Data Sheet 1_Long-term tuina can inhibit the occurrence of gastroparesis by protecting gastrointestinal function in diabetic rats.docx

BackgroundDiabetic 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.
MethodsTwenty 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.
ResultsRandom 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.
ConclusionsLong-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.

背景
糖尿病胃轻瘫（Diabetic gastroparesis, DGP）是糖尿病（Diabetes mellitus, DM）晚期常见并发症。本研究旨在探讨长期推拿（tuina）对糖尿病大鼠胃肠（Gastrointestinal, GI）功能及糖尿病胃轻瘫发生的保护作用。
方法
将20只健康雄性SD大鼠随机分为四组：正常对照组（NC）、糖尿病模型组（DM）、糖尿病+假推拿组（DM + GT）以及糖尿病+真推拿组（DM + TT）。采用链脲佐菌素联合高脂高糖饮食造模6周以构建糖尿病模型。糖尿病+真推拿组接受推拿治疗（tuina therapy，每次20 min，每周5次），持续6周。每周检测随机血糖、胃排空率及小肠推进率。通过苏木精-伊红（Hematoxylin and eosin, HE）染色评估胃窦及回肠病理变化。采用聚合酶链式反应（Polymerase Chain Reaction, PCR）与蛋白质印迹（Western blot）检测Ca²⁺-Mg²⁺-ATP酶、柠檬酸合酶（CS）活性，以及神经元型一氧化氮合酶（neuronal nitric oxide synthase, nNOS）、钙调蛋白（calmodulin, CaM）、肌球蛋白轻链激酶（myosin light chain kinase, MLCK）的mRNA与蛋白表达水平。采用酶联免疫吸附试验（Enzyme Linked Immunosorbent Assay, ELISA）检测5-羟色胺（5-hydroxytryptamine, 5-HT）含量。通过免疫荧光染色分析Piezo2与5-羟色胺4受体（5-hydroxytryptamine 4 receptor, 5-HT4R）的表达情况，以观察推拿（tuina）对糖尿病大鼠胃肠功能的保护作用。
结果
随机血糖检测结果显示，正常对照组血糖维持正常水平，其余各组血糖均高于16.7 mmol/L。糖尿病模型组大鼠胃排空率与小肠推进率降低，且胃窦及回肠组织出现病理性损伤。与糖尿病模型组相比，糖尿病+真推拿组的胃排空率与小肠推进率显著改善，组织损伤程度明显减轻。糖尿病+真推拿组大鼠胃窦组织中CaM与MLCK的mRNA及蛋白表达、回肠组织中nNOS、CaM与MLCK的mRNA及蛋白表达均显著升高。回肠组织中Ca²⁺-Mg²⁺-ATP酶与CS酶活性亦有所提升，提示胃肠功能得到增强。进一步分析显示，糖尿病+真推拿组大鼠回肠组织中Piezo2与5-HT4R的mRNA表达，以及Piezo2、5-HT与5-HT4R的蛋白表达均上调，回肠中Piezo2与5-HT的免疫荧光强度亦显著增强。上述结果表明，推拿（tuina）对胃肠功能的保护作用与Piezo2离子通道的表达密切相关。
结论
长期推拿（tuina）可保护糖尿病大鼠的胃肠功能，抑制糖尿病胃轻瘫的发生，其作用机制可能与Piezo2/5-HT通路有关。