Oxypeucedanin hydrate alleviates rheumatoid arthritis by inhibiting the TLR4-MD2/NF-κB/MAPK signaling axis

Abstract

Rheumatoid arthritis (RA) is an idiopathic and chronic autoimmune disease for which there are currently no effective treatments. Oxypeucedanin hydrate (OXH) is a natural coumarin known for its potent anti-inflammatory properties. However, further investigations are needed to determine its therapeutic efficacy in treating RA. In this study, we evaluate the anti-inflammatory activity of OXH by treating LPS-induced RAW264.7 macrophages. Our results show that OXH treatment reverses the changes in iNOS, COX-2, IL-1β, IL-6, and TNF-α levels. Additionally, OXH reduces ROS production. Further analysis reveals that OXH suppresses the activation of the NF-κB/MAPK pathway. CETSA results show that OXH competes with LPS for binding to the TLR4/MD2 complex. MST experiments demonstrate the specific affinity of OXH for the TLR4/MD2 complex, with a Kd value of 33.7 μM. Molecular docking analysis suggests that OXH binds to the pocket of the TLR4/MD2 complex and interacts with specific amino acids, such as GLY-343, LYS-388, and PHE-345. Molecular dynamics simulations further confirm this conclusion. Finally, we investigate the potential of OXH in treating RA using a collagen-induced arthritis (CIA) model in rats. OXH effectively ameliorates the symptoms of CIA, including improving body weight, reducing swelling and redness, increasing talus volume, and decreasing bone erosion. OXH also decreases the mRNA levels of pro-inflammatory factors in synovial tissue. Transcriptome enrichment analysis and western blot analysis confirm that OXH suppresses the NF-κB/MAPK pathway, which is consistent with our in vitro findings.

1. Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease. Although the exact cause remains unknown, it is believed to be affected by both environmental and genetic factors. The primary clinical manifestations of RA include joint injury and synovitis [1,2]. RA has a global prevalence of approximately 0.1% and is more common in females than in males [3]. The pathogenic mechanism of RA is complicated and is related to bone and cartilage erosion as well as synovial cell fibrosis and proliferation [4,5]. Current clinical treatment options for RA include non-steroidal anti-inflammatory drugs, glucocorticoids, and disease-resistant anti-rheumatic agents. However, these therapeutic agents often cause adverse reactions, including gastrointestinal reactions, organ damage, and allergic reactions [6,7]. Additionally, certain biological agents, such as TNF-α antagonists, may increase the risk of tumor infection [8]. Therefore, there is an urgent need to discover effective compounds with fewer side effects and better outcomes. Traditional Chinese medicine (TCM), with its potential for multiple components, multiple targets, effectiveness, and few side effects, has attracted increasing attention from researchers who are seeking active ingredients [9,10].

According to previous research [11], RA is associated with major pathological features, including synovial hyperplasia and inflammatory infiltration. This occurs when a significant number of immune cells, such as macrophages and T cells, accumulate within synovial tissue and the joint cavity during the development of RA. Due to inflammation, numerous inflammatory factors, such as TNF-α, IL-6 and IL-1β, are released. Simultaneously, these inflammatory factors stimulate synovial cells to produce chemokines, thereby attracting circulating immune cells to the affected site and worsening joint inflammation [12]. Moreover, the inflammatory response further intensifies as synovial cells proliferate in this environment. Therefore, targeting immune cells or synovial cells has emerged as a candidate strategy for treating RA [13].

The Toll-like receptor 4 and myeloid differentiation factor 2 (TLR4-MD2) complex and its associated pathways are important for inflammation progression [14]. The TLR4-MD2 complex is primarily expressed on the cytoplasmic membrane of immune cells such as monocytes, macrophages, and dendritic cells. It is activated by various substances, such as lipopolysaccharide (LPS) extracted from gram-negative bacteria, intracellular peptides, glycoproteins, and endotoxins [15]. Such activation triggers downstream pathways, such as the NF-κB pathway, leading to inflammation [16]. Oxypeucedanin hydrate (OXH) is a furan coumarin commonly found in various anti-inflammatory herbs, including Notopterygium incisum, Angelica dahurica [17], and Whiteflower Hogfennel. OXH was previously suggested to suppress NO synthase production [18]. Howeve