Analysis of the therapeutic effect of eleutheroside E, a natural lignan glycoside, on knee osteoarthritis and its interaction with MMPs
Osteoarthritis (OA) is a degenerative disease characterized by abnormal metabolism of articular cartilage and destruction of joint bone structure. In severe cases, it can lead to loss of joint function and has become an important cause of labor loss and disability in people over 50 years old. Among them, the incidence of knee osteoarthritis is the highest. According to the World Health Organization, there are currently about 190 million patients with osteoarthritis worldwide. With the aging society, the number of OA patients is increasing year by year, which brings great economic burden and mental pressure to patients’ families. At present, the treatment of osteoarthritis mainly relies on non-specific drugs to alleviate clinical symptoms. Non opioid analgesics (acetaminophen, central analgesics) and nonsteroidal anti-inflammatory drugs are taken orally to alleviate the symptoms of arthritis patients, but they cannot fundamentally cure osteoarthritis. At the same time, these drugs have many adverse reactions in the cardiovascular and gastrointestinal systems, limiting their scope of application. Therefore, there is an urgent need to develop an innovative OA treatment drug with a new mechanism of action and strong efficacy in clinical practice. Eleutheroside E (EE) is a unique lignan glycoside component in Acanthopanax senticosus (Figure 1). Our research team found in previous studies that EE has good anti-inflammatory effects, but so far, there have been no international research reports on the application of EE in the treatment of OA. Therefore, based on previous work, this study established a rabbit knee osteoarthritis model using anterior cruciate ligament transection (ACLT). For the first time, EE was selected as the therapeutic drug and administered through intra-articular injection for therapeutic intervention. The therapeutic and protective effects of EE on experimental rabbit knee osteoarthritis were evaluated at the animal level. Furthermore, molecular docking technology and molecular dynamics simulation methods will be used to analyze the interaction sites and binding modes between EE and MMPs, clarify the binding mode of EE and MMPs complexes, and lay the foundation for the development of a novel candidate therapeutic drug for osteoarthritis from natural sources.

The basic components of articular cartilage are cartilage matrix, chondrocytes, and water. The cartilage matrix is mainly composed of proteoglycans and collagen, and chondrocytes are the main source of cartilage matrix degradation and metabolism reactions. Under normal physiological conditions, a dynamic balance is maintained between the degradation and synthesis of cartilage components. The occurrence of osteoarthritis (OA) lesions is mainly due to the progressive destruction of bone, cartilage, and even extracellular matrix (ECM) of intra-articular ligaments and tendons caused by some related mediators or factors, resulting in local softening, abrasion, and structural damage of articular cartilage. In recent years, scholars have found that matrix metalloproteinases (MMPs) are closely related to bone and joint injuries and disease activity in OA patients, playing a crucial role in the pathological process of cartilage matrix and chondrocyte destruction. MMPs are zinc protease enzymes that can degrade extracellular matrix (ECM) and almost all components of ECM. During the occurrence of osteoarthritis, chondrocytes and synovial cells secrete excessive matrix metalloproteinases (MMP-3 and MMP-9), breaking the balance between matrix metalloproteinases tissue inhibitory factor (MMP-IMP) and causing irreversible degradation of ECM. This leads to swelling of articular cartilage, decreased resistance to external forces, aggravated intra-articular inflammatory reactions, and ultimately loss of joint function. The cartilage damage caused by overexpression of MMPs has become one of the hotspots in the study of the pathogenesis of OA. Therefore, finding MMPs inhibitors has become a new strategy for the development of drugs for treating OA.

Traditional Chinese medicine is an important component of the treasure trove of Chinese medicine. Due to its wide range of sources, rich chemical composition, and low incidence of adverse reactions, it has attracted increasing attention from domestic and foreign researchers. Currently, attempting to screen and discover new small molecule therapeutic drugs targeting disease targets from traditional Chinese medicine has become a research hotspot. Acanthopanax senticosus (Rupr. et Maxim.) Harms, also known as ginseng or thorn cane, is a precious medicinal plant mainly produced in Heilongjiang, Jilin, Liaoning, Hebei, the Russian Far East, and Hokkaido, Japan. It has a long history of medicinal use in China. The preliminary research of the research group found that eleutheroside E (EE) is one of the main active ingredients in Acanthopanax senticosus. Currently, there are few reports on the pharmacological effects of EE, mainly for reducing blood sugar, improving postmenopausal osteoporosis, anti central fatigue and memory impairment, and reducing cerebral ischemia-reperfusion injury. Our previous research found that EE can significantly inhibit inflammatory factors and joint cartilage damage. Through literature search and investigation, there are currently no research reports on the application of EE in the treatment of OA internationally.

Therefore, this article is the first to study the therapeutic effect of EE on osteoarthritis, evaluating the therapeutic and protective effects of EE on experimental rabbit knee osteoarthritis at the animal level, providing experimental evidence for the prevention and treatment of osteoarthritis with EE. This study used the classic anterior cruciate ligament transection (ACLT) method to construct a rabbit knee osteoarthritis model. EE-20 and EE-60 were injected into the joint cavity for therapeutic intervention, once a week for 5 consecutive weeks. The effects of EE-20 on inflammatory cytokines and joint pathological changes in the rabbit knee osteoarthritis model were studied, and the preventive and therapeutic effects of EE on the rabbit knee osteoarthritis animal model were observed. The experimental results showed that EE can significantly improve the infiltration of inflammatory cells, fibrous tissue proliferation, and cartilage surface damage in osteoarthritis sites. Studies have shown that MMP-3 and MMP-9 can accelerate the damage of extracellular matrix in the cartilage structure of OA joints, promote the speed of cartilage destruction, and thus advance the progression of OA. In addition, IL-1 β is also a key factor in the production of 0A. IL-1 β can induce the production of nitric oxide (NO) and cyclooxygenase (COX) in synovial cells, while cyclooxygenase-2 can promote the secretion of nitric oxide and prostaglandin E2 (PGE2). PGE2 is a multifunctional inflammatory mediator that can increase the production of MMPs and other catabolites, thereby affecting the structure and function of joint tissue and accelerating the destruction of joint structure. Therefore, we further used ELISA detection method to analyze and determine the levels of inflammatory mediators (IL-1 β and PGE2) and matrix metalloproteinases MMP-3 and MMP-9 in the joint fluid of OA model animals after EE intervention. The results showed that EE treatment intervention could significantly reduce the levels of inflammatory mediators (IL-1 β and PGE2) and matrix metalloproteinases MMP-3 and MMP-9 in joint fluid (P<0.001), indicating that EE has a significant anti osteoarthritis effect.

At present, molecular docking technology and molecular dynamics simulation have become important tools for exploring the binding mode between proteins and receptors, and have significant application value in the field of new drug research. This study further utilized molecular docking technology and molecular dynamics simulation to analyze and explore the interaction sites and binding modes between EE and MMPs. The hydroxyl group on the EE sugar ring can form hydrogen bonds with the amino acids in the MMP-3 and MMP-9 receptors in the grooves of the catalytic sites of MMP-3 and MMP-9, which play an important role in ligand binding. In addition, the EE ligand benzene ring can also form hydrophobic stacking interactions with hydrophobic residues in MMP-3 and MMP-9, which can stabilize their binding. Molecular dynamics simulation experiments show that the main contributions to the binding process of EE with MMP-3 and MMP-9 complexes come from van der Waals potential energy and electrostatic interactions, and the two systems have similar binding modes. Among them, the total binding energy of EE and MMP-3 is lower, while EE has a stronger binding ability to MMP-3. This result is consistent with the trend of the ELISA detection experiment results mentioned above. The above data provides a key theoretical basis for future research on the molecular mechanism of EE’s anti MMPs effects and the development of novel MMPs inhibitors based on lignin glycosides with a nuclear structure.

In summary, intra-articular injection of EE can significantly improve inflammatory cell infiltration, fibrous tissue proliferation, and cartilage surface damage in osteoarthritis sites, and reduce the levels of inflammatory mediators (IL-1 β and PGE2) and matrix metalloproteinases MMP-3 and MMP-9 in joint fluid, indicating that EE has a significant anti osteoarthritis effect. In addition, further molecular docking techniques and molecular dynamics experiments have found that EE ligands can bind to the grooves of the catalytic sites of MMP-3 and MMP-9. Multiple hydroxyl groups on the sugar ring of EE ligands can form hydrogen bonds with multiple amino acids in the MMP-3 and MMP-9 receptors, which play an important role in ligand binding. The results of this study will lay the foundation for the development of a novel candidate therapeutic drug for osteoarthritis from natural sources.