Function and molecular mechanism of physcion in regulating bone homeostasis

Abstract

BACKGROUND: Although physcion has been shown to have protective effects against osteoporosis, the exact mechanism is not fully understood. OBJECTIVE: Through multidimensional analysis of the regulatory effect of physcion on the AKT signaling pathway, the molecular mechanism of its regulation on osteoclast induced differentiation and osteogenic function induced differentiation is revealed. METHODS: (1) RAW264.7 cells and C3H10T1/2 cells were cultured in vitro and subsequently exposed to 0, 10, 20, 30, 40, 50, and 60 µmol/L physcion, respectively. The cytotoxicity of physcion was detected by cell counting kit-8 assay. (2) RAW264.7 cells and C3H10T1/2 cells were treated with different concentrations (0, 20, 40 µmol/L) of physcion during osteoclast and osteoblast differentiation, respectively. Differentiation ability was assessed by qPCR, Western Blot, and alkaline phosphatase staining. (3) Network pharmacology was used to analyze the regulation of physcion on osteoclast differentiation and related signaling pathways, and molecular docking was performed for target proteins. (4) Western Blot was used to verify the phosphorylation level of AKT in the downstream target signaling pathway AKT axis regulated by physcion. RESULTS AND CONCLUSION: (1) At concentrations of 0-60 µmol/L, cell viability in all groups was greater than 90%, indicating no significant cytotoxicity. (2) Physcion significantly inhibited the expression of osteoclast differentiation-related genes, with Acp5, CTSK, DC-STAMP, and Nfatc1 showing downregulation, but had no significant effect on osteoblast differentiation-related genes COL1A1, Runx2, OSX expression or alkaline phosphatase staining intensity. (3) Network pharmacology and molecular docking suggested that physcion affects osteoclast differentiation and regulates the PI3K-AKT pathway, with a binding energy of -10.72 kJ/mol to AKT1, indicating strong binding activity. (4) During osteoclast differentiation, the p-AKT/AKT ratio in RAW264.7 cells increased (n=3, P=0.0063), while physcion decreased this ratio. These findings indicate that physcion inhibits osteoclast differentiation by regulating the AKT signaling pathway, thereby modulating bone homeostasis.

1. Introduction

Bone structure formation is primarily achieved through processes such as intramembranous ossification and endochondral ossification, which require the coordinated action of various functional cells and the matrix within the bone microenvironment to maintain skeletal balance [1]. Once bone homeostasis is disrupted, it can lead to bone-related diseases such as osteopetrosis and osteoporosis [2-3]. Osteoporosis is closely linked to bone metabolic imbalance, characterized by decreased bone density and disrupted trabecular microarchitecture, often leading to severe complications like femoral neck fractures in advanced stages [4-5]. Osteoporosis is highly prevalent in postmenopausal women, the elderly, and diabetic patients, with its incidence rising annually, significantly impacting patients' quality of life and imposing substantial burdens on healthcare systems and society [6-7]. Consequently, various pharmacological treatments have been developed, such as alendronate, risedronate, and denosumab, which aim to inhibit osteoclastogenesis to alleviate the severity and symptoms of osteoporosis [8-10]. However, these chemical drugs have adverse reactions and toxic side effects in clinical use [11].

Recently, research on traditional Chinese medicine in the prevention and treatment of osteoporosis has deepened, demonstrating unique advantages in bone disease management. The concept of 'holistic regulation and eliminating pathogens while supporting vital energy' aligns closely with the biological connotation of bone homeostasis regulation [12-13]. According to the TCMSP database, physcion is an anthraquinone compound extracted from the classic blood-activating and stasis-resolving herb rhubarb, and is also widely present in traditional Chinese medicines such as Salvia miltiorrhiza, wolfberry, and Morinda officinalis, which have effects of 'unblocking meridians and nourishing bones' and 'supplementing vital energy' [14-16]. Physcion possesses multiple pharmacological properties, including antibacterial activity against Escherichia coli and anticancer potential against human cervical cancer HeLa cells [17-18]. Additionally, physcion has neuroprotective effects, preventing cerebral ischemic injury and protecting neurons from damage [19]. Studies have shown that physcion can prevent lipid accumulation in white adipose tissue and liver, thereby alleviating diet-induced obesity and related complications such as hyperlipidemia and hepatic steatosis [20]; physcion also improves diabetic osteoporotic damage [21-22]. However, the specific mechanism of physcion in bone metabolism regulation remains unexplored.

Based on this, the present study elucidates the functional differences of physcion in osteoclasts and osteoblasts, and combined with network pharmacology, deeply explores the specific mechanism of physcion as a therapeutic agent for osteoporosis, aiming to provide more effective strategies for the treatment of osteoporosis.