| Objectives The abnormality of structure and function of interstitial cell of Cajal(ICC)is closely related to Gastrointestinal(GI)motility disorder that plays a crucial part in FD pathology.We established FD model rats as our research objectives to explore the effect of electroacupuncture(EA)at zusanli(ST36)on ICC in FD pathogenesis.Meanwhile,AMPK inhibitor Compound C was also selected to study the possible mechanism of EA in the regulation of ICC autophagy through AMPK/ULK1 signaling pathway in FD rats.Methods 82 adult male SD rats were randomly allocated to control group(n=12)and model building group(n=70)after one week of adaptive feeding.The rats of model building group were suffered stimulations combining tail-clamping,irregular feeding and gastric perfusion of 0℃ saline for 2 weeks.Then 60 of 70 rats were randomly selected and divided into model group,EA group,sham EA group,inhibitor group,EA plus inhibitor group,respectively,with 12 rats in each group.The rats in EA group were treated with EA at ST36,After piercing at ST36,then HANS-EA apparatus was connected with needle handle,and adjust EA waveform to continuous wave with 2Hz,1 m A.once a day for 7 days.Rats of sham EA group were employed with special Streitberger device as a placebo acupuncture needle.The needle was designed with a blunt intercalated needle.When blunt needle piercing skin,it could be automatically retract into needle sleeve,and then fixed with adhesive plaster using rubber ring in the corresponding position,the course is same with EA group.Rats in inhibitor group were intraperitoneally injected with Compound C at a dose 20 mg/kg once daily for 7 days.Rats in EA plus inhibitor group were injected with Compound C before EA intervention,and the dosage of inhibitor and EA parameters set were same with inhibitor group and EA group,respectively.During the intervention period,rats in control group,FD group and inhibitor group were restricted and fixed as same as EA group to ensure the consistency of experimental conditions.The general behavior and weight of each group rats was recorded.After intervention period ended,4 rats from each group were selected for EGG test after fasting but not yield to water for 24 hours.The residue rats were gavaged with nutritive semisolid paste(2ml/100 g)、ink(1ml/100g)in sequence,respectively.Half an hour after gavage,rats were treated with 10% chloral hydrate(0.35ml/100g)by intraperitoneal injection.The gastric residual rate and small intestinal transit rate were measured.The characteristics of pathomorphological variation of gastric antrum and duodenum were investigated with HE staining.The expressions of c-kit、Cx43、LC3、Beclin1、p62、p-AMPK、AMPK、p-ULK1、ULK1、p-LKB1、LKB1、Ca MKK2 in gastric antrum and duodenum tissues of each group rats were measured by western blot.RT-q PCR was used to detect m RNA expression of c-kit、Cx43、Beclin1 in gastric antrum and duodenum tissues.Double-labelling immunofluorescence was used to detect the expression and localization of c-kit/LC3、c-kit/Beclin 1 in gastric antrum and duodenum.The ultrastructure characteristics of ICC in GI tract were observed by transmission electron microscopy.Results 1.Effect of EA on behavior changes and GI motility in FD rats Pathomorphological changes in gastric antrum and duodenal tissues of in each group rats using HE staining: the color of gastric mucosa was a little white in model group and sham EA group,and the morphology of each layer of tissue was normal under the microscope,and no organic changes such as ulcer and hemorrhage were observed.(1)EA intervention can improve the general behavior of FD rats There were no significant difference in weight between rats in each groups at period of adaptive feeding(P>0.05).After the FD model was established,the weight of rats were significantly decreased in model group,EA group,sham group,inhibitor group and EA+ inhibitor group when compared to control group(all P<0.01),and there were no statistical difference among five groups(P>0.05).After intervention ended,compared with control group,the weight of rats in model group and sham EA group decreased significantly(all P<0.01);Compared with model group,the weight of rats increased significantly in EA group,inhibitor group and EA + inhibitor group(all P<0.01);The weight of rats was increased in EA+ inhibitor group when comparing to EA group and inhibitor group(all P<0.01);There was no significant difference in weight between EA group and inhibitor group(P>0.05).(2)EA intervention can promote GI motility in FD rats Changes of gastric residual rate in each group: the gastric residual rate in model group and sham EA group was up-regulated than that in control group(all P<0.01);Compared with model group,the gastric residual rate in EA group,inhibitor group and EA+ inhibitor group was down-regulated significantly(all P<0.01);The gastric residual rate of EA+ inhibitor group was lower than EA group and inhibitor group(P<0.05,P<0.01).There was no significant difference in gastric residual rate between EA group and inhibitor group(P>0.05).Changes of intestinal transit rate in each group: the intestinal transit rate in model group and sham EA group was down-regulated than in control group(all P<0.01);Compared with model group,the intestinal transit rate in EA group,inhibitor group and EA+ inhibitor group was up-regulated significantly(all P<0.01);The intestinal transit rate of EA+ inhibitor group was higher than EA group,inhibitor group(P<0.05,P<0.01,);There was no significant difference in intestinal transit rate between EA group and inhibitor group(P>0.05).Changes of gastric electric rhythm in each group: the main frequency and main duty of EGG slow wave in model group and sham EA group was decreased than control group(all P<0.01);Comparing to model group,the main frequency of EGG slow wave in EA group,inhibitor group and EA+ inhibitor group was increased significantly(P<0.01,P<0.05,P<0.01),and the main duty of EGG slow wave in EA group,inhibitor group and EA+ inhibitor group was increased significantly(all P<0.01);The main duty of EGG slow wave of EA+ inhibitor group was increased than EA group and inhibitor group(P<0.01,P<0.05);There was no significant difference in the main frequency of EGG slow wave between EA group,inhibitor group and EA+ inhibitor group(all P>0.05).2.Effect of EA on GI interstitial cell of cajal in FD rats(1)The expression of c-kit and Cx43 was detected by western blot The expression of c-kit protein in gastric antrum and duodenum tissues of rats in each group was as follows: compared with control group,the model group and sham EA group showed decreased c-kit in gastric antrum and duodenum(all P<0.01);Compared with model group,the expression of c-kit in gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly increased(all P<0.01);Compared with EA group,the expression of c-kit in gastric antrum and duodenum tissues in inhibitor group was significantly decreased(P<0.01,P<0.05);Compared with inhibitor group,the expression of c-kit in gastric antrum and duodenum tissues in EA+ inhibitor group was significantly increased(all P<0.01);There was no significant difference between model group and sham EA group as well as between EA group and EA+ inhibitor group(all P>0.05).The expression of Cx43 protein in gastric antrum and duodenum tissues of rats in each group was as follows: compared with control group,the model group and sham EA group showed decreased Cx43 in gastric antrum and duodenum(all P<0.01);Compared with model group,the expression of Cx43 in gastric antrum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly increased (all P<0.01),the expression of Cx43 in duodenum tissues was also increased(P<0.01,P<0.05,P<0.01);Compared with EA group,the expression of Cx43 in gastric antrum tissues in inhibitor group was significantly decreased(P<0.05);Compared with inhibitor group,the expression of Cx43 in gastric antrum tissues in EA+ inhibitor group was significantly increased(P<0.05);There was no significant difference between model group and sham EA group as well as between EA group and EA+ inhibitor group(all P>0.05).(2)The expression of c-kit m RNA and Cx43 m RNA was detected by q PCR The expression of c-kit m RNA in gastric antrum and duodenum tissues of rats in each group was follows: compared with control group,the model group and sham EA group showed decreased c-kit m RNA in gastric antrum and duodenum(all P<0.01);Compared with model group,the expression of c-kit m RNA in gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly increased(all P<0.01);Compared with EA group,the expression of c-kit m RNA in gastric antrum and duodenum tissues in inhibitor group was significantly decreased(all P<0.01);Compared with inhibitor group,the expression of c-kit m RNA in gastric antrum and duodenum tissues in EA+ inhibitor group was significantly increased(all P<0.01);There was no significant difference between model group and sham EA group as well as between EA group and EA+ inhibitor group(all P>0.05).The expression of Cx43 m RNA in gastric antrum and duodenum tissues of rats in each group were as follows: compared with control group,the model group and sham EA group showed decreased Cx43 m RNA in gastric antrum and duodenum(all P<0.01);Compared with model group,the expression of Cx43 m RNA in gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly increased(all P<0.01);Compared with EA group,the expression of Cx43 m RNA in gastric antrum and duodenum tissues in inhibitor group was significantly decreased(all P<0.01);Compared with inhibitor group,the expression of Cx43 m RNA in gastric antrum and duodenum tissues in EA+ inhibitor group was significantly increased(P<0.05);There was no significant difference between model group and sham EA group as well as between EA group and EA+ inhibitor group(all P>0.05)3.Effect of EA on ICC autophagy in FD rats(1)The ultrastructure changes of ICC was observed by transmission electron microscopy ICC cells in control group were spindle shaped or stellate shaped,The nuclear werelarge with a dense band of marginal heterochromatin,numerous mitochondria,abundant intermediate filaments,smooth and rough endoplasmic reticulum,Golgi complexes in cytoplasm and processes,a large number of caveolae were frequently observed along the plasma membrane edge,incomplete basal lamina was also seen,autophagosomes were rare,these cells have more than 2 long processes in different directions,and formed close gap junctions with neighboring ICC and GI smooth muscle cells(SMC),ICCs were also found in close proximity of enteric nerve bundle and formed a pre-and post-synaptic junctions;The model group and EA group showed a damage ultrastructure of ICC,with abnormal shape nuclear,cytoplasm dissolution and vacuolations,swelling mitochondria,with inner carinulae absent.And rough endoplasmic reticulum were decreased,autophagosomes were significantly increased,the processes of ICC were ruptured,with reduced gap junction with SMC;The mitochondria swelling of ICC cells in EA group,inhibitor group and EA+ inhibitor group was not serious,with rare autophagosomes and normal processes,gap junctions and synaptic junctions were observed between ICC,SMC and nerve terminals.(2)The expression of LC3Ⅱ/LC3Ⅰ、Beclin1、p62 was detected by western blot The expression of LC3Ⅱ/LC3Ⅰin gastric antrum and duodenum tissues of rats in each group was as follows: compared with control group,the model group and sham EA group showed increased LC3Ⅱ/LC3Ⅰin gastric antrum and duodenum(all P<0.01);Compared with model group,the expression of LC3Ⅱ/LC3Ⅰin gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly decreased(all P<0.01);Compared with EA group,the expression of LC3Ⅱ/LC3Ⅰin duodenum tissues in inhibitor group was significantly increased(P<0.01);Compared with inhibitor group,the expression of LC3Ⅱ/LC3Ⅰin gastric antrum and duodenum tissues in EA+ inhibitor group was significantly decreased(P<0.05,P<0.01);There was no significant difference between model group and sham EA group as well as between EA group and EA+ inhibitor group(all P>0.05).The expression of autophagy associated gene-Beclin 1 in gastric antrum and duodenum tissues of rats in each group was as follows: compared with control group,the model group and sham EA group showed increased Beclin 1 in gastric antrum and duodenum(all P<0.01);Compared with model group,the expression of Beclin 1 in gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly decreased(all P<0.01);Compared with inhibitor group,the expression of Beclin 1 in gastric antrum and duodenum tissues in EA+ inhibitor group was significantly decreased(P<0.01,P<0.05);There was no significant difference between model group and sham EA group,between EA group and inhibitor group as well as between EA group and EA+ inhibitor group(all P>0.05).The expression of p62 in gastric antrum and duodenum tissues of rats in each group was as follows: compared with control group,the model group and sham EA group showed decreased p62 in gastric antrum and duodenum(all P<0.01);Compared with model group,the expression of p62 in gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly increased(all P<0.01);Compared with EA group and inhibitor group,the expression of p62 in gastric antrum and duodenum tissues in EA+ inhibitor group was significantly increased(P<0.05,P<0.01);There was no significant difference between model group and sham EA group as well as between EA group and inhibitor group(all P>0.05).(3)The expression of Beclin 1 m RNA was detected by q PCR The expression of Beclin 1 m RNA in gastric antrum and duodenum tissues of rats in each group was as follows: compared with control group,the model group and sham EA group showed increased Beclin 1 m RNA in gastric antrum and duodenum(all P<0.01);Compared with model group,the expression of Beclin 1 m RNA in gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly increased(all P<0.01);Compared with EA group,the expression of Beclin 1 m RNA in duodenum tissues in inhibitor group was significantly increased(P<0.05);Compared with inhibitor group,the expression of Beclin 1 m RNA in gastric antrum and duodenum tissues in EA+ inhibitor group was significantly decreased(all P<0.05);There was no significant difference between model group and sham EA group as well as between EA group and EA+ inhibitor group(all P>0.05)(4)Effect of EA on expression of c-kit/LC3 in FD rats Immunofluorescence staining showed that c-kit staining was positive for red fluorescence,and LC3 staining was positive for green fluorescence.The results showed that the relative expression of c-kit in gastric antrum and duodenum tissues in model group and sham EA group was significantly down-regurated than in control group(all P<0.01);Compared with model group,the relative expression of c-kit in gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly up-regulated(all P<0.01);there was no significant difference between EA group,inhibitor group and EA+ inhibitor group(all P>0.05).Compared to control group,the relative expression of LC3 in gastric antrum and duodenum tissues in model group and sham EA group was significantly increased(all P<0.01);Compared to model group,the relative expression of LC3 in gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly decreased(all P<0.01);compared to inhibitor group,the relative expression of LC3 in gastric antrum in EA+ inhibitor group was significantly decreased(P<0.05);there was no significant difference between EA group and inhibitor group(all P>0.05).(5)Effect of EA on expression of c-kit/Beclin 1 in FD rats Immunofluorescence staining showed that c-kit staining was positive for red fluorescence,and Beclin1 staining was positive for green fluorescence.The results showed that the relative expression of c-kit in gastric antrum and duodenum tissues in model group and sham EA group was significantly decreased than in control group(all P<0.01);Compared with model group,the relative expression of c-kit in gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly increased(all P<0.01);there was no significant difference between EA group,inhibitor group and EA+ inhibitor group(all P>0.05).Compared to control group,the relative expression of Beclin 1 in gastric antrum and duodenum tissues in model group and sham EA group was significantly increased(all P<0.01);Compared to model group,the relative expression of Beclin 1 in gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly decreased(all P<0.01);compared to inhibitor group,the relative expression of LC3 in duodenum tissues in EA+ inhibitor group was significantly decreased(P<0.05);there was no significant difference between EA group and inhibitor group(all P>0.05).4.Effect of EA on AMPK/ULK1 signaling pathway in FD rats.The expression of p-AMPK、AMPK in gastric antrum and duodenum tissues of rats in each group was as follows: compared with control group,model group and sham EA group showed increased p-AMPK/AMPK in gastric antrum and duodenum(all P<0.01);Compared with model group,the expression of p-AMPK/AMPK in gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly decreased(all P<0.01);Compared with EA group,the expression of p-AMPK/AMPK in duodenum tissues in inhibitor group was significantly increased(P<0.01);Compared with inhibitor group,the expression of p-AMPK/AMPK in gastric antrum and duodenum tissues in EA+ inhibitor group was significantly decreased(P<0.05,P<0.01);There was no significant difference between model group and sham EA group as well as between EA group and EA+ inhibitor group(all P>0.05).The expression of p-ULK1、ULK1 in gastric antrum and duodenum tissues of rats in each group was as follows: compared with control group,the model group and sham EA group showed increased p-ULK1/ULK1 in gastric antrum and duodenum(all P<0.01);Compared with model group,the expression of p-AMPK/AMPK in gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly decreased(all P<0.01);Compared with inhibitor group,the expression of p-ULK1/ULK1 in gastric antrum and duodenum tissues in EA+ inhibitor group was significantly decreased(P<0.05,P<0.01);There was no significant difference between model group and sham EA group as well as between EA group and EA+ inhibitor group(all P>0.05).The expression of p-LKB1、LKB1 in gastric antrum and duodenum tissues of rats in each group was as follows: there was no significant difference in the expression of p-LKB1/LKB1 in gastric antrum and duodenum tissues between six groups(all P>0.05).The expression of Ca MKK2 in gastric antrum and duodenum tissues of rats in each group was as follows: compared with control group,model group and sham EA group showed increased Ca MKK2 in gastric antrum and duodenum(all P<0.01);Compared with model group,the expression of Ca MKK2 in gastric antrum and duodenum tissues in EA group,inhibitor group and EA+ inhibitor group was significantly down-regulated(all P<0.01);Compared with EA group,the expression of Ca MKK2 in gastric antrum and duodenum tissues in EA+ inhibitor group was significantly decreased(all P<0.05);Compared with inhibitor group,the expression of Ca MKK2 in gastric antrum and duodenum tissues in EA+ inhibitor group was significantly decreased(all P<0.01);There was no significant difference between model group and sham EA group as well as between EA group and inhibitor group(all P>0.05).Conclusions(1)In FD rats,the gastric emptying was delayed and EGG slow waves was decreased,the numbers of ICC cells in the gastric antrum and duodenum tissues were decreased,the ICC network and ultrastructure was impaired.(2)EA can promote GI motility,gastric emptying,and gastric electric rhythm,and alleviate general conditions of FD rats.(3)EA can inhibit excessive autophagy of ICC,recover the ICC cells function of GI tract,and the combination of AMPK inhibitor and EA has a cooperative effect,The effect of EA on ICC was associated with AMPK signaling pathway.(4)EA can recover damaged structure and function of ICC in FD rats through mediating AMPK/ULK1 autophagy signaling pathway,which may be a possible mechanism of EA effect on FD rats. |
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