A total of 20 healthy SPF male Sprague-Dawley rats, aged 8 weeks, weighing 180–220 g, were provided by Sichuan Research Institute of Biomedical Industry and Technology [production license number: SYXK (Sichuan) 2020-199]. The animals were raised in the Sichuan Institute of Biomedical Industry Technology. The ambient temperature was 20–25°C, lights on at 8:00–20:00, the relative humidity was 45%–60%, and the animals were free to drink and eat. All animal feeding and experimental procedures comply with the Guidelines for the Management and Use of Laboratory Animals. The trial protocol of this study was implemented with reference to ARRIVE guidelines (32). This project passed the ethical review of the Animal Ethics Committee of Sichuan Research Institute of Biomedical Industry Technology (No. 2021-07-15).

After 1 week of adaptive feeding, the rats were randomly assigned to four groups using a random number table method: the normal control group (NC group), the diabetes mellitus group (DM group), the diabetes mellitus + gentle touch group (DM + GT group), and the diabetes mellitus + tuina treatment group (DM + TT group), with 5 rats in each group.

The modeling method of Srinivasan was used (33). The DM group, DM + GT group, and DM + TT group were fed a high-fat and high-sugar diet (60% fat, 20% protein, and 20% carbohydrate) and 5% sucrose water for 2 weeks. Two weeks later, streptozotocin (STZ) (35 mg/kg) was injected intraperitoneally after 12 h of fasting. The rats in the NC group were fed a normal diet and intraperitoneally injected with sodium citrate buffer (1 mL/kg). Blood samples were collected from the tail vein of the rats 72 hours after the injection to measure random blood glucose levels. The DM model was successfully established when the random blood glucose levels reached ≥16.7 mmol/L. The DM, DM + GT, and DM + TT groups were fed a high-fat and high-sugar diet and 5% sucrose water for 6 weeks. Random blood glucose was measured once a week during this period, and rats with random blood glucose < 16.7 mmol/L or dead rats were excluded ( Figure 1 ).

Successful DGP modeling criteria (34): Random blood sugar is not less than 16.7 mmol/L. There were significant differences in hair color, behavior, mental state, and stool characteristics between the two groups. The semisolid gastric emptying rate and small intestinal propulsion rate in the DM group were lower than those in the NC group.

After successful establishment of the DM model, rats in the DM + TT group received tuina therapy. The rats were immobilized using a custom-made rat tuina restraining device. Abdominal kneading: the operator’s right index finger and middle finger are naturally close together, and the rest of the palms are naturally flexed. The finger thread surface was attached to both sides of Shenque point (CV8), and clockwise rhythmic ring kneading was performed, with a frequency of 80 times/min, and the operation time was 10 min. Kneading Pishu (BL20): the operator’s right index finger and middle finger are naturally close together, and the rest of the palms are naturally flexed. The center of the finger thread surface was attached to the Pishu point (between the two ribs under the 12th thoracic vertebra, 6 mm away from the dorsal midline), the frequency of clockwise rhythmic ring kneading was 80 times/min, and the operation time was 10 min. The tuina therapy will be administered 5 times a week for a total of 30 sessions ( Figure 1 ).

The DM + GT group, which serves as the sham tuina control, used a fine brush to perform gentle combing of localized hair areas. The fixing method and the position, frequency, and time of carding operation are consistent with the DM + TT group. The NC group and the DM group were fixed.

All data collectors, testers, and analysts in this study were blinded to the grouping. The experiment was carried out at 9:00 a.m. on days 18, 26, 33, 40, 47, 54, and 61. Blood was collected from the tail of the rat while it was in a conscious state. Random blood glucose values were measured and recorded using a blood glucose meter and blood glucose test strips (ACCU-CHEK Performa, Roche Diagnostic Products Shanghai Co., Ltd., Shanghai, China).

After 6 weeks of tuina intervention, the rats were fasted for 24 h and fed 3 mL of semi-solid nutritional paste containing carbon powder (carboxymethylcellulose sodium 10 g, milk powder 16 g, starch 8 g, white sugar 8 g, activated carbon 2 g, dissolved in distilled water 250 mL mixed to make 300 mL, approximately 300 g of black semi-solid paste). Thirty minutes later, the whole stomach was weighed with intraperitoneal injection of 3% sodium pentobarbital at 5 mL·kg⁻¹ under anesthesia. The stomach was cut along the greater curvature and rinsed thoroughly with 0.9% NaCl solution, the net weight of the stomach was weighed, and the gastric emptying rate was calculated. Gastric emptying rate = [1 − (total weight of stomach − net weight of stomach) ÷ weight of char powder semi-solid nutritional paste] ×100%. The total length of the small intestine (pylorus to ileocecal junction) and the propulsion length of the semi-solid nutritional paste containing charcoal powder were measured, and the propulsion rate of the small intestine was calculated. Intestinal propulsion rate = advancement distance of black paste measured from pylorus as starting point/total intestinal distance of small intestine ×100%.

Fresh gastric antral and ileal tissues were obtained and fixed in 4% paraformaldehyde solution (G1101, servicebio, Wuhan, China) for 24 h. The embedding machine (JB - P5, Wuhan Junjie Electronics Co., Ltd., Wuhan, China) was used for paraffin embedding. Sections (4 μm) were made on a microtome (HM325, Thermo Fisher Scientific, Waltham, MA, USA) and stained with hematoxylin and eosin (HE). The specimens were examined under a light microscope (400×), and the images were collected and analyzed.

The ileal segment tissue was harvested and homogenized by adding ninefold homogenization medium, and the supernatant was used for quantification by BCA (BL521A, biosharp, Beijing, China). In accordance with the ATP enzyme kit (A016-1-1, Nanjing Jiancheng Bioengineering Research Institute Co., LTD., Nanjing, China) and citrate synthase (CS) kit (E-BC-K178-M, Elabscience Biotechnology Co., Ltd, Wuhan, China) operating instructions, biochemical detection analysis of Ca²⁺-Mg²⁺-atpase and CS enzyme activities is performed.

Gastric antrum and ileal segment tissues were harvested for total RNA extraction using Trizol reagent. Synthetic cDNA was obtained using the AccuRT Genomic DNA Removal kit (G492, Applied Biological Materials Inc, Richmond, Canada) standard. Real-time quantitative PCR assays were performed using EvaGreen Express 2X qPCR MasterMix-No Dye (G891, Applied Biological Materials Inc., Richmond, Canada) on a fully automated medical PCR analysis system (Shanghai Hongshi Medical Technology Co., Ltd., Shanghai, China). Primer design methods and primer sequences are shown in Table 1 .

Apply an adequate amount to the gastric antrum and ileum segment organization, using RIPA (BL504A biosharp, Beijing, China) pyrolysis liquid. Centrifuge for 10 minutes for the total protein solution. Protein concentrations were determined using the enhanced BCA protein kit (BL507A, biosharp, Beijing, China). Equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were blocked with 5% skim milk for 1 h at room temperature. Primary antibodies Piezo2 (1:500, PA5-72976, Thermo Fisher Scientific, Waltham, MA, USA), 5-HT4R (1:1,000, DF3503, Affinity Biosciences Pty Ltd, Melbourne, Australia), neuronal nitric oxide synthase (nNOS) (1:1,000, AF6249, Affinity Biosciences Pty Ltd, Melbourne, Australia), calmodulin (CaM) (1:1,000, ab45689, abcam, Cambridge, UK), myosin light chain kinase (MLCK) (1:500, AF5314, Affinity Biosciences Pty Ltd, Melbourne, Australia), and β-Actin (1:5,000, AF7018, Affinity Biosciences Pty Ltd, Melbourne, Australia) were incubated overnight at 4°C. The specimens were incubated with the secondary Goat Anti-Rabbit IgG (H + L) HRP (1:10,000, GAR0072, Multisciences, Hangzhou, China) for 1 h at room temperature. In the chemidoc™ XRS imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) on the image.

Add a ninefold homogenate culture medium preparation to the ileal tissue. After centrifugation, the supernatant was extracted for quantification by BCA (BL521A, biosharp, Beijing, China). The content of 5-HT in ileum tissue was determined according to the instruction from the 5-HT ELISA kit (E-EL-0033c, Elabscience Biotechnology Co., Ltd, Wuhan, China). The OD values of each well were measured at a wavelength of 450 nm using a microplate reader.

The ileal segment tissue was fixed with 4% paraformaldehyde (G1101, servicebio, Wuhan, China) for 24 h. Embedding paraffin was acquired after ileal cross-sectional slices of the organization. Antigen repair was performed using citrate antigen repair solution (G1219, servicebio, Wuhan, China). The cells were incubated with 5% goat serum for 30 min. Primary antibodies Piezo2 (1:50, PA5-72976, Thermo Fisher Scientific, Waltham, MA, USA) and 5-HT (1:200, sc-58031, Santacruz Biotech, Texas, USA) were added and incubated overnight at 4°C. They were washed three times in phosphate-buffered saline (PBS) (G4202, biosharp, Beijing, China). The cells were incubated with secondary antibodies Cy3-conjugated Goat Anti-Rabbit IgG H&L (1:200, GB21303, servicebio, Wuhan, China) and FITC-conjugated Goat Anti-Rabbit IgG H&L (1:200, GB22303, servicebio, Wuhan, China) for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamino-2-phenylindole (G1012, servicebio, Wuhan, China). Images were observed and collected under an ICX41 fluorescence microscope (Ningbo Sunny Instrument Co., Ltd., Ningbo, China) (400×).

IBM SPSS 26.0 software (IBM Statistics, Armonk, NY, USA) was used for statistical analysis. GraphPad Prism 10 Software (GraphPad Software, Boston, MA, USA) was used for plotting. Measurement data were expressed as mean ± SEM. One-way analysis of variance (ANOVA) was used to test the differences between groups. If the data were normally distributed, followed by the Bonferroni post-hoc test was used for pairwise comparisons. Otherwise, Kruskal–Wallis test was performed, followed by the Tukey or Dunn test for pairwise comparisons. Repeated measurement data were analyzed by repeated-measures ANOVA. A p-value of less than 0.05 is considered statistically significant.

During the long-term tuina treatment, random blood glucose detection was carried out once a week. The results showed that the rats in the DM group, DM + GT group, and DM + TT group were fed a high-fat and high-sugar diet for 6 weeks after the establishment of the DM model. The random blood glucose value of rats was maintained above 16.7 mmol/L, which met the basic conditions for the preparation of DGP GI dysfunction. There was no significant decrease in the DM + TT group compared with the DM group, which may be related to the continuous feeding of a high-fat and high-sugar diet ( Figure 2 ).

In order to observe the protective effects of long-term tuina on GI function in DM rats. After completing 30 tuina treatments, we measured the gastric emptying rate and the small bowel propulsion rate. Compared with the NC group, the gastric emptying rate ( Figure 3A ) and the small intestinal propulsion rate ( Figure 3B ) in the DM group were significantly decreased (p < 0.01). Compared with the DM group, the levels in the DM + TT group were significantly increased (p < 0.01). The DM + TT group was superior to the DM + GT group (p < 0.01). The results showed that long-term tuina could significantly protect gastric emptying and small intestinal propulsion in DM rats.

To observe the effect of long-term tuina on the morphology of GI tissue in DM rats, we performed HE staining of tissue from the antrum and ileum ( Figure 4 ). There were no obvious pathological changes in the gastric antrum and ileum in the NC group and the DM + TT group. In the DM group, gastric mucosal hemorrhage, necrosis and exfoliation of mucosal epithelial cells, cell vacuolization, and inflammatory cell infiltration were observed. The structure of intestinal mucosa was destroyed, the apical structure of villi was dissolved, and the epithelial cells were necrotic and exfoliated. Vacuolization of gastric epithelial cells and edema of submucosa were observed in the DM + GT group. Local exfoliation of villous epithelial cells and histolysis were observed in the intestinal mucosa.

CaM and MLCK molecules play an important role in regulating the contraction behavior of GI smooth muscle through the CaM/MLCK pathway (35). In order to further verify the regulatory effect of long-term tuina on gastric motility, antral tissue was assayed for CaM and MLCK mRNA and protein by RT-qPCR ( Figures 5A, B ) and WB ( Figures 5C–E ). Compared with the NC group, the mRNA and protein expressions of CaM and MLCK in the gastric antrum tissue of the DM group were significantly decreased (p < 0.01). Compared with the DM group, the expression of CaM protein in the DM + GT group was significantly increased (p < 0.05), and the expression of CaM and MLCK mRNA and protein in the DM + TT group was significantly increased (p < 0.01). Compared with the DM + GT group, the mRNA and protein expressions of CaM and MLCK in the DM + TT group were increased (p < 0.01).

nNOS is involved in the maintenance of normal intestinal peristalsis. To verify the regulatory effect of long-term tuina on small intestinal motility, we examined the expression of nNOS, CaM, and MLCK molecules (mRNA: Figures 6A–C ) (protein: Figures 6D–G ) in ileal tissues. Compared with the NC group, the mRNA and protein expressions of nNOS, CaM, and MLCK in the DM group were significantly decreased (p < 0.01). Compared with the DM group, the protein expression of nNOS and MLCK in the DM + GT group was significantly increased (p < 0.01, p < 0.05). The mRNA and protein expressions of nNOS, CaM, and MLCK in the DM + TT group were significantly increased (p < 0.01). Compared with the DM + GT group, the mRNA and protein expressions of nNOS, CaM, and MLCK in the DM + TT group were significantly increased (p < 0.01).

Ca²⁺-Mg²⁺-ATPase and CS enzyme activities are involved in energy metabolism in the regulation of GI motility. In order to further verify the regulatory effect of long-term tuina on small intestinal motility, we examined the Ca²⁺-Mg²⁺-ATPase ( Figure 7A ) and CS enzyme ( Figure 7B ) activities in ileal tissues. Compared with the NC group, the activities of Ca²⁺-Mg²⁺-ATPase and CS enzyme in the ileum tissue of the DM group were significantly decreased (p < 0.01). Compared with the DM group, the activities of Ca²⁺-Mg²⁺-ATPase and CS enzyme in the DM + GT group and DM + TT group were significantly increased (p < 0.01). Compared with the DM + GT group, the activities of Ca²⁺-Mg²⁺-ATPase and CS enzyme in the DM + TT group were higher (p < 0.01).

To verify whether the protection of GI function by long-term tuina is related to the expression of Piezo2 and 5-hydroxytryptamine 4 receptor (5-HT4R) molecules, we examined Piezo2 and 5-HT4R mRNA expression in ileum tissue ( Figures 8A,B ). Compared with the NC group, the expression of Piezo2 and 5-HT4R mRNA in the DM group was significantly decreased (p < 0.01, p < 0.05). Compared with the DM group, the DM + TT group’s Piezo2, 5-HT4R mRNA expression was significantly increased (p < 0.01). Compared with the DM + GT group, the mRNA expression of Piezo2 and 5-HT4R in the DM + TT group was higher (p < 0.01, p < 0.05).

To further verify whether long-term tuina protection of GI function is related to the expression of Piezo2, 5-HT, and 5-HT4R, which are important molecules regulating Piezo2/5-HT signaling pathway, we used WB, ELISA, and immunofluorescence staining techniques for detection. The results showed that compared with the NC group, the protein expression of Piezo2, 5-HT, and 5-HT4R in the ileum tissue of the DM group was significantly decreased (p < 0.01) ( Figures 8C–F ). Compared with the DM group, the expression of 5-HT4R protein in the DM + GT group was significantly increased (p < 0.01), and the expression of Piezo2, 5-HT, and 5-HT4R protein in the DM + TT group was significantly increased (p < 0.01). Compared with the DM + GT group, the protein expressions of Piezo2, 5-HT, and 5-HT4R in the DM + TT group were higher than those in the DM + GT group (p < 0.01, p < 0.05).

Immunofluorescence staining results showed that compared with the NC group, the DM group’s ileum tissue, Piezo2, and 5-HT positive fluorescent expression reduced significantly (p < 0.01) ( Figures 9A, B ). Merge images showed that Piezo2 and 5-HT co-expression fluorescence was significantly reduced ( Figure 9 ). Compared with the DM group, the positive fluorescence expression of Piezo2 and 5-HT in the DM + TT group was significantly increased (p < 0.01). Merge images showed that the co-expression fluorescence of Piezo2 and 5-HT was significantly increased. Compared with the DM + GT group, the positive fluorescence expression of Piezo2 in the DM + TT group was higher than that in the DM + GT group (p < 0.01).