Mechanism of epothilone B improving spinal cord microcirculation after spinal cord injury in rats

Abstract

BACKGROUND: Animal experiments have found that epothilone B can remodel blood microcirculation and reduce tissue scar formation after spinal cord injury, but the specific mechanism remains unclear. OBJECTIVE: To clarify the mechanism by which epothilone B improves spinal cord microcirculation after spinal cord injury. METHODS: Fifty Sprague-Dawley rats were randomly divided into a sham-operated group (n=10), a spinal cord injury group (n=20), and an epothilone B group (n=20). The sham-operated group underwent only laminectomy at T10, while the other two groups were subjected to laminectomy at T10 followed by spinal cord contusion. Immediately after modeling, the epothilone B group received intraperitoneal injections of epothilone B solution, while the other two groups received corresponding solvents. At corresponding time points after modeling, motor function was assessed using Basso-Beattie-Bresnahan (BBB) score, inclined plane test, and open field test; blood flow recovery in the posterior median spinal cord vessels was detected by laser speckle flow imaging; hematoxylin-eosin staining was used to evaluate the overall spinal cord tissue; Western blot was used to detect Toll-like receptor 4 and nuclear factor κB protein expression; immunofluorescence staining was used to detect vascular endothelial growth factor receptor 2 and Toll-like receptor 4 expression in spinal cord injury tissue. RESULTS AND CONCLUSION: (1) The BBB score in the epothilone B group was higher than that in the spinal cord injury group at 14 and 28 days after modeling (P < 0.05), the inclined plane test angle was greater than that in the spinal cord injury group at 28 days after modeling (P < 0.05), and the open field test moving distance was greater than that in the spinal cord injury group at 14 and 28 days after modeling (P < 0.05), indicating that epothilone B improved motor function in rats with spinal cord injury. Laser speckle flow imaging at 28 days after modeling showed that epothilone B increased blood flow recovery in spinal cord injury rats. Hematoxylin-eosin staining at 28 days after modeling showed that the spinal cord cavity area in the epothilone B group was smaller than that in the spinal cord injury group (P < 0.05). Western blot at 5 days after modeling showed that the expression of Toll-like receptor 4 and nuclear factor κB protein in the spinal cord injury group was higher than that in the sham-operated group and epothilone B group (P < 0.05). Immunofluorescence staining at 5 days after modeling showed that the expression of vascular endothelial growth factor receptor 2 in the spinal cord injury group was lower than that in the sham-operated group and epothilone B group (P < 0.05), while the expression of Toll-like receptor 4 was higher than that in the sham-operated group and epothilone B group (P < 0.05). (2) These results indicate that epothilone B may reduce local inflammation after spinal cord injury by regulating the Toll-like receptor 4 and nuclear factor κB pathway, ensuring the regeneration of vascular endothelial cells in spinal cord tissue, thereby promoting the reconstruction of blood microcirculation.

1. Introduction

Spinal cord injury (SCI) has a distinct pathological process characterized by a biphasic pattern comprising primary mechanical injury and secondary pathological injury [1-5]. Biomechanical trauma causing three-dimensional spinal instability can directly lead to structural damage such as neural tissue tearing and axonal transection, with the degree of injury positively correlated with impact kinetic energy. Within 24-72 hours after this phase, a cellular inflammatory molecular cascade is initiated, characterized by: (1) disruption of cellular integrity in the injured area, releasing endogenous ligands for Toll-like receptor 4 (TLR4) such as heat shock proteins and extracellular matrix degradation components, which activate downstream nuclear factor κB (NF-κB) via classical pathways, leading to increased expression of interleukins and tumor necrosis factor α, and inducing neutrophil infiltration [6-7]; (2) neutrophil infiltration and M1 microglial activation forming an inflammatory microenvironment, further increasing secretion of interleukin-1 and tumor necrosis factor α, promoting TLR4 and NF-κB pathway expression, creating a positive feedback loop [7-8]; (3) the inflammatory environment leading to ischemia and hypoxia, mitochondrial damage, decreased intracellular ATP levels, and generation of reactive oxygen species and free radicals, triggering calcium overload and inducing apoptosis [9]; (4) free radical chain reactions causing lipid peroxidation, resulting in myelin sheath disintegration [10]. This vicious cycle ultimately leads to delayed neuronal apoptosis and irreversible neurological deficits [10]. Long-term clinical practice indicates that the majority of primary injuries in SCI patients result from diseases or accidents, making clinical prevention and intervention difficult. Notably, a series of effective clinical interventions have been developed targeting the secondary injury process, which can slow the progression of secondary injury through multi-target mechanisms, thereby achieving protective treatment for residual neurological function.

Clinical observations show that neurological function repair and complication prevention in SCI patients involve dual intervention pathways: direct repair strategies centered on reconstructing neural signal transmission, and indirect protective mechanisms by blocking secondary injury cascades [11-12]. Evidence-based medicine has confirmed that three types of interventions can be used for direct repair: (1) surgical decompression to relieve mechanical compression on neural tissue; (2) application of neurotrophic factors combined with physical therapy to promote axonal regeneration; (3) transplantation of exogenous neural precursor cells to reconstruct neural conduction pathways. In the field of indirect protection, regulatory strategies involve three key links: blocking the inflammatory cascade through anti-inflammatory therapy, using vascular