Myrtenal and β-caryophyllene oxide screened from Liquidambaris Fructus suppress NLRP3 inflammasome components in rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the synovium. Untreated RA can cause progressive joint destruction, leading to disability, poor quality of life, and increased mortality. Approximately 1% of the world’s population is affected by RA. Onset of disease usually occurs between the ages of 30 and 50, with a higher incidence in women [1‐4]. Fibroblast-like synoviocytes (FLS) and inflammatory cells, such as macrophages and T cells, produce proinflammatory cytokines, such as IL-1β and TNF-α, which play key roles in the pathogenesis of RA [5].

In the innate immune system, sentinel immune cells sense the invasion of pathogens and initiate the body’s inflammatory defense response, thereby playing a role in resisting pathogen infection. Nucleotide-binding, oligomerization domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) gene polymorphisms have been reported to be associated with susceptibility, disease activity and anti-tumour necrosis factor (TNF) treatment response in RA. RA is a heterogeneous disease entity with a wide range of immunopathological mechanisms and various manifestations, including articular and extra-articular manifestations. In this regard, NLRP3 inflammasome activation is one of the most notable pathological mechanisms that contribute to the pathogenesis of RA [6]. It was reported that the NLRP3 inflammasome is activated in the synovium of RA mice, and treatment with a selective NLRP3 inhibitor can reduce the symptoms of arthritis and cartilage destruction, indicating that targeting the NLRP3 inflammasome with small molecule chemical inhibitors may be a potential therapeutic strategy for the treatment of RA [7‐9]. In 2002, the concept of the inflammasome was first proposed by the Tschopp experimental team [10]. Inflammasomes are large multimeric protein complexes, mainly present in innate immune cells that attack pathogens. The three main components of the inflammasome include sensor Nod-like receptors (NLRs), an adaptor protein called ASC and an effector caspase which can hydrolyze and activate pro-IL-1β and pro-IL-18. NLRs are a family of pattern recognition receptors in the cytoplasm that recognize pathogen-related molecular patterns (PAMPs) or damage-related molecular patterns (DAMPs). These patterns are indicative of invading pathogens, prompting NLRs to trigger an innate immune response. Before the inflammasome is activated, the PAMP/DAMP signal triggers the innate immune cells to transcriptionally upregulate the expression of IL-1β and inflammasome sensors. When the triggered cells are stimulated by additional PAMPs/DAMPs, the inflammasome complex assembles and initiates a proteolytic cascade, leading to the hydrolysis and release of IL-1β and IL-18. This usually leads to an inflammatory form of programmed cell death called pyroptosis [10‐16].

Liquidambaris Fructus (LF), the infructescence of Liquidambar formosana, has been used to treat joint pain and some gynecological diseases in Traditional Chinese Medicine [17]; however, a lack of pharmacological evidence limits the applications of LF in modern clinics. It has been reported that LF shows significant anti-inflammatory effects on xylene-induced ear swelling in mice as well as carrageenan-induced paw edema in rats [18]. LF treatment may decrease the level of inflammatory cytokines in serum, including TNF-α, IL-1β and IL-6, while also increasing the levels of the anti-inflammatory cytokine IL-10. This anti-inflammatory effect is linked to the suppression of COX-2, iNOS and NF-κB p65 [18], indicating LF is a potential crude drug in the treatment of some inflammatory diseases. As joint pain is a common symptom of RA, and it is also the major indication for LF. This study aims to explore the protective effect of LF on RA and to identify its active ingredients while also elucidating the underlying molecular mechanism of action of LF.

In Traditional Chinese Medicine, LF has been used to treat joint pain and some gynecological diseases [17]. As joint pain is a common symptom of RA, in this study we explored the anti-RA potential of LF using an AIA rat model. The results showed that LF could alleviate paw swelling, bone destruction, synovial inflammation and cartilage erosion in AIA rats, and the efficacy of PEL was superior to that of EEL and WEL. Triterpenoids are generally considered the main components of LF; however, according to our previous study, triterpenoids exist in EEL and not PEL [39]. The components of PEL are monoterpenoids and sesquiterpenoids (Fig. 5A). Our lab is currently investigating the bioactive chemical ingredients of LF, and in a previous study we investigated the anti-breast cancer effects and mechanisms of action of the triterpenoids in LF [40]. This study supports the traditional utility of LF in the treatment of gynecological diseases. However, this study showed that monoterpenoids and sesquiterpenoids play a more important role than triterpenoids in the anti-RA effect of LF. According to some Traditional Chinese Medicine literature, such as Bencao Gangmu Shiyi (1765 AD), LF was burned and the resulting smoke inhaled to treat joint pain, indicating that the active ingredients are volatile components [41].

As one of the most important multimeric protein complexes, NLRP3 inflammasome activation leads to increased cleavage of caspase-1 and downstream IL-1β production, and plays a pathological role in many diseases including diabetes, atherosclerosis and cardiovascular diseases [42‐44]. Recently, studies have revealed the significance of the NLRP3 inflammasome in innate and adaptive immune responses, and activation of the NLRP3 inflammasome pathway has been implicated in many autoimmune diseases, such as systemic lupus erythematosus, systemic sclerosis, inflammatory bowel disease and RA [45, 46]. It has been reported that the NLRP3 inflammasome is activated in the synovium of RA mice, and that treatment with a selective NLRP3 inhibitor can reduce the symptoms of arthritis and cartilage destruction [7‐9]. Therefore, in this study we evaluated the effects of LF on the expression of IL-1β, caspase-1 p20 and NLRP3 in the ankle joint synovial tissue of AIA rats, and quantified the serum levels of inflammatory cytokines IL-1β and TNF-α. Among the 3 extracts, PEL showed the strongest inhibition on the activation of the NLRP3 inflammasome pathway, which was consistent with the morphological and histopathological results. In addition, network pharmacology analysis also showed that many potential targets of components of PEL, including MEK1, ERK2, JNK1, p38 MAPK, Src, JAK2 and caspase-1, were closely related to the NLRP3 inflammasome pathway. The ERK signaling pathway is an important upstream regulator of the NLRP3 inflammasome. p38 MAPK plays an important role in proinflammatory cytokine synthesis. It has been reported that the activation of p38 MAPK is required for NLRP3-mediated IL-1β secretion in cultured human monocyte - derived macrophages [31]. It has also been reported that inhibition of ERK strongly suppresses NLRP3 expression both in 3 T3-L1 adipocytes induced by TNF-α and in LS14 preadipocytes induced by calcium-sensing receptors [32]. MEK1 activates ERK and functions specifically in the ERK cascade, acting as an essential component of MAPK signal transduction. It has been reported that when THP-1 macrophages are stimulated with oxidized low-density lipoprotein, the MEK/ERK pathway is activated and the expression of NLRP3 inflammasome-related proteins is upregulated [33]. Conversely, MEK inhibitors have been shown to decrease TNF-α induced NLRP3 expression in 3 T3-L1 cells [32]. Taken together, this evidence indicates that components of PEL probably suppress NLRP3 expression by inhibiting the MAPK signal transduction pathway, which might play a major role in the anti-RA effect of LF (Fig. 10). Other components’ targets of LF, including Src and JAK2, are also important regulators of the NLRP3 inflammasome. Cellular responses to TNF are initiated by the TNF receptor [47]. It has been reported that the type 1 TNF receptor (TNFR1) could form complexes with Src or JAK2, and then play a role in activating p38 MAPK, JNK1, Akt and NF-κB [30]. Activation of TNFR1 increased the IL-1β expression in THP-1 macrophages, which could be impeded by inhibitors of Src and JAK2 [30]. Moreover, Src can also activate TNF-α converting enzyme and then lead to increased TNF-α release and p38 MAPK activation in myogenic precursor cells [29]. These lines of evidence suggest that components of PEL are likely to exert their inhibitory effects on caspase-1 expression by inhibiting Src and JAK2, resulting in inhibition of the TNF signaling pathway. After the NLRP3/ASC/pro-caspase-1 protein complex assembles, pro caspase-1 self-cleaves into the activated form, and the activated caspase-1 releases IL-1β precursors into the extracellular cleavage activation. These predicted mechanisms of the anti-RA effect of LF are shown in Fig. 10.

The molecular docking study showed that components of PEL could bind with protein targets including MEK1, ERK2, JNK1, p38 MAPK, Src, JAK2 and caspase-1 as their inhibitors. Among the components, myrtenal and β-caryophyllene oxide exhibited superior binding affinity with these targets, especially MEK1 and p38 MAPK. Since fibroblast-like synoviocytes in the synovial intimal lining play a key role in the pathogenesis of RA by producing cytokines that perpetuate inflammation and proteases that contribute to cartilage destruction [36], they were used to verify the anti-RA potential of myrtenal and β-caryophyllene oxide. Our results showed that these two components could suppress the release of IL-1β and TNF-α and the expression of NLRP3 and caspase-1 p20, similar to the effect of PEL, indicating that they were the active ingredients of LF in the treatment of RA. Given the critical function of NLRP3 activation and induction of IL-1β in the pathogenesis of diseases such as gout, these mechanisms of action suggest that LF could have utility in other diseases as well [48‐50].

PEL can alleviate paw swelling, bone destruction, synovial inflammation and cartilage erosion in AIA rats. These effects are associated with inhibition of the NLRP3 inflammasome activation. Inhibition of the upstream MAPK signaling pathway and the TNF signaling pathway is also likely to be involved. Two components, myrtenal and β-caryophyllene oxide, screened from PEL, showed a similar inhibitory effect on the release of IL-1β and TNF-α and the expression of NLRP3 and caspase-1 in RA-FLS, indicating they might be the major active ingredients of LF in the treatment of RA.