| Objectives This study takes functional dyspepsia(FD),which has a high prevalence rate,a large impact and consumes a large amount of health and medical resources,as the entry point.It not only combined with the characteristics of gastric motility disorder as the main pathophysiological basis of FD,but also based on the premise that interstitial cell of Cajal(ICC)is gastric slow-wave pacemaker cell and micro RNA(NA)can regulate cell proliferation,differentiation,autophagy and other cellular effects by regulating target genes and target signaling pathways.Based on this,the study aims to investigate the mechanism of NA regulated ICC in the process of Electroacupuncture(EA)treatment of FD rats to improve their gastric motility.Methods A total of 18 SPF healthy adult male Sprague Dawley rats were randomly divided into control group,model group and EA group after adaptive feeding for 1 week,with 6 rats in each group.Except for the control group,the other two groups of rats were given multi-factor stress intervention to construct FD models,and the modeling period was 14 days.After the model was successfully built,ST36 and LR3 were intervened with EA,and the intervention period was 10 days.During the EA intervention period,the general conditions of rats in each group were observed,and the weight,diet and water intake of rats were measured.After EA intervention,the rats were gavaged with nutrient semi-solid paste(2m L/100g).After30 minutes,the rats were sequentially anesthetized by intraperitoneal injection of Pelltobarbitalum Natricum(5mg/100g),and the rats were dissected for sampling.To calculate the gastric remnant rate of rats,the total stomach weight and net stomach weight of each rat were weighed.After weighing the stomach,part of the gastric antrum was removed.Hematoxylin-eosin staining was used to observe the morphological structure in gastric antrum of rats.Transmission electron microscope was used to observe the ultrastructure of ICC in gastric antrum tissue of rats.Western blotting was used to detect the expression level of c-kit protein in gastric antrum tissue of rats.The NA sequencing was used to screen the differentially expressed NA before and after EA intervention in FD rats,and verify the targeting relationship between the differentially expressed -X and c-kit genes(KIT) by luciferase testing.Real-time PCR was used to detect the expression levels of c-kit mRNA and differential -X in gastric antrum tissue of rats.A total of 30 SPF healthy adult male Sprague Dawley rats were randomly divided into control+NC,model+NC,EA+NC,EA+-X inhibitor and EA+-X agomir after adaptive feeding for 1 week,with 6 rats in each group.Except for the control+NC,the other groups of rats were given multi-factor stress intervention to construct FD models,and the modeling period was 14 days.After the model was successfully built,ST36 and LR3 were intervened with EA,and the intervention period was 10 days.During the period,the constructed agomir and inhibitor -X vectors were injected into corresponding groups of rats through tail vein injection.During the EA intervention period,the general conditions of rats in each group were observed.After EA intervention,the gastric remnant rate was calculated by the same method.Part of the gastric antrum was removed,and hematoxylin-eosin staining was used to observe the morphological structure in gastric antrum of rats.Western blotting was used to detect the expression levels of CX43,SCF,c-kit,p-Raf/Raf,p-Erk/Erk protein in gastric antrum tissue of rats.Real-time PCR was used to detect the expression levels of CX43,SCF,c-kit,Raf,Erk mRNA and -X in gastric antrum tissue of rats.Results1.Effect of EA intervention on FD rats and screening the differentially expressed NA before and after EA intervention(1)Establishment of FD rat model: It was observed that the body weight,diet and water intake of rats decreased after modeling.The hair is withered and disorderly,yellow and shedding;Loose stools with a strong smell of acid and rot.The active degree was weakened,the arrest reaction was slow,and there was no aggression and defense basically.Rats were mentally tired and listless and in poor condition.Hematoxylin-eosin staining was used to observe the morphology and structure of gastric antrum of rats.No obvious organic lesions were found.(2)Effect of EA on general behavior of FD rats: Before modeling(0d),there were no statistically significant in body weight,diet and water intake among the three groups(P>0.05).After modeling(14d),the body weight,diet and water intake in model group and EA group decreased,there were no statistically significant between the two groups(P>0.05).Compared with control group,the differences were statistically significant(P<0.05).After EA intervention(24d),the weight,diet,and water intake of rats in EA group and control group increased significantly,and model group also increased.However,the differences among the three groups were statistically significant(P<0.05).(3)Effect of EA on gastric motility of FD rats: The gastric remnant rate of rats in model group and EA group was significantly higher than that in control group,while the gastric remnant rate in EA group was significantly lower than that in model group.The differences among the three groups were statistically significant(F=185.637,P<0.001).(4)Effect of EA on ICC in gastric antrum tissue of FD rats:Observation under transmission electron microscope showed that the ICC in control group was fusiform or irregular polygon,the nucleus was large and oval,the periphery of the nucleus was heterochromatic dense band.The cytoplasm is less,there are abundant mitochondria and other organelles in the cytoplasm,and autophagosomes are rare.The ultrastructure of ICC in model group was destroyed,the nucleus was larger.The number of organelles in the cytoplasm was significantly reduced and there were obvious autophagosomes.The ICC in EA group was fusiform or irregular polygon,with larger nuclei,more organelles in the cytoplasm,less swelling of mitochondria,and less obvious autophagosome structure.Western blotting showed that the expression level of c-kit protein in the gastric antrum tissue in model group and EA group was significantly lower than that in control group,while the expression level of c-kit protein in EA group was significantly higher than that in model group.The differences among the three groups were statistically significant(F=556.283,P<0.001).(5)Screening the differentially expressed NA before and after EA intervention in FD rats: A total of 18 NAs were differentially expressed of FD rats before and after EA intervention,13 NAs were down-regulated and 5 NAs were up-regulated.Through the analysis of Ensembl and Target Scan database,it is found that only -221-3p has complementary site with the 3’UTR of c-kit gene(KIT).(6)Verification of the targeting relationship between -221-3p and c-kit genes: In the c-kit 3’UTR wild-type(WT)vector plasmid group,the luciferase activity in -221-3p mimic group was significantly inhibited.Compared with -221-3p nc group,the difference was statistically significant(t=24.595,P<0.001).In the c-kit 3’UTR mutant-type(MUT)vector plasmid group,there was no significant difference in the luciferase activity ratio between the two group(t=-0.254,P>0.05).The expression level of c-kit mRNA in gastric antrum tissue of rats in model group and EA group was significantly lower than that in control group,but the expression level of -221-3p in gastric antrum tissue of rats was significantly higher than that in control group.The expression level of c-kit mRNA in gastric antrum tissue of rats in EA group was significantly higher than that in model group,but the expression level of -221-3p in gastric antrum tissue of rats was significantly lower than that in model group.The differences among the three groups were statistically significant(Fc-kit mRNA=285.762,Pc-kit mRNA<0.05;F-221-3p=342.916,P-221-3p<0.05).2.The mechanism of -221-3p mediated EA in the treatment of FD rats(1)Establishment of FD rat model: It was observed that the body weight,diet and water intake of rats decreased after modeling.The hair is withered and disorderly,yellow and shedding;Loose stools with a strong smell of acid and rot.The active degree was weakened,the arrest reaction was slow,and there was no aggression and defense basically.Rats were mentally tired and listless and in poor condition.Hematoxylin-eosin staining was used to observe the morphology and structure of gastric antrum of rats.No obvious organic lesions were found.(2)Mi R-221-3p targeted regulation of c-kit gene and its effect on c-kit mRNA expression: The expression level of c-kit mRNA in gastric antrum tissue of rats in Model+NC group and EA+NC group was significantly lower than that in Control+NC group(P<0.001),while the expression level of -221-3p in gastric antrum tissue of rats was significantly higher than that in Control+NC group(P<0.001).The expression of c-kit mRNA in gastric antrum tissue of rats in EA+NC group was significantly higher than that in Model+NC group(P<0.05),while the expression of -221-3p in gastric antrum tissue of rats was significantly lower than that in Model+NC group(P<0.05).Compared with EA+NC group,the expression of c-kit mRNA was significantly increased in EA+-221-3p inhibitor group(P<0.001),while the expression of -221-3p was significantly decreased(P<0.05).Compared with EA+NC group,the expression of c-kit mRNA was significantly decreased in EA+-221-3p agomir group(P<0.05),while the expression level of -221-3p was significantly increased(P<0.05).The differences were statistically significant.(3)Effect of -221-3p mediated EA on gastric motility in FD rats: The gastric remnant rate of rats in Model+NC group and EA+NC group was significantly higher than that in Control+NC group(P<0.001).The gastric remnant rate of rats in EA+NC group was significantly lower than that in Model+NC group(P<0.001).Compared with EA+NC group,The gastric remnant rate of rats in EA+-221-3p inhibitor group was significantly decreased(P<0.001),while EA+-221-3p agomir group was significantly increased(P<0.001).The differences were statistically significant.(4)Change of ICC quantity in gastric antrum tissue of rats in each group: The expression level of c-kit protein in gastric antrum tissue of rats in Model+NC group and EA+NC group was significantly lower than that in Control+NC group(P<0.001).The expression level of c-kit protein in gastric antrum tissue of rats in EA+ NC group was significantly higher than that in Model+NC group(P<0.05).Compared with EA+NC group,the expression level of c-kit protein in gastric antrum tissue of rats in EA+-221-3p inhibitor group was significantly increased(P<0.001),while EA+-221-3p agomir group was significantly decreased(P<0.001).The differences were statistically significant.(5)Change in the expression of gap junction protein Cx43 between ICC and ICC and between ICC and SMC in gastric antrum tissue of rats in each group: The expression of CX43 in the protein and mRNA levels in gastric antrum tissue of rats in Model+NC group and EA+NC group were significantly lower than that in Control+NC group(P<0.001).The expression of CX43 in the protein and mRNA levels in gastric antrum tissue of rats in EA+NC group were significantly higher than that in Model+NC group(P<0.001).Compared with EA+NC group,the expression of CX43 in the protein and mRNA levels in gastric antrum tissue of rats in EA+-221-3p inhibitor group was significantly increased(P<0.001),while EA+-221-3p agomir group was significantly decreased(P<0.001).The differences were statistically significant.(6)Change of protein expression related to SCF/c-kit and Raf/Erk signaling pathways in gastric antrum tissue of rats in each group: The expression of SCF,c-kit,Raf and Erk in the protein and mRNA levels in gastric antrum tissue of rats in Model+NC group and EA+NC group were significantly lower than that in Control+NC group(P<0.05).The expression of SCF,c-kit,Raf and Erk in the protein and mRNA levels in gastric antrum tissue of rats in EA+NC group were significantly higher than that in Model+NC group(P<0.05).Compared with EA+NC group,the expression of SCF,c-kit, Raf and Erk in the protein and mRNA levels in gastric antrum tissue of rats in EA+-221-3p inhibitor group was significantly increased(P<0.05),while EA+-221-3p agomir group was significantly decreased(P<0.05).The differences were statistically significant.Conclusions1.The multi-factor stress intervention method of Guo’s tail-clamping stimulation method combined with irregular diet method combined with ice saline gavage method can successfully construct the FD rat model.2.FD model rats showed significant decrease in body weight,food and water intake,delayed gastric emptying,the morphology and structure of ICC in gastric antrum tissue were abnormal and the number of ICC was reduced.3.EA intervention can improve the general behavior of FD rats,reduce gastric remnant and enhance gastric motility.The possible mechanism is that EA can improve the morphology and structure of ICC and increase the amount of ICC in gastric antrum tissue.4.A total of 18 NAs differentially expressed of FD rats before and after EA intervention were screened.Among them,only -221-3p has complementary site with the 3’UTR of c-kit gene(KIT),showing a direct targeting relationship.5.Mi R-221-3p targeted c-kit gene,and negatively regulated the expression of c-kit mRNA.6.Interfering with -221-3p can inhibit its targeted regulation of c-kit gene and up-regulate the expression of c-kit mRNA.By activating the SCF/c-kit and Raf/Erk signaling pathway, it can promote the proliferation of ICC and increase the expression of gap junction protein Cx43 in gastric antrum tissue,and enhance the intercellular communication in gastric antrum tissue between ICC and ICC and between ICC and SMC,thus enhancing gastric motility.It plays a role in the process of EA treatment of FD. |
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