Mechanism by which Yougui Pill inhibits pyroptosis of chondrocytes in rats with knee osteoarthritis

Abstract

BACKGROUND: Yougui Pill is derived from Jingyue Quanshu. Studies have confirmed that Yougui Pill is highly effective in treating patients with knee osteoarthritis, but its mechanism of action remains unclear. OBJECTIVE: To explore the potential molecular mechanism of Yougui Pill in improving knee osteoarthritis in rats. METHODS: Forty-eight SPF-grade Sprague-Dawley rats were randomly divided into four groups: blank control group, model group, Yougui Pill group and celecoxib group. The latter three groups were subjected to modified Hulth method for surgical modeling of knee osteoarthritis. After wound healing, rats were driven for 8 weeks. After modeling, the celecoxib group was given celecoxib suspension by gavage, the Yougui Pill group was given Yougui Pill decoction by gavage, and the sham operation group and model group were given equal volume of normal saline, once daily for 4 weeks. Hematoxylin-eosin staining, toluidine blue staining, and safranin O-fast green staining were used to observe the pathological changes of rat cartilage tissue; transmission electron microscopy was used to observe the ultrastructure of rat chondrocytes; ELISA was used to detect the levels of interleukin-18, interleukin-1β, and tumor necrosis factor-α in rat serum; western blot was used to detect the protein expression of PI3K, AKT, NF-κB, p-PI3K, p-AKT, p-P65, NLRP3, GSDMD, GSDMD-N, Caspase1, Cleaved-Caspase1, interleukin-18, and interleukin-1β in rat cartilage tissue. RESULTS AND CONCLUSION: Compared with the blank group, the model group showed severe destruction of cartilage edge, cartilage tissue defect, thinning and disordered arrangement of cartilage layer cells, subchondral bone hyperplasia, disordered tide line, severe structural damage of chondrocytes, formation of pyroptotic bodies, significantly increased serum levels of tumor necrosis factor-α, interleukin-18, and interleukin-1β (P < 0.05), and significantly increased protein expression of p-PI3K, p-AKT, p-P65, NLRP3, GSDMD-N, Cleaved-Caspase1, interleukin-18, and interleukin-1β in cartilage tissue (P < 0.01). Compared with the model group, the cartilage structure of rats in the Yougui Pill group and celecoxib group tended to be normal, with deeper cartilage staining, thicker cartilage, more complete chondrocyte membrane, significantly decreased serum levels of tumor necrosis factor-α, interleukin-18, and interleukin-1β (P < 0.05), and significantly decreased protein expression of p-PI3K, p-AKT, p-P65, NLRP3, GSDMD-N, Cleaved-Caspase1, interleukin-18, and interleukin-1β in cartilage tissue (P < 0.01). Compared with the celecoxib group, the Yougui Pill group showed more regular arrangement of chondrocytes, smoother articular cartilage surface, significantly thickened cartilage layer, relatively complete tide line, relatively complete chondrocyte membrane, significantly decreased serum levels of tumor necrosis factor-α, interleukin-18, and interleukin-1β (P < 0.05), and significantly decreased protein expression of p-PI3K, p-AKT, p-P65, NLRP3, GSDMD-N, Cleaved-Caspase1, interleukin-18, and interleukin-1β in cartilage tissue (P < 0.01). These results indicate that Yougui Pill can improve the inflammatory response of chondrocytes in rats with knee osteoarthritis, and the mechanism may be related to inhibiting the activation of PI3K/AKT/NF-κB pathway, thereby regulating NLRP3/Caspase1/GSDMD pathway-mediated pyroptosis.

1. Introduction

Knee osteoarthritis is a highly disabling degenerative joint disease characterized by chronic pain and limited joint mobility, affecting the entire knee joint, including articular cartilage, synovium, and surrounding muscles. Pathologically, it manifests as degeneration of cartilage and synovium, inflammatory damage, and muscle atrophy. Epidemiological surveys show that the number of new cases of knee osteoarthritis has increased by 128.72% in the past decade. In terms of treatment, surgical methods mainly include arthroscopic surgery and knee replacement, while pharmacological treatment primarily relies on non-steroidal anti-inflammatory drugs to relieve joint pain, but cannot reverse the progression of knee osteoarthritis. Therefore, it is crucial to seek effective drug treatments that can delay the development and progression of knee osteoarthritis.

Pyroptosis is a programmed cell death mediated by inflammasome activation and Gasdermin proteins, characterized by cell membrane perforation, cell swelling, and release of large amounts of inflammatory cytokines (such as IL-1β and IL-18). Studies have shown that pyroptosis plays a key role in the pathogenesis of knee osteoarthritis by inducing chondrocyte death, promoting inflammatory responses, and matrix degradation.

According to traditional Chinese medicine theory, the kidney governs bones, and kidney essence can transform into marrow to nourish bones. Deficiency of kidney yin leads to marrow depletion and bone malnutrition, while deficiency of kidney yang fails to warm and nourish, causing tendon contracture and pain. Yougui Pill, derived from Jingyue Quanshu, is a formula that warms and tonifies kidney yang while supplementing marrow and essence, aiming to treat both yin and yang. It is used to relieve pain and provide nutrients for bones. This study aims to explore the potential molecular mechanism of Yougui Pill in improving knee osteoarthritis in rats.