| Objectives Visceral hypersensitivity(VH)is one of the main pathophy siological bases of functional dyspepsia(FD).Enterochromaffin cell(EC)is an important neuroendocrine cell in intestinal mucosa epithelium,serves as the mechanical and chemical sensory organ of the gastrointestinal tract.The synthesized and secreted 5-HT plays an important role in gastrointestinal motility and secretion,especially visceral sensation.PIEZO2,a mechanically-sensitive ion channel,was found to express in EC in recent years and participates in the regulation of visceral sensitivity(VS)by sensing mechanical stimuli and promoting EC secretion of 5-HT.And Exchange protein1 directly activated by c AMP,One of the downstream effector proteins of c AMP,could selectively enhance PIEZO2-mediated mechanical transduction.In this study,FD rats were used as the research object,took into account the fact that 5-HT is involved in the regulation of gastrointestinal sensitivity,we aimed to investigate the effect and mechanism of electroacupuncture(EA)at Zusanli acupoints on FD,in order to preliminary clarify the mechanism of EA on FD that EA could improve VH by inhibiting EPAC1/PIEZO2 axis and reducing the number of EC in gastric fundus as well as the secretion of 5-HT,thus providing theoretical basis for treatment of FD by means of EA.methods Fifty SPF male SD rats aged 7 days were randomly divided into control group,model group,EA group,EPAC1 inhibitor group(inhibitor group)and EA+EPAC1 inhibitor group(EA+inhibitor group)after 3 days of adaptive feeding,with 10 rats in each group.The rats in the normal group were given0.2ml 2% sucrose solution through intragastric administration,and the other four groups were given 0.2ml 0.1% iodoacetamide(IA)and 2% sucrose solution through intragastric administration combined with the compound factors of adult clipping stimulation and irregular diet to establish FD rat model.Rats in the EA group received EA intervention,"Zusanli" acupoints on both hind limbs of rats were selected,electrodes of Han's acupoint nerve stimulator was connected after acupuncture,dilatational wave was selected with a frequency of 2/100 Hz and current intensity of 1m A.Each treatment was 30 min,once a day,and continuous treatment was conducted for 10 days.Rats in inhibitor group were given ESI-09(10mg/kg)through intraperitoneal injection,once a day,for 10 consecutive days.Rats in the EA+inhibitor group received intraperitoneal injection of ESI-09 with the same dose and intervention course as those in the inhibitor group.30 minutes after inhibitor injection,the rats were given EA intervention with the same method and treatment course as those in the EA group.During the intervention period,rats in the control group,model group and inhibitor group underwent the same fixation operation as those in the EA group,and rats in the control group,model group and EA group were intra-peritoneally injected with 0.9% Na CL as control to ensure the consistency of experimental conditions in each group.Before and after intervention,the general situation of rats in each group was observed and scored.Body weight,3h food intake,mechanical pain threshold(MPT)and abdominal withdrawal reflex(AWR)were measured and scored,Rats in each group were subject to Open Field Test(OFT)and Forced Swimming Test(FST).After the intervention,rats in each group were fasted without water for 24 h and given 2% pentobarbital sodium(5mg/100g)through intraperitoneal injection.Anesthetized rats were sampled successively.The serum,gastric fundus and spinal dorsal horn tissues of rats in each group were collected and preserved according to testing requirements.Hematoxylin-eosin(HE)staining was used to observe the morphology and structure of gastric fundus.Immunohistochemical technique(IHC)was used to observe the number of EC in gastric fundus of rats in each group.The levels of 5-HT in serum,gastric fundus and spinal dorsal horn were determined by enzyme-linked immunosorbent assay(ELISA).The protein expression levels of EPAC1,PIEZO2,5-HT3 R in gastric fundus and c-fos in spinal dorsal horn were detected by Western blot(WB).Real-time quantitative polymerase chain reaction(RT-q PCR)was used to detect the gene expression levels of EPAC1,PIEZO2,5-HT3 R in the gastric fundus and c-fos in spinal dorsal horn.The co-localization of 5-HT/EPAC1 and 5-HT/PIEZO2 in the gastric fundus of rats in each group was detected by immunofluorescence double-standard method.Results1.EA can improve the general condition of FD rats,increase body weight and3 h food intake,reduce visceral sensitivity and improve depression.1.1 General situation and grading(1)General situation After modeling,the hair of rats was tarnished,messy and sparse.The rats were listless and huddled together,with decreased activity,significant weight loss,significant decrease in food intake,stool were loose and yellowish green,sour,smelly;After intervention,hair,mental activity,food intake,body weight and feces of rats in normal group were normal as before;The mental state of the model group was slightly improved,the hair color was still yellow and dull,the food intake and body weight were basically unchanged,and the feces were often thin and loose with sour smell.Rats in Inhibitors group,EA group and EA+inhibitor group had better mental state,the hair gradually regained its luster,activity degree and sensitivity and appetite were gradually improved,food intake,body weight of rats in above groups though had larger difference with the control group,but the growth rate of body weight were close to normal group,most stool were forming with no taste,occasionally became soft and smelt sour.(2)General situation scoringBefore intervention,compared with the control group,the general situation score of the other four groups was significantly lower(all P <0.01),and the difference was statistically significant,and there was no difference among the four groups(P >0.05).After intervention,compared with the control group,the general condition score of the model group was still significantly lower(P <0.01),and there was no difference between the other three groups(P >0.05),and the scores of the three groups were similar to that of the control group(all P <0.01),and the scores of the EA group,inhibitor group and EA+inhibitor group were significantly higher than that of the model group(all P <0.01).There was no significant difference among the three groups(P >0.05).1.2 body qualityBefore intragastric administration,there was no difference in body weight among the five groups(P >0.05);Before clamping and intervention,compared with control group,the body weight of the other four groups significantly decreased(all P <0.01),and there was no difference among the four groups(P >0.05).After intervention,compared with the control group,the body weight of the other four groups was still significantly lower(P <0.01),and the difference was statistically significant.The body weight of the EA group,inhibitor group and EA+inhibitor group was significantly higher than that of the model group(P <0.01,P <0.05,P<0.01).There was no difference in body weight between EA group and inhibitor group(P >0.05),and the body weight of EA+inhibitor group was significantly higher than that of EA group and inhibitor group(all P<0.05).1.3 3h food intakeBefore intervention,compared with control group,the 3h food intake of the other four groups significantly decreased(all P <0.01),and the difference was statistically significant,and there was no difference among the four groups(P >0.05).After intervention,compared with the control group,the 3h food intake of the other four groups were still significantly lower(P <0.01),and the difference was statistically significant.The 3h food intake of the EA group,inhibitor group and EA+inhibitor group were significantly higher than that of the model group(all P <0.01),and there was no difference in the 3h food intake between the EA group and inhibitor group(P >0.05).The 3h food intake of rats in the EA+inhibitor group was significantly higher than that in the EA group and the inhibitor group(P <0.05,P <0.01).1.4 Visceral sensitivity(1)Plantar mechanical pain threshold(MPT)Before intervention,compared with the control group,MPT in the plantar of the other four groups significantly decreased(all P <0.01),and there was no difference among the four groups(P >0.05).After intervention,compared with the control group,the MPT of the model group,EA group and inhibitor group were still significantly lower(P<0.01),and the difference was statistically significant,while the MPT of the EA+inhibitor group was similar to that of the control group(P >0.05).The plantar MPT of EA group,inhibitor group and EA+inhibitor group were significantly higher than that of model group(P <0.01,P <0.05,P <0.01),the plantar MPT of EA group was significantly higher than that of inhibitor group(P <0.05,P <0.01),and the plantar MPT of EA+inhibitor group was significantly higher than that of EA group and inhibitor group(P <0.05,P <0.01).(2)AWR scoringAt 20 mm Hg,the AWR scores of the other four groups were higher than those of the control group before and after intervention,but the differences was not statistically significant(P >0.05),and there was no statistical difference on the AWR scores between the four groups(P >0.05).At 40 mm and 60 mmHg,the AWR scores of the other four groups were significantly higher than that of the control group before intervention(all P<0.01),and there was no difference among the four groups(P>0.05).After intervention,compared with the control group,the AWR score of the model group was still significantly higher(P <0.01),and the difference was statistically significant.The AWR score of the EA group,inhibitor group and EA+inhibitor group were similar to that of the control group(all P >0.05),and there was no difference among the three groups(P >0.05).AWR score of EA group,inhibitor group and EA+inhibitor group was lower than that of model group(P <0.05,P <0.05,P <0.01),and the difference was statistically significant.At 80 mmHg,the AWR scores of the other four groups were significantly higher than that of the control group before intervention(all P <0.01),and the difference was statistically significant,but there was no significant difference in AWR score among the four groups(P >0.05).After intervention,AWR score of model group was higher than that of the other four groups,but the difference was not statistically significant(P >0.05).1.5 Depressed mood(1)Open field test(OFT)Before intervention,compared with the control group,the total exercise distance and residence time of OFT in the central region of the remaining four groups significantly shortened(all P <0.01),and there was no statistical difference among the four groups(P >0.05).After intervention,compared with the control group,the total exercise distance and central area residence time of rats in the model group,EA group and inhibitor group were significantly shorter(all P <0.01),and the differences were statistically significant.The total exercise distance and central area residence time of rats in the EA+inhibitor group were similar to those in the control group(P >0.05).The total movement distance and central area residence time of rats in the EA group,the inhibitor group and the EA+inhibitor group were longer than those in the model group(P <0.05,P <0.05,P <0.01),and the differences were statistically significant.The total movement distance and central area residence time of rats in the EA group were similar to those in the inhibitor group(P >0.05).The total movement distance and central area residence time of rats in the EA+inhibitor group were longer than those in the EA group and the inhibitor group(all P <0.05),and the differences were statistically significant.(2)Forced swimming test(FST)Before intervention,compared with control group,FST immotility time of rats in the other four groups were significantly longer(all P <0.01),and the difference was statistically significant,and there was no statistical difference among the four groups(P >0.05).After intervention,compared with the control group,the FST immovability time of rats in the other four groups was prolonged(P <0.01,P <0.01,P <0.01,P <0.05),and the difference was statistically significant.The FST immovability time of rats in the EA group,inhibitor group and EA+inhibitor group was significantly shorter than that in the model group(all P<0.01),with significant statistical difference.The FST immobility time of the EA group was similar to that of the inhibitor group(P >0.05),and the FST immobility time of the EA+inhibitor group was shorter than that of the EA group and the inhibitor group(all P <0.05),the difference was statistically significant.1.6 Gastric fundus histomorphology(HE staining)The structure and morphology of gastric fundus were intact,and the boundaries of mucosa,submucosa,muscle and serosal layer were clear.The mucosa of the control group,the EA group,the inhibitor group and the EA+inhibitor group were intact and normal in shape,with neat and close arrangement of glands and abundant blood vessels in the submucosa.The mucosa of the model group was relatively intact and normal in shape,but the arrangement of glands was slightly loose and disorder.There were no obvious organic changes in gastric fundus such as hyperemia,edema,ulcer or erosion.2.EA can reduce EC quantity in gastric fundus and 5-HT level in serum,gastric fundus and spinal dorsal horn of FD rats2.1 EC numberCompared with control group,the number of EC in gastric fundus mucosa in model group significantly increased(P <0.01),and the difference was statistically significant.Compared with the model group,EC number in the EA group,inhibitor group and EA+inhibitor group decreased(P <0.05,P<0.05,P <0.01),and there was no statistical difference in EC number among the three groups(P >0.05).2.2 5-HT levelCompared with control group,the levels of 5-HT in serum,fundus of stomach and dorsal horn of spinal cord in the other four groups significantly increased(all P<0.01),and the differences were statistically significant.Compared with model group,the levels of 5-HT in serum,gastric fundus and dorsal horn of spinal cord in EA group,inhibition group and EA+inhibitor group significantly decreased(all P <0.01),and the differences were statistically significant.Compared with EA group and inhibitor group,the levels of 5-HT in serum,gastric fundus and dorsal horn of spinal cord in EA+inhibitor group significantly decreased(all P<0.01),with statistically significant differences.The levels of 5-HT in serum,gastric fundus and dorsal horn of spinal cord were similar between EA group and inhibitor group(P >0.05),and the differences were not statistically significant.3.The protein and gene expression levels of 5-HT3 R in the fundus of the gastric and c-fos in spinal dorsal horn of FD rats were decreased by EA(1)5-HT3 R protein level in gastric fundusCompared with control group,the protein expression level of 5-HT3 R in model group,EA group and inhibitor group were significantly higher than that in control group(all P <0.01),and the difference was statistically significant.The protein expression level of 5-HT3 R in EA+inhibitor group was similar to that in control group(P >0.05).Compared with model group,the protein expression level of 5-HT3 R in gastric fundus of EA group,inhibition group and EA+inhibitor group decreased(P <0.05,P <0.01,P <0.01),with statistical difference.Compared with the EA group and the inhibitor group,the protein expression level of 5-HT3 R in gastric fundus in the EA+inhibitor group further decreased(P <0.01,P <0.05),and the difference was statistically significant.(2)c-fos protein expression level in spinal dorsal hornCompared with control group,the protein expression level of c-fos in model group,EA group and inhibitor group were significantly higher than that in control group(all P<0.01),and the difference was statistically significant.The protein level of c-fos in EA+inhibitor group was similar to that in control group(P >0.05).Compared with model group,the protein expression level of c-fos in the spinal dorsal horn of EA group,inhibition group and EA+inhibitor group decreased(P <0.05,P <0.05,P <0.01),with statistical difference.Compared with the EA group and the inhibitor group,the protein expression level of c-fos in the spinal dorsal horn of rats in the EA+inhibitor group further decreased(P<0.01,P<0.05,respectively),and the difference was statistically significant.(3)Gene expression level of 5-HT3 R in gastric fundusCompared with control group,the gene expression level of 5-HT3 R in the gastric fundus of the other four groups was higher(P <0.01,P <0.01,P <0.01,P <0.05),and the difference was statistically significant.Compared with model group,the gene expression level of 5-HT3 R in gastric fundus of EA group,inhibition group and EA+inhibitor group significantly decreased(all P<0.01),with significant statistical difference.Compared with the EA group and the inhibitor group,the gene expression level of 5-HT3 R in gastric fundus in the EA group further decreased(all P<0.05),and there was no statistical difference between the EA group and the inhibitor group.(4)c-fos gene expression level in spinal dorsal hornCompared with control group,c-fos gene expression level in model group,EA group and inhibitor group was higher than that in control group(P <0.01,P <0.05,P <0.05,respectively),and the difference was statistically significant.Compared with model group,the gene expression level of c-fos in spinal dorsal horn of rats in EA group,inhibition group and EA+inhibitor group significantly decreased(all P<0.01),with significant statistical difference;The levels of EA group,inhibitor group and EA+inhibitor group were similar(all P >0.05).4.The protein and gene expression levels of EPAC1 and PIEZO2 and the co-expression of 5-HT/EPAC1 and 5-HT/PIEZO2 in the fundus of FD rats were decreased by EA4.1 EPAC1 protein expression level in gastric fundusCompared with control group,the protein expression level of EPAC1 in gastric fundus of model group,EA group and inhibitor group significantly increased(P <0.01,P <0.05,P <0.05),and the difference was statistically significant.The protein expression level of EPAC1 in EA+inhibitor group was similar to that in control group(P >0.05).Compared with model group,EPAC1 protein expression level in gastric fundus of EA group,inhibition group and EA+inhibitor group significantly decreased(all P<0.01),with significant statistical difference;Compared with EA group and inhibitor group,the protein expression level of EPAC1 in gastric fundus of EA+inhibitor group significantly decreased(P <0.01,P <0.05),and the difference was statistically significant.The levels of EA group and inhibitor group were similar(P >0.05).4.2 Protein expression level of PIEZO2 in gastric fundusCompared with the control group,the protein expression of PIEZO2 in the model group and the inhibitor group increased(P <0.01 and P <0.05,respectively),and the differences were statistically significant.The levels of PIEZO2 protein in the EA group and the EA+inhibitor group were similar to that in the control group(P >0.05).Compared with the model group,the protein expression of PIEZO2 in the gastric fundus of the rats in the EA group,inhibitor group and EA+inhibitor group significantly decreased(P<0.01),and the difference was statistically significant.Although the level of EA group and EA+inhibitor group was lower than that of inhibitor group,the difference was not statistically significant(P >0.05).4.3 EPAC1 gene expression level in gastric fundusCompared with normal group,EPAC1 gene expression level in gastric fundus of control group,EA group and inhibitor group significantly increased(all P <0.01),the difference was statistically significant,and the level of EA+inhibitor group was similar to that of control group(P >0.05).Compared with model group,EPAC1 gene expression level in gastric fundus of EA group,inhibition group and EA+inhibitor group significantly decreased(all P <0.01),with significant statistical difference;Compared with EA group and inhibitor group,EPAC1 gene expression level in gastric fundus of EA+inhibitor group further decreased(P <0.01,P <0.05),and the difference was statistically significant.There was no significant difference between EA group and inhibitor group(P >0.05).4.4 PIEZO2 gene expression level in gastric fundusCompared with the control group,PIEZO2 gene expression in the model group,the EA group and the inhibitor group increased(P <0.01,P <0.05 and P<0.05,respectively),and the differences were statistically significant.The PIEZO2 gene expression in the EA+inhibitor group was similar to that in the control group(P >0.05).Compared with the model group,PIEZO2 gene expression in the gastric fundus of the rats in the EA group,inhibitor group and EA+inhibitor group significantly decreased(all P<0.01),with a statistically significant difference.Although the level of EA+inhibitor group was lower than that of EA group and inhibitor group,the difference was not statistically significant(all P>0.05).4.5 Co-expression of 5-HT/EPAC1 in gastric fundusThe red fluorescence was 5-HT and the green fluorescence was EPAC1.(1)5-HT positive cell rateCompared with control group,the rate of 5-HT positive cells in gastric fundus of model group,EA group and inhibitor group significantly increased (all P <0.01),and the difference was statistically significant.Compared with model group,the 5-HT positive cell rate in EA group,inhibitor group and EA+inhibitor group significantly decreased(all P<0.01),and the difference was statistically significant.Compared with the EA group and the inhibitor group,the 5-HT positive cell rate in the EA+inhibitor group further decreased(P<0.01,P<0.05),with statistical difference.The levels of EA group and inhibitor group were similar(P>0.05).(2)EPAC1 positive cell rateCompared with control group,the positive rate of EPAC1 in model group and EA group was higher(P<0.01,P<0.05),and the difference was statistically significant.Compared with model group,the positive rate of EPAC1 in EA group,inhibitor group and EA+inhibitor group significantly decreased(all P<0.01),and the difference was statistically significant.Compared with the EA group,the rate of EPAC1 positive cells in the EA+inhibitor group further reduced(P<0.05),and the difference was statistically significant.The levels of EA group and inhibitor group were similar(P>0.05).4.6 Expression of 5-HT/PIEZO2 in gastric fundusThe red fluorescence is 5-HT and the green fluorescence is PIEZO2.(1)5-HT positive cell rateCompared with control group,the 5-HT positive cell rate in model group,EA group and inhibitor group significantly increased(all P<0.01),and the difference was statistically significant.Compared with model group,the 5-HT positive cell rate in EA group,inhibitor group and EA+inhibitor group decreased(P<0.05,P<0.05,P<0.01),and the difference was statistically significant.Compared with EA group and inhibitor group,the 5-HT positive cell rate in EA+inhibitor group further decreased(all P <0.05),and the difference was statistically significant.The levels of EA group and inhibitor group were similar(P >0.05).(2)PIEZO2 positive cell rateThe PIEZO2 positive rate of the model group was significantly higher than that of the control group(P <0.01),and the difference was statistically significant.Compared with the model group,the PIEZO2-positive cell rates in the EA group,the inhibitor group and the EA+inhibitor group decreased(P<0.05,P<0.05 and P<0.01,respectively),and there was no statistical difference among the three groups(P>0.05).Conclusion1.The general condition of FD rats became worse,body weight decreased,3h food intake decreased,visceral sensitivity increased,and depression occurred.2.EA at "Zusanli" acupoints can improve the general situation of FD rats,increase body weight and 3h food intake,reduce visceral sensitivity and improve depression.3.EA at "Zusanli" acupoints can reduce the number of EC in the gastric fundus,the level of 5-HT in serum,gartric fundus and spinal dorsal horn,and the protein and gene expression levels of EPAC1,PIEZO2,5-HT3 R in the gastric fundus and c-fos in spinal dorsal horn and the co-expression of5-HT/EPAC1 and 5-HT/PIEZO2 in the gartric fundus.The combination of EPAC1 inhibitor ESI-09 and EA has a synergistic effect.4.By suppressing EPAC1/PIEZO2 axis,EA at "Zusanli" acupoints decreased the number of EC and the secretion of 5-HT in the gastric fundus,thereby improving the visceral sensitivity of FD rats,which is the possible mechanism of EA in the treatment of FD. |
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