Therapeutic Effects of Gualou Guizhi Antispasmodic Granules on Spasticity and PANoptosis in a Rat Model of Spinal Cord Injury

Summary

This study evaluates the effects of Gualou Guizhi Antispasmodic Granules (GLGZ) on spasticity induced by spinal cord injury (SCI) using behavioral, biomechanical, imaging, and histological methods and explores PANoptosis-related mechanisms.

Abstract

Muscle spasticity after spinal cord injury (SCI) severely impacts quality of life, with limited effective treatments. Gualou Guizhi Antispasmodic Granules (GLGZ), a hospital-developed traditional Chinese medicine (TCM) formulation, can relieve spasms. This study aimed to assess GLGZ's efficacy in SCI-induced spasticity and its association with PANoptosis (a novel programmed cell death involving apoptosis, necroptosis, and pyroptosis).

Male Sprague-Dawley rats weighing 180-200 g were used. SCI was induced via the Allen weight-drop method. The successful model rats were randomly assigned to a model group or one of the GLGZ dose groups: high, medium, or low. Sham rats underwent laminectomy without spinal cord injury. Interventions lasted 4 weeks. After the intervention, limb motor function was evaluated using the Basso-Beattie-Bresnahan (BBB) scale. Femoral muscle stiffness was assessed via ultrasound shear wave elastography (SWE), while spinal cord integrity was determined with magnetic resonance imaging (MRI). Hematoxylin-eosin (HE), Nissl, and Luxol fast blue staining were performed to observe histopathological alterations. Multiplex immunofluorescence staining was used to label apoptosis-associated speck-like protein containing a CARD (ASC, green), Caspase-8 (orange), and receptor-interacting serine/threonine-protein kinase 3 (RIPK3, yellow) to assess PANoptosis.

GLGZ improved BBB scores dose-dependently: sham (21.00 ± 0.00), model (5.8 ± 1.1), high-dose (10.6 ± 1.3), medium-dose (8.5 ± 1.2), low-dose (7.1 ± 1.0). SWE showed reduced muscle stiffness: model (6.68 ± 0.74 m/s), high-dose (2.19 ± 0.36 m/s, P < 0.001), medium-dose (3.04 ± 0.45 m/s, P = 0.002), low-dose (4.08 ± 0.65 m/s, P = 0.031) vs model. As compared with the model group (3.65 ± 1.03 mm²), GLGZ dose-dependently reduced the spinal cord lesion size in all dosage groups (all P < 0.05), (1.33 ± 0.79 mm² in high-dose, 1.94 ± 0.67 mm² in medium-dose, 2.50 ± 0.79 mm² in low-dose group. Histology revealed GLGZ preserved neurons and promoted myelin repair. GLGZ dose-dependently reduced ASC, Caspase-8, and RIPK3 expression.

Introduction

Muscle spasm is a common complication of spinal cord injury (SCI), severely impacting patients' quality of life. Its pathological process includes initial mechanical injury, secondary inflammation, oxidative stress, apoptosis, and impaired nerve regeneration, among others^1,2. Current treatments (surgical decompression, pharmacotherapy, rehabilitation) have limited efficacy, with long-term medication causing side effects like drug resistance³. Therefore, exploring novel mechanisms and treatments is critical.

Recent studies have identified PANoptosis, a pro....

Protocol

Animal modeling
This study was approved by the Ethics Committee of the Second Affiliated Hospital of Fujian University of Traditional Chinese Medicine (Approval No. FJPSPH-IAEC2024114) and conducted in compliance with animal welfare guidelines. Male Sprague-Dawley rats weighing 180-200 g were housed under controlled conditions at 22-26°C and 40-70% humidity. SCI was induced by the Allen weight-drop method¹⁰: 3-4% isoflurane inhalation anesthesia, midline incision at T10, exposure of T9-T11 segments, laminectomy (dura mater intact), and impact (2 mm tip, 0.5 m/s speed, 1.3 mm depth, 1 s duration). Sham rats underwent ....

Results

BBB score
Sham group BBB score was 21.00 ± 0.00, significantly higher than that of the model (5.8 ± 1.1, P < 0.001). GLGZ dose-dependently increased BBB scores: high-dose (10.6 ± 1.3, P < 0.001), medium-dose (8.5 ± 1.2, P = 0.003), low-dose (7.1 ± 1.0, P = 0.028) vs model (Figure 1).

Shear wave elastography analysis of rectus femoris stiffness
Shear wave elastography (SWE) can reflect changes in muscle stiffness thro.......

Discussion

This study systematically evaluated the ameliorative effects of GLGZ on muscle spasticity in rats with SCI and explored its relationship with neuroprotection and PANoptosis. The results indicated that GLGZ could improve BBB scores and reduce muscle stiffness in SCI rats in a dose-dependent manner. The study also found that the mechanism of action of GLGZ may be related to neuroprotection, myelin repair, and inhibition of PANoptosis. Currently, clinical pharmacological treatments for post-SCI muscle spasticity mainly alle.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anhydrous ethanol	240403A1		Dehydration
Anhydrous ethanol	241101A1		Staining
Antifluorescence attenuated film coating agent (containing DAPI)	MA0221		Immunofluorescence Staining
ASC Antibody	Bs-6741R		PANoptosis analysis (1:300)
Caspase-8 Antibody	13423-1-AP		PANoptosis analysis (1:400)
Fluorescence microscope	NIKON		ECLIPSECi-L
Fully automatic digital glass slide scanner	GCell-60		Slide scanner
hematoxylin-Eosin kit	R23718		Hematoxylin-Eosin (HE) Staining
Image Analysis Software	NIH		ImageJ 1.53k
Luxol Fast Blue	R23193		Luxol Fast Blue-Cresyl Violet Staining
MRI Scanner	Bruker		7.0T
Multiple Fluorescence Staining Kit	240522		Immunofluorescence Staining
Myeloid staining kit	G3245		Myeloid staining
Neutral  balsam	MB9899		Mounting after staining
Nissl staining kit	R23093		Nissl Staining
RIPK3 Antibody	Bs-3551R		PANoptosis analysis (1:400)
SPSS 22.0			statistical analysis software
Sprague-Dawley rats	Hangyi Biotechnology company in Fuzhou
Tartaric acid, sodium tripolyphosphate	10019418		Staining
Ultrasound System	SIEMENS Acuson		Linear array probe (18L6)
xylene	240904A2		Staining