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

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.

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.

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 through color mapping and numerical values. The left image shows the quality control chart, where green indicates good image quality; the right image displays the muscle stiffness color scale, with higher stiffness shown as red or yellow areas and lower stiffness as blue or green areas. Sham SWV was 1.27 ± 0.31 m/s. Model SWV (6.68 ± 0.74 m/s) was significantly higher (P < 0.001). GLGZ reduced SWV: 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 (Figure 2). Visual inspection revealed a blue-dominant color in the high-dose group, whereas the model group showed red or yellow.

Spinal cord MRI examination
T2-weighted fat-suppressed MRI revealed no spinal cord deformation or abnormal signals in the sham-operated group. The model group exhibited localized spinal cord thinning with mixed signal intensity in the lesion area (indicating inflammatory response, necrotic tissue, and glial scar formation), with a lesion area of 3.65 ± 1.03 mm². GLGZ significantly reduced spinal cord lesion size across all dosage groups (all P < 0.05) in a dose-dependent manner: The high-dose group (1.33 ± 0.79 mm², P < 0.001) exhibited near-normal signal intensity, the medium-dose group (1.94 ± 0.67 mm², P = 0.004) showed moderate improvement, while the low-dose group (2.5 ± 0.79 mm², P = 0.035) demonstrated only mild alleviation (Figure 3).

HE staining
HE staining revealed an intact spinal cord structure in the sham-operated group, characterized by evenly distributed and neatly arranged nerve cells, with no cavities or necrotic tissue (Figure 4). In the model group, spinal cord tissue exhibited damage and structural disorganization, significant reduction of neuronal cells, blurred boundaries between gray and white matter, vacuolation, necrosis, extensive inflammatory cell infiltration, and glial scar formation. In the high-dose GLGZ group, the damaged areas of spinal cord tissue were significantly reduced, the structure was partially restored, and the neuronal arrangement became more regular. The medium-dose GLGZ group still exhibited inflammatory cell infiltration and necrotic areas, accompanied by more glial scars; however, these were improved compared to the model group, although the recovery was less pronounced than in the high-dose group. The low-dose GLGZ group exhibited higher structural disorganization, with persistent vacuolation and necrotic areas, but overall conditions were better than those of the model group.

Nissl staining
Nissl staining was primarily used to observe neuronal structure, particularly the distribution and morphology of Nissl bodies (which represent rough endoplasmic reticulum involved in protein synthesis). In the sham-operated group, neurons were abundant and arranged in an orderly manner within the tissue. The neuronal nuclei were large and round, with clearly visible nucleoli, and Nissl bodies were evenly distributed (Figure 5). In contrast, the model group exhibited a significant reduction in neuronal count, with some neurons showing atrophy or degenerative changes. Cell boundaries became blurred and indistinct. The number of Nissl bodies markedly decreased, and their staining intensity weakened, with complete absence of Nissl bodies in some neurons. The tissue structure was severely disorganized, and the gaps between neurons significantly widened. In the high-dose GLGZ group, neuronal numbers increased significantly, and cell body sizes approached normal conditions. Nuclei were clearly visible with distinct nucleoli. The number of Nissl bodies recovered substantially, showing relatively even distribution and deep blue staining. The degree of tissue disorganization was significantly improved. The medium-dose GLGZ group showed an increase in neuronal count compared to the model group, though it remained lower than that of the high-dose group. The number and distribution of Nissl bodies exhibited partial recovery, and tissue arrangement disorder was somewhat alleviated. However, enlarged interneuronal gaps and mild structural disorganization were still observable. In the low-dose GLGZ group, neuronal numbers increased slightly compared to the model group; however, most neurons remained atrophic, with blurred cell contours. The improvement in the number and distribution of Nissl bodies was limited, and staining remained faint. The tissue structure continued to show significant disorganization.

Luxol Fast Blue-Cresyl Violet staining for myelin integrity assessment
Luxol fast blue selectively binds to phospholipid components in the myelin sheath, staining myelinated areas a characteristic dark blue. Cresyl violet is used as a counterstain for nuclei and other tissue structures, effectively enhancing overall contrast. Sham-operated group: The myelin structure was intact without damage, showing uniform and consistent staining with intense dark blue coloration and regular distribution, clearly demonstrating typical morphological features of normal spinal cord myelin (Figure 6). Model group: Significant myelin damage was observed, with markedly reduced blue staining intensity and disorganized staining distribution. Clearly visible demyelinated areas indicated compromised myelin integrity. GLGZ intervention groups: the effects of myelin repair showed a dose-dependent trend. The high-dose GLGZ group exhibited myelin staining depth similar to the sham-operated group, with darker staining, significantly reduced demyelinated areas, and well-restored myelin integrity. In contrast, the low-dose GLGZ group exhibited noticeably lighter staining intensity, numerous white vacuoles within the field of view, and limited myelin repair, with persistent structural abnormalities.

Immunofluorescence staining of PANoptosis
The multiplex immunofluorescence staining results demonstrated the expression and distribution of target proteins (ASC, Caspase-8, and RIPK3) in each group. Nuclei were stained blue, ASC appeared green, Caspase-8 was orange, and RIPK3 was yellow (Figure 7).

Quantitative analysis using ImageJ showed that the proportion of triple-positive cells in the sham-operated group (Sham) was 2.53 ± 0.87%. In contrast, this proportion significantly increased to 40.75 ± 4.26% in the model group (Model, P < 0.05). GLGZ dose-dependently inhibited PANoptosis: the proportion of triple-positive cells decreased to 7.03 ± 1.55% in the high-dose GLGZ group (P < 0.05 vs Model); both the medium-dose (15.71 ± 2.84%) and low-dose (28.77 ± 3.64%) GLGZ groups exhibited significant reductions (both P < 0.05 vs Model), albeit with less pronounced effects than the high-dose group.

DATA AVAILABILITY
Raw data will be uploaded to FigShare (10.6084/m9.figshare.30995272) for public access.

Figure 1. BBB scores. Statistical bar graph of BBB scores (n = 10/group). Data are mean ± SD. One-way ANOVA with LSD post-hoc test. *P < 0.05 vs Sham; #P < 0.05 vs Model. Please click here to view a larger version of this figure.

Figure 2. Ultrasonic shear wave elastography of the rectus femoris. (A) Sham-operated group, (B) Model group, (C) High-dose group, (D) Medium-dose group, (E) Low-dose group. In each figure, the left panel is the quality control image, and the right panel is the elastic color-coded image. The circles in the figures indicate sampling sites within the region of interest, and the median shear wave velocity (Vs Median) is shown. (F) Statistical bar graph of shear wave velocity in the rectus femoris of each group. (n= 10/group). Data are presented as mean ± standard deviation. *P < 0.05 vs Sham-operated group, #P < 0.05 vs Model group. Abbreviation: SWV = shear wave velocity. Please click here to view a larger version of this figure.

Figure 3. Spinal cord coronal MRI images. (n = 3/group). (A) Sham (no abnormal signals); (B) Model; (C) High-dose GLGZ; (D) Medium-dose GLGZ; (E) Low-dose GLGZ. Please click here to view a larger version of this figure.

Figure 4. HE staining of spinal cord tissue. (×40, n = 10/group). (A) Sham; (B) Model; (C) High-dose GLGZ; (D) Medium-dose GLGZ; (E) Low-dose GLGZ. Scale bar = 200 µm. Please click here to view a larger version of this figure.

Figure 5. Nissl staining of neuronal structure. (×200) (A) Sham; (B) Model; (C) High-dose GLGZ; (D) Medium-dose GLGZ; (E) Low-dose GLGZ. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 6. Luxol Fast Blue-Cresyl Violet staining. (×100) (A) Sham (intact myelin); (B) Model (demyelination); (C) High-dose GLGZ (myelin repair); (D) Medium-dose GLGZ; (E) Low-dose GLGZ. Scale bar = 200 µm. Please click here to view a larger version of this figure.

Figure 7. Multiplex immunofluorescence staining. (×200). Nuclei were stained blue, ASC was visualized in green, Caspase-8 in orange, and RIPK3 in yellow. The expression and distribution of these target proteins (ASC, Caspase-8, and RIPK3) suggest the occurrence of PANoptosis. (A) Sham (low expression); (B) Model (high expression); (C) High-dose GLGZ (reduced expression); (D) Medium-dose GLGZ; (E) Low-dose GLGZ. Scale bar = 100 µm. Abbreviations: ASC = Apoptosis-associated Speck-like protein containing a CARD; RIPK3 = Receptor-Interacting Protein Kinase 3. Please click here to view a larger version of this figure.

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 alleviate symptoms without addressing the core pathological issues of spinal cord injury. This study, through animal experiments, confirmed that GLGZ not only effectively improves the symptoms of muscle spasticity but also targets the core pathological processes of spinal cord injury for repair. This provides experimental evidence for the use of GLGZ in treating spinal cord injury.

The results demonstrated that GLGZ significantly improved the Basso-Beattie-Bresnahan (BBB) scores in SCI rats. Compared to the model group, the high-, medium-, and low-dose groups showed increases in BBB scores by 82.76%, 46.55%, and 22.41%, respectively, indicating a dose-dependent effect. These results suggest that GLGZ effectively enhances limb motor function in SCI rats. Quantitative assessment of rectus femoris stiffness using shear wave elastography further confirmed that GLGZ reduced muscle stiffness in a dose-dependent manner. Compared to the model group (6.68 ± 0.74 m/s), SWV were significantly reduced in all dose groups: the low-dose group (4.08 ± 0.65 m/s), the medium-dose group (3.04 ± 0.45 m/s), and the high-dose group (2.19 ± 0.36 m/s). This technique, by measuring tissue elasticity (Young's modulus), can capture subtle changes in muscle stiffness, addressing the subjectivity limitations of traditional muscle tone assessment methods such as the Modified Ashworth Scale. These findings are consistent with the results of Cho KH et al., who suggested that ultrasound imaging could serve as a valuable non-invasive tool for assessing stiffness in spastic muscles and may help identify histopathological changes associated with spinal cord injury.

This study, through MRI and histological analysis, found that GLGZ significantly alleviated inflammatory responses in the spinal cord injury area, reduced tissue necrosis, increased the number of neurons and Nissl bodies, and promoted myelin repair. Hematoxylin and eosin (H&E) staining results visually demonstrated a significant reduction in inflammatory cell infiltration in the injured area, indicating that GLGZ effectively inhibited the aggregation and activation of inflammatory cells. Nissl staining showed improved integrity of neuronal structures and an increased number of Nissl bodies, suggesting enhanced metabolic and functional states of neurons. Luxol fast blue-cresyl violet staining revealed that myelin morphology tended to normalize, indicating that GLGZ played an active role in promoting myelin regeneration. These results collectively suggest that GLGZ may protect neuronal survival, promote myelin regeneration, and ultimately improve neural conduction function by inhibiting local inflammatory cascades in the spinal cord and reducing damage to neurons and nerve fibers caused by inflammatory factors⁹.

These findings are consistent with studies on GLGZ promoting neurological recovery and neurogenesis after focal cerebral ischemia/reperfusion⁹. The results demonstrate the potential of GLGZ in improving muscle spasticity and motor function recovery after spinal cord injury, which aligns closely with its efficacy in "softening tendons and relieving spasms"¹². This also fully reflects the unique advantage of traditional Chinese medicine compounds in improving SCI pathological damage through multiple targets and pathways⁶. GLGZ is derived from the classic formula Trichosanthes and Cinnamon Twig Decoction from the "Synopsis of the Golden Chamber," composed of various medicinal herbs such as Trichosanthes kirilowii, cinnamon twig, white peony root, licorice, and ginger¹³. Among them, cinnamon twig has the effects of warming yang, relieving surface symptoms, dispersing cold, and activating collaterals. It promotes local blood circulation and improves muscle blood supply, thereby effectively alleviating muscle spasms caused by poor qi and blood flow¹⁴.

Trichosanthes kirilowii exhibits strong moistening, drying, and anti-swelling effects, which help to relax muscles and relieve spasms. White peony root can "nourish blood and soften tendons," alleviating muscle tension and spasms caused by blood deficiency or qi stagnation by improving blood circulation and reducing muscle tension. Licorice harmonizes the effects of various herbs and alleviates excessive excitability of the central nervous system by modulating neurotransmitters. Ginger warms yang, disperses cold, promotes blood circulation, enhances blood flow, and further reduces spastic symptoms. Red dates regulate the spleen and stomach, enhance physical strength, and nourish qi and blood. These herbs work together to harmonize yin and yang, promote blood circulation, remove blood stasis, soften tendons, and nourish yin to promote fluid production. Our results indicated the improvement effects of GLGZ on the histopathological morphology of spinal cord tissue, further revealing its potential mechanisms for SCI.

Multiplex immunofluorescence staining results showed that the expression levels of ASC, Caspase-8, and RIPK3 proteins in the spinal cord tissue of the model group were significantly elevated, with corresponding fluorescence intensities also markedly enhanced, indicating that the PANoptosis pathway may have been activated. PANoptosis is a recently recognized form of programmed cell death that plays a key role in neural cell damage following SCI¹⁵. Excessive activation of PANoptosis promotes the formation of inflammasomes via ASC, activates apoptotic signaling through Caspase-8, and mediates necroptosis via RIPK3, resulting in massive death of neurons and glial cells¹⁶. This disrupts and further deteriorates the homeostasis of the spinal cord microenvironment, thereby exacerbating neurological damage. After GLGZ intervention, the fluorescence intensities of these proteins showed a dose-dependent decreasing trend, indicating that GLGZ can block the PANoptosis process. By inhibiting ASC activity, GLGZ blocks inflammasome assembly, thereby reducing the release of inflammatory factors and mitigating damage to neural cells caused by neuroinflammation¹⁷. Simultaneously, GLGZ can inhibit the expression of Caspase-8 and RIPK3, reducing apoptosis and necrosis of neural cells and protecting the integrity of spinal cord function. The mechanism by which GLGZ regulates neural repair through inhibiting the PANoptosis pathway is unique, providing an important theoretical basis for further understanding the therapeutic effects of GLGZ.

This study provided preliminary evidence that GLGZ has the potential to ameliorate muscle spasticity in rats with SCI. The mechanism may be related to anti-inflammation, myelin repair and the inhibition of PANoptosis. There are three critical procedural steps. For SCI modeling, strict control of impact parameters (speed, depth, duration) and postoperative analgesia/bladder care ensured model consistency and reduced mortality. In SWE, muscle relaxation and standardized ROI placement avoided false stiffness measurements. Immunofluorescence utilized citrate buffer (pH 6.0) for antigen retrieval (optimizing antibody binding) and sequential labeling (mitigating fluorochrome cross-reactivity).

This protocol integrates behavioral (BBB), biomechanical (SWE), imaging (MRI), and histological assessments, thereby overcoming the subjectivity inherent in traditional spasticity scales (e.g., Modified Ashworth Scale). SWE provides quantitative data on muscle spasticity, while multiplex immunofluorescence simultaneously detects three PANoptosis markers, offering a comprehensive mechanistic view. The methodological limitation of this study is that neither the upstream regulatory mechanisms of PANoptosis nor its interactions with other cell death pathways were explored. SWE can be utilized for longitudinal clinical monitoring of spasticity in future methodological applications.

Troubleshooting strategies were implemented as follows: For inconsistent SWV measurements, animals were placed on their left side to alleviate muscle tension. To minimize motion artifacts in MRI, respiratory gating was combined with short repetition time (TR) and echo time (TE) parameters. During histological staining, over-differentiation was prevented by microscopically monitoring color development, while insufficient staining was corrected by extending the staining duration. In summary, GLGZ significantly improved muscle spasticity in SCI rats, which may be related to its promotion of spinal cord pathological repair and inhibition of PANoptosis. Our study provides new insights for developing drugs that combine symptom relief with neuroprotective functions.