| Objective:In this study,slow transit constipation(STC)rats were used as the research carrier,and the imbalance of autophagy in interstitial cells of cajal(ICC)in colon was used as the starting point to observe the differences in the expression of icc labeling protein c-kit and autophagy related proteins Beclin1,LC3 and p62 in colon of STC model rats treated with electro-acupuncture and sham electro-acupuncture.Combined with the changes of the time of the first black stool,the number of feces in 24 hours,the propulsive rate of small intestine,the morphology of colon and the ultrastructure of colon ICC,the mechanism of electro-acupuncture at large intestine Back-shu and Front-mu points on STC rats was deeply discussed,so as to provide some research evidence for the clinical application of acupuncture in the treatment of gastrointestinal diseases.Methods:40 male SD rats were randomly divided into blank group(n = 10)and STC group(n = 30).The STC model was established by intragastric administration of compound diphenoxylate,and the effect of the model was evaluated 14 days later.After successful modeling,the rats in the STC group were randomly divided into model group(n = 10),electro-acupuncture group(n = 10)and sham electro-acupuncture group(n = 10).In the electro-acupuncture group,Tianshu(ST 25),Dachangshu(BL 25)on each side about alternate acupuncture for30 minutes,once a day,a course of five treatments,and rest for two days between two courses.In the sham electro-acupuncture group,the acupoints of the electro-acupuncture group were shallowly needled(skin can be broken)and the electrodes were clamped without electrification,and the rest were the same as the electro-acupuncture group.The rats in the blank group and model group were fixed by the same method,but they did not receive electro-acupuncture treatment after fixation.At the end of the second course of treatment,the excretion time of the first black stool and the number of feces in 24 hours were calculated;the small intestine and proximal colon tissues were taken to detect the intestinal propulsion rate;the colon morphology and ICC ultrastructure were observed by digital scanning microscope and transmission electron microscope;the expression levels of c-kit,Beclin1,LC3 II and p62 in colon tissue were detected by western blot method;the co-localization expression of c-kit,Beclin1 and LC3 was detected by immunofluorescence double labeling method.Results:1.Gastrointestinal transmission function test:Compared with the blank group,the first black stool excretion time of the model group was significantly longer,the number of 24-hour feces decreased and the intestinal propulsion slowed down significantly,the difference was statistically significant(p<0.01).Compared with the model group,the electro-acupuncture group significantly shortened the excretion time of the first black stool,increased the number of fecal particles in 24 hours and accelerated the propulsion of the small intestine,and the difference was statistically significant(p<0.05).There was no significant difference in the time of excretion of the first black stool,the number of fecal particles in 24 hours and the propulsion of small intestine in the sham electro-acupuncture group(p>0.05).Compared with the sham electro-acupuncture group,the time of excretion of the first black stool in the electro-acupuncture group was significantly longer than that in the sham electro-acupuncture group(p<0.05).2.Observation of colonic histomorphologyIn the blank group,the colonic mucosa was intact,the glands were abundant and arranged neatly,there was no edema and hyperemia,the number of myenteric plexus in the muscular layer was more,and the nerve cells were arranged neatly.In the model group,the integrity of colonic mucosa was slightly poor,the arrangement of glands was irregular,there was no edema and congestion,the muscle layer became thinner,the number of myenteric plexus decreased,the arrangement of nerve cells was disordered,and vacuole-like changes were seen.The pathological changes in the electro-acupuncture group were slighter than those in the model group,and there was no sexual difference between the electro-acupuncture group and the blank control group.The morphological changes in the sham electro-acupuncture group were similar to those in the model group.3.ICC ultrastructure observation showed that the morphology and structure of ICC cells in the blank group was normal,but there was slight swelling of mitochondria in the cytoplasm of a few cells,and a small amount of secondary lysosomes and autophagy were also seen.Under electron microscope,the structure of ICC cells in the model group was abnormal,most of the mitochondria in the cytoplasm had swollen,the crest was broken,dissolved or even disappeared,forming vacuoles of different sizes,part of the rough endoplasmic reticulum was obviously dilated like a sac,and obvious autophagy could be seen;the morphology and structure of some ICC cells were normal,a few mitochondria were slightly swollen and golgi complex could be seen.The structure of ICC cells in the electro-acupuncture group was normal,but most of the mitochondria in the cytoplasm were slightly swollen,and a small amount of autophagy and primary lysosomes could be seen,which showed a close connection with the nerve endings.In the sham electro-acupuncture group,ICC cells were normal,but a few mitochondria in the cytoplasm were slightly swollen,and a small amount of autophagy and primary lysosomes were occasionally seen.4.The expression level of c-kit,Beclin1,l C3 II and p62 protein in colon tissueCompared with the blank group,the expression of c-kit protein in the colon of the model group was significantly decreased,while the expression of Beclin-1 and LC3 II protein was significantly increased,and the expression of p62 protein was significantly lower than that of the blank group(p<0.01).Compared with the model group,the expression of c-kit protein was significantly increased,the expression of Beclin-1 protein was significantly decreased and the expression of p62 protein was significantly increased in the colon of rats in the electro-acupuncture group(p<0.01);The expression of c-kit,Beclin-1,LC3 II and p62 protein in sham electro-acupuncture group had no obvious change,and the difference was not statistically significant(p>0.05).Compared with the sham electro-acupuncture group,the expression of c-kit protein was significantly increased,the expression of Beclin-1 protein was significantly decreased,and the expression of p62 protein was significantly increased in the electro-acupuncture group(p<0.01).5.Average fluorescence intensity of c-kit+Beclin1 and c-kit+LC3 in colon of rats in each groupCompared with the blank group rats,the protein content of c-kit+Beclin1 and c-kit+LC3in colon tissue of model group rats increased,and the difference was statistically significant(p<0.01).Compared with the model group rats,the contents of c-kit+Beclin1 and c-kit+LC3(photosynthetic light)protein in colon tissue of rats in electro-acupuncture group decreased significantly(p < 0.01).The changes of c-kit+Beclin1 and c-kit+LC3(photosynthetic light)protein contents in colon tissue of rats in sham electro-acupuncture group were not obvious,and the difference was not statistically significant(p>0.05).Compared with sham electro-acupuncture group,the protein content of c-kit+Beclin1 and c-kit+LC3 in colon tissue of electro-acupuncture group was significantly lower(p<0.01).Conclusion:1.The intestinal transport function of STC rats is slow,the number and structure of ICC in colon are damaged and there is excessive autophagy.2.Compared with the sham electro-acupuncture group,electro-acupuncture at large intestine Back-shu and Front-mu points could promote the recovery of the number and structure of colonic ICC and improve the intestinal motility disturbance of STC rats.3.electro-acupuncture at large intestine Back-shu and Front-mu points may play a therapeutic role on ICC by down-regulating the expression of Beclin1 and LC3 II,and up-regulating the expression of P62,so as to inhibit excessive autophagy of ICC,promote the regeneration and repair of ICC,and improve intestinal transport function. |
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