Mechanism of electroacupuncture in ameliorating mild duodenal inflammation in rats with functional dyspepsia through regulation of the Hippo/YAP pathway

To clarify the pathway by which electroacupuncture (EA) at “Taichong” (LR3) and “Zusanli” (ST36) alleviates low-grade duodenal inflammation in functional dyspepsia (FD) rats via regulating the Hippo/Yes-associated protein (YAP) signaling pathway.

A total of 21 male SD rats were enrolled and randomly divided into the blank group, the model group, and the EA group (n = 7). The FD model was established through a combined induction strategy involving simultaneous administration of forced swimming, tail-clamping stimulation, and food restriction/overeating protocols, with continuous intervention lasting 21 days. Rats in the EA group received EA stimulation at LR3 and ST36, once daily for 20 min,6 consecutive days followed by 1-day rest each course, with 2 courses administered in total. The general physical conditions and body weight of rats in each group were monitored throughout model establishment and treatment. Upon completion of intervention, the 3-h food intake, gastric emptying rate and small intestinal transit rate of rats were measured. HE staining was performed to observe pathological changes in duodenal mucosa. ELISA was adopted to detect serum content of motilin (MTL) and gastrin (GAS). Western blot was used to quantify the protein expressions of YAP, transcriptional coactivator with PDZ-binding motif (TAZ), phosphorylated (p)-YAP and p-TAZ in duodenal tissues. Immunohistochemistry was applied to determine the positive expression of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α(TNF-α) in duodenum. Immunofluorescence staining was conducted to detect the positive expression of Occludin and zonula occludens-1(ZO-1) in duodenal tissues. Quantitative real-time PCR was utilized to test the mRNA expression levels of YAP, TAZ, B-cell lymphoma-2(Bcl-2) and Bcl-2-associated X protein (Bax) in duodenum. TUNEL staining was carried out to assess the apoptotic rate of duodenal epithelial cells.

Compared with the blank group, rats in the model group exhibited severe lethargy, manifested as narrowed eyes, curled recumbency, dry and messy fur, and loose stools; the body weight,3-h food intake, gastric emptying rate and small intestinal transit rate were markedly decreased (P<0.05, P<0.01); massive inflammatory infiltration was observed in the lamina propria of duodenal tissues; serum MTL and GAS concentrations were significantly reduced (P<0.01); the protein expression ratios of p-YAP/YAP and p-TAZ/TAZ in duodenum were notably elevated (P<0.01); the positive expression of IL-1β, IL-6 and TNF-α in duodenal tissues was up-regulated (P<0.01). The mean fluorescence intensity of Occludin and ZO-1 in duodenum was remarkably lowered (P<0.01). The mRNA level of Bax in duodenal tissues was significantly increased (P<0.01), while the mRNA expressions of YAP, TAZ and Bcl-2 were distinctly down-regulated (P<0.01). The apoptotic rate of intestinal epithelial cells was markedly higher (P<0.01). After EA intervention, relative to the model group, rats in the EA group showed improved mental state, enhanced locomotor activity, glossy fur and formed stools, the inflammatory infiltration in the duodenal lamina propria was alleviated, and all the indicators mentioned above were reversed (P<0.01, P<0.05).

EA at LR3 and ST36 can effectively relieve symptoms of FD, improve the general physical status and suppress low-grade duodenal inflammation in FD rats. Its therapeutic mechanism may be associated with regulating the Hippo/YAP signaling pathway.