| Objective Gastrointestinal motility disorder is the main pathophysiological basis of functional dyspepsia.In this study,FD rats and mTOR(-/-)KO rats were used as research objects,electroacupuncture and mTOR inhibitor were used as intervention measures to explore the effects of electroacupuncture on gastrointestinal motivity in FD rats,and to study the possible mechanism of electroacupuncture activating mTOR pathway to regulate Ghrelin in FD rats.Methods Sixty SD rats were randomly selected,half male and half female,aged 10 weeks,with a body mass of about 200.MTOR(-/-)KO rat F0 generation,SPF grade,1 male,2 female,cage propagation,after breedingfor formal experiment homozygous 10.Sixty SD rats were randomly divided into 6 groups: normal group,FD model group,FD+ drug group,FD+ EA group,FD+ EA + mTOR agonist group,and FD+ EA + mTOR inhibitor group.Twenty mTOR(-/-)KO rats were randomly divided into mTOR(-/-)KO + FD model group and mTOR(-/-)KO + FD + EA treatment group.The FD rat model was constructed after 14 days of continuous intervention using clip-tail stimulation + irregular diet.In the drug group,0.02 g /m L cisapili was administered to rats at a dose of 2 m L/100 g by intragastric administration once per day,6 times per week,for 14 days consecutively for 2 weeks.Electric acupuncture intervention group rats "st 36",vertical Pierce bilateral 0.3-0.5 inches "st 36" acupuncture points,improving fast plug twist to needle like fish bait under heavy acerbity feeling after connect with Hans electroacupuncture apparatus,continuous wave,frequency of 4 Hz,strength 2 m A,no obvious discomfort in rat limb and ostracism for degrees,each stimulus 20 min,1 times a day,six times a week,for 2 weeks intervention,a total of 14 days.Agonist group: Intraperitoneal injection of L-leucine(0.45g/kg)once per day,6times per week,continuous treatment for 2 weeks,a total of 14 days after the use of electric acupuncture zusanli treatment.Inhibitor group,intraperitoneal injection of rapamycin(1 mg/kg)was performed once per day,6 times per week.After continuous treatment for 2 weeks for a total of 14 days,electroacupuncture was used to stimulate “zusanli ”therapy.At the same time,the rats in the normal group were also bound and fixed(same as the electroacupuncture group),but electro-acupuncture intervention was not given.The general conditions of rats in each group were observed and scored before and after inter-vention;The gastric residual rate and small intestinal propulsive rate were determined;The tissues of hypothalamus,antrum and small intestinewere observed by HE staining;Real-time RT-PCR and Western Blot were used to detect the m RNA and phosphorylation expressions of Ghrelin,GOAT,pre-Ghrelin,P-P70S6 K,mTOR m RNA,mTOR,P70S6 K,P4E-BP1 and pe IF4 E in each group;The localization of mTOR/Ghrelin and P70S6K/Ghrelin was observed by immunofluorescence method.Results1.After 14 days of modeling,most of the rats showed symptoms of indigestion,such as less diet and activity,loss weight,tired lying in piles,tired expression,depressed mood,messy and dull hair,loose and loose stools and odors.2.Daily behavioral status: after the electroacupuncture group and the drug group FD rats were stimulated with cisapril and electroacupuncture,the activity and food intake of the rats were increased,and the hair gradually regained its luster and smoothness,and the condition of the rats in the electroacupuncture group was improved more significantly.3.Gastrointestinal dynamic changes:Compared with the normal group,the gastric residual rate in the model group was significantly increased(P < 0.01),and the small intestinal propulsion rate was significantly decreased(P < 0.05).Compared with the model group,the gastric residual rate of FD rats in the drug group,the electroacupuncture group,the agonist group and the inhibitor group was significantly reduced(P < 0.05),and the small intestinal propulsion rate was significantly increased(P < 0.05).Compared with the electroacupuncture group,the gastric residual rate of the agonist group was significantly increased and the small intestinal propulsion rate was significantly decreased(P < 0.05),but the difference between the electroacupuncture group and the inhibitor group was not statistically significant(P > 0.05).The small intestinepropulsion rate of the electroacupuncture group and the drug group was significantly increased(P < 0.01,P < 0.01),and the small intestine propulsion rate of the electroacupuncture group was higher than that of the drug group,but the difference between the two groups was not statistically significant.4.Histomorphological changes(HE):From the pathological changes of the hypothalamus,compared with the normal group,the number of neurons in the FD model group was reduced,the size and morphology of cells were changed,and the nuclei were hyperchromatic and vacuolated.EA therapy can significantly improve the pathological changes in FD model rats.However,on the basis of EA treatment,the use of mTOR agonists and inhibitors led to the aggravation and reduction of hypothalamic lesions,respectively.EA therapy had no significant effect on the pathological changes of hypothalamus in mTOR(-/-)KO FD model rats.In terms of the pathological changes of gastric antrum and small intestine,the gastric mucosa of FD model group was thinned and ruptured,and the gastric matrix was congested.Intestinal epithelial cells are edema,focal necrosis,abscess,and mass inflammatory cell infiltration.The gastrointestinal mucosa of the rats in each group(EA or EA + drug)was basically normal,with only mild hyperemia and inflammatory cell infiltration.Again,the epithelial cells are more or less structurally normal.However,the effect of EA on the gastrointestinal pathological changes of mTOR(-/-)KO FD model rats was not obvious.5.Real-time PCR results showed that:Compared with the normal group,the expression levels of Ghrelin and GOAT m RNA in the stomach and small intestine of the model group were significantly down-regulated(P<0.01,P<0.01).Compared with themodel group,the expressions of Ghrelin and GOAT m RNA in the electroacupuncture group and the drug group were significantly up-regulated(P<0.01,P<0.01).Compared with the drug group,the expression levels of Ghrelin and GOAT m RNA in the stomach and small intestine of the rats in the electroacupuncture group were up-regulated,but the difference of Ghrelin m RNA in the stomach and small intestine of the rats in the two groups was statistically significant(P<0.01),while the difference of GOAT m RNA in the two groups was not significant(P>0.05).Compared with the normal group,the expression level of Ghrelin m RNA in the stomach and small intestine of the model group was significantly down-regulated(P<0.05),while the expression level of mTOR m RNA in the model group was significantly up-regulated(P<0.05).Compared with the model group,the expression level of Ghrelin m RNA in the stomach and small intestine of the electroacupuncture group and the inhibitor group was significantly up regulated(P<0.05),while the expression level of mTOR m RNA in the electroacupuncture group and the inhibitor group was significantly down-regulated(P<0.05).Compared with the electroacupuncture group,the expression level of Ghrelin m RNA in the stomach and small intestine of the rats in the agonist group was significantly decreased(P<0.05),while the expression level of mTOR m RNA in the agonist group was significantly increased(P<0.05).The expression level of mTOR m RNA in the stomach and small intestine of the inhibitor group decreased significantly(P<0.05).Compared with the normal group,the m RNA levels of mTOR and p70S6 K in the hypothalamus of FD model rats were significantly increased,while those of Ghrelin,4EBP1 and e IF4 E were significantly decreased(All P<0.01).However,EA treatment significantly reduced them RNA levels of mTOR and p70S6 K,and increased the m RNA levels of Ghrelin,4EBP1 and e IF4E(all P<0.01).Compared with THE FD + EA group,mTOR inhibitors further promoted the therapeutic effect of EA to a certain extent,while mTOR agonists were the opposite(All P< 0.01).Surprisingly,EA treatment significantly increased the m RNA expression of Ghrelin in mTOR(-/-)KO FD rats(P<0.01),but had no effect on the downstream target expression of mTOR signaling pathway.Compared with the normal group,the m RNA levels of mTOR and its downstream targets(4EBP1,e IF4 E,p70S6K)in the gastric antrum and small intestine of FD model rats were significantly increased,while the m RNA levels of Ghrelin were significantly decreased(All P<0.01).EA treatment significantly reduced the m RNA levels of mTOR and its downstream targets(4EBP1,e IF4 E,p70S6K)in FD model rats,while the m RNA levels of Ghrelin were increased(All P<0.01).Compared with FD + EA group,mTOR inhibitors further promoted the effect of EA treatment on all indicators(except Ghrelin)to a certain extent,while mTOR agonists were on the contrary(all P<0.01).Similarly,EA treatment significantly increased Ghrelin m RNA expression in gastric antrum and small intestine in rats with mTOR(-/-)KO FD model(P<0.01),but had no effect on downstream targets of mTOR signaling pathway(4EBP1,e IF4 E and p70S6K).6.Western blot results showed that:The expression levels of Ghrelin and GOAT proteins in the gastric antrum and small intestine of the model group were significantly decreased compared with that of the normal group(P<0.01,P<0.01).Compared with the model group,the expressions of Ghrelin and GOAT proteins in the stomach and small intestine of the rats in the electroacupuncture group and the drug group were significantlyincreased(P<0.01,P<0.01).Compared with the drug group,the expression levels of Ghrelin and GOAT protein in the gastric antrum and small intestine of the rats in the electroacupuncture group were increased,but the differences of Ghrelin protein expression levels in the gastric antrum and small intestine of the rats in the two groups were not statistically significant(P>0.05),while the differences of GOAT protein expression levels in the gastric antrum and small intestine of the rats in the two groups were statistically significant(P<0.01).Compared with the normal group,the expression level of pre-ghrelin protein in the stomach of the model group was significantly decreased(P<0.05),while the expression level of p-P70S6 K protein was significantly increased(P<0.05).Compared with the model group,the expression level of pre-ghrelin protein in the stomach of FD rats in the electroacupuncture group,agonist group and inhibitor group was significantly increased(P<0.05),the expression level of p-P70s6 K protein in the stomach of FD rats in the agonist group was significantly increased(P<0.05),while the expression level of P-P70s6 K protein in the inhibitor group was significantly decreased(P<0.05).Compared with the electroacupuncture group,the expression of pre-ghrelin protein in the stomach of rats in the agonist group was significantly decreased(P<0.05),while the expression of p-P70S6 K protein was significantly up-regulated(P<0.05).The expression level of pre-ghrelin protein in the stomach of the inhibitor group was significantly increased(P<0.05),while the expression level of p-P70S6 K protein was significantly decreased(P<0.05).Compared with the normal group,activation of typical mTOR signaling pathway and low Ghrelin protein expression were observed in FD model rats in three types of tissues,which mainly represented highexpression of P-mTOR and phosphorylated downstream targets(P-4EBP1,P-eif4 E p-P70S6K)(P<0.01).Accordingly,EA treatment significantly increased Ghrelin protein levels while decreased mTOR signal activation and its downstream target expression levels(P<0.01).Compared with THE FD + EA group,mTOR inhibitors further promoted the therapeutic effect of EA to a certain extent,while mTOR agonists were the opposite(All P<0.01).Surprisingly,EA treatment significantly increased ghrelin expression in rats with mTOR(-/-)KO FD model(P<0.01),but had no effect on the activation of downstream targets of mTOR signaling pathway(P-4EBP1,P-EIF4 E,p-P70S6K).7.Dual-standard immunofluorescence results showed that:The results showed that the proportion of mTOR positive cells in the gastric antrum and small intestine tissues of the model group was higher than that of the normal group,and the proportion of Ghrelin positive cells was lower than that of the normal group,indicating that modeling could promote the expression of mTOR protein in the gastric antrum and small intestine tissues of the rats and inhibit the expression of Ghrelin protein.The proportion of mTOR positive cells in the gastric antrum and small intestine of the rats in the electroacupuncture group was lower than that in the model group,and the proportion of Ghrelin positive cells was higher than that in the model group,indicating that the electroacupuncture could inhibit the expression of mTOR protein in the gastric antrum and small intestine of the rats and promote the expression of Ghrelin protein.M TOR inhibitors + electric acupuncture group rats mTOR positive cells in the gastric antrum and intestine tissue number ratio is higher than normal group is lower than the model group rats,Ghrelin positive cells number ratio is far lower than the model group rats,indicating mTOR inhibitorscan promote gastric antrum of rats and intestinal tissue of mTOR protein expression,inhibition of the expression of Ghrelin protein,called mTOR agonist + electric acupuncture group achieve the opposite effect;The proportion of mTOR positive cells in the gastric antrum and small intestinal tissues of rats in the mTOR inhibitor + electroacupuncture group was higher than that in the electroacupuncture group,and the proportion of Ghrelin positive cells was lower than that in the electroacupuncture group,indicating that the inhibitory effect of electricity on the expression of mTOR protein in the gastric antrum and small intestinal tissues of rats and the promotion effect of electricity on the expression of Ghrelin protein could be blocked by mTOR inhibitors.Compared with mTOR(-/-)KO+FD+EA group,the number of Ghrelin positive cells in the gastric antrum and small intestinal tissues of rats was significantly increased,indicating that electroacupuncture treatment could increase the secretion of Ghrelin in the gastric antrum and small intestinal tissues of rats.Rat gastric and duodenal tissue P70S6K/Ghrelin immunofluorescence double targets as a result,the model group rats gastric antrum and the duodenum in the organization P70S6 K positive cells number ratio is higher than normal group of rats,Ghrelin positive cells was less than normal group of rats,that building can promote rat P70S6 K protein expression in gastric antrum and the duodenum tissue,inhibit the expression of Ghrelin protein.The proportion of P70S6 K positive cells in the gastric antrum and duodenum of the rats in the electroacupuncture group was lower than that of the model group,and the proportion of Ghrelin positive cells was higher than that of the model group,indicating that the electroacupuncture could inhibit the expression of P70S6 K protein in the gastric antrum and duodenum of therats and promote the expression of Ghrelin protein.The proportion of P70S6 K positive cells in the gastric antrum and duodenum of the rats in the mTOR inhibitor + electroacupuncture group was higher than that of the model group,and the proportion of Ghrelin positive cells was lower than that of the model group,indicating that mTOR inhibitor can promote the expression of P70S6 K protein in the gastric antrum and duodenum of the rats,but the expression of Ghrelin protein was not affected.MTOR agonists act contrary to mTOR inhibitors;Compared with the mTOR(-/-)KO+FD+EA group,the number of Ghrelin positive cells in the gastric antrum and small intestine of rats was significantly increased,but there was no significant difference in the number of P70S6 K positive cells,indicating that electroacupuncture treatment could significantly improve the expression of Ghrelin in the gastric antrum and small intestine of rats with mTOR(-/-)KO+FD model,but had no effect on the downstream target P70S6 K of mTOR signaling pathway.Conclusion1.Combined with daily behavior,gastrointestinal motility and histomorphological observation results,FD rat model can be constructed by continuous intervention of clip-tail stimulation + irregular diet for 14 days.2.Electroacupuncture can effectively improve the daily behavioral status and gastrointestinal motility of FD rats,and electroacupuncture intervention is safe and will not cause organic gastrointestinal lesions.3.Electroacupuncture can promote the expression of Ghrelin,GOAT,P70S6 K,4EBP1,e IF4 E protein and m RNA in the gastric antrum and small intestinal tissues of FD rats,and inhibit the expression of mTOR protein and m RNA in the gastric antrum and small intestinal tissues,which may be the therapeutic mechanism of electricity against FD.4.Electricity for FD rats daily behavior at the state and the improvement of the gastrointestinal function can be called mTOR inhibitors block,electricity for FD rats Ghrelin in the gastric antrum and intestine tissue protein and m RNA expression of promoting effect can be mTOR inhibitors block,shows that the intervention of the electricity for FD rats and the effect may be through the mTOR pathways play a role,mTOR(-/-)KO further confirms the corollary of rats. |
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