Inhibition of Toll-Like Receptor 4 Signaling Pathway Accelerates the Repair of Avascular Necrosis of Femoral Epiphysis through Regulating Macrophage Polarization in Perthes Disease

Background: Legg-Calvé-Perthes disease (LCPD) is still a refractory disease in children's orthopedics. With the introduction of the concept of "osteoimmunology", the immune-inflammatory mechanisms between bone and immune system have become a research focus of LCPD. However, few studies have reported on the pathological role of inflammation-related receptors such as toll-like receptors (TLRs) as well as immune cells such as macrophages in LCPD. This study was for investigating the mechanism of TLR4 signaling pathway on the direction of macrophage polarization and the repair of avascular necrosis of femoral epiphysis in LCPD.

Methods: With GSE57614 and GSE74089, differentially expressed genes were screened. Through enrichment analysis and protein-protein interaction network, the functions of TLR4 were explored. Furthermore, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), hematoxylin & eosin (H&E) staining, micro-CT, tartrate-resistant Acid Phosphatase (TRAP) dyeing and western blotting were performed for determining the influences of TAK-242 (a TLR4 Inhibitor) on the repair of avascular necrosis of femoral epiphysis in rat models.

Results: Totally 40 co-expression genes were screened as well as enriched in TLR4 signaling pathway. Immunohistochemistry and ELISA analyses certified that TLR4 facilitated macrophage polarization toward the M1 phenotype and prevented macrophage polarization toward the M2 phenotype. Besides, the results of H&E and TRAP staining, micro-CT, and western blotting showed that TAK-242 can inhibit osteoclastogenesis and promote osteogenesis.

Conclusion: Inhibition of TLR4 signaling pathway accelerated the repair of avascular necrosis of femoral epiphysis by regulating macrophage polarization in LCPD.

Bioinformatics analysis; Legg-Calvé-Perthes disease; Macrophages; TAK-242; Toll-like receptor 4.