| Background:Chronic transit constipation is a common clinical digestive disease,also belongs to the common type of functional constipation.Studies have shown that,on the basis of the gut brain axis theory,PI3K/AKT/mTOR signaling pathway regulates the autophagy process of colon and brain tissues of STC,which is a key mechanism for the occurrence and development of STC and an important target for treatment.Traditional Chinese medicine can play an important role in the treatment of STC.This experimental study further clarified the clinical efficacy of Jiawei Tongbian Decoction in the treatment of STC.At the same time,explore its mechanism of action.Objective:From the perspective of PI3K/AKT/mTOR autophagy pathway,the clinical efficacy evaluation and specific mechanism of Jiawei Tongbian decoction on STC were explored,so as to better understand the theory of entero-brain co-therapy and give play to the unique advantages of traditional Chinese medicine.Methods:1.Clinical trialsSixty-eight STC patients were randomly divided into moxabide group(n=31)and Jiawei Tongbian Decoction group(n=32).Through the use of primary efficacy indicators,secondary efficacy indicators,laboratory observation indicators to clarify the Jiawei Tongbian Decoction intervention 4w after the clinical efficacy evaluation of STC patients.The total clinical response rate,PAC-SYM scale score,ZUNG’s Anxiety and depression Scale score,Quality of Life Scale(PAC-QOL)score,Pittsburgh Sleep Quality Scale(PSQI)score and colon transport function index were analyzed.The expressions of brain gut peptide 5-HT,SP,NPY and CGRP were detected by ELISA.1.Animal experiment1.1 A total of 70 healthy male SD rats were selected.After successful replication of STC rat model,the rats were divided into blank control group,natural recovery group,moxabide group,fluoxetine hydrochloride group,Jiawei Tongbian Decoction high-dose,medium-dose and low-dose groups according to random number table method.Four weeks after drug intervention,to observe the general situation of rats,the determination of the rats’ body quality,eating and drinking,determination of rat excrement and water content,total 24 hours rats 30 minutes of small intestine propulsion rate at the end of the carbon and the total number of colonic fecal retained,rats colon muscle activity,evaluation in depression rats behavioral science,including determination of rat kuang,sugar water preference index and tail suspension experiment,The subcellular structure of the colon and brain tissues of rats was observed by transmission electron microscopy.The expressions of brain gut peptides 5-HT,SP,NPY and CGRP in serum,colon and brain tissues of rats were determined by ELISA.Western Blot was used to detect the protein levels of PI3K/AKT/mTOR signaling pathway in colon and brain tissues of rats.2.2 A total of 60 healthy male SD rats were selected.After the STC rat model was successfully replicated,the rats were divided into blank group,model group,moxabide group,fluoxetine hydrochloride group,Jiawei Tongbian Decoction high-dose group and signal pathway inhibitor group according to random number table method,with 10 in each group.After 4 weeks of drug intervention,the hypothalamus and colon tissues of rats were stained with HE,and the subcellular structures of colon and brain tissues were observed by transmission electron microscopy.Western Blot was used to detect the expression of PI3K/AKT/mTOR signaling pathway and autophagy related proteins in rats.The mRNA expression of PI3K/AKT/mTOR pathway and autophagy related genes in rats were detected by Q-PCR..Result:1.Clinical trialsCompared with moxabide group,the recovery rate of Jiawei Tongbian Decoction group((P<0.05),and the recovery rate(P<0.01),total effective rate increased((P<0.05),,total PAC-SYM score was significantly decreased(P<0.01),individual PAC-SYM score was significantly decreased(P<0.01),SAS and SDS scores were significantly decreased(P<0.01),PAC-QOL and PSQI scores were significantly decreased(P<0.01).After 48h,the colon transport index was significantly increased(P<0.01),the contents of 5-ht and SP were significantly increased(P<0.01),and the contents of NPY and CGRP were significantly decreased(P<0.01).2.Animal experiment2.1(1)The general conditions of the rats were observed and the body weight,diet and water intake of the rats were determined.Compared with blank control group,the general condition of natural recovery group was worse,body weight,diet and water intake decreased significantly(P<0.01).Compared with the natural recovery group,the changes of body weight,diet and water intake in moxabide group and fluoxetine hydrochloride group were slow(P<0.05),while the changes of body weight,diet and water intake in Jiawei Tongbian Decoction group were significantly increasing(P<0.01),and presented a dose-dependent(P<0.01).(2)The total amount and water content of feces at 24h,the small intestine carbon end-advance rate and the total amount of feces retained in colon at 30min,and the colonic myoelectric activity of rats were measured.Compared with blank control group,the total fecal amount and water content in the natural recovery group decreased significantly at 24h,the small intestine end carbon advance rate decreased significantly at 30min,the total amount of retained feces increased significantly,the slow wave contraction frequency of colon decreased,the amplitude increased,and the variation coefficient of frequency and amplitude increased.Compared with natural recovery group,fluoxetine hydrochloride group rat excrement and water content,total 24 h rats for 30 min at the end of the small intestine carbon propulsion rate and the total number of colonic fecal retained,rats colon muscle no obvious change was found in all electrical activity,moser will benefit group,Jiawei Tongbian Decoction group can obviously increase the rat 24h total waste,improve water content of excrement and urine,promote carbon propulsion rate at the end of the small intestine,Reduce the total amount of colonic stool retention,change colonic muscle electrical activity to promote colonic peristalsis.(3)The depression-like behavior of rats was evaluated,including open-field,sugar-water preference index and tail suspension immovability test.Compared with blank control group,the central region residence time of rats in natural recovery group was shortened,the total travel distance was reduced,the number of crossing the grid was reduced,the sugar water preference index was decreased,and the tail suspension immobility time was increased.Compared with the natural recovery group,there was no significant change in depression-like behavior in the moxabide group,the residence time in the central region of the fluoxetine hydrochloride and Jiawei Tongbian Decoction groups was significantly prolonged,the total travel distance and the number of crossing the grid were significantly increased,the sugar water preference index was increased,and the tail suspension immobility time was significantly decreased.(4)The subcellular structure of colon and brain tissue was observed by HE staining and transmission electron microscopy.The expressions of brain gut peptides 5-HT,SP,NPY and CGRP in serum,colon and brain tissues of rats were determined by ELISA.Western Blot was used to detect the protein levels of PI3K/AKT/mTOR signaling pathway in colon and brain tissues of rats.Compared with blank control group,the colon and brain tissues of rats in natural recovery group showed obvious pathological damage,increased autophagosomes,and the contents of brain-gut peptides 5-HT and SP in serum and colon tissues decreased significantly,while the contents of NPY and CGRP increased significantly.The contents of brain-gut peptides 5-HT and NPY in brain tissues decreased significantly,while the contents of SP and CGRP increased significantly.The phosphorylation levels of PI3K,AKT and mTOR decreased significantly.Compared with the natural recovery group,HE staining observed that the pathological lesion of the colon tissue of the mosabide group was relieved,and the pathological lesion of the brain tissue of the fluoxetine hydrochloride group was relieved,but no significant changes were observed in the autophagosomes of the two groups.ELISA showed that the contents of 5-HT and SP in serum and colon of the mosabide group were increased,while the contents of NPY and CGRP were decreased.The contents of 5-HT,SP,NPY and CGRP in brain tissue did not change significantly.ELISA was used to determine the contents of 5-HT and NPY in fluoxetine hydrochloride group,while the contents of SP and CGRP in fluoxetine hydrochloride group were increased,the contents of 5-HT and SP in serum were increased,while the contents of NPY and CGRP in colon tissue were decreased.Western Blot analysis showed no significant changes in PI3K/AKT/mTOR signaling pathway protein expression in colon and brain tissues of moxabide group and fluoxetine hydrochloride rats.In the Jiawei Tongbian Decoction group,the pathological damage of colon and brain tissue was significantly repaired,the number of autophagosomes was significantly reduced,the contents of brain intestinal peptides 5-HT,SP,NPY and CGRP were regulated,and the phosphorylation levels of PI3K,AKT and mTOR in brain and colon tissues were significantly increased.2.2Compared with the blank group,the colon and brain tissues of model group showed pathological lesions by HE staining,and the number of autophagosomes was observed by transmission electron microscopy.Western Blot and Q-PCR detected decreased expression of PI3K/AKT/mTOR signaling pathway,increased expression of autophagy related molecules LC3 and Beclinl,and decreased expression of P62 in colon and brain tissues.Compared with model group,no significant changes were observed in moxabide group,fluoxetine hydrochloride group and signaling pathway inhibitor group.In the Jiawei Tongbian Decoction group,pathological damage of colon and brain tissues was significantly repaired,autophagosome production was significantly reduced,PI3K/AKT/mTOR signaling pathway expression was significantly up-regulated,autophagy-related molecules LC3 and Beclinl expression was significantly down-regulated,and P62 expression was significantly up-regulated.Conclusion:1.STC can induce the formation of depression,and STC has pathological features of synchronous intestinal brain injury.Jiawei Tongbian Decoction can play a synchronous gut-brain protective effect and improve the intestinal and depressive symptoms of STC,while moxabide and fluoxetine hydrochloride only have a single target improvement effect.2.Based on the theory of the gut axis,Jiawei Tongbian Decoction can activate the PI3K/AKT/mTOR signaling pathway,inhibit the autophagy level of colon and brain tissues,regulate the expression of brain intestinal peptide,enhance the intestinal transport function and anti-depression effect. |
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