| ObjectivesGastrointestinal dysmotility is the basic pathophysiological of FD.ENS is called "intestinal brain",which can regulate the movement,secretion,absorption,sensation and blood circulation of the digestive system without relying on the central nervous system.EGCs,a main component of ENS,plays a key role in gastrointestinal homeostasis.GDNF release a wide range of factors accounting for the development,survival and differentiation of peripheral neurons and also plays an important role in the survival of mature neurons after birth.GDNF signals through a multicomponent receptor system,composed of the RET(rearranged during transfection)receptor and one of the four GFR?(1-4)(GDNF family)receptors.Activation of the receptor complex in neurons can lead to modifification of various signaling events,including PI3 K,ERK.It has been shown that Phospho-ERK1/2(p ERK1/2)levels increased in the gasstric in FD rats compared to controls.In our study,were used the FD model rat as model,and observed the change of ENS and GDNF downstream of ERK signaling pathways in FD rats,investigated the effect of Electroacupuncture on ENS and GDNF downstream MEK/ERK1/2 signaling pathway.The U0126,one if ERK inhibitor,was used to Trying to illustrate the possible mechanism of EA on FD via regulating ENS and ERK signaling pathway.MethodsIn part 1,40 SD(Sprague-Dawley)rats were randomly allocated to normal group(MG,n=10)and FD group(n=10).The FD group was allocated to FD2 weeks group(FD2G),FD 3 weeks group(FD3G),FD4 weeks group(FD4G)according to the course of disease,with 10 rats in each group.Rats in the FD group received tail-stimulation(30 min/time,2 times/day)and irregular diet(alternate eating)for 2 weeks,3 weeks and 4 weeks respectively to building model.After the model was established,the rat gastric antrum and duodenum tissues were taken.The levels of PGP9.5,s100?,GFAP,GDNF and p ERK in gastric antrum and duodenum were detected by Western Blotting(WB).The ultrastructure of EGCs was observed by transmission electron microscopy.In the remaining experiments,we obtained 60 SD rats,which were randomly allocated to normal group(NG,n=10)and modeling group(50).Rats in modeling undergoing modeling for 2 weeks.40 rats were randomly select from 50 rats which were build and identified as FD model,and allocated to model group(MG),electroacupuncture group(EAG),inhibitor group(IG),electroacupuncture and inhibitor group(EAIG),with 10 rats in each group.Rats in the EAG were received electroacupuncture at Zusanli(ST36)and Taichong(LR3).The electroacupuncture parameters were continuous wave,2Hz,1 m A.1 time a day for 10 consecutive days.Rats in the IG were intraperitoneally injected with U0126 0.3 mg/kg once daily.Rats in the EAIG was injected with U0126 30 minutes before the electroacupuncture,and then give the electroacupuncture.The general state of the rats was observed,and the rats were weighed.After the electroacupuncture intervention,the nutritive semi-solid paste was administered at a rate of 1g/100 g.After 30 min,the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate(0.35 m L/100 g).Open the abdominal cavity,perform gastric emptying and small intestinal transit.The morphological changes of gastric antrum and duodenum were observed by HE staining.Immunofluorescence staining was used to detect the level and overall distribution of s100? protein in gastric antrum and duodenum.WB was used to detect PGP9.5,GFAP,s100?,GDNF,GFR?1,p MEK and p ERK proteins in rat gastric antrum and duodenum.Expression of PGP9.5,GFAP,s100?,GDNF m RNA levels in rat gastric antrum and duodenum were detected by PCR.results1.Changes of ENS and GDNF/ERK signaling pathways in FD ratsCompared to NG,The protein expression of PGP9.5 in gastric antrum and duodenum were significantly increase in FD2 G,FD3G,FD4G;The level of PGP9.5 in gastric antrum and duodenum in FD4 G was less than that in FD2 G,FD3G(both P<0.05).Compared to FD2 G,the protein expression of s100? in gastric antrum and duodenum was decreased in FD3 G.Compared to NG,The protein expression of s100? in gastric antrum and duodenum were significantly increase in FD2 G,FD3G,FD4 G.The protein expression of s100? in gastric antrum and duodenum was the highest in FD4 G,which was significantly higher than that in FD2 G,FD3G.The protein expression of s100? in FD3 G was significantly higher than that in FD2 G.Compared to NG,The protein expression of GFAP in gastric antrum and duodenum were significantly increase in FD2 G,FD3G,FD4 G.Compred to FDG2,the level of GFAP protein in gastric antrum and duodenum were lower than in FD3 G and FD4 G.Compared to FD3 G,the protein expression of GFAP in gastric antrum and duodenum was increased in FD4 G.Transmission electron microscopy showed ultrastructural changes of EGCs in gastric antrum and duodenum of each group.Transmission electron microscopy showed the normal EGCs cell structure around the nerve fibers in the normal group.The cells were irregularly star-shaped,the shape of the nucleus was irregular,the chromatin was often chromatin,the heterochromatin was less,and there were many sizes around the nucleus.A non-myelinated nerve fiber axon with a circular or elliptical shape,a small amount of mitochondria near the axon,and a neurofilament distributed in the axon,and fewer other organelles in the cytoplasm.In the FD model,the damaged EGCs ultrastructure was observed,the heterochromatin in the nucleus of EGCs cells increased,the chromatin was concentrated,the mitochondria were swollen,and the endoplasmic reticulum expanded.Compared to NG,the expression of GDNF and p MEK in gastric antrum and duodenum were significantly increased in FD 2G,FD3 G,and FD4 G.Compared to FD2 G,the expressions of GDNF and p MEK in the antrum and duodenum were significantly higher in the FD3 G and FD4 G.Compared to the FD3 G,The expressions of GDNF and p ERK in gastric antrum and duodenum were significantly increased in FD4 G.2.Effects of electroacupuncture on morphological changes and gastrointestinal kinetics in FD ratsThere was no significant difference in weight between the all groups before the model establishment,which was basically the same(P>0.05).After the model was established,the weights of the rats were significantly decreased in the MG,EAG,IG and EAIG.The MG,EAG,IG and EAIG were did not show difference.After different interventions in each group,the weight of rats in MG was significantly lower than in the NG.Compaired to MG,the weight of the rats were increased significantly in EAG,IG,EAIG.The weight of rats in the EAIG was higher than that in EAG and IG.There was no significant difference between the EAG and IG.The rate of gastric residual in each group was compared.The rate of the MG was significantly higher than that in the NG.Compared to NG,the rate of gastric residual was decreased significantly in the EAG,IG,EAIG.The rate of gastric residual in the EAIG was lower than in EAG,IG.While there was no significant difference between the EAG and IG.The rate of small intestine propulsion in the MG was significantly lower than that in the NG.Compare to MG,the rate of small intestine propulsion were significantly higher in the EAG,IG,EAIG.Compared to EAG,there was no significant difference in the IG.Compare to EAIG,the rate of small intestine propulsion was decreased in EAG,IG.Morphological changes of gastric antrum and duodenum were compared between the groups.No organic changes were found in the stomach and duodenum tissues of the rats in each group.The color of the gastric mucosa in the MG was observed by the naked eye.There was no hemorrhage or inflammatory reaction in the gastric mucosa in the NG,EAG,IG,EAIG.3.Effect of electroacupuncture on ENS in FD ratsCompared to NG,the expression of PGP9.5 in the gastric antrum and duodenum in the MG was significantly decreased.Compared to MG,the expression of PGP9.5 in gastric antrum and duodenum was significantly increased in EAG,IG,EAIG.The level of PGP9.5 in gastric antrum and duodenum was significantly higher than that in EAG and IG.The level of PGP9.5 in the EAG was higher than that in IG.Immunofluorescence staining showed that s100? staining was red fluorescence,mainly distributed in the mucosal layer and muscle layer of gastric antrum and duodenum.The MG was more strongly stained,especially the mucosal layer fluorescent staining was significantly increased by immunofluorescence photographs.Statistical analysis of mean optical density showed that the mean optical density of s100? in gastric antrum and duodenum was significantly higher in the MG than in the NG.Compared to MG,the mean optical density of s100? in gastric antrum and duodenum was significantly decreased in EAG,IG,EAIG.There was no significant difference between the EAG and IG.Compared to EAIG,the mean optical density of s100? in the gastric antrum and duodenum was significantly lower in the EAG and IG.At the same time,WB analysis was performed on the gastric antrum and duodenum.The expression of s100? protein in the gastric antrum and duodenum in MG was significantly higher than that in the NG.Compared to MG,the expression of s100? protein in the gastric antrum and duodenum was decreased in EAG,IG,EAIG.There was no significant difference between the EAG and IG.Compared to EAIG,the expression of s100? in the gastric antrum and duodenum was significantly lower in the EAG and IG.The level of GFAP in the gastric antrum and duodenum in the MG were higher than those in the NG.Compared to MG,the level of GFAP in the gastric antrum and duodenal tissues was decreased in EAG,IG,EAIG.The levels of GFAP in EAIG was lower than in EAG and IG.There was no significant difference between EAG and IG.The expression of PGP9.5 m RNA in the antrum and duodenum in MG were significantly lower than those in NG.Ccompared to MG,the level of GFAP in the gastric antrum and duodenal was increased in EAG,IG,EAIG.The expression of PGP9.5 m RNA in gastric antrum and duodenum in EAIG was significantly higher than that in EAG and IG.The level of PGP9.5 m RNA in gastric antrum and duodenum in EAG was significantly increased than in IG.The expression of s100? m RNA in the gastric antrum and duodenum in MG was significantly higher than that in NG.Compared to MG,the expression of s100? m RNA in the gastric antrum and duodenum was lower in EAG,IG,EAIG.The expression of s100? m RNA in the gastric antrum and duodenum in EAG was lower than in IG,but higher than in EAIG.Compared to IG,The expression of s100? m RNA in EAG was significantly decreased.The expression of GFAP mRNA in the gastric antrum and duodenum in MG was significantly higher than in NG;Compared to MG,the expression of GFAP m RNA in gastric antrum and duodenum was significantly lower in EAG,IG,EAIG.Compared to EAIG,the expression of GFAP m RNA in gastric antrum and duodenum was higher in EAG and IG.The expression of GFAP m RNA in gastric antrum and duodenum in IG was higher than that in EAG4.The effect of electroacupuncture on GDNF and receptor GFR?1 in FD rats.The positive staining of GDNF is a brown-yellow positive reaction product.In the NG,the brown-yellow positive reaction products were observed in the mucosa and muscle layer of the gastric antrum,mainly distributed in the mucosal layer.The brown-yellow products of the duodenum were mainly distributed in the mucosa.The brown-yellow positive reaction particles in the gastric antrum and duodenum in MG was increased significantly.The statistical analysis of the mean optical density expressed by each group showed that the mean optical density of GDNF in gastric antrum and duodenum in MG was significantly higher than that in NG.Compared to the MG,the mean optical density of GDNF in gastric antrum and duodenum was significantly decreased in EAG,IG,EAIG.The mean optical density of GDNF in EAIG was lower than that in EAG and IG.there was no significant difference between EAG and the IG.At the same time,we used WB to analyze the expression of GDNF protein in gastric antrum and duodenum of rats in each group.The expression of GDNF protein in the gastric antrum and duodenum in MG was the highest,compared to the other groups.there was no significant difference between EAG and the IG.The level of GDNF protein in gastric antrum and duodenum in eaig were significantly higher than in EAG and IG.The level of GFR?1 protein in the gastric antrum and duodenum in MG was significantly higher than in the NG.Compared to MG,the level of GFR?1protein in the gastric antrum and duodenum was significantly decreased in the EAG,IG,EAIG.The level of GFR?1 in EAIG was significantly lower than that in EAG and IG.There was no significant difference between EAG and IG.The level of RET protein in the gastric antrum and duodenum in MG was significantly higher than in the NG.Compared to MG,the level of RET protein in the gastric antrum and duodenum was significantly decreased in the EAG,IG,EAIG.The level of RET in EAIG was significantly lower than that in EAG and IG.The level of RET protein in the duodenum was decreased in EAG than in IG,and the level of RET protein in the gastric antrum was no significant difference between EAG and IG.It was found that the level of GDNF m RNA in the gastric antrum and duodenum in MG was significantly higher than in NG.Compared to MG,the level of GDNF m RNA in the gastric antrum and duodenum was significantly decreased in the EAG,IG,EAIG.The level of GDNF m RNA in gastric antrum and duodenum in EAIG were lower than those in EAG and IG.There was no statistically significant difference between the EAG and IG(all P>0.05).5.The effect of electroacupuncture on the MEK/ERK signaling pathway.Compared to NG,the expression of p MEK in the gastric antrum and duodenum was significantly up-regulated in MG.Compared to MG,the expression of p MEK in the gastric antrum and duodenum was decreased in EAG,IG,and EAIG.The level of p MEK protein in the gastric antrum and duodenum in EAIG was significantly decreased than in EAG,IG.The level of p MEK protein in gastric antrum and duodenum in IG was lower than in EAG.The level of p ERK1/2 protein in the gastric antrum and duodenum in MG was significantly increased.Compared to MG,the level of p ERK1/2 protein in the gastric antrum and duodenum was significantly lower in EAG,IG,EAIG.The expression of p ERK1/2 protein in gastric antrum and duodenum in EAIG was lower than that in EAG and IG.Compared to IG,the level of p ERK1/2 in the gastric antrum and duodenum in was increased in EAG.conclusions(1)In the FD model,the neurons in the gastric antrum and duodenum were decreased.EGCs abnormally proliferate and activate,and ultrastructure is impaired.(2)Electroacupuncture increased the weight of FD rats and promoted gastrointestinal motility.(3)Electroacupuncture can promote the recovery of damaged ENS,and the combination of ERK signaling pathway inhibitor and electroacupuncture has a superposition effect.The recovery effect of electroacupuncture on ENS is related to ERK signaling pathway.(4)Electroacupuncture inhibited the activation of MEK/ERK1/2signaling pathway and promoted the recovery of damaged ENS in FD rats.It is one of the possible mechanisms of electroacupuncture intervention in FD rats. |
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