Mechanistic Study On The Function Of Cilengitide In Inhibiting Osteoclast Bone Resorption And The Establishment Of Nano-Delivery System

Background: Bone homeostasis is a dynamic balance maintained in the skeleton between osteoblasts,which regulate the bone formation,and osteoclasts,which regulate bone resorption.When this process is disrupted,such as increased osteoclast bone resorption activity leads to osteoporosis.Osteoclasts adhere to the bone matrix through integrins,and the osteoclast membrane is located in a closed area near the bone surface to form a wrinkled membrane that forms an acidic microenvironment by releasing protons and proteases.Currently,bisphosphonates,calcitonins and estrogen replacement therapies used to inhibit osteoclast bone resorptive function are clinically effective,but their application is limited by their drug side effects.Therefore,it is important to find new therapeutic agents for osteoclast-related diseases.Recent studies have shown that osteoclasts adhere to the bone matrix through integrins to form a sealed zone,and a folded membrane composed of osteoclast cell membranes near the bone surface releases protons and proteases to form an acidic microenvironment.Therefore,in this study,acid-sensitive nanomaterials loaded with the anti-integrin drug Cilengitide were designed for osteoclast integrin protein and acidic microenvironment and their effects were verified.Objects: The aim of this study was to investigate the impact of Cilengitide on RANKL-induced osteoclast differentiation and adhesion,and to elucidate its molecular mechanism.Additionally,we employed acid-sensitive nanomaterials to augment Cilengitide’s effectiveness in inhibiting RANKL-induced osteoclast differentiation and bone resorption,based on the acidic microenvironment of osteoclast bone absorption.These findings offer a theoretical foundation for the use of Cilengitide in treating osteoporosis.Methods: The model used involved inducing differentiation of bone marrow-derived macrophages into osteoclasts using RANKL.To evaluate the effects of Cilengitide on osteoclast generation and bone resorption capacity,Tartrate-resistant acid phosphatase(TRAP)staining assay,actin ring(F-actin)staining assay,and bone resorption lacunae assay were employed.Western blot assay,real-time fluorescence quantitative PCR(RT-PCR),and cell adhesion assay were used to investigate the impact of Cilengitide on pathways associated with osteoclast formation and adhesion.Furthermore,acid-sensitive biomaterials were developed based on the effects of Cilengitide on osteoclasts,and their effects on osteoclast differentiation and bone resorption were assessed using the above-mentioned experiments.Results: Cilengitide was found to inhibit the generation of RANKL-induced osteoclasts,particularly in the late stage of osteoclast differentiation.Furthermore,Cilengitide prevented the formation of osteoclast F-actin rings and bone resorption,as well as significantly reducing osteoclast adhesion to bone matrix proteins.The mechanism of action involves inhibition of FAK/Src signaling downstream of integrin αvβ3,leading to a reduction in osteoclast adhesion and bone resorption.Finally,the combination of acid-sensitive nanomaterials and Cilengitide was found to have a synergistic effect,resulting in a significant reduction in osteoclast formation,F-actin production,and bone resorption during osteoclast differentiation.Conclusion: The findings suggest that Cilengitide has the potential to inhibit osteoclast adhesion and bone resorption by blocking the αvβ3-mediated FAK/Src signaling pathway.Moreover,the use of acid-sensitive nanomaterials appears to enhance the inhibitory effect of Cilengitide,providing a promising basis for future research and clinical applications.