Effects of Biling Weitong granules (荜铃胃痛颗粒) on gastrointestinal motility and gastrointestinal hormones levels in rats with functional dyspepsia

Abstract

OBJECTIVE:

To investigate the effects of Biling Weitong granules (荜铃胃痛颗粒) on gastrointestinal motility and serum mouse motilin/human vasoactive intestinal peptide (MTL/VIP) enzyme-linked immunosorbent assay kit levels in functional dyspepsia (FD) rat models, and explore its therapeutic mechanisms against FD to provide further clinical application data.

METHODS:

Sixty Sprague-Dawley rats were randomized into blank group (BG) and model group (MG). FD was induced in MG via30-d chronic unpredictable stress. After successful modeling, the model rats were divided into MG BG, Biling Weitong granules low/middle/high-dose (BL/BM/BH), and trimebutine maleate (TM) groups (n = 7/group) for 14-d treatments. At the end of the gavage, General parameters (body weight, food intake), gastrointestinal motility indices (gastric emptying and small intestinal propulsion rates), serum MTL/VIP levels, and gastric antrum histopathology were analyzed using hematoxylin and eosin (HE) staining.

RESULTS:

After 30 d of modeling, compared with the BG group, rats in the MG group presented FD-like symptoms such as lethargy and irritability, along with significantly decreased body weight and food intake (P < 0.05). The gastric emptying rate and small intestinal propulsion rate were significantly reduced (P < 0.01), and the serum MTL content significantly decreased (P < 0.01). Serum VIP levels were significantly increased (P < 0.01). After 14 d of treatment, compared with the MG group, rats in the BL, BM, BH, and TM groups exhibited improved general condition, with significantly increased body weight and food intake (P < 0.05); significantly faster gastric emptying rate and small intestinal propulsion rate (P < 0.01); and significantly higher serum MTL content (P < 0.01). The serum VIP levels were significantly decreased (P < 0.01). There were no significant differences in body weight or food intake between the BM group and BH group (P > 0.05). Consistently, there were no significant differences in gastric emptying rate, small intestinal propulsion rate, or serum MTL and VIP contents between the BH and TM groups (P > 0.05). HE staining revealed no marked changes in the gastric antrum tissues of the BG, MG, or drug groups.

CONCLUSION:

Biling Weitong granules can modulate the levels of the gastrointestinal hormones MTL and VIP in FD rats, promote gastric emptying and small intestinal propulsion, improve the gastrointestinal motility and mental status of FD rats, and alleviate the symptoms of FD, accounting for its efficacy against FD.

1. INTRODUCTION

Functional dyspepsia (FD) is a chronic functional gastrointestinal disorder with a high global prevalence, characterized by upper abdominal pain that cannot be attributed to any organic, systemic, or metabolic disease.¹ Common clinical manifestations include upper abdominal pain or burning, nausea, vomiting, postprandial fullness, early satiety, loss of appetite, and belching.² FD typically exhibits a high incidence rate, a prolonged course, and a high recurrence rate, significantly impairing patient quality of life. Epidemiological studies indicate that the global incidence of FD ranges from 7% to 40%, with an annual increase of 1%.³ According to the Rome IV criteria, FD can be divided into epigastric pain syndrome (EPS) and postprandial distress syndrome (PDS), and both can exist simultaneously.⁴ The etiology and pathogenesis of FD are complex and the pathogenesis is still unclear. Current hypotheses in Western Medicine suggest the involve-ment of various factors, including gastroduodenal dysfunction, intestinal immune dysfunction, abnormal regulation of the brain-gut axis, visceral hypersensitivity, Helicobacter pylori (HP) infection, and mental stress.^5-8 Notably, the brain-gut axis affects signal transmission between the host and the gut microbiota.⁹ Alterations in the central nervous system can affect gastrointestinal function via the brain-gut axis. The brain-gut peptides related to FD include serum mouse motilin (MTL) and human vasoactive intestinal peptide (VIP). MTL stimulates gastrointestinal motility and promotes gastrointestinal motility, whereas VIP inhibits gastric emptying and gastric acid secretion.¹⁰ Dysregulation of the brain-gut axis has also been established as one of the main causes of FD.

At present, the clinical management of FD emphasizes symptomatic relief, including antacids, gastrointestinal function modulators, prokinetic agents, and the eradication of HP infection. TM can relieve FD symptoms such as epigastric pain, epigastric burning, nausea, and vomiting by relaxing the gastrointestinal smooth muscle and promoting peristalsis.¹¹ However, symptom recurrence is common after drug withdrawal. In recent years, Traditional Chinese Medicine (TCM), guided by dialectical treatment and holistic concepts, has shown unique advantages in the comprehensive treatment of FD, mainly improving the clinical symptoms, reducing recurrence, and improving quality of life. Biling Weitong granules (荜铃胃痛颗粒) can promote Qi circulation and blood circulation and relieve stomach pain. Approved by the State Medical Products Administration in 2016, it is currently widely used in patients with epigastric pain caused by Qi stagnation and blood stasis and chronic gastritis exhibiting these syndromes. According to the Expert Consensus on Traditional Chinese Medicine Diagnosis and Treatment of Functional Dyspepsia (2017), Biling Weitong granules is indicated for patients with epigastric pain, highlighting its significance in the treatment of FD.¹² This study aimed to investigate the potential mechanisms of Biling Weitong granules in the treatment of FD from the perspective of gastrointestinal hormones.

Therefore, this study established rat models of FD, followed by treatment with Biling Weitong granules for 14 d to explore the mechanisms underlying the therapeutic effects on gastrointestinal motility and the MTL and VIP levels, thereby providing preclinical data to support future clinical research.

2. MATERIALS AND METHODS

2.1. Experimental animals

Sixty specific pathogen-free (SPF) 4-week-old Sprague-Dawley (SD) rats (30 males and 30 females) with a mean weight of [(80 ± 20) g] were used. The rats were obtained from Liaoning Changsheng Biotechnology (Benxi, China). (Certificate of quality: No. 21072623010084-3412, License number: SCXK (Liao) 2020-0001). The animals were kept in a standard animal facility maintained at 18-22 ℃ with 50%-60% relative humidity for 7 d and adequate lighting and ventilation. All rats were fed a regular diet with free access to water, and bedding was changed once every 3 d on average. Only rats exhibiting no observable abnormalities were included in the experiment. All animal handling and disposal during the experimental process were conducted according to SPF laboratory requirements. This study was approved by the Laboratory Animal Ethics Committee of First Affiliated Hospital of Qiqihar Medical College (ethics number: QMU-AECC-2022-78).

2.2. Drugs and reagents

Biling Weitong granules (Taizhou, China) mainly composed of Bichengqie (Fructus Litseae), Chuanlianzi (Fructus Toosendan), and Dahuang (Radix Et Rhizoma Rhei Palmati). Trimebutine maleate tablets (Datong, China) were used in this study. The rat MTL and VIP enzyme-linked immunosorbent assay (ELISA) detection kits were purchased from Shanghai Yanzun Biological Technology (Shanghai, China). The sodium carboxy-methyl cellulose was purchased from Shanghai McLean Biological Technology (Shanghai, China). The 10% chloral hydrate solution was purchased from Wuhan Qianstranger Technology (Wuhan, China). The 4% paraformaldehyde fixative solution was purchased from Shanghai McLean Biotechnology (Shanghai, China). The xylene was purchased from Tianjin Kaitong Chemical Reagent (Tianjin, China). The hematoxylin and eosin staining (HE) solution was purchased from Beijing Langeke Technology (Beijing, China). The absolute ethanol was purchased from Tianjin Fuyu Fine Chemical (Tianjin, China). The neutral gum was purchased from Sinopdrug Group Chemical Reagent (Beijing, China).

2.3. Main equipment

A GRT22-1 high-speed freezing centrifuge was used from Beijing Times Beili Centrifuge (Beijing, China). An EUK2-20TF laboratory ultrapure water machine was used from Nanjing Oukai Environmental Technology (Nanjing, China). A F0RMA 900 SERIES ultralow temperature refrigerator was used from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA). A VIP 6-E2 tissue dehumidifier was used from Sakura Finnetek (Sakura Finetek, Torrance, CA, USA). A HistoCore Arcadia H paraffin embedding machine and an RM 2245 rotary microtome were used from Leica Microsystems (Shanghai, China). A BGZ-246 electric air drying oven was used from Shanghai Bosun Industrial (Shanghai, China). A TS2R-C-AL inverted fluorescence phase contrast microscope was used from Nikon Corporation (Nikon Corporation, Tokyo, Japan). A Tecan Safire 2 microplate reader was used from Tecan Group (Tecan Group, Maennedorf, Switzerland). A LAC 214 electronic balance was used from Changshu Bailing Balance Instrument (Changshu, China). A ZHWY-200D constant temperature shaking incubator was used from Shanghai Zhicheng Analytical Instrument Manufacturing (Shanghai, China).

2.4. Animal grouping

After adaptive feeding for 1 week, the rats were randomly divided into 2 groups by random number table method: BG group (n = 10) and MG group (n = 50). After the model was established, five rats were randomly selected from each group to verify whether the model succeeded. During the modeling period, because the rats were irritable, they tore each other and caused eight rats to be injured, so they were excluded from the experiment. Finally, forty-two rats with successful modeling were randomly divided into 6 groups by random number table method: BG group, MG group, BL group, BM group, BH group and TM group (n = 7 rats/group).

2.5. Establishment of the FD rat model

At the end of conditioning, the rats were randomly divided into the BG group and the MG group. The BG group was fed a conventional basic diet (fish meal, meat and bone meal, corn, soybean meal, flour, or wheat bran) with free access to drinking water. The MG group was fed a high-fat diet (basic diet, cholesterol, lard) with free access to drinking water. To prevent habituation to single stimuli, the FD rat model was established by a chronic unpredictable stress,^13-17 which consisted of: noise stimulation for 11 h (10 kHz, 2 min duration, and repeated at an interval of 15 min), flickering light stimulation for 11 h (2 Hz, 2 min duration, and 15 min-interval), circadian reversal stimulation for 24 h, stimulation with an ice water bath twice a day for 5 min each, tail clip stimulation (The distal 1/3 of the rat tail was clamped and wrapped with gauze to prevent skin damage (if there was a scratch in the skin or tail, it was disinfected with 0.5% iodophor to prevent infection); periods of induced aggression twice daily for 30 min, including rage, scream, and fighting) and restraint stimulation (3 h/d in a barbed-wire cage of size 33.5 cm × 26 cm × 8 cm divided into 12 compartments to restrict movement). The sequence of these stressors was determined using an orthogonal experiment design to ensure no repetition within the next two days. The stimulation lasted for 30 d. The general characteristics of the rats, food intake, and changes in body weight before and after modeling and treatment were recorded.

2.6. Drug preparation and intervention

Rat clinical equivalent doses were calculated according to the body surface coefficient of humans and animals. The BM groups received Biling Weitong granules at a clinical equivalent dose of 1.35 g/kg. The BL groups received 0.5 times the equivalent dose (0.68 g/kg), and the BH groups received 2 times the equivalent dose (2.70 g/kg). TM tablets were pulverized into powder and prepared at a clinically equivalent dose of 0.03 g/kg, which were separately formulated into 20 mL solutions with distilled water, with a fixed gavage volume of 2 mL for each. The rats in the BG and MG groups were given 2 mL of distilled water by gavage. Except for the BG group and MG group, the other groups were gavaged once a day at a fixed time for 14 d.

For the preparation of semisolid paste for the determination of gastric emptying and the small intestinal propulsion rate, 3 g of sodium carboxymethylcellulose, 3 g of white sugar, 5 g of milk powder, 3 g of starch, and 1 mL of carbon ink were mixed thoroughly. The final volume was adjusted with 100 mL of water, yielding a black semisolid paste. A dose of 3 mL·kg^-1·d^-1 was administered by oral gavage.

2.7. Observation indicators and methods

2.7.1. Observations of general characteristics, body weight, and food intake

The mental status, activity level, fur condition, tongue appearance, auricle features, and fecal consistency of the two groups were observed and recorded every day. The body weights of the rats in the two groups were measured and recorded at 08:30 every 5 d. The rats in the two groups were given a pre-weighed quantity of food at 08:00 every morning, and the remaining food was weighed at 08:00 the next morning. The average daily food intake per rat was calculated by dividing the difference between the provided and remaining food weights by the number of rats in the group.

2.7.2. Determination of the gastric emptying rate and small intestinal propulsion rate

After the last drug administration, each rat was given water ad libitum and fasted for 24 h. Each rat was fed 3 mL of a black semisolid paste by oral gavage. Forty minutes later, 10% chloral hydrate solution was given by intraperitoneal injection for anesthesia. After induction of anesthesia, the abdominal cavity was opened quickly, and approximately 4 mL of blood was collected from the abdominal aorta for use. The cardia and pylorus of each rat were ligated, and the whole stomach was removed. After drying with filter paper, the whole stomachs of the rats were weighed. After weighing, the rats were cut along the greater curvature of the stomach with surgical scissors, and the contents of the stomach were cleaned with 0.9% sodium chloride injection. After washing, the stomach was wiped dry and weighed, and the net weight of the stomach was calculated. The entire small intestine from the pylorus to the ileocecal junction was cut, and the excised small intestine was placed on the experimental table and gently straightened. The length of the intestine was measured, and the total length of the small intestine was denoted L. The distance traveled by the carbon ink within the small intestine, from the pylorus to the carbon ink front, was measured and denoted L₀.

The gastric emptying rate and small intestinal propulsion rate were calculated via the following formulas:

Gastric emptying rate (%) = [1－ (total gastric weight － net gastric weight)/gavage volume] × 100%

Small intestinal propulsion rate (%) = L₀/L × 100%

2.7.3. Hematoxylin and eosin (HE) staining assessment of gastric antrum tissue

Rat gastric and duodenal tissues were fixed in a 4% paraformaldehyde solution, embedded in paraffin, and sectioned. HE staining was performed according to the manufacturer’s instructions, with results observed and photographed under an inverted fluorescence microscope at × 100 and × 200 magnification. Abnormal pathological changes, such as tissue degeneration, necrosis, and inflammatory infiltration, were observed according to the morphology of the gastric antrum tissue.

2.7.4. The contents of MTL and VIP in the serum were determined via ELISA

The rat blood samples were left at room temperature for 1 h and then centrifuged at 3500 r/min and 4 ℃ for 15 min in a GTR 22-1 high-speed refrigerated centrifuge. The supernatant was collected and stored at －80 ℃. Serum MTL and VIP were detected via ELISA. Before testing, the serum was thawed at room temperature, and the MTL and VIP kits were removed from the 4 ℃ refrigerator and incubated at room temperature for 20 min. All operations were performed according to the instructions of the ELISA kit. The absorbance (OD) value of each well was measured with a Tecan Safire 2 microplate reader at a wavelength of 450 nm, and the average OD value of the tested samples was substituted into the MTL and VIP standard curves to determine the content of MTL and VIP.

2.8. Statistical analysis

All data were analyzed using SPSS 17.0 (IBM Corp., Armonk, NY, USA) software. The normally distributed data were expressed as the mean ± standard deviation ($\overline{x}\pm s$), and t tests were used for pairwise comparisons. Analysis of variance was used for comparisons between multiple groups, followed by the least significant difference post hoc test where appropriate. A P-value < 0.05 was statistically significant, and a P-value < 0.01 indicated very high statistical significance.

3. RESULTS

3.1. Observation of the general characteristics of rats in each group

Throughout the modeling period, rats in the BG group exhibited normal behavior, characterized by alertness, activity, docile temperament, glossy fur, a pink tongue, light pink auricles, and firm, yellow-brown feces without a foul odor. In contrast, rats in the MG group displayed signs of depression, including lethargy, irritability, huddling in cage corners, arched backs, piloerection, dull and yellowing fur, a dark and bluish-purple tongue, white auricles, and large, soft, malodorous feces with high water content. MG group rats exhibited heightened responses to tail clip application, displaying aggression and fighting. During ice water immersion, these rats showed intense reactions, defecation, soft and malodorous feces, and a fearful demeanor. Following 14 d of treatment, BG group rats maintained their normal behavior, with glossy fur, activity, and dry feces. MG group rats remained depressed, with dry yellow fur, drowsiness, and soft, malodorous feces. Rats in the treatment groups exhibited varying degrees of mood stabilization, improved fur gloss, increased activity, and progressively drier feces.

3.2. Changes in the food intake and body weight of the rats in each group

Before modeling, there was no significant difference in body weight between the BG group and the MG group (P > 0.05). After modeling, the body weights of the rats in both groups increased significantly (P < 0.01). Compared with the BG group, the body weights of rats in the MG group increased slowly and decreased significantly (P < 0.05) (Table 1). The changes figure in body weight before and after modeling are shown in the attachment. Compared with the MG group, the body weights of the BL group were significantly higher (P < 0.05), while the BM, BH, and TM groups exhibited extremely significantly higher body weights (P < 0.01). While the TM group and BL exhibited significantly different body weights (P < 0.05), the body weights of the BM group and the BH group remained comparable (P > 0.05) (Table 2). The body weight changes figure between groups are shown in the attachment. Before modeling, there was no significant difference in food intake between the BG group and the MG group (P > 0.05). During the modeling period, food intake in the BG group gradually increased, and decreased in the MG group. Compared with the BG group, food intake in the MG group was significantly lower (P < 0.01) (Figure 1). Compared with the MG group, the BL, BM, BH, and TM groups presented significant increases in food intake after drug intervention (P < 0.05). Compared with the TM group, food intake in the BL group was significantly different (P < 0.05), while food intake between the BM group and the BH group remained comparable (P > 0.05) (Figure 1). Table data on changes in food intake of the rats in each group are shown in the attachment.

Table 1.

Changes in body weight of rats in each group (g, $\overline{x}\pm s$)

Group	n	Before modeling	After modeling
BG	5	99±8	180±28a
MG	5	99±8	136±11ab

Notes: BG group: fed only with conventional basic diet and free access to drinking water; MG group: fed a high-fat die and free access to drinking water; BG group: blank group; MG group: model group. t tests were used for pairwise comparisons. Compared with before modeling, ^aP < 0.01; compared with the BG group, ^bP < 0.05.

Table 2.

Changes of body weight of rats in each group (g, $\overline{x}\pm s$)

Group	n	Body weight
BG	7	312±38
MG	7	221±16a
BL	7	244±16bc
BM	7	266±29d
BH	7	298±31d
TM	7	283±31d

Notes: BG and MG groups: fed only with 2 mL distilled water; BL group: treated with 0.68 g/kg; BM group: treated with 1.35 g/kg; BH group: treated with 2.70 g/kg; TM group: treated with 0.03 g/kg; BG group: blank group; MG group: model group; BL group: Biling Weitong granules low-dose group; BM group: Biling Weitong granules middle-dose group; BH group: Biling Weitong granules high-dose group; TM group: trimebutine maleate group. t tests and analysis of variance were used for comparisons. Compared with the BG group, ^aP < 0.01; compared with the MG group, ^bP < 0.05, ^dP < 0.01; compared with the TM group, ^cP < 0.05.

Figure 1. Comparison of food intake of rats in each group.

BG and MG groups: fed only with 2 mL distilled water; BL group: treated with 0.68 g/kg; BM group: treated with 1.35 g/kg; BH group: treated with 2.70 g/kg; TM group: treated with 0.03 g/kg; BG group: blank group; MG group: model group; BL group: Biling Weitong granules low-dose group; BM group: Biling Weitong granules middle-dose group; BH groups: Biling Weitong granules high-dose group; TM group: trimebutine maleate group. t tests and analysis of variance were used for comparisons. Data are presented as mean ± standard deviation (n = 7). Compared with the MG group, ^aP < 0.05; compared with the TM group, ^bP < 0.05; compared with the BG group, ^cP < 0.05.

3.3. Comparison of the gastric emptying rate and small intestinal propulsion rate among the rats in each group

Compared with the BG group, the gastric emptying rate of the MG group was significantly lower (P < 0.01) (Figure 2A).The changes figure in the gastric emptying rates in each group are shown in the attachment. Compared with the MG group, the gastric emptying rates of the BL, BM, BH, and TM groups were significantly higher after drug intervention (P < 0.01). Compared with the TM group, the gastric emptying rates of the BL group and BM group were significantly different (P < 0.01), while no significant difference was observed with the BH group (P > 0.05) (Figure 2A). Table datas on changes in the gastric emptying rates in each group are shown in the attachment. Compared with the BG group, the small intestinal propulsion rate of the MG group decreased significantly (P < 0.01) (Figure 2B). The changes figure in the small intestinal propulsion rates in each group are shown in the attachment. Compared with the MG group, the small intestinal propulsion rates of the BL, BM, BH, and TM groups were significantly greater (P < 0.01). Compared with the TM group, the small intestinal propulsion rates in the BL group and BM group were significantly different (P < 0.01), while there was no significant difference with the BH group (P > 0.05) (Figure 2B). Table datas on changes in the small intestinal propulsion rate in each group are shown in the attachment.

Figure 2. Comparison of gastric emptying rates A and small intestinal propulsion rates B between groups of rats.

A: comparison of gastric emptying rates; B: small intestinal propulsion rates. BG and MG groups: treated by gavage at a dose of 3 mL·kg^-1·d^-1. BL group: treated with 0.68 g/kg. BM group: treated with 1.35 g/kg. BH group: treated with 2.70 g/kg; TM group: treated with 0.03 g/kg; BG group: blank group; MG group: model group; BL group: Biling Weitong granules low-dose group; BM group: Biling Weitong granules middle-dose group; BH group: Biling Weitong granules high-dose group; TM group: trimebutine maleate group. t tests and analysis of variance were used for comparisons. Data are presented as means ± standard deviations (n = 7). Compared with the BG group, ^aP < 0.01; compared with the MG group, ^bP < 0.01; compared with the TM group, ^cP < 0.01.

3.4. Comparison of the serum MTL and VIP contents of rats in each group

Compared with the BG group, the serum MTL content in the MG group was significantly lower (P < 0.01), and the VIP content in the MG group was significantly higher (P < 0.01) (Figure 3). The changes figure in serum MTL and VIP contents in the two groups are shown in the attachment. Compared with the MG group, the contents of MTL and VIP in the BL, BM, BH and TM groups were comparable. Consistently, the TM and BH groups exhibited comparable serum MTL and VIP levels (Figure 3) Table datas on changes in serum MTL and VIP contents in each group are shown in the attachment.

Figure 3. Comparison of serum MTL A and VIP B contents of rats in each groups.

A: MTL level; B: VIP level. BG, MG, BL, BM, BH, TM groups: prepared according to 50 μL (25 μL serum +25 μL dilution). BG group: blank group; MG group: model group; BL group: Biling Weitong granules low-dose group; BM group: Biling Weitong granules middle-dose group; BH group: Biling Weitong granules high-dose group; TM group: trimebutine maleate group. t tests and analysis of variance were used for comparisons. Data are presented as mean ± standard deviation (n = 7). Compared with the BG group, ^aP < 0.01; compared with the MG group, ^bP < 0.01; compared with the TM group, ^cP < 0.01

3.5. Pathological observation of the gastric antrum tissues of the rats in each group via HE staining

Histopathological analysis revealed that the antrum tissue of the BG, MG, BL, BM, BH, and TM groups exhibited normal findings with regularly arranged epithelial cells and glands, and no pathological changes, such as edema, inflammatory infiltration, or congestion, were observed. The results revealed no structural changes in the gastric antrum tissues of the rats in the MG, BL, BM, BH, and TM groups (Figure 4).

Figure 4. Gastric antrum tissues of rats in each group.

A: expression of the gastric antrum tissues × 100; B: expression of the gastric antrum tissues × 200; A1, B1: BG group; A2, B2: MG group; A3, B3: BL group; A4, B4: BM group; A5, B5: BH group; A6, B6: TM group. BG and MG groups: treated by gavage at a dose of 3 mL·kg^-1·d^-1; BL group: treated with 0.68 g/kg; BM group: treated with 1.35 g/kg; BH group: treated with 2.70 g/kg; TM group: treated with 0.03 g/kg; BG group: blank groups; MG group: model group; BL group: Biling Weitong granules low-dose group; BM group: Biling Weitong granules middle-dose group; BH group: Biling Weitong granules high-dose group; TM group: trimebutine maleate group. Dyeing method of all pictures are hematoxylin and eosin staining.

4. DISCUSSION

FD is a chronic functional gastrointestinal disease that is common worldwide and characterized by upper abdominal discomfort. At present, the incidence of this disease is increasing annually in China. FD is characterized by a high recurrence rate and a long course of disease, which reduces patient quality of life. Current modern medicine approaches primarily focus on symptomatic relief, often accompanied by adverse reactions and a high rate of relapse following drug cessation. TCM, guided by dialectical treatment and holistic principles, offers a complementary approach. TCM treatment modalities include herbal medicine, individualized prescriptions, and Chinese patent medicine. Biling Weitong granules is a Chinese patent medicine developed by academician Tung Chie-hua based on long-term clinical experience and three major theories. Biling Weitong granules are widely used to promote Qi and blood circulation and relieve stomach pain. It is typically indicated in patients with epigastric pain and chronic gastritis caused by Qi stagnation and blood stasis. While Biling Weitong granules has not been proposed for the treatment of FD, expert consensus suggests its potential utility, providing a new option for the treatment of FD.

Based on studies conducted in China and abroad, the pathogenesis of FD is the result of the interaction and mutual influence of many factors. From the perspective of Western medicine, FD is related to multiple factors, such as gastroduodenal dysfunction, gastrointestinal motility changes, intestinal immune dysfunction, and abnormal regulation of the brain-gut axis.^8,18,19 TCM posits that Qi, representing Yang, and blood, representing Yin, are interdependent. Qi facilitates blood biochemistry and circulation, while adequate blood volume supports Qi generation and physiological functions.²⁰ Qi stagnation causes stasis of Qi and blood, resulting in pain. It is common in the liver, spleen, stomach, and other viscera and is associated with emotional disorders, abdominal distention and pain, less food, and anorexia.²¹ The spleen, stomach, and liver play pivotal roles in digestion, storage, and dispersal, respectively, and are central to Qi and blood production. Spleen dysfunction may lead to gastric discomfort, flank distension, nausea, vomiting, loose stools, and other symptoms. Unfavorable liver Qi flow can disrupt emotional balance and exacerbate the development of FD.²² Qi stagnation, blood stasis and gastric disturbance constitute the basis of FD pathogenesis. Both single and multiple stressor models are employed in FD research, with careful selection of the methodology being crucial. Studies have shown that four factors, including auditory and photoelectric stimulation, tail clamping, restraint, and ice/water bath, have significant effects on the modeling results.²³ A high-fat diet and chronic unpredictable stimulation can simulate three different blood-stasis syndrome states, namely, Qi stagnation, turgid phlegm, and both, causing emotional damage and aggravating the blood-stasis state of rats.¹⁸ Multi-stressor models typically incorporate physical factors (tail clamping, an ice-water bath, restraint, sound and photoelectric, etc.), diet (high fat and high sugar), and environmental factors (day and night reversal and lighting changes, etc.).¹⁴ To better mimic clinical FD symptoms, the chronic unpredictable stress model provides a strong basis for FD model establishment.

According to the Rome IV criteria, FD can be divided into EPS and PDS, neither of which is not necessarily related to the human diet. However Chinese medicine thinks the occurrence of FD is related to spleen and stomach weakness, emotional disturbances, poor diet, and six exogenous pathogens.²⁴ In the Huang Di Nei Jing, it is said that "there is no apparatus for ascending and descending in and out".²⁵ The ascending and descending of Qi machinery is orderly, and people are at peace.²⁶ FD is related to Qi stagnation for a long time, the obstruction of the stomach's blood flow, and the stomach's blood stasis. A high-fat diet is a key factor causing Qi stagnation and blood stasis.²⁷ Therefore, in this study, according to the idea of depression and anger for a long time, wood stagnation by soil, Qi stagnation, and blood stasis, a high fat diet was superimposed and adopted, and chronic unpredictable stress, such as sound and light stimulation, tail clamping stimulation, ice-water bath stimulation, 24-h circadian reversal, and restraint stimulation, were more consistent with the etiology and pathogenesis of FD. A study revealed that a long-term high-fat diet could affect gastrointestinal motility and hormone secretion in rats, increasing gastrointestinal sensitivity, slowing gastric emptying, and ultimately leading to FD symptoms.²⁸ Sound and light stimulation and 24-h circadian reversal can induce anxiety and tension in rats and increase visceral sensitivity. Tail pinch stimulation can cause emotions such as surprise, fear, and anger in rats. It can also cause symptoms such as reduced food intake and slow gastric emptying, which domestic scholars commonly used to establish FD rat models.²⁹ Chronic restraint stress can cause struggle, helplessness, anxiety, depression, and other psychological conditions by limiting rat activity, which is similar to the development of chronic physical and mental diseases.³⁰ Ice water immersion allows cold stimuli to invade the body, causing cold and Qi stagnation in rats, aggravating Qi and blood stasis, and resulting in FD symptoms.³¹

Therefore, after modeling in this study, compared with the BG group, the MG group presented FD-like characteristics, such as lethargy, irritability, irritability, full fur, and soft malodorous feces. The body weight and food intake of MG rats were significantly decreased (P < 0.05), and the gastric emptying rate and small intestinal propulsion rate exhibited extremely significant decreases (P < 0.01). In the BG group and MG group, there was no obvious inflammatory infiltration or pathological changes in the gastric antrum tissue, which was consistent with the characteristics of FD. MTL in the MG group significantly decreased (P < 0.01), while VIP levels significantly increased (P < 0.01). Model validation revealed that the rats in the MG group developed mental depression, irritability, loss of appetite, slow gastrointestinal peristalsis, gastric emptying, and slow intestinal propulsion, which indicated that the FD model was successfully established.

Drug intervention was given after model verification. The general condition, body weight, food intake, gastric emptying rate, small intestinal propulsion rate, pathological changes in the gastric antral tissue, and serum MTL and VIP contents of the Biling Weitong granules rats were observed to explore the effects of the Biling Weitong granules on FD. Biling Weitong granules carried out modern pharmacological analysis of each Chinese medicine in Biling Weitong granules prescription through the emperors and ministers: Bichengqie (Fructus Litseae) in the Fructus Litseae has the effects of warming and dissipating cold, promoting Qi and relieving pain, antibacterial and anti-inflammatory.³² Chuanlianzi (Fructus Toosendan) has the effect of soothing the liver and relieving heat.³³ Yanhusuo (Rhizoma Corydalis Yanhusuo) has the effect of promoting Qi, promoting blood circulation and relieving pain and anti-inflammation.³⁴ The above three are the best medicines. Huanglian (Rhizoma Coptidis) has can clear heat, dry dampness and reduce fire and detoxification.³⁵ Wuzhuyu (Fructus Evodiae Rutaecarpae) has the effects of dispersing cold and relieving pain, soothing Qi under the liver, anti-inflammatory and analgesic. They are official medicines, and their combination is the Zuojin pill prescription in Dan Xi Xin Fa.³⁶ Foshou (Fructus Citri Sarcodactylis) has the functions of soothing the liver, regulating Qi, and reconciling depression. Xiangfu (Rhizoma Cyperi) and Xiangyuan (Fructus Citri Medicae) have the functions of soothing the liver and regulating Qi, neutralizing phlegm, antibacterial and anti-inflammatory.^37,38 Dahuang (Radix Et Rhizoma Rhei Palmati) clears heat and reduces fire, promotes blood circulation and removes blood circulation and removing blood stasis, reducing down and attacking accumulation.³⁹ These four were adjuvants. Haipiaoxiao (Endoconcha Sepiellae) and Walengzi (Concha Arcae) are ladyship medicines, that make acid and relieve pain, remove pain, remove phlegm and blood stasis, and soften firmness, and dispersing knots.^40,41 Biling Weitong granules is believed to exert its therapeutic effects through a synergistic mechanism of its constituent herbs, aiming to soothe the liver and stomach, regulate Qi, promote blood circulation, neutralize acidity, and alleviate pain. Compared with the MG group, rats in the BL group, BM group, BH group, and TM group exhibited improved general condition, characterized by alertness, activity, glossy fur, docile temperament, and firm, yellow-brown feces without malodor, indicating that Biling Weitong granules improved the characteristics of FD rats, such as lethargy, irritability, and soft and smelly feces. Compared with the MG group, the BL, BM, BH, and TM groups presented significant increases in body weight and food intake (P < 0.05). Compared with the TM group, there were no significant differences in body weight or food intake between the BM and BH groups (P > 0.05), indicating that Biling Weitong granules improved FD symptoms in FD rats, with weight loss and loss of appetite.

Of course, the role of gastrointestinal hormones in gastrointestinal motility is crucial. The decrease in the gastric emptying rate and small intestinal propulsion rate in FD rats is related to dysregulated gastrointestinal hormone secretion in vivo, with MTL and VIP being important indicators of gastrointestinal diseases. MTL is an isolated polypeptide gastrointestinal hormone distributed in motilin cells of the duodenal epithelium.⁴² Reduced MTL levels are associated with FD symptoms such as postprandial fullness, nausea, and vomiting.⁴³ VIP is a gastrointestinal hormone and neurotransmitter of the nonadrenergic and noncholinergic neuroinhibitory systems, which exist in the central nervous system and enteric nervous system.⁴⁴ Elevated VIPs can slow gastrointestinal peristalsis, delay gastric emptying, and aggravate FD symptoms such as insomnia, dreaminess, and mental depression.¹⁰ Biling Weitong granules regulates Qi, blood, cold and heat by the ruler and the subjects, and then unobstructs Qi, cleans the heart fire and reduces the liver fire, reduces the stomach heat and reduces the Qi from time to time, so as to relieve pain, clear the liver and reduce fire, reduce regurgitation and antiemetic, soothe the liver and regulate Qi, dispel phsputum, reduce acid and relieve pain.^32-41 Thus, the content of VIP is reduced, the content of MTL is increased, the gastrointestinal motility is promoted, and the clinical symptoms of FD are relieved. After the drug intervention, compared with the MG group, the gastric emptying rate, the intestinal transit rate, and the serum MTL content in the BL, BM, BH, and TM groups were significantly greater (P < 0.01), and the serum VIP levels were significantly lower (P < 0.01). Compared with the TM group, there were no significant differences in the gastric emptying rate, small intestinal propulsion rate, serum MTL, or VIP content in the BH group (P > 0.05), indicating that Biling Weitong granules promoted gastrointestinal peristalsis and increased the gastric emptying rate and small intestinal propulsion rate in FD rats by promoting the secretion of MTL and inhibiting the release of VIP. Histopathological analysis revealed no major changes in the gastric antrum tissues in the MG, BL, BM, BH, and TM groups.

Overall, our findings suggest that Biling Weitong granules can improve gastrointestinal motility in FD rats, increase gastric emptying and small intestine propulsion rates, elevate serum MTL levels and reduce serum VIP levels, suggesting a potential therapeutic effect on FD, offering novel research avenues and expanding treatment options for this condition, while also establishing a foundation for future clinical investigations.

Deficiencies and prospects: Due to time and resource limitations, only two gastrointestinal hormones, MTL and VIP, were studied in this study. In terms of safety, this study has improved the detection of liver and kidney function, and we believe that these problems will be solved in the future through continuous efforts and exploration. In order to further improve the accuracy and reliability of the study, future research should consider adding experiments such as¹³ C-octanoate breath test or scintigraphy and additional indicators such as gastrin, neuropeptide Y, and somatostatin should also be considered for a comprehensive assessment.

In conclusion, Biling Weitong granules can modulate the levels of the gastrointestinal hormones MTL and VIP in FD rats, promote gastric emptying and small intestinal propulsion, improve gastrointestinal motility and mental status, and alleviate the symptoms of FD, accounting for its efficacy against FD.

Funding Statement

Supported by the Heilongjiang Provincial Health Commission Project: Effectiveness and safety evaluation of Biling Weitong Granules for Treating Functional Dyspepsia (No. 20220303030646)