Exploring the Mechanism of Medicated Thread Moxibustion of Zhuang Medicine in Diabetic Gastroparesis Rats Based on the Enteric Nervous System

ABSTRACT

Background

Diabetic gastroparesis (DGP) is a common complication of diabetes mellitus, for which Western medicine treatments are associated with adverse effects and limitations. Enteric nervous system (ENS) injury is a core mechanism underlying this condition. Although Medicated Thread Moxibustion of Zhuang Medicine shows significant therapeutic efficacy, the specific mechanism by which it regulates the ENS to improve DGP remains unclear.

Methods

A DGP rat model was established by intraperitoneal injection of streptozotocin (STZ) (60 mg/kg). The experimental animals were divided into five groups: control group, model group, mosapride citrate group, non‐meridian and non‐acupoint group, and Yangming meridian acupoint group. The Yangming meridian acupoint group received Medicated Thread Moxibustion of Zhuang Medicine at bilateral ST21, ST30, ST36, and ST40. Blood glucose, body weight, gastric emptying rate, and small intestinal propulsion rate were measured in each group. Hematoxylin–eosin (HE) staining was used to observe the histomorphology of the antrum and duodenum. Western blotting, immunofluorescence, and immunohistochemistry were performed to detect markers of enteric neurons (PGP9.5, ChAT, and neuronal nitric oxide synthase [nNOS]) and enteric glial cells (EGCs) (S100β, glial fibrillary acidic protein [GFAP], and Glial‐cell line‐derived neurotrophic factor [GDNF]).

Results

Following treatment with Medicated Thread Moxibustion of Zhuang Medicine at the Yangming meridian acupoints, DGP rats exhibited reduced blood glucose levels, restored body weight, increased gastric emptying rate and small intestinal propulsion rate, and ameliorated pathological damage in the gastric antrum and duodenum. Moreover, the expression levels of PGP9.5, ChAT, nNOS, S100β, GFAP, and GDNF were elevated. In addition, the glucose‐lowering effect of this therapy was superior to that of mosapride citrate.

Conclusion

Medicated Thread Moxibustion of Zhuang Medicine at the Yangming meridian acupoints effectively restores gastrointestinal motility in DGP rats by upregulating the expression of marker proteins of enteric neurons and EGCs, thereby ameliorating the morphological damage to both cell types.

Key Points

• Medicated Thread Moxibustion of Zhuang Medicine effectively protects and repairs the damaged enteric nervous system (ENS) in diabetic gastroparesis (DGP) rats, and its mechanism of action may involve the regulation of neurotransmitter secretion and the restoration of neuron‐ and enteric glial cells‐related protein expression.

• This study is the first to link Medicated Thread Moxibustion of Zhuang Medicine to ENS repair in DGP, bridging traditional medical practice with modern neurogastroenterology.

• This study provides robust preclinical evidence for the therapeutic potential of Medicated Thread Moxibustion of Zhuang Medicine in DGP, supporting its clinical application.

This study systematically evaluates the therapeutic effect of Medicated Thread Moxibustion of Zhuang Medicine on Yangming meridian acupoints by establishing a diabetic gastroparesis (DGP) rat model and explores whether it improves gastrointestinal motility by repairing enteric nervous system (ENS) damage.

1. Introduction

Gastroparesis is a gastrointestinal motility disorder characterized by symptoms such as nausea, vomiting, bloating, early satiety, postprandial fullness, and epigastric discomfort [1]. Epidemiological studies indicate that diabetes mellitus is the most common cause of gastroparesis, accounting for 57.4% of all cases [2]. Meanwhile, diabetic gastroparesis (DGP) is the most symptomatic type of gastroparesis [3], with a significantly higher mortality rate and increased living costs for patients compared to other types of gastroparesis [4]. Existing pharmacological therapies, such as prokinetic drugs and glucose control medications, have limitations, and surgical treatments face restrictions due to risks and indications. Therefore, exploring safe and effective novel therapeutic approaches is of great clinical significance for improving the prognosis of DGP patients.

Recent studies have highlighted the crucial role of the enteric nervous system (ENS) in physiological processes such as gastrointestinal motility, secretion, and immune regulation [5] The ENS is composed of a large number of enteric neurons and enteric glial cells (EGCs), which interact with each other to participate in regulating various physiological functions, such as gastrointestinal motility and sensation. Research has shown that chronic hyperglycaemia affects the structural and functional characteristics of intestinal neurons, leading to reduced cell viability of enteric neurons and EGCs, oxidative stress, and inflammatory responses, ultimately causing cell damage or apoptosis [6, 7]. This ENS damage disrupts the normal neural regulation of the gastrointestinal tract, leading to motility disorders and playing a key role in the development of DGP. However, there are still many gaps in current research on how to repair damaged ENS to improve DGP. Notably, gastric emptying, a precisely coordinated physiological process, depends on the coordination of rhythmic relaxation of the pyloric sphincter in the gastric antrum and the peristalsis of the proximal duodenum [8]. Most existing studies focus on the pathological mechanisms of the gastric antrum tissue, and the role of the duodenum in delayed gastric emptying in DGP has not been fully elucidated.

Medicated Thread Moxibustion of Zhuang Medicine, an intangible cultural heritage of China, has a unique theoretical system and rich clinical application experience. In clinical practice, Medicated Thread Moxibustion of Zhuang Medicine has been widely applied to gastrointestinal diseases, skin diseases, and pain‐related disorders [9, 10, 11, 12]. Given the pivotal role of the ENS in the pathogenesis of DGP, and the unique advantages of Medicated Thread Moxibustion of Zhuang Medicine in regulating bodily functions, this study aims to establish a rat model of DGP to systematically investigate the therapeutic effects of Medicated Thread Moxibustion of Zhuang Medicine on DGP rats, and to deeply analyze its potential mechanisms of action based on the ENS. This research is expected not only to reveal the scientific essence of Medicated Thread Moxibustion of Zhuang Medicine in treating DGP and provide a solid theoretical foundation for its clinical application but also to open up new avenues for the treatment of DGP, bearing significant clinical guiding significance.

2. Materials and Methods

2.1. Experimental Animals

A total of 70 healthy SPF‐grade male Sprague–Dawley (SD) rats, with body weights ranging from 220 to 250 g, were selected for this study. These rats were provided by Hunan Slack Jingda Laboratory Animal Co. Ltd. (License No. SCXK [Xiang] 2019–0004). The experimental animals were housed in the Animal Experiment Center of Guangxi University of Chinese Medicine and underwent adaptive feeding under standard environmental conditions: the relative humidity in the laboratory was controlled at (55 ± 5)%, the temperature was maintained at (20 ± 2) °C, the light cycle was a 12‐h light–dark alternation, and free access to water and ordinary feed was provided. The experimental protocol was approved by the Animal Experiment Ethics Committee of Guangxi University of Chinese Medicine (GXTCMU‐EC KS20250000‐204), and all experimental operations were strictly performed according to animal ethics guidelines.

2.2. DGP Model Establishment

Refer to previous studies, the DGP model was established by intraperitoneal injection of streptozotocin (STZ) [13, 14]. Rats in the model preparation group were fasted for 12 h (with free access to water), and fasting blood glucose was measured. A 1% STZ solution was prepared by diluting STZ in 0.1 mmol/L sodium citrate buffer (pH 4.5) at 4°C. After weighing, the rats were intraperitoneally injected with the STZ solution at a dose of 60 mg/kg. Random blood glucose was measured again 72 h later, and rats with blood glucose ≥ 16.7 mmol/L were considered successfully modeled for diabetes. Rats with successful diabetes modeling were continuously raised for an additional 8 weeks. Blood glucose was measured weekly during this period, and rats with blood glucose ≤ 16.7 mmol/L were excluded. After 8 weeks, three rats were selected from the control group and the model preparation group to measure the small intestinal propulsion rate and gastric emptying rate. The model was evaluated by combining the blood glucose levels and the general conditions of all rats. The criteria for successful DGP rat modeling were as follows:

(a). Random blood glucose ≥ 16.7 mmol/L; (b). Obvious differences in general conditions between the rats and normal rats; (c). Three rats were randomly selected from the control group and the model preparation group, respectively, to measure the gastric emptying rate and small intestinal propulsion rate. Compared with the control group, the model preparation group showed a significant decrease (p < 0.05).

2.3. Grouping

A total of 70 SD rats were divided into a control group (13 rats) and a model preparation group (57 rats) using a random number table method. After 1 week of adaptive feeding, the DGP model was established in the model preparation group via intraperitoneal injection of STZ, while the control group was injected with an equal volume of citrate buffer solution. During the modeling period, seven rats in the model preparation group died, and two rats failed to meet the blood glucose criteria. To determine model success, three rats were randomly selected from the control and model preparation groups for detecting gastric emptying and small intestinal propulsion rates. After confirming successful modeling, the remaining 45 rats in the model preparation group were randomly divided into the model group (Model), mosapride citrate group (MC), non‐meridian and non‐acupoint group (NMNA), and Yangming meridian acupoint group (YMA), with 10 rats in each group. The remaining five rats were set as replacement reserves for experimental loss. If any rat in a group died accidentally or had missing data during the experiment, replacements would be supplemented from the reserve group according to random principles (Figure 1A).

FIGURE 1.

(A) Experimental timeline diagram; (B) Simplified schematic of Medicated thread moxibustion of Zhuang medicine intervention in DGP rats. DGP, diabetic gastroparesis; MC, mosapride citrate group; NMNA, non‐meridian and non‐acupoint group; YMA, Yangming meridian acupoint group.

2.4. Intervention Methods

After successful DGP modeling, the rats in the model preparation group, divided into four groups, were subjected to a 3‐week intervention as follows:

YMA: Rats in this group received Medicated Thread Moxibustion of Zhuang Medicine at bilateral Liangmen (ST21), Qichong (ST30), Zusanli (ST36), and Fenglong (ST40). They were fed normally and received distilled water by gavage at a dose of 10 mL/kg body weight once daily for three consecutive weeks.

Specific operation of Medicated Thread Moxibustion of Zhuang Medicine: Rats were restrained in a supine position. Type II Medicated Thread Moxibustion of Zhuang Medicine thread with a diameter of 0.7 mm was used. The thread was first twisted tightly and straightened. The thread was held between the thumb and index finger of the right hand, with a 1–2 cm thread end exposed. The thread end was ignited, and after the flame extinguished, a bead‐like spark appeared at the thread end. It was quickly pressed onto bilateral Liangmen (ST21, the navel is located at the lower 1/4 of the line connecting the upper border of the sternal manubrium and the pubic symphysis. Liangmen is at the intersection of the midline between the navel and xiphoid process and the midclavicular line, approximately 5 mm lateral to the umbilicus). Qichong (ST30, at the upper border of the pubic symphysis, approximately 5 mm lateral to the umbilicus), Zusanli (ST36, posterolateral to the knee joint of the hind limb, 5 mm straight below the fibular head), and Fenglong (ST40, posterolateral to the knee joint, at the midline of the fibula, approximately 7 mm below the fibular head). Removing the thread end from the acupoint after the spark extinguished counted as 1 time. Moxibustion was performed at 0, 10, and 20 min after binding, with 3× per acupoint per session, alternating between bilateral acupoints (Figure 1B).

NMNA: Operations were the same as those for the YMA group, but moxibustion was applied to “non‐meridian and non‐acupoint” sites. The non‐acupoint sites for ST21 and ST30 were located 2.5 mm laterally from the acupoints (at the inner 3/4 and outer 1/4 of the horizontal line connecting the anterior midline and the nipple). The nonacupoint sites for ST36 and ST40 were located 5 mm laterally from the acupoints, bilaterally.

MC: Rats were gavaged with mosapride citrate suspension at a dose of 10 mL/kg body weight. All interventions were performed once daily for 3 consecutive weeks.

2.5. Blood Glucose

Blood glucose concentration was detected by caudal vein blood sampling. Blood glucose was measured every 2 weeks during modeling and once a week during the intervention treatment.

2.6. Body Weight

The body weight of rats in each group was measured using an electronic scale before modeling, after modeling, and after intervention.

2.7. Gastric Emptying Rate

The complete stomach was incised along the lesser curvature, and the gastric contents were rinsed with distilled water and made up to 20 mL. Then 20 mL of 0.5 mol/L NaOH was added, mixed thoroughly, and left to stand for 2 h. Five milliliters of the supernatant was taken, mixed with 0.5 mL of 20% trichloroacetic acid to remove proteins, centrifuged at low temperature (4°C, 3500 r/min, 20 min), and the supernatant was collected to measure the absorbance value at 560 nm (OD1). Additionally, 2 mL of 50 mg/dL phenol red solution was used to measure the absorbance of standard phenol red (OD2). The gastric emptying rate was calculated as (1−OD1/OD2) × 100%.

2.8. Small Intestinal Propulsion Rate

The dissected small intestine was spread straight on a low‐temperature operating table. The total length from the pylorus to the ileocecal valve (A) was measured with a ruler. The terminal position of the small intestine stained with phenol red was observed, and a small incision was made at this position. A small amount of 0.5 mol/L NaOH solution was dropped—if the solution turned purple, it indicated the phenol red arrival site. NaOH solution was dropped before and after this area to determine the actual position reached by phenol red. The distance from the pylorus to the red‐stained terminal of phenol red (a) was measured. The small intestinal propulsion rate (%) was calculated as (a/A) × 100%.

2.9. Hematoxylin–Eosin (HE) Staining

Tissues were harvested, dehydrated, embedded into wax blocks, and sectioned at a thickness of 3 μm. Sections were mounted on slides and baked at 65°C overnight. For routine dewaxing to water, slides were placed on a rack and sequentially immersed in dewaxing solution I, dewaxing solution II, and dewaxing solution III for 10 min each, followed by anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 min each, then rinsed with running water for 2 min. Nuclei were stained with hematoxylin for 5 min, rinsed with running water until clear, differentiated with hydrochloric acid alcohol solution for 2–3 s, washed with water for 1 min, and blued by rinsing with tap water. Nuclear staining was observed under a microscope. Cytoplasm was stained with eosin for 1–2 min, followed by dehydration in 75% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 1 min each, anhydrous ethanol III for 5 min, and clearing in clearing solution I, clearing solution II, and clearing solution III for 5 min each. Sections were coverslipped with neutral gum and examined under an optical microscope.

2.10. Western Blot (WB) Analysis for Detecting PGP9.5, ChAT, nNOS, S100β, GFAP, and GDNF Protein Expression in Gastric and Duodenal Tissues

Gastric antrum and duodenal tissues from each group of rats were fully ground, mixed with appropriate high‐efficiency RIPA lysis buffer, and subjected to protein quantification using a BCA protein concentration assay kit. After protein gel preparation, electrophoresis, membrane transfer, and overnight blocking, primary antibodies (GAPDH antibody at 1:40000 dilution, glial fibrillary acidic protein (GFAP) antibody at 1:10000, ChAT antibody at 1:3000, neuronal nitric oxide synthase (nNOS) antibody at 1:2500, PGP9.5, GDNF, and S100β antibodies at 1:2000) were added and incubated at 4°C on a shaker. The membranes were washed three times for 15 min each, followed by secondary antibodies (goat anti‐mouse IgG or goat anti‐rabbit IgG, both at 1:8000 dilution). Protein bands were visualized by gel imaging, and grayscale values were analyzed using Image J software.

2.11. Immunofluorescence Double Staining for Detecting nNOS, ChAT, S100β, and GFAP Protein Expression in Gastric and Duodenal Tissues

Gastric antrum and duodenal tissues from each group were dehydrated, embedded into wax blocks, and sectioned. Sections were placed in 3% citric acid repair solution, subjected to high‐pressure antigen retrieval for 10 min, and slowly cooled to room temperature. 3% hydrogen peroxide was added for 20 min at room temperature, followed by BSA blocking solution for 20 min. After removing the blocking solution, primary antibody (nNOS antibody at 1:100 dilution, S100β antibody at 1:150 dilution) was added, and sections were incubated at 4°C overnight. The next day, after returning to room temperature, sections were washed three times with PBS for 5 min each, followed by HRP‐conjugated anti‐rabbit secondary antibody in the dark for 50 min at room temperature and PBS washing. A TSA staining working solution was prepared by mixing 1 mL of Tyramide diluent containing 0.03‰ H₂O₂ with 2 μL of IF488‐Tyramide, and 100 μL was added to each section for 10 min in the dark at room temperature, followed by PBS washing. Microwave treatment was performed, and the steps were repeated for the second primary antibody (ChAT antibody at 1:150 dilution, GFAP antibody at 1:120 dilution), secondary antibody, and SA (IF594‐Tyramide) labeling, with microwave treatment to remove primary and secondary antibodies. Nuclei were counterstained with DAPI, and sections were coverslipped for microscopic observation and image acquisition under appropriate fluorescent channels.

2.12. Immunohistochemistry (IHC) for Detecting GDNF Protein Expression in Gastric and Duodenal Tissues

Gastric antrum and duodenal tissues were dehydrated, paraffin‐embedded, sectioned, and baked. After dewaxing to water and antigen retrieval, sections were washed three times with PBS for 5 min each, followed by blocking of endogenous peroxidase and another three PBS washes. Primary antibody (Anti‐GDNF antibody at 1:100 dilution) was added and incubated at 4°C overnight, followed by three PBS washes at room temperature on a shaker. Goat Anti‐Rabbit IgG H&L (HRP)‐conjugated secondary antibody was applied evenly, incubated at 37°C for 30 min, and washed three times with PBS. Sections were developed, counterstained with hematoxylin, dehydrated, coverslipped, and observed under an optical microscope.

2.13. Statistical Analysis

Data were processed using SPSS 27.0 software, and statistical graphs were generated with GraphPad Prism 10.1. Measurement data are presented as mean ± standard deviation (x̄ ± s). The Shapiro–Wilk test was used to analyze normal distribution; non‐normally distributed data were analyzed by the Kruskal–Wallis nonparametric test. For normally distributed data with homogeneous variance, one‐way ANOVA was used for intergroup comparison, followed by LSD for multiple comparisons. For non‐normally distributed data or heterogeneous variance, rank sum tests were applied. Repeated measures data across different time points were analyzed by repeated measures ANOVA if satisfying normality, homogeneous variance, and sphericity; otherwise, adjustments were made using the Greenhouse–Geisser or Lower‐bound method. Statistical significance was set at p < 0.05 with α = 0.05.

3. Results

3.1. Blood Glucose

To evaluate the changes in blood glucose levels, experimental animals were continuously monitored. From the successful establishment of the diabetes model (week 0) to the completion of the DGP model (week 8), all animals subjected to STZ induction exhibited significantly elevated blood glucose levels, which remained in a hyperglycemic state. Starting from week 9, blood glucose levels decreased following treatment with Medicated Thread Moxibustion of Zhuang Medicine at the Yangming meridian acupoints (p < 0.05); while a reduction in blood glucose was also observed after western medicine intervention, the difference was not statistically significant compared with the model group (p > 0.05) (Figure 2A).

FIGURE 2.

(A) Changes in blood Glucose levels among groups of rats (𝑥¯ ± s，n = 10/group); (B) Changes in body weight of rats in each group (𝑥¯ ± s，n = 10/group). Statistical significance: **p < 0.01 vs. control group; ^## p < 0.01 vs. model group; ^ΔΔ p < 0.01 vs. Yangming meridian acupoint group. DGP, diabetic gastroparesis; MC, mosapride citrate group; Model, model group; NMNA, non‐meridian and non‐acupoint group; YMA, Yangming meridian acupoint group.

3.2. Body Weight

We assessed changes in body weight before modeling, after successful establishment of the DGP model, and following intervention. Body weight decreased significantly after DGP model induction (p < 0.01). Treatment with Medicated Thread Moxibustion of Zhuang Medicine at the Yangming meridian acupoints, as well as western medicine intervention, led to an increase in body weight (p < 0.01) (Figure 2B).

3.3. Comparison of Gastric Emptying Rate and Small Intestinal Propulsion Rate Among Rat Groups

In DGP model rats, both gastric emptying rate and small intestinal transit rate were significantly decreased (p < 0.01). Following intervention with Medicated Thread Moxibustion of Zhuang Medicine at the Yangming meridian acupoints or mosapride citrate, these parameters were significantly increased (p < 0.01), with no significant difference observed between the two intervention groups (Figure 3).

FIGURE 3.

Changes in the gastric emptying rate and small intestinal propulsion rate of rats in each group(𝑥¯ ± s, n = 10/group). Statistical significance: **p < 0.01 vs. control group; ^## p < 0.01 vs. model group; ^ΔΔ p < 0.01 vs. Yangming meridian acupoint group. MC, mosapride citrate group; Model, model group; NMNA, non‐meridian and non‐acupoint group; YMA, Yangming meridian acupoint group.

3.4. Comparison of Tissue Pathological Changes Among Rat Groups

In the DGP model rats, smooth muscle tissue exhibited pathological damage, characterized by disrupted cell structure, incomplete cell membranes, irregularly shaped nuclei, pale or translucent cytoplasm with vacuolar changes, and disorganized arrangement. Following intervention with Medicated Thread Moxibustion of Zhuang Medicine at the Yangming meridian acupoints or mosapride citrate, the pathological damage was markedly alleviated: cell morphology approached normal, cell membrane structure was largely intact, cytoplasmic vacuolation was notably reduced, and cell arrangement became orderly, with the degree of cell–cell junction integrity approaching that observed in the control group (Figure 4).

FIGURE 4.

Comparative pathological morphology of the stomach and duodenum in each group of rats (HE staining, ×200). Black arrows indicate vacuolar changes. MC, mosapride citrate group; Model, model group; NMNA, non‐meridian and non‐acupoint group; YMA, Yangming meridian acupoint group.

3.5. Protein Expression of PGP9.5, ChAT, and nNOS in Antrum and Duodenal Tissues of Each Group

In the antrum and duodenum, compared with the control group, the protein expression of PGP9.5, ChAT, and nNOS in the model group significantly decreased (p < 0.01). When compared with the model group, the protein expression of PGP9.5, ChAT, and nNOS in the MC and YMA significantly increased (p < 0.01), while there was no statistically significant difference in the NMNA (p > 0.05). Compared with the YMA, the protein expression of PGP9.5, ChAT, and nNOS in the MC showed no significant difference (p > 0.05) (Figure 5A).

FIGURE 5.

(A) Protein expressions of PGP9.5, ChAT and nNOS in antrum and duodenal tissues of each group (𝑥¯ ± s, n = 5/group); (B) Expression levels of ChAT and nNOS in the antrum and duodenal of rats in each group (immunofluorescence, ×200, 𝑥¯ ± s, n = 3/group). Statistical significance: **p < 0.01 vs. control group; ^# p < 0.05, ^## p < 0.01 vs. model group; vs. YMA group, ^Δ p < 0.05, ^ΔΔ p < 0.01. ChAT, choline acetyltransferase; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; MC, mosapride citrate group; Model, model group; NMNA, non‐meridian and non‐acupoint group; nNOS, neuronal nitric oxide synthase; PGP9.5, protein gene product 9.5; YMA, Yangming meridian acupoint group.

3.6. Average Immunofluorescence Intensity of ChAT and nNOS in the Antrum and Duodenum of Rats in Each Group

In the antrum tissue and duodenum, compared with the control group, the average immunofluorescence intensity of ChAT and nNOS in the model group significantly decreased (p < 0.01); compared with the model group, the average immunofluorescence intensity of ChAT and nNOS in the MC and YMA significantly increased (p < 0.05 or P<0.01), while the NMNA showed no statistically significant difference (p > 0.05); compared with the YMA, the average immunofluorescence intensity of ChAT and nNOS in the NMNA significantly decreased (p < 0.05 or p < 0.01), among which in the duodenum the ChAT average immunofluorescence intensity in the MC increased (p < 0.05), and the remaining differences were not statistically significant (p > 0.05) (Figure 5B).

3.7. Protein Expression of GFAP, S100β, and GDNF in Gastric and Duodenal Tissues of Each Group

In the antrum and duodenum, compared with the control group, the protein expression of GFAP, S100β, and GDNF in the model group significantly decreased (p < 0.01). When compared with the model group, the protein expression of GFAP, S100β, and GDNF in the MC and YMA significantly increased (p < 0.01), while there was no statistically significant difference in the NMNA (p > 0.05). Compared with the YMA, the protein expression of GFAP, S100β, and GDNF in the MC showed no significant difference (p > 0.05) (Figure 6).

FIGURE 6.

Expression of S100β, GFAP and GDNF proteins in the gastric antrum of rats in each group (𝑥¯ ± s，n = 5/group). Statistical significance: **p < 0.01 vs. control group; ^# p < 0.05, ^## p < 0.01; ^Δ p < 0.05 vs. model group; ^ΔΔ p < 0.01 vs. Yangming meridian acupoint group. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GDNF, glial‐cell line‐derived neurotrophic factor; GFAP, glial fibrillary acidic protein; MC, mosapride citrate group; Model, model group; NMNA, non‐meridian and non‐acupoint group; S100β, S100 calcium‐binding protein beta subunit; YMA, Yangming meridian acupoint group.

3.8. Immunofluorescence of GFAP and S100β in Gastric and Duodenal Tissues of Each Group

Compared with the control group, the average immunofluorescence intensity of GFAP and S100β in the stomach and duodenum of rats in the model group significantly decreased (p < 0.01). When compared with the model group, the average immunofluorescence intensity of GFAP and S100β in the stomach and duodenum of rats in the MC and YMA significantly increased (p < 0.05 or p < 0.01). Compared with the YMA, the average immunofluorescence intensity of GFAP and S100β in the stomach and duodenum of rats in the NMNA significantly decreased (p < 0.05 or p < 0.01). The average immunofluorescence intensity of gastric GFAP in the MC significantly decreased (p < 0.05), while no significant differences were observed in other indicators (p > 0.05) (Figure 7A).

FIGURE 7.

(A) Expression levels of GFAP and S100β in gastric and duodenal tissues of each group (immunofluorescence, ×200, 𝑥¯ ± s, n = 3/group). (B) Expression levels of GDNF in gastric and duodenal tissues of each group (immunohistochemistry, ×200, 𝑥¯ ± s, n = 5/group). Statistical significance: **p < 0.01 vs. control group; ^# p < 0.05, ^## p < 0.01 vs. model group; ^Δ p < 0.05, ^ΔΔ p < 0.01 vs. Yangming meridian acupoint group. AOD, average optical density; GFAP, glial fibrillary acidic protein; MC, mosapride citrate group; Model, model group; NMNA, non‐meridian and non‐acupoint group; S100β, S100 calcium‐binding protein beta subunit; YMA, Yangming Meridian Acupoint group.

3.9. Effects of GDNF Protein Expression in Gastric and Duodenal Tissues of Each Group

Compared with the control group, the expression of GDNF in the antrum and duodenum of rats in the model group significantly decreased (p < 0.01). When compared with the model group, the expression of GDNF in the antrum and duodenum of rats in the MC and the YMA significantly increased (p < 0.05 or p < 0.01). Compared with the YMA, the expression of GDNF in the antrum and duodenum of rats in the NMNA significantly decreased (p < 0.05), while there was no significant difference in the MC (p > 0.05) (Figure 7B).

4. Discussion

In recent years, diabetes mellitus and its complications have emerged as major global health issues. Studies indicate that approximately 643 million people (11.3% of the global population) will have diabetes by 2030, and this number is projected to rise to 783 million (12.2% of the population) by 2045 [15, 16]. As one of the common chronic complications of diabetes, DGP is caused by autonomic neuropathy and metabolic disorders induced by long‐term hyperglycemia [17]. Gastroparesis delays gastric emptying, leading to postprandial blood glucose fluctuations, while persistent hyperglycemia further exacerbates gastroparesis symptoms, forming a vicious cycle that significantly reduces patients' quality of life and increases hospitalization risks [18]. The primary treatments for DGP involve pharmacological and surgical interventions, yet these methods may cause side effects such as drowsiness, cardiac complications, and extrapyramidal disorders [19]. In contrast, Medicated thread moxibustion of Zhuang medicine demonstrates unique advantages in DGP treatment due to its simple operation, definite efficacy, and high safety. Previous studies have shown that Medicated thread moxibustion of Zhuang medicine improves gastrointestinal motility in DGP rats through multiple mechanisms: first, it upregulates the levels of anti‐inflammatory factors such as IL‐6, IL‐8, and TNF‐α [20]; second, it activates the Nrf2/HO‐1 signaling pathway to enhance antioxidant capacity [14]; third, it inhibits cell apoptosis by regulating the expression of Caspase‐3, bax, and Bcl‐2 [21]. As a key inhibitory neurotransmitter in the ENS, nitric oxide (NO) suppresses gastric smooth muscle contraction, while nNOS, as the rate‐limiting enzyme for NO synthesis, plays a decisive role in regulating ENS function. Studies have confirmed that restoring nNOS‐mediated gastric emptying in DGP rats can effectively regulate key factors such as TNF‐α, Nrf2, and Caspase‐3, further verifying the central role of nNOS in the pathological mechanism of DGP [22]. Additionally, abnormal activation of EGCs can induce gastrointestinal inflammatory responses and exacerbate ENS functional damage [23], further highlighting the critical role of ENS in the pathological process of DGP. Damage to the structure and function of ENS is a key factor leading to intestinal motility disorders, and its abnormalities directly affect DGP progression [24]. Thus, ENS occupies a core position in the pathological mechanism of DGP. However, the regulatory mechanism of Medicated thread moxibustion of Zhuang medicine on ENS remains unclear and requires further exploration.

The intestine, with its vast neural network system known as the “ENS,” is referred to as the “second brain.” The ENS extends from the esophagus to the anus, forming a continuous network of ganglia and nerve bundles, primarily located in two major plexuses: the myenteric plexus and the submucosal plexus. The myenteric plexus controls gastrointestinal motility by regulating the contractility of the longitudinal and circular muscles [25]. Situated between the longitudinal and circular muscle layers, it is distributed along the entire gastrointestinal tract. Enteric neurons sense chemical changes and mechanical stimuli induced by food in the intestine, integrate and process this information, and secrete neurotransmitters to regulate various intestinal physiological activities. Protein gene product 9.5 (PGP 9.5), a neuron‐specific protein, is expressed in neurons [26]. In the ENS, acetylcholine (ACh) acts as an excitatory neurotransmitter, while NO serves as an inhibitory neurotransmitter, synergistically regulating intestinal physiological functions. Similar to nNOS, choline acetyltransferase (ChAT) is a key enzyme catalyzing ACh synthesis from acetyl‐CoA and choline. These neurotransmitters and their synthetic enzymes are crucial for maintaining normal intestinal motility and secretion. Studies have shown that nNOS expression is significantly reduced in a diabetic mouse model of gastroparesis, and increasing nNOS content effectively reverses gastroparesis symptoms [22, 27]. Additionally, research has found that both nNOS‐labeled and ChAT‐positive neurons decrease in the myenteric plexus of diabetic mice, suggesting that changes in the number of nNOS‐ and ChAT‐positive neurons are closely associated with diabetic gastrointestinal motility disorders. Under hyperglycemia, EGCs in the mouse duodenum undergo oxidative stress, accumulating excessive intracellular reactive oxygen species [28], which damage mitochondrial function, activate apoptosis‐related signaling pathways, and ultimately induce cell apoptosis [6]. GFAP and S100β are important structural components of EGCs, with GFAP expression being dynamic and varying with EGC status and subtypes [29]. Researchers have improved gastrointestinal motility by supplementing GFAP through neural stem cell transplantation [30]. GDNF, a neurotrophic factor secreted by EGCs [31], plays a key role in optimizing neuronal function, similar to all glial cells [32]. GDNF binds to GDNF family receptors (GFRα), interacts with tyrosine kinase receptor molecules, leading to phosphorylation of their specific substrates, thereby activating the AKT and MEK signaling pathways in cells, inhibiting enteric neuron apoptosis, protecting the ENS, and further improving gastrointestinal motility [33, 34]. In rats with gastrointestinal motility disorders, the study found that the regeneration of lost enteric neurons was induced through the GDNF and PI3K/Akt signaling pathways, thereby improving gastrointestinal motility [35]. Another study [36] indicated that modulating the GDNF/GFRα1/PI3K/Akt signaling pathway can increase the number of EGCs, thereby enhancing gastric emptying rate and small intestine propulsion rate in rats. Therefore, the recovery of enteric neurons and glial cells is closely related to the improvement of gastrointestinal motility.

In this study, based on the theory of “treatment for flaccidity aims at YangMing meridian” and preliminary research, four acupoints were selected for Medicated thread moxibustion of Zhuang medicine: ST36, ST21, ST40, and ST30. ST36, the lower He‐Sea point of the stomach meridian, specifically treats abdominal disorders and promotes gastric qi generation. ST21, an acupoint of the stomach meridian, harmonizes the stomach and intestines and resolves food stagnation. ST40, the collateral point of the Stomach Meridian, connects the spleen meridians and stomach meridians, regulates the ascending‐descending function of the spleen‐stomach, and relieves gastrointestinal symptoms such as vomiting and abdominal distension. ST30, the intersection point of the Foot‐Yangming Meridian and Chong Meridian, can fundamentally supplement and irrigate the Yangming meridian's qi and blood. The combination of these four acupoints not only considers the interconnections between the spleen‐stomach and the Chong Meridian‐Yangming Meridian but also effectively stimulates Yangming's function of irrigating qi and blood to nourish the gastric muscles. Results showed that compared with the control group, DGP model rats exhibited persistent hyperglycemia, decreased body weight, gastric emptying rate, and small intestinal propulsion rate. Pathological findings revealed damaged tissue structures of the gastric antrum and duodenum, with cellular edema and disordered arrangement. After intervention with Medicated thread moxibustion of Zhuang medicine or mosapride citrate, rats showed increased body weight, gastric emptying rate, and small intestinal propulsion rate, with significantly improved pathological damage in the antrum and duodenum. Although the blood glucose level in the MC trended downward from the baseline, there was no significant difference compared with the model group, suggesting it may promote recovery from blood glucose disorders by restoring gastric emptying, thereby affecting blood glucose values. In contrast, Medicated thread moxibustion of Zhuang medicine exhibited more pronounced effects on promoting the recovery of body weight and blood glucose levels. Notably, the reduction in blood glucose levels further promoted the improvement of gastrointestinal motility to a certain extent, with the two factors mutually reinforcing each other, forming a virtuous circle. Consistent with previous studies [14, 20, 21, 37], these effects may be attributed to its multi‐pathway and multi‐target mechanisms of action. In conclusion, the findings of this study indicate that Medicated Thread Moxibustion of Zhuang Medicine at the Yangming meridian acupoints effectively improves gastrointestinal motility in rats with DGP.

To further investigate whether the effect of Medicated thread moxibustion of Zhuang medicine is related to the regulation of ENS injury, this study observed changes in PGP9.5, ChAT, nNOS, S100β, GFAP, and GDNF in the stomach and duodenum. The results showed that the contents of PGP9.5, nNOS, ChAT, GFAP, S100β, and GDNF were significantly reduced in DGP rats, indicating that DGP rats suffered from not only enteric neuron damage but also EGC damage. Long‐term hyperglycemia induced by diabetes causes degeneration and necrosis of nerve fibers, disrupts normal metabolism and signal transduction of enteric neurons, and directly leads to gastrointestinal motility imbalance.

The concurrent loss of nNOS and ChAT in DGP rats fundamentally disrupts the excitatory‐inhibitory regulatory framework of the ENS. The loss of nNOS inhibitory neurons impairs receptive relaxation of the gastric fundus and induces pyloric sphincter spasm, whereas the loss of ChAT excitatory neurons abolishes antral propulsive contractions. The combined deficiency of both neuronal subtypes further leads to a complete interruption of the gastric peristaltic reflex arc, rendering the stomach neither able to relax for food accommodation nor capable of contracting to complete emptying, thereby resulting in delayed gastric emptying.

Immunofluorescence showed simultaneous reduction of GFAP and S100β in the myenteric plexus of the gastric antrum and duodenum, suggesting a decrease in the function or number of EGCs. IHC revealed reduced GDNF in the stomach and duodenum, further indicating potential weakening of neuroprotection, abnormal synaptic regulation, or repair disorders. Medicated thread moxibustion of Zhuang medicine promoted the repair of enteric neurons and glial cells, consistent with previous findings that electroacupuncture improves gastrointestinal function by repairing nerve cells [38]. Our preliminary study verified that Medicated thread moxibustion of Zhuang medicine and electroacupuncture have the same effect on DGP rats [39], thus further validating the reliability of this study's conclusions.

ENS injury, as the core pathological mechanism of DGP, involves multiple pathological responses. Oxidative stress damages ENS neurons and glial cells, reduces the activity of nNOS, and disrupts gastrointestinal motility regulation [32]. Abnormal ENS function impairs the integrity of the interstitial cells of Cajal network, leading to pacing dysfunction and exacerbating gastric emptying delay [40]. Meanwhile, oxidative stress‐mediated smooth muscle cell apoptosis and fibrosis weaken gastrointestinal contractility, forming a vicious cycle with ENS injury [41]. As an important intracellular quality control mechanism, mitochondrial autophagy maintains intracellular homeostasis and ensures normal ENS function by removing damaged mitochondria [42]. Increased microbiota promotes the proliferation and survival of myenteric plexus neurons, thereby increasing neuronal density [43]. Autonomic neuropathy further aggravates motility disorders by interfering with sympathetic/parasympathetic regulation of ENS [44]. Macrophages mediate neuroinflammatory responses by secreting inflammatory factors, affecting the functional status of EGCs and neurons [45]. After immune activation, inflammatory factors (such as TNF‐α) penetrate into ENS, inducing neuroinflammation and exacerbating oxidative stress injury [46], collectively participating in the pathological process of DGP. This study confirms that Medicated thread moxibustion of Zhuang medicine may improve gastrointestinal motility in DGP rats by repairing ENS neuron and glial cell damage, providing a new target for clinical treatment. Future research could further explore the interaction between ENS and various pathological responses to deepen the understanding of DGP pathogenesis and intervention strategies of Medicated Thread Moxibustion of Zhuang Medicine, as illustrated in Figure 8.

FIGURE 8.

Schematic diagram illustrating the mechanism of mzedicated Thread Moxibustion of Zhuang Medicine in improving gastrointestinal motility in DGP rats. Akt, protein kinase B; ChAT, choline acetyltransferase; EGCs, enteric glial cells; GDNF, glial‐cell line‐derived neurotrophic factor; GFAP, glail fibrillary acidic protein; GFRα, GDNF family receptor; MEK, mitogen‐activated protein kinase; nNOS, neuronal nitric oxide synthase; PGP9.5, Protein gene product 9.5; PI3K, phosphatidylinositol 3‐kinase; RET, rearranged during transfection; S100β, S100 calcium‐binding protein beta subunit; ST21, Liangmen; ST30, Qichong; ST36, Zusanli; ST40, Fenglong.

Additionally, the mutual influence between ENS and the central nervous system (CNS) has gradually become a research hotspot, especially in the fields of CNS diseases such as Parkinson's disease, Alzheimer's disease, and stroke [47, 48, 49]. This study confirms that Medicated Thread Moxibustion of Zhuang Medicine exerts positive regulatory effects on ENS by modulating neurotransmitter secretion and improving neuronal and glial cell functions. Given the connection between ENS and CNS, this regulatory effect is expected to have potential therapeutic effects on CNS diseases. This provides a theoretical basis for expanding the application of Medicated Thread Moxibustion of Zhuang Medicine to treat other diseases associated with neural regulatory disorders, thus broadening its therapeutic scope and enhancing clinical applicability.

This study has several limitations. First, due to experimental constraints, some indicators were assessed in only three rats per group, and the relatively small sample size may limit statistical power and the generalizability of the findings. Second, to minimize the potential confounding effects of cyclic estrogen and progesterone fluctuations on gastric motility, EGC function, and neurotransmitter signaling, only male SD rats were used in this study; future investigations will include female rats at different estrous stages to determine whether the observed alterations in the ENS exhibit sex‐specific patterns. Finally, although this study demonstrated that Medicated Thread Moxibustion of Zhuang Medicine upregulated GDNF expression in the gastric antrum and duodenum, the downstream signaling cascade of GDNF has not been directly validated by experimental data herein. The precise molecular mechanisms underlying ENS repair remain to be further elucidated through pathway‐specific inhibition experiments and comprehensive phosphorylation profiling in future studies.

5. Conclusion

In conclusion, this study shows that Medicated Thread Moxibustion of Zhuang Medicine at the Yangming meridian acupoints repairs ENS injury by regulating ENS neurotransmitter secretion and improving neuronal and glial cell functions. This regulation not only restores the structure and functional morphology of cells and tissues but also plays a dual role in regulating gastrointestinal motility and blood glucose levels in DGP rats.

Author Contributions

Yushan Fan served as the supervising investigator, responsible for study design and technical guidance. Hui Xu was the primary executor of this study, conducting the experiments, data collection, statistical analysis, and drafting the original manuscript. Yujun He participated in experimental operations and data collation, reviewed the results, and approved the final version of the manuscript. Fangzhi Zhang, Zibin Wang, and Yu Wu contributed to experimental procedures and data analysis.

Funding

This study is supported by the National Natural Science Foundation of China (No. 82260983) and the 2024 Innovation Project of Guangxi Graduate Education (No. YCBZ2024148).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.