Long-term tuina can inhibit the occurrence of gastroparesis by protecting gastrointestinal function in diabetic rats

Abstract

Background: Diabetic gastroparesis (DGP) is a common complication in the later stage of diabetes mellitus (DM). The aim of this study was to investigate the protective effect of long-term tuina on gastrointestinal (GI) function and the occurrence of DGP in diabetic rats.

Methods: Twenty healthy male SD rats were randomly divided into four groups: NC, DM, DM + GT, and DM + TT. DM was induced with streptozotocin and a high-fat, high-sugar diet for 6 weeks. The DM + TT group received tuina therapy (20 min/session, 5 times/week) for 6 weeks. Weekly random blood glucose, gastric emptying rate, and small intestinal propulsion rate were measured. Hematoxylin and eosin (HE) staining assessed gastric antrum and ileum pathology. Ca²⁺-Mg²⁺-ATPase and CS activities, and neuronal nitric oxide synthase (nNOS), calmodulin (CaM), and myosin light chain kinase (MLCK) mRNA and protein expressions were evaluated by PCR and Western blot. 5-HT content was measured by ELISA. Piezo2 and 5-HT4R expressions were analyzed by immunofluorescence staining to observe tuina's protective effect on GI function in DM rats.

Results: Random blood glucose measurements showed normal levels in the NC group, while other groups remained above 16.7 mmol/L. The DM group exhibited reduced gastric emptying and small intestinal propulsion rates, along with gastric antrum and ileum damage. The DM + TT group showed significant improvements in gastric emptying and intestinal propulsion rates, and reduced tissue damage compared to the DM group. In the DM + TT group, mRNA and protein expressions of CaM and MLCK in antrum tissue, and nNOS, CaM, and MLCK in ileum tissue, were significantly increased. Activities of Ca²⁺-Mg²⁺-ATPase and CS enzymes in ileum tissue were also elevated, indicating enhanced GI function. Further analysis showed increased mRNA expressions of Piezo2 and 5-HT4R, and protein expressions of Piezo2, 5-HT, and 5-HT4R in the ileum tissues of the DM + TT group. Immunofluorescence intensity of Piezo2 and 5-HT in the ileum was also heightened. These results suggest that tuina's protective effect on GI function is related to the expression of Piezo2 ion channels.

Conclusions: Long-term tuina protects the GI function of DM rats and inhibits the occurrence of DGP, which might be related to the Piezo2/5-HT pathway.

Keywords: Piezo2/5-HT pathway; diabetes mellitus; diabetic gastroparesis; gastrointestinal function; long-term tuina.

Figure 1

Experimental protocol. After 7 days of adaptive feeding, the rats in the DM group, DM + GT group, and DM + TT group were fed a high-fat and high-sugar diet for 62 days, and the rats in the NC group were fed a conventional diet. Fifteenth day STZ injection solution. Random blood glucose was measured every 7 days starting from day 18. After the DM model was successfully established, tuina treatment was started on the 19th day, 5 times a week for 6 weeks, for a total of 30 times. The 63rd day based measurement.

Figure 2

Groups of rats at different time points of random blood glucose. Data are expressed as mean ± standard deviation, n = 5. NC, normal control group; DM, diabetes mellitus group; DM + GT, diabetes mellitus + gentle touch group; DM + TT, diabetes mellitus + tuina treatment group.

Figure 3

Comparison of gastric emptying rate and small intestinal propulsive rate in each group. (A) Gastric emptying rate. (B) Small intestinal propulsive rate. Data are expressed as mean ± standard deviation, n = 5. NC, normal control group; DM, Diabetes mellitus group; DM + GT, Diabetes mellitus + gentle touch group; DM + TT, Diabetes mellitus + tuina treatment group.

Figure 4

HE staining results of gastric antrum and ileum of rats in each group (400×). (A) Gastric antrum tissue. (B) Ileum tissue. NC, Normal control group; DM, Diabetes mellitus group; DM + GT, Diabetes mellitus + gentle touch group; DM + TT, Diabetes mellitus + tuina treatment group.

Figure 5

mRNA and protein expression of CaM and MLCK in gastric antrum tissues of rats in each group. (A, B) Gastric antrum tissue CaM and MLCK mRNA expression. (C–E) Expression of CaM and MLCK protein in gastric antrum tissue. Data are expressed as mean ± standard deviation, n = 5. NC, Normal control group; DM, Diabetes mellitus group; DM + GT, Diabetes mellitus + gentle touch group; DM + TT, Diabetes mellitus + tuina treatment group. CaM, Calmodulin; MLCK, Myosin light chain kinase.

Figure 6

Expression of nNOS, CaM, and MLCK mRNA and protein in the ileum tissue of rats in each group. (A–C) Expression of nNOS, CaM, and MLCK mRNA in ileum tissue. (D–G) Protein expression of nNOS, CaM, and MLCK in ileum tissue. Data are expressed as mean ± standard deviation, n = 5. NC, Normal control group; DM, Diabetes mellitus group; DM + GT, Diabetes mellitus + gentle touch group; DM + TT, Diabetes mellitus + tuina treatment group. nNOS, Neuronal nitric oxide synthase; CaM, Calmodulin; MLCK, Myosin light chain kinase.

Figure 7

Comparison of ileum tissue enzyme activity between rat groups. (A) Activities of Ca²⁺-Mg²⁺-ATPase. (B) Activities of CS enzyme. Data are expressed as mean ± standard deviation, n = 5. NC, Normal control group; DM, Diabetes mellitus group; DM + GT, Diabetes mellitus + gentle touch group; DM + TT, Diabetes mellitus + tuina treatment group.

Figure 8

Ileum tissue Piezo2, 5-HT, and 5-HT4R protein expression in rat groups. (A, B) Expression of Piezo2 and 5-HT4R mRNA in ileum tissue. (C) Expression of 5-HT protein was detected by ELISA. (D–F) Western blotting was used to detect the expression of Piezo2 and 5-HT4R protein. Data are expressed as mean ± standard deviation, n = 5. NC, Normal control group; DM, Diabetes mellitus group; DM + GT, Diabetes mellitus + gentle touch group; DM + TT, Diabetes mellitus + tuina treatment group. Piezo2, The mechanically sensitive ion channel Piezo2; 5-HT, 5-hydroxytryptamine; 5-HT4R, 5-hydroxytryptamine 4 receptor.

Figure 9

Immunofluorescence staining of Piezo2 and 5-HT in ileum tissue of rats in each group (400×). (A) Immunofluorescence staining. (B) Average optical density of Piezo2. (C) Average optical density of 5-HT. Data are expressed as mean ± standard deviation, n = 5. NC, Normal control group; DM, Diabetes mellitus group; DM + GT, Diabetes mellitus + gentle touch group; DM + TT, Diabetes mellitus + tuina treatment group. Piezo2, The mechanically sensitive ion channel Piezo2; 5-HT, 5-hydroxytryptamine.