Mechanism of Gugutou Huaisiyu Capsule in alleviating peripheral pain sensitization in rats with steroid-induced osteonecrosis of the femoral head

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. After 7 days of modeling, the low- and high-dose Chinese medicine groups were administered 0.33 and 0.67 g/kg of Gugutou Huaisiyu Capsule decoction by gavage, respectively, while the blank control and model groups received normal saline, once daily for 8 weeks. After the last administration, Micro-CT was used to analyze the microstructure of the femoral head cancellous bone; Von-Frey test was used to detect mechanical pain threshold; ELISA was used to detect serum levels of pain mediators calcitonin gene-related peptide (CGRP) and substance P; multiplex immunofluorescence staining and real-time quantitative PCR were used to detect protein and gene expression of tyrosine kinase receptor A (TrkA), CGRP, and neuronal marker β3-tubulin in the femoral head and dorsal root ganglion. RESULTS AND CONCLUSION: (1) Micro-CT and pain behavior tests showed that compared with the blank control group, the model group had significant destruction of the femoral head cancellous bone microstructure, decreased bone mineral density and bone volume fraction, increased trabecular separation, and decreased mechanical pain threshold. Compared with the model group, the low- and high-dose Chinese medicine groups had significantly improved trabecular structure, increased bone mineral density and bone volume fraction, decreased trabecular separation, and increased mechanical pain threshold. (2) ELISA showed that the levels of CGRP and substance P in the model group were significantly higher than those in the blank control group, while the levels in the low- and high-dose Chinese medicine groups were lower than those in the model group. (3) Multiplex immunofluorescence staining and real-time quantitative PCR showed that compared with the blank control group, the protein and gene expression of CGRP, TrkA, and β3-tubulin in the femoral head and dorsal root ganglion were increased in the model group; compared with the model group, the expression of these proteins and genes was decreased in the low- and high-dose Chinese medicine groups. (4) These results indicate that Gugutou Huaisiyu Capsule can increase the mechanical pain threshold, inhibit abnormal neuronal activation, and alleviate pain sensitization in rats with steroid-induced osteonecrosis of the femoral head, and the mechanism may be related to the inhibition of TrkA.

1. Introduction

Osteonecrosis of the femoral head is a refractory orthopedic disease characterized by collapse of the femoral head [1-2]. Its causes are diverse, mainly including glucocorticoid use, alcoholism, hyperlipidemia, and genetic susceptibility, among which steroid-induced osteonecrosis of the femoral head accounts for the highest proportion [3-4]. Pain is the main symptom of osteonecrosis of the femoral head and the primary reason for patients to seek medical attention [5]. The author's team previously conducted a comprehensive analysis of factors affecting pain in osteonecrosis and demonstrated that pain sensitization is an independent risk factor for aggravating pain in patients with osteonecrosis of the femoral head [6].

Tyrosine kinase receptor A (TrkA) is a specific high-affinity receptor for nerve growth factor. Its activation in the dorsal root ganglion leads to neuronal sensitization and increased excitability, inducing pain sensitization, and maintaining and amplifying chronic pain [7-10]. In injured or inflamed environments, damaged cells release nerve growth factor, which binds to and activates TrkA. On one hand, it activates downstream signaling pathways such as PI3K/Akt and MAPK/ERK, promoting phosphorylation and activation of ion channels; on the other hand, the nerve growth factor/TrkA complex is retrogradely transported to the dorsal root ganglion, promoting the expression of pain mediators such as calcitonin gene-related peptide (CGRP), leading to peripheral pain sensitization [11-14]. Current studies have shown that inhibiting TrkA can effectively alleviate pain sensitization in animal models of various chronic bone diseases such as arthritis and rheumatoid arthritis [15-18]. Therefore, TrkA may be an important target for clarifying the mechanism of pain sensitization in steroid-induced osteonecrosis of the femoral head.

In traditional Chinese medicine theory, steroid-induced osteonecrosis of the femoral head belongs to the category of "bone erosion". Its root cause is deficiency of the liver and kidney, and blood stasis blocking the collaterals. Gugutou Huaisiyu Capsule, a hospital preparation, has the effects of nourishing the liver and kidney, promoting blood circulation, and relieving pain. Previous clinical and experimental studies have shown that it can effectively relieve pain in patients with osteonecrosis of the femoral head and delay the progression of bone necrosis by regulating inflammatory factors such as interleukin-1β and tumor necrosis factor-α. However, whether it can alleviate pain sensitization and its specific mechanism remain unclear. This study aims to investigate the mechanism of Gugutou Huaisiyu Capsule in alleviating peripheral pain sensitization in rats with steroid-induced osteonecrosis of the femoral head, focusing on the TrkA signaling pathway, to provide a theoretical basis for clinical application.