Abstract: BACKGROUND: Previous studies have demonstrated that Gugutou Huaisiyu Capsule effectively alleviate pain in patients with osteonecrosis of femoral head; however, the mechanism of action remains unclear.
OBJECTIVE: To investigate the mechanism by which Gugutou Huaisiyu Capsule mitigate peripheral pain sensitization in rats with steroid-induced osteonecrosis of the femoral head.
METHODS: Thirty-two Sprague-Dawley rats were randomly assigned to four groups: blank control, model, low-dose Gugutou Huaisiyu Capsule, and high-dose Gugutou Huaisiyu Capsule groups with eight rats in each group. Steroid-induced femoral head necrosis rat models were established via intraperitoneal injection of lipopolysaccharide combined with gluteal injection of methylprednisolone in the later three groups. Seven days post-modeling, rats in the low- and high-dose Gugutou Huaisiyu Capsule groups received intragastric administration of 0.33 g/kg and 0.67 g/kg of the decoction, respectively, while the blank control and model groups received intragastric administration of saline once daily for 8 consecutive weeks. Following the final administration, trabecular bone microarchitecture in the femoral head was assessed using micro-CT analysis. The mechanical pain thresholds were measured with the Von Frey test. Serum levels of pain mediators calcitonin gene-related peptide and substance P were detected using ELISA. Multiplex immunofluorescence staining and real-time quantitative PCR were used to detect the protein and gene expression of tyrosine kinase receptor A, calcitonin gene-related peptide, and neuronal marker β3-tubulin in the femoral head and dorsal root ganglion.
RESULTS AND CONCLUSION: (1) Micro-CT and pain behavioral tests revealed that compared with the blank control group, significant destruction of trabecular bone microarchitecture in the femoral head, decreased bone density and bone volume fraction, increased trabecular separation, and reduced mechanical pain threshold are found in the model group. Compared with the model group, trabecular structure significantly improved, bone mineral density and bone volume fraction significantly increased, trabecular separation significantly decreased, and mechanical pain threshold significantly elevated in the low- and high-dose Gugutou Huaisiyu Capsule groups. (2) ELISA analysis revealed calcitonin gene-related peptide and substance P levels were significantly increased in the model group compared with the blank control group, while calcitonin gene-related peptide and substance P levels were significantly decreased in the low- and high-dose Gugutou Huaisiyu Capsule groups compared with the model group. (3) Multiplex immunofluorescence staining and real-time quantitative PCR revealed that compared with the blank control group, protein and gene expression of calcitonin gene-related peptide, tyrosine kinase receptor A, and β3-tubulin were significantly elevated in the femoral head and dorsal root ganglion in the model group. Compared with the model group, protein and gene expression of calcitonin gene-related peptide, tyrosine kinase receptor A, and β3-tubulin were significantly decreased in the femoral head and dorsal root ganglion in both low- and high-dose Gugutou Huaisiyu Capsule groups. (4)The findings suggest that Gugutou Huaisiyu Capsule can elevate the mechanical pain threshold, suppress abnormal neuronal activation, and alleviate pain sensitization in rats with steroid-induced osteonecrosis of the femoral head. This mechanism may involve the inhibition of tyrosine kinase receptor A.

Key words: steroid-induced osteonecrosis of the femoral head, Gugutou Huaisiyu Capsule, tyrosine kinase receptor A, pain sensitization, osteonecrosis, tissue construction