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 programmed cell death that integrates apoptosis (caspase-dependent), necroptosis (RIPK3/MLKL-dependent), and pyroptosis (caspase-8/GSDMD-dependent)⁴ as a key pathological mechanism of secondary injury in SCI. Abnormal activation of (ASC, caspase-8, and RIPK3) induces neuron loss and glial scar formation, exacerbating spasticity⁵. Targeting PANoptosis has thus emerged as a promising strategy to interrupt SCI's secondary injury cascade⁴.

Traditional Chinese medicine (TCM) has gained attention for neurological diseases due to multi-target effects (e.g., anti-inflammation, antioxidant stress)⁶. Gualou Guizhi antispasmodic Granules (GLGZ) is derived from Zhang Zhongjing's Synopsis of the Golden Chamber and optimized as a hospital preparation⁷. Its components include Trichosanthes kirilowii (Tianhuafen), Cinnamomum cassia (Guizhi), Paeonia lactiflora (Baishao), Glycyrrhiza uralensis (Gancao), jujube, and ginger⁸. Preliminary studies have shown GLGZ alleviates spasms after central nervous system injury by regulating neurotransmitters and inhibiting inflammation⁹. However, its effects on SCI-induced spasticity and potential association with PANoptosis remain unelucidated.

Unlike single-target conventional drugs, GLGZ's multi-component nature aligns with SCI's complex pathological network -- addressing inflammation, neuronal loss, myelin damage, and potentially PANoptosis simultaneously. This study hypothesizes that GLGZ ameliorates muscle spasticity in SCI rats by promoting spinal cord pathological repair (reducing inflammation, preserving neurons, repairing myelin) and inhibiting PANoptosis. By verifying this hypothesis, we aim to provide experimental evidence for GLGZ as a promising therapy that integrates symptom relief and core pathological repair for SCI.

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 laminectomy without impact. Inclusion criteria: BBB score < 8 at 24 h post surgery; exclusion criteria: infection, spinal cord transection. Postoperative care: analgesia (buprenorphine, 0.1 mg/kg s.c., q12h for 3 days), bladder expression (q6h for 1 week), and sterile wound care. The successful model rats were randomly assigned to a model group, and high- (2.32 g/kg, n = 10), medium- (1.16 g/kg, n = 10), low-dose (0.58 g/kg, n = 10) GLGZ groups.

Motor function analysis
Basso, Beattie, and Bresnahan (BBB) scoring was performed blindly. The rats were observed in an open field for 5 min, scoring hindlimb joint movement, weight-bearing, gait, and coordination.

Shear wave elastography for measuring rectus femoris stiffness
A Doppler ultrasound system with a linear array probe (18L6) was used. Rats were placed in lateral recumbency (muscle relaxed). SWE was performed with the probe positioned at a 90° angle to the muscle, at a depth of 1-2 cm. The region of interest (ROI) was set to 0.5 × 0.5 cm, and ten frames were acquired per measurement. The final stiffness value represented the average of three valid measurements. Shear wave velocity (SWV) was recorded; green quality control indicated valid images¹¹.

MRI examination
MRI was performed using a 7.0T scanner with a T2weighted fatsuppressed sequence and an animalspecific coil. Acquisition parameters were as follows: repetition time (TR) 3,800 ms, echo time (TE) 72 ms, slice thickness 1 mm, voxel size 0.1 × 0.1 × 1 mm, matrix 256 × 256, field of view (FOV) 25.6 × 25.6 mm, four signal averages, and respiratory gating. Lesion area was measured using image processing software.

Histological staining
HE staining was performed through sequential deparaffinization, rehydration, hematoxylin staining for 5 min, rinsing in running water, differentiation in 1% hydrochloric acid-ethanol for 30 s, eosin staining for 2 min, dehydration, clearing, and mounting. As a visual checkpoint, stained sections showed blue nuclei and red cytoplasm.

Nissl staining was conducted by deparaffinization, rehydration, incubation in 0.1% toluidine blue at 50-60 °C for 30 min, followed by rinsing with distilled water, ethanol differentiation, dehydration, clearing, and mounting. As a visual checkpoint, dark blue Nissl bodies were clearly observed in the sham group.

Luxol fast blue-cresyl violet staining was performed by deparaffinization, rehydration, incubation in Luxol fast blue at 60 °C for 2 h, rinsing with 95% ethanol, differentiation in lithium carbonate for 30 s, followed by 70% ethanol differentiation, cresyl violet counterstaining for 10 min, dehydration, clearing, and mounting. The working concentration of cresyl violet was 0.1%. As a visual checkpoint, dark blue myelin was observed in the sham group.

Multiplex immunofluorescence staining was performed beginning with deparaffinization, rehydration, and antigen retrieval using citrate buffer (pH 6.0) at 95 °C for 20 min. Sections were treated with 3% hydrogen peroxide for 10 min, followed by blocking with 5% BSA at 37 °C for 1 h. Primary antibodies were incubated overnight at 4 °C (ASC: 1:500; Caspase-8: 1:400; RIPK3: 1:500). After washing with PBS-T (0.1% Tween-20, 3 x 5 min), sections were incubated with HRP-labeled secondary antibodies (1:1000) at 37 °C for 1 h. Fluorochrome labeling was then applied: YTR520 Plus for ASC, TYR570 Plus for Caspase8, and TYR690 Plus for RIPK3 (30 min), followed by DAPI nuclear staining for 5 min. Slides were mounted, and imaging was conducted using a fluorescence microscope set at 500 ms exposure, gain 1.0, magnification ×200, with filter sets matching the respective fluorochromes.

For xylene and ethanol, all operations were performed in a fume hood, and gloves and goggles were worn during use. For staining reagents, direct skin contact was strictly avoided. All waste reagents were disposed of in designated hazardous waste containers. For biohazard materials, thorough autoclave sterilization was conducted prior to final disposal to prevent potential biological contamination.

Statistical analysis
One-way ANOVA with LSD post-hoc test was used for group comparisons. P < 0.05 was considered statistically significant.

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