Cinnamaldehyde suppresses NLRP3 derived IL-1β via activating succinate/HIF-1 in rheumatoid arthritis rats

https://doi.org/10.1016/j.intimp.2020.106570Get rights and content

Highlights

  • CA alleviated inflammation of rheumatoid arthritis in rats by suppressing IL-1β.

  • CA reduced HIF-1α expression by limiting succinate aggregation and GPR91 activation.

  • CA inhibited NLRP3 expression through Succinate/GPR91&HIF-1α for IL-1β suppression.

Abstract

Cinnamaldehyde (CA) is an essential component of cinnamon (Cinnamomum cassia Presland), which is often used as a flavoring condiment in beverages, pastries, perfumes, etc. Cinnamon is also used as herbal medicine in China and Southeast Asia to treat rheumatoid arthritis. However, the molecular mechanism is unclear. In this study, we aim to investigate its anti-inflammatory effects against Rheumatoid arthritis (RA) using activated macrophages (Raw246.7) in vitro and adjuvant arthritis rats (AA) in vivo. The results demonstrated that CA significantly reduced synovial inflammation in AA rats, possibly due to suppression of the expressions of pro-inflammatory cytokines, especially the IL-1β. Further investigation found that CA also suppressed the activity of HIF-1α by inhibiting the accumulation of succinate in cytoplasm. As we know, the reduction of HIF-1α nucleation slows down IL-1β production, because HIF-1α activates the expression of NLRP3, which is involved in the assembly of inflammasome and processing of IL-1β. In addition, CA also inhibited the expression of the succinate receptor GPR91, which in turn inhibited the activation of HIF-1α. In conclusions, our results suggested that CA might be a potential therapeutic compound to relieve rheumatoid arthritis progress by suppressing IL-1β through modulating succinate/HIF-1α axis and inhibition of NLRP3.

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis and hyperplastic synovial tissue, and ultimately leads to destructions of cartilage, joint and bone [1], [2], [3]. The Macrophages are dominant cells involved in the synovial inflammation and a major contributor to the cytokine-rich pro-inflammatory environment. In addition, they are also actively involved in inflammatory infiltrations, joint destruction, and various kinds of comorbidity [4], [5]. Thus, inhibiting macrophage-mediated inflammation may be a potential strategy for RA treatment.

Macrophages are critically involved in the pathogenesis of RA. Not only do they produce a variety of pro-inflammatory cytokines and chemokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), but they also contribute to the cartilage and bone destruction in RA through multiple mechanisms [6], [7], [8], [9], [10]. IL-1β is critically involved in synovial inflammatory response and synovial fibrosis in RA [11]. It is well known that succinate is an intermediate metabolite of the Tricarboxylic acid cycle in energy metabolism [12], [13]. Succinate accumulation has been detected in synovial fluid from RA patients [14], and a metabolic profiling study defined succinate as a marker metabolite of RA, which is an important feature of RA being different from other inflammatory arthropathies [15]. As an inflammatory signal, succinate induces IL-1β via HIF-1α [16]. The accumulation of succinate creates a local hypoxic inflammatory environment. In response, HIF-1α acts as a hypoxia sensor to regulate the cellular response to adapt to the hypoxia environment. Simultaneously acting as a transcription factor, HIF-1α regulates a range of genes involved in inflammation, angiogenesis, mitochondrial function [17], [18], [19]. In macrophages of RA, the accumulation of succinate leads to the stabilization and activation of HIF-1α, which enters the nucleus to initiate the transcription and translation of IL-1β [16], [20], [21], [22]. Furthermore, succinate reaches the extracellular milieu after necrosis or pathological damage of inflamed tissue [23]. In the inflammatory environment, macrophages recycle extracellular succinate via GPR91 to up-regulate a HIF-1α–dependent innate pathway, which ultimately potentiates IL-1β production [24]. NLRP3 inflammasome belongs to the NLR family, and consists of NLRP3, apoptosis-associated speck-like protein containing (ASC), and caspase-1 [25], [26], [27]. NLRP3 recruits ASC and caspase-1 to form inflammasome and processes the precursors of IL-1β, which is critical for IL-1β maturation and release [28], [29], [30], [31]. Based on above speculation, reducing the IL-1β production and release induced by the succinate- HIF-1α axis in macrophages may be a potential way for the treatment of synovitis in RA.

Cinnamaldehyde (CA) (Fig. 1) is an active component of Cinnamomum cassia Presl, which is a tropical aromatic evergreen tree of the Lauraceae family, and is commonly used in traditional Chinese medicine [32]. Cinnamon is obtained from the bark of young tree branches, and is widely used all around the world for its fragrance and spicy flavor, especially in India, and was also used by the ancient Romans. In western countries, cinnamon is also used as a flavoring agent for confectionery, pastries, and meat, and as a spice for cosmetics. Cinnamon has been used to treat RA in China for nearly 2000 years. Recent pharmacological studies also suggested that cinnamon had a good therapeutic effect on RA. Similarly, cinnamaldehyde has been shown to have potential effects in anti-inflammatory and treatment of RA. According to reports, Cinnamaldehyde significantly inhibits joint disease in experimental arthritis animals. And Cinnamaldehyde could not only significantly reduce the IL-6 content of inflammatory mediator TNF-α in peripheral monocytes of RA patients [33], but also inhibit the release of IL-1β and matrix MMP-13 from synovial fibroblasts in arthritic patients [34], [35]. Cinnamaldehyde could significantly reduce the levels of TNF-α, IL-6 and IL-1β in peripheral blood of collagen-induced RA rat, and significantly increase the content of anti-inflammatory factor IL-10, exerting systemic anti-inflammatory effects [36]. Based on this, the aim of this study was to investigate the new mechanism of cinnamaldehyde in inhibiting the release of IL-1β against synovial inflammation.

Section snippets

Drugs and reagents

Cinnamaldehyde (14371-10-9) was bought from Herbest Bio-Pharmaceutical Co. Ltd (Baoji, China). Dexamethasone acetate (161231) was obtained from Xianju Pharmaceutical Co. Ltd (Zhejiang, China). Lipopolysaccharides (LPS,032M4082V) and complete freund's adjuvant (CFA, F5881) were purchased from sigma (America). Adenosine 5′-triphosphate disodium salt (ATP, S0714A) and Oltipraz (HY-12519) were purchased from Meilun biotechnology Co. Ltd (Dalian, China). HPLC-grade methanol was from Chron chemical

The anti-inflammatory effect of cinnamaldehyde

To investigate the actions of CA, we firstly cultured Raw246.7 cells in vitro and stimulated them with LPS + ATP to activated macrophages. Then we detected anti-inflammatory effects of CA on macrophages. The results showed that CA significantly inhibited levels of IL-1β released by macrophages (12.5 μM, P < 0.05; 25 μM, P < 0.01; Fig. 2A). Moreover, it showed that CA had significant inhibitory effect on TNF-α and NO (6.25 μM, P < 0.05; 12.5 μM and 25 μM, all P < 0.01, Fig. 2B, C). Next, we

Discussion

In the present study, we demonstrated that CA could suppress the release of IL-1β, and CA treatment could attenuate synovial inflammation in adjuvant arthritis (AA) rat. And we found that the expression of NLRP3 was reduced by CA. Moreover, our findings suggested that CA could block succinate-induced HIF-1α and GPR91 activation in vitro and in vivo. Thus, this study suggested that CA conferred inhibitory effects on release of IL-1β by blocking NLRP3 and succinate-induced HIF-1α, GPR91

CRediT authorship contribution statement

Panwang Liu: Conceptualization, Investigation, Data curation, Visualization, Writing - original draft. Jie Wang: Methodology, Validation, Formal analysis, Investigation, Data curation. Wen Wen: Methodology, Validation, Formal analysis, Data curation, Writing - review & editing. Ting Pan: Investigation, Writing - review & editing. Huan Chen: Data curation, Writing - review & editing. Ying Fu: Methodology, Validation. Fushun Wang: Formal analysis, Writing - review & editing. Jason H. Huang:

Declaration of Competing Interest

All authors declare that there are no conflicts of interest.

Acknowledgement

This study was supported by the grants from the project supported by National Natural Science Foundation of China, China (81773972, 81273900).

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    Panwang Liu, Jie Wang and Wen Wen contribute equally to this paper.

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