The Mechanism Of Electroacupuncture Regulating Ghrelin In Rats With Functional Dyspepsia Via AMPK

Objectives To observe the effects of electroacupuncture on the behavior,histomorphology and gastrointestinal kinetics of FD rats,and to explore the possible mechanism of electroacupuncture activation of AMPK pathway in regulating Ghrelin in rats with FD.Methods Twelve SD rats were randomly selected and included in the normal group.The rat model of FD was established by tail-clamping stimulation plus irregular diet for 14 days.The FD rats were randomly divided into model group,electroacupuncture group,AMPK inhibitor group and AMPK inhibitor + electroacupuncture group,12 rats per group.Rats in the EA group were treated with EA at Zusanli once daily for 10 consecutive days.The AMPK inhibitor group was intraperitoneally injected with Compound C at a dose(20 mg/kg)once daily for 10 consecutive days.The AMPK inhibitor + EA group was intraperitoneally injected with Compound C.Two hours after the injection,EA was used,and the intervention was performed once a day for 10 consecutive days.At the same time,the rats in the normal group,the model group and the AMPK inhibitor group were restrained and fixed(same as EA group),but no EA intervention was given.Before and after the intervention,the general condition of the rats in each group were observed and scored.The sucrose consumption,the horizontal movement and the vertical movement of the Open-Field were observed.The gastric residual rate and intestinal propulsion rate were measured.HE staining was used to observe the gastric antrum.and small intestine tissues.RT-PCR was used to detect the expression of AMPK,TSC2,Rheb,m TOR and Ghrelin m RNA in gastric antrum and small intestine of each group.The expressions of AMPK,TSC2,Rheb,m TOR and Ghrelin in gastric antrum and small intestine were detected by Western blot.Double immunofluorescence staining was used to detect the quantification and localization of AMPK/m TOR and m TOR/Ghrelin proteins in gastrointestine of rats in each group.Results1.Fourteen days after the establishment of the model,most rats suffered from dyspepsia such as less diet and activity,weight loss,tired lying in piles,fatigue,low mood,less reaction when restrained,weak calls,dull hair and shallow stool.2.Daily Behavioral Status:Before intervention,there was no difference in the general condition score,sugar and water consumption,horizontal and vertical movement times between model group,EA group,AMPK inhibitor group,AMPK inhibitor + EA group,which were lower than those of normal group.After intervention,the general situation score,sugar and water consumption,horizontal and vertical movement times of rats in model group,AMPK inhibitor + EA group did not change significantly compared with those before intervention.The general situation score,sugar and water consumption,horizontal and vertical movement times of rats in EA group were higher than those before intervention and model group.The general situation score,sugar and water consumption,horizontal and vertical movement times of rats in AMPK inhibitor group were lower than those before intervention and model group.The general situation score,sugar and water consumption,horizontal and vertical movement times of rats in AMPK inhibitor+ EA group were lower than those in EA group.3.Gastrointestinal Dynamics: Compared with the normal group,the gastric residual rate in the model group increased,and the small intestinal propulsion rate decreased.Compared with the model group,the gastric residual rate in EA group was lower,and the small intestinal propulsion rate was higher.Compared with model group,The AMPK inhibitor group had higher gastric residual rate and lower small intestinal propulsion rate.Compared with EA group,AMPK inhibitor + EA group increased gastric residual rate and decreased small intestinal propulsion rate.4.Histopathological changes(HE): No organic lesions were found in the gastric antrum and small intestine of rats in normal group,model group,electro-acupuncture group,AMPK inhibitor + EA group under high power microscopy.Only in the AMPK inhibitor group,partial edema in the submucosa of stomach and small intestine and a small amount of inflammatory cell infiltration were observed.5.RT-PCR results showed that the expression of AMPK,TSC2 and Ghrelin m RNA in gastric antrum and small intestine tissue of model group was significantly lower than that of normal group,while the expression of Rheb and m TOR m RNA was significantly increased.Compared with the model group,the expression of AMPK,TSC2 and Ghrelin m RNA in gastric antrum and small intestine of EA group was significantly up-regulated,while the expression of Rheb and m TOR m RNA was significantly down-regulated.Compared with the model group,the expression of AMPK,TSC2 and Ghrelin m RNA in gastric antrum and small intestine of rats in AMPK inhibitor group was significantly decreased,while the expression of Rheb and m TOR m RNA was significantly increased.Compared with EA group,the expression of AMPK,TSC2 and Ghrelin m RNA in gastric antrum and small intestine of rats in AMPK inhibitor + EA group was significantly down-regulated,while the expression of Rheb and m TOR m RNA was significantly up-regulated.6.Western blot results showed that the expression of AMPK,TSC2 and Ghrelin protein in gastric antrum and small intestine of model group was significantly lower than that of normal group,while the expression of Rheb and m TOR protein was significantly increased.Compared with the model group,the expression of AMPK,TSC2 and Ghrelin protein in gastric antrum and small intestine of EA group was significantly increased,while the expression of Rheb and m TOR protein was significantly decreased.Compared with the model group,the expression of AMPK,TSC2,Ghrelin protein in gastric antrum and small intestine tissue of rats in AMPK inhibitor group was significantly down-regulated,while the expression of Rheb and m TOR protein was significantly up-regulated.Compared with EA group,the expression of AMPK,TSC2 and Ghrelin protein in gastric antrum and small intestine of rats in AMPK inhibitor + EA group was significantly down-regulated,while the expression of Rheb and m TOR protein was significantly up-regulated.7.The results of double immunofluorescence staining showed that AMPK/m TOR and m TOR/Ghrelin were co-localized in the gastric antrum and duodenum of rats.There was a negative correlation between AMPK and m TOR cell count,and a negative correlation between m TOR and Ghrelin cell count.Compared with the normal group,the number of AMPK positive cells in the gastric antrum and duodenum of the model group was significantly decreased,the number of m TOR positive cells was significantly increased,and the number of Ghrelin positive cells was significantly decreased.Compared with the model group,the number of AMPK positive cells in the gastric antrum and duodenum tissues of the EA group was significantly increased,the number of m TOR positive cells was significantly decreased,and the number of Ghrelin positive cells was significantly increased.Compared with the model group,the number of AMPK positive cells in the antrum and duodenum tissues of the AMPK inhibitor group was significantly decreased,the number of m TOR positive cells was significantly increased,and the number of Ghrelin positive cells was significantly decreased.Compared with the EA group,the number of AMPK positive cells in the antral and duodenal tissues of the AMPK inhibitor + EA group was significantly decreased,the number of m TOR positive cells was significantly increased.and the number of positive cells was significantly decreased.Conclusions1.According to the results of daily behavior,gastrointestinal motility and histomorphology,the rat model of FD can be established by tail-clamping stimulation plus irregular diet for 14 days.2.EA can effectively improve the daily behavioral status and gastrointestinal motility of FD rats,and EA intervention is safe and will not cause organic lesions of the gastrointestinal tract.3.EA can promote the expression of AMPK,TSC2,Ghrelin protein and m RNA in gastric antrum and small intestine of FD rats,and inhibit the expression of Rheb,m TOR protein and m RNA,which may be the therapeutic mechanism of EA for FD.4.The effect of EA on daily behavior and gastrointestinal motility of FD rats can be blocked by AMPK inhibitors.The effect of EA on AMPK protein and m RNA expression in gastric antrum and small intestine of FD rats can be blocked by AMPK inhibitors.These results suggest that electroacupuncture may play an important role in the intervention of FD rats through AMPK pathway.