et al., 2006). The activated FLS promotes the clinical symptoms and processes of RA (Ospelt et al., 2004). The excessive proliferation and invasion of FLS has been reported to be closely related to the pathogenesis of RA, effective inhibiting of cytokines and proteases through blocking or attenuating the activation of FLS might be another strategy for clinical therapeutics of RA (Xu et al., 2017).

In the present study, we investigated the therapeutic effects of SO against C II-induced RA in rats, and further revealed the anti-arthritic mechanisms of SO on IL-1β–induced human fibroblast-like synovial cells (SW982 and MH7A). Our results suggested that SO effectively alleviated the C II-induced RA through inhibiting of multi-target–mediated the synovial hyperplasia and inflammation.

Rheumatoid arthritis (RA) is a chronic disease that eventually leads to joint damage and bone erosion. The severe disabling risk of RA has brought a huge economic burden to society. With the innovation of drug development technology, RA treatments from the non-steroidal anti-inflammatory agents (NSAIDs) to biologics have obtained some achievements on improving the life quality of patients. However, due to the toxicities to liver and kidney of chemical drugs and the high price of biologics, a large number of people would prefer to choose the traditional medicines as their long-term drug treatment approaches. In such a circumstance, it is essential to conduct a systemic and scientific evaluation including the quality control, pharmacology, and pharmacokinetics on the traditional herb medicine, which could help such a group of patients to rationally use the traditional herb medicine.

Sigesbeckia orientalis L. (SO) is a plant source of Sigesbeckiae Herba (SH) which has been traditionally used for rheumatism in China since Tang dynasty. Because of the long history of use for this medicine, Chinese people would conventionally use the herb medicine to treat RA as a single medicine or a formula. In the past years, we have made a systemic identification on the active components and a preliminary evaluation on the pharmacokinetics. An additional therapeutic and mechanical demonstration of SH on RA animal model is beneficial to contribute the rational use of this herb medicine. Hong et al. reported that SO attenuated λ-carrageenan–induced paw edema and LPS-induced systemic inflammation in mice (Hong et al., 2014), which indicated the potential of SO on RA. At present study, we investigated the anti-arthritic effects of SO extract (SOE) on collagen type II (C II)-induced rheumatoid arthritis (CIA). As shown in Figure 1A, the body weight of rats between the control and SOE was no difference, which indicated the SOE was non-toxicity to rats. Moreover, SOE attenuated the RA symptom with the reduced clinical scores (Figure 1B).

Generally, continuous dysfunction of immune system and local inflammatory infiltration to the joints would be the main causes of RA. Production of abnormal autoantibodies usually leads to the activation of T/B lymphocytes and the interaction with macrophages to produce multiple inflammatory cytokines. These cytokines further act on the synovial membrane and cartilage, thus initiating excessive proliferation and invasion of synovial fibroblasts to the articular cavity and producing a lot of proteases and arachidonic acid metabolites, which finally leads to the joint damage and bone erosion. CD4⁺CD25⁺FOXP3⁺ T lymphocytes are the critical regulatory T cells (Tregs) to maintain the function of immune system and be reported to involve in the RA development process. Lu et al. reported that stimulation of Kirenol (one of the active compounds of SOE) on the CD4⁺CD25⁺Foxp3⁺ Tregs altered potent antiarthritic effect against CIA. Therefore, we detected the effects of SOE on the Tregs, our results show that the proportion of CD4⁺CD25⁺FOXP3⁺ T lymphocytes in the spleen of CIA rats was much lower than those of vehicle rats, which could be upregulated by treating with SOE (Figures 3A, B). C-reactive protein (CRP) is a ring-shaped pentameric protein existed in plasma with response to inflammation (Mortensen, 2001). The concentration of CRP has been reported to be dramatically increased in the peripheral blood in RA patients (Kowalski et al., 2008). As shown in Figure 2A, SOE effectively reduced the increase of CRP level in CIA rats, which might be correlated with the rebalanced immune system (Figures 3A, B). Moreover, SOE ameliorated the swelling of hind paw induced by C II (Figure 4A), which were consistent with the decreased expression of IL-6 and IL-1β and increased IL-10 in the joint muscles (Figures 2B–D). In addition, after treatment with SOE, the proliferation of synovial cells was alleviated, the number of infiltrated inflammatory cells and proteases were significantly decreased (Figures 4C, D), and the damages of bone or cartilage were also obviously improved (Figure 4B).

The significant inhibitory effects of SOE on synovial hyperplasia and inflammation in rats inspired us to further investigate the biological mechanisms of SOE against RA using the human fibroblast-like synoviocytes (FLS). The most commonly used cell lines on studying synovitis in RA is the human synovial sarcoma cell line (SW982) and a human rheumatoid arthritis synovial cell line (MH7A), which have been successfully used to investigate the effects of drugs and phytochemicals (Yamazaki et al., 2003; Chang et al., 2014; Khansai et al., 2017).

In consistent with the in vivo results, SOE reduced the expression of pro-inflammatory cytokines and chemokines on SW982 and MH7A synovial cells in the presence or absence of IL-1β induction (Figures 5A–H), the SOE also alleviated the IL-1β–induced proliferation, migration, and invasion in the human SW982 synovial cell line (Figures 6B–I). Besides, SOE did not affect the expression of COX-1 but reduced the expression of COX-2 and NLRP3 (Figure 7A) which were pivotal regulators of inflammation in RA. t COX-2 activity is known to be upregulated in the RA synovium (Woods et al., 2003). The selective COX-2 inhibitors such as Rofecoxib and Celecoxib have emerged as an important option in the treatment of RA (Sundy, 2001). Zhang et al. reported that extent of synovial NLRP3 expression was correlated with the clinical severity of arthritis and radiological scores (Zhang et al., 2016). SOE also reduced the expression of MMP9 while increased the expression of MMP2 in IL-1β–induced SW982 cells (Figure 7A), Xue et al. reported that endogenous MMP-2 or MMP-9 contribute to RA synovial fibroblast survival, proliferation, migration, and invasion, with MMP-9 having more potent effects. More importantly, MMP-9 stimulates RA synovial fibroblast-mediated inflammation and degradation of cartilage, whereas MMP-2 inhibits these parameters (Xue et al., 2014). Unlike MMP-2 KO mice, MMP-9 KO mice showed reduced levels of Antibody-Induced Arthritis, indicating that MMP-9 enhances arthritis in this model (Itoh et al., 2002). Our results revealed that SOE rebalanced the expression of MMP-2 and MMP-9 induced by IL-1β in synovial SW982 cell, this might be one of the reason that SOE inhibited the synovial inflammation and degradation of cartilage in the joint of CIA rat.

According to our previous results from computational predictions, RA was frequently involved in multiple signal pathways (Wang et al., 2019). The interactions of these pathways were associated with pathological mechanisms of RA. Based on the current reports and references, modulation on several signal pathways have been reported to be successful approaches to treat RA. Among them, the transcription factor NF-κB has been well recognized as a potential therapeutic target (Roman-Blas and Jimenez, 2006), and a pivotal regulator of inflammation, hyperplasia, and tissue destruction in rheumatoid arthritis (Makarov, 2001). Several previous studies have revealed that AP-1 (c-Fos and c-Jun) are highly expressed in the RA synovium (Kinne et al., 1995; Dooley et al., 1996). AP-1 binding sites have been identified in the promoter region of all matrix metalloproteinases (MMPs) (Benbow and Brinckerhoff, 1997), which indicates AP-1 might play a pivotal role in the transcriptional activation of MMPs. Selective inhibition of c-Fos/AP-1 resolves arthritis in a preclinical model of the disease by inhibiting the production of arthritis upstream of inflammatory cytokine and MMPs (Aikawa et al., 2008). Upstream of NF-κB and AP-1, mitogen-activated protein kinases (MAPKs) are highly active in chronic synovitis and also involved in the regulation of MMP expression (Schett et al., 2000). Three MAPK families including p38, extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) seem to be involved in the activation of synovial cells in RA (Meyer and Pap, 2005). MAPKs have been implicated in transducing inflammation and joint destruction and therefore are key molecular targets for therapeutic intervention of RA (Thalhamer et al., 2008). In previous reports, SOE have been reported to inhibit the activation of NF-κB and the phosphorylation of MAPKs on LPS-induced RAW264.7 macrophages to attenuate inflammation (Hong et al., 2014; Linghu et al., 2020b). Leocarpinolide B, an active compound from SH, significantly inhibited the NF-κB activation therefore ameliorating the inflammation and stress oxidant (Linghu et al., 2020a). Kirenol, one of the main compounds of SH, has been shown to inhibit the phosphorylation of NF-κB in cytokines-treated primary RA-synovial fibroblasts (Wu et al., 2019). Therefore, we investigated the effects of SOE on these three most important signal pathways. As shown in Figures 8B–D. our results showed that SOE blocked the activation of NF-κB by reducing the degradation of IκBα, the phosphorylation, and the translocation of NF-κB p65 in IL-1β–induced human synovial SW982 cells. Also, SOE decreased the expression of AP-1 (Figure 8A) and the phosphorylation of MAPKs (Figure 7B).

In summary, SO is traditional herb medicine with a long history of application. The culture recognition, low price, and safety of this herbal medicine still attracted a lot of patients and widely used in modern society. Based on the previous study on the quality control and pharmacokinetics, this present research evaluated the anti-arthritic effects and mechanisms of SOE on treating RA. Results from the in vivo and in vitro studies suggested that therapeutic effects of SOE on RA. The mechanisms of action would mainly be the rebalancing of immune system, inhibition of synovial hyperplasia and inflammation through targeting MAPKs/NF-κB/AP-1 signal pathways, suggesting the multi-targets of SO on RA treatment. Our work suggested that SOE would be an effective therapeutic drug on RA, but the doctors and patients should pay more attention on the additive effects on targets when taking a combination use of western drugs (i.e. NSAIDs, selective COX-2 inhibitors) and this herb medicine.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the Animal Research Ethics Committee of the University of Macau, Macau Special Administrative Region, China.

HY and K-GL conceived and designed the study. K-GL, SX, GZ, TZ, WXiong, and MZ conducted the experiments. X-CS, WXu, ZB, and YW provided the technical support and advices for the study. K-GL wrote the manuscript, and HY revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by grants from the National Natural Science Foundation of China [NSFC, No. 81470170], the Science and Technology Development Fund of Macau SAR [File No. 0096/2019/A2], the Research Committee of the University of Macau [MYRG2017-00178-ICMS and MYRG2018-00043-ICMS], and the international Cooperation Department of National Administration of traditional Chinese Medicine [GZYYGJ2019042].

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.