| Purpose:Through network pharmacology technology,the biological processes related to Huayu Tongbian Decoction and slow transmission constipation(STC)were preliminatively explored,including PI3K/AKT/mTOR signaling pathway.In vivo experiments were conducted to detect and observe the effects of Huayu Tongbian Decoction on Cajal interstitial cells of colon and the regulation of autophagy related proteins in rats with slow transit constipation induced by compound diphenoxate,and to explore the mechanism of Huayu Tongbian Decoction on STC rats based on PI3K/AKT/mTOR signaling pathway.Material and method:Paper Ⅰ To explore the mechanism of Huayu Tongbian Decoction in the treatment of slow transit constipation based on network pharmacologyThe effective ingredients of 13 medicines in Huayu Tongbian Decoction were searched in THE TCMSP database respectively,based on the screening conditions of oral bioavailability(OB)≥30%and drug-like(DL)≥0.18 The results of TCMSP were supplemented by Batman-TCM and SymMap,combined with Uniprot database,and the gene target of the effective ingredient was obtained."Slow transit constipation" was retrieved using Genecards and OMIM databases respectively,and the retrieval results were combined to eliminate repeated targets and obtain all gene targets of Slow transit constipation.The intersection of all drug targets and slow-transport constipation targets was obtained using R language(R version4.1.1).The above intersection targets were imported into STRING database to obtain protein interaction network(PPI),and the TCM regulation network diagram of "Huayu Tongbian Decoction-active component-slow transport constipation" was obtained by Cytoscape 3.7.1.All data were imported into R language for module analysis and overall GO biological process and KEGG pathway enrichment analysis.Paper Ⅱ Experimental study on the treatment of slow-transmission constipation with Huayu Tongbian DecoctionExperiment 1:Evaluation of STC rat model and autophagic changes in colonic cellsAfter weighing and numbering,24 SD rats were randomly divided into 4 groups,including blank control group(n=6),model groupI(n=6),model groupII(n=6)and model groupⅢ(n=6).At the beginning of modeling,the blank control group was given 0.1 mL/10g distilled water by intragastric administration once a day.Model groupⅠwas given 25mg/(Kg·d)compound diphenoxate,model groupⅡwas given 50mg/(Kg·d)compound diphenoxate,model groupⅢwas given 75mg/(Kg·d)compound diphenoxate,once a day,for 14 days,to create slow transport constipation model.After modeling,normal water feeding was continued for 7 days to consolidate the experimental slow transport constipation model.The rats were weighed once a week during gavage.Mental state,activity,hair and defecation of rats were observed daily.Observe and record the fecal moisture content and the time of first black stool before and after modeling and 7 days after modeling.The death number of rats in each group was calculated.After the end of the first black stool time,fasting without water for 24h,1%pentobarbital sodium 50 mg/kg full anesthesia,open the abdominal cavity and cut the middle of the colon about 3cm in two sections,one section was directly fixed in 4%paraformaldehyde,and the other section was frozen in 1.5mLEP tube at-80℃.The pathological morphological changes of the colon were observed by HE staining,and the expression of ICCs-specific c-kit and autophagy signature protein Beclin-1 in the colon of each group was detected by western blot.Experiment 2:The effect of Huayu Tongbian Decoction on Cajal interstitial cells in the colon of rats with slow-transmission constipationThe weight numbers of 72 SD rats were included in the Excel table,and 12 rats were divided into blank control group by random number table method,and the remaining 60 rats were modeled according to the optimal modeling concentration of compound diphenoxate 50mg/(Kg d)obtained from the experiment in Part Ⅰ.The modeling was conducted for 14 days.After the successful modeling was determined by weight and fecal moisture content,According to the grouping method of experiment 1,60 rats were randomly divided into model control group,Huayu Tongbian Decoction low-dose group(referred to as low-dose group,followed by the same treatment of medium and high dose),Huayu Tongbian Decoction medium-dose group,Huayu Tongbian Decoction high-dose group,and Western medicine control group(moxabide group),with 12 rats in each group.According to the body surface area between human and rats,the dose of gavage was 42.3g/(kg·d)in the high-dose group,21.15g/(kg·d)in the medium-dose group and 10.575g/(kg·d)in the low-dose group.The dose volume was calculated as 0.1ml/l0g,and the dose volume was 3ml/day according to the average body weight of rats.The dose of mosapride citrate in western medicine control group was 1.35mg/(kg·d)by the same method.During the treatment,the model control group and the blank control group were given equal volume of distilled water.All the experimental groups were given normal food and water and gavage once a day from 9:00 a.m.to 10:00 a.m.for 14 consecutive days.The weight of the rats before modeling,after modeling and after treatment was observed and recorded,and the mental state,exercise,body hair gloss,defecation volume and dryness of the rats were observed and recorded daily.The fecal water content before and after modeling and the carbon end advancing rate after treatment were observed.The death number of rats in each group was counted and the cause of death was recorded and analyzed.After the last intragastric treatment,the patient was fasted without water for 24h,gavaged with 8%carbon and 10%Arabian gel suspension according to 0.1ml/10g,and fully anesthetized with 1%pentobarbital sodium 50 mg/kg 30min later.The abdominal cavity was opened and the incision was taken from the pylorus at the bottom of the stomach to the top of the anal canal at the end of the rectum.The total length of the intestine and the length of the lowest end of the intestine from the pylorus to the black stool formed by the end-carbon suspension were measured,and the value was recorded by photographing.After measurement,about 3cm of the middle part of the colon was quickly cut out and residual feces,urine connective tissue and other materials in the intestinal lumen were extruded.After washing with PBS buffer solution 1×,it was divided into three sections.The first section was pre-fixed in pre-cooled 2.5%glutaraldehyde,the second section was fixed directly in 4%paraformaldehyde at room temperature,and the third section was frozen in 1.5MLEP tube at-80℃.The fecal moisture content and carbon propulsive rate of rats in each group were calculated and compared.The expression of c-kit in colon tissues was detected by immunofluorescence,and the expression of ICCs-related c-kit,HCN1 and HCN2 proteins in colon of rats in each group was detected by western blot.The calculation method of end-carbon propulsion was as follows:end-carbon propulsion distance(cm)/total intestinal length(cm)×100%.Experiment 3:Based on PI3K/AKT/mTOR signaling pathway,the effect of Huayu Tongbian Decoction on colon cell autophagy in STC rats was investigatedGroup modeling,drug administration and specimen collection methods were the same as experiment 2.Protein western blot was used to detect the protein expressions of p-mTOR,p-AKT and p-PI3K related proteins in THE PI3K/AKT/mTOR signaling pathway.The expression of autophagy related landmark proteins Beclin-1,Atg5,LC3B and P62,and the protein bands were obtained,and the gray values were measured to compare the relative gray values.The expression of Belinl and P62 in colon tissue was detected by immunohistochemistry.The expression of LC3B was detected by immunofluorescence.The mRNA expressions of mTOR,Beclin-1,Atg5,LC3B and P62 were detected by RT-PCR.Results:1 To explore the mechanism of Huayu Tongbian Decoction in the treatment of slow transit constipation based on network pharmacologyA total of 223 compounds satisfying the conditions were obtained by screening.Among them,Atractylodes contains seven,Pinellia tuber contains 13,Radix bupleuri contains 17,Dried tangerine or orange peel contains five,Red paeony root contains 29,Ligusticum wallichii contains seven,Angelica contains two,Licorice contains 92,Radix Ophiopogonis contains two,Dried rehmannia root contains two,Semen persicae contains 23,Almond contains 19,Fructus Aurantii contains five.A total of 1379 targets were obtained,and 254 targets were obtained after deleting duplicate values.3985 disease-related genes were obtained after the duplication was removed and combined with gene targets of slow transport constipation diseases.The intersection of drug targets and disease-related genes was used to obtain 185 common drug-disease targets.After uploading 185 target genes to String platform,94 core targets can be obtained.After R Version calculation,enrichment analysis of KEGG pathway was obtained,and the first 20 were screened out.They include PI3K-Akt signaling Pathway,mTOR Signaling Pathway,autophagy-animal,etc.(1)During the adaptive feeding stage,24 rats were in good mental condition,with more activities,smooth and bright fur,harmonious stool,and large and full fecal pellets.After the start of modeling,the rats in each model group showed poor mental condition,less activity,less feeding,yellowish and lusterless fur,unfavorable urination,small,dry and hard feces,and slow weight gain.(2)In the first week of modeling,no rats died in model group Ⅰ and Ⅱ,and one rat died in model group Ⅲ;in the second week,no rats died in model group Ⅰ and Ⅱ,and one rat died in model group Ⅲ;there was no death in the blank control group during the whole 14-day modeling period,and the mortality rate of model group Ⅲ was as high as 33.333%.(3)Compared with the blank control group,the three model groups showed slow growth in body mass,reduced number of defecation,reduced fecal water content,and longer time to first black defecation(p<0.05),which were statistically different;compared with model group Ⅰ,the changes in body mass,number of defecation,fecal water content,and time to first black defecation were more significant in model group Ⅱ and model group Ⅲ(p<0.05),which were statistically different;and the changes in body mass,number of defecation,fecal water content,and time to first black defecation between model group(p>0.05),indicating that there were differences in constipation symptoms between model groups Ⅱ and Ⅲ,but the differences were not significant.(4)After seven days of normal feeding at the end of modeling,the cross-sectional comparisons within each experimental group revealed that in model group Ⅰ,there was a difference in stool water content and time of first black stool on the 14th day of modeling and 7 days after the completion of modeling(p<0.05),indicating that the intestinal function of model group Ⅰ had recovered;while in model group Ⅱ and model groupⅢ,there were changes in stool water content and time of first bowel movement in the respective group comparisons but the changes were not significant(p>0.05).(5)HE staining results showed that there were no obvious pathological changes in the blank group;a little inflammatory cell infiltration in the colon of model groupⅠ;more inflammatory cell infiltration and swelling of the lamina propria in the colon of model groupⅡ;a large number of inflammatory cell infiltration and swelling of the lamina propria in the colon of model groupⅢ,and more lymph nodes were seen in the mucosa.(6)Western Blot detected the expression of c-kit,a protein related to the specific expression of Cajal mesenchymal cells,and Beclin-1,a signature protein of cellular autophagy,and the results showed that the expression of c-kit gradually decreased and that of Beclin-1 gradually increased with the increase of the administered dose,and the comparison between groups(p<0.01)was statistically significant.The increase in the expression of c-kit and Beclin-1 was statistically significant,demonstrating that there is Caial interstitial cell injury in STC model rats,and ICCs play an important role in intestinal motility as "intestinal pacemaker",and the occurrence of STC is closely related to the injury and decrease of ICCs.The increase of autophagy signature protein Beclin-1 indicates that the decrease and damage of ICCs are synchronized with the change of cellular autophagy,suggesting that STC may have a reduced number of impaired morphology due to excessive autophagy of ICCs,which affects colonic function and leads to the development of slow-transit constipation.3 Effect of Huayu Tongbian Decoction on Cajal interstitial cells of colon in rats with slow transit constipation(1)Before the preparation of the model,72 rats were in good mental condition,active,with smooth and bright fur,harmonious stool,large and full feces,and small differences in body weight among the groups,with no statistical differences(p>0.05);after the start of the modeling,12 rats in the blank control group showed no significant changes in various conditions,and 60 rats in the modeling group successively showed poor mental condition,less activity and piling up,reduced food intake,yellowish and lusterless fur,unfavorable urine,small and dry feces,and slow body weight growth.During the gavage process,it was also obvious that the rats in each group were more irritable than the blank control group,and struggled significantly when grasping the back of the rats,and some rats showed aggressive behaviors.(2)Compared with the blank control group,the rats in the model control group were easily provoked,and their body weight gain was significantly reduced(p<0.01),the Cajal interstitial cells were severely damaged,the number of c-kit expression was significantly reduced(p<0.01),and the expression ability of c-kit,HCN1 and HCN2 protein was significantly decreased(p<0.01);(3)After 14 days of treatment,compared with the model(3)After 14 days of treatment,compared with the model control group,the general condition of the rats in the treatment group improved and their body weight increased(p<0.05),which was statistically different;(4)The observation of carbon end propulsion rate was calculated:compared with the blank control group,the carbon end propulsion rate of the rats in the model group decreased significantly(p<0.01);compared with the model group,there were differences between the medium and high dose groups of Huayu Tongbian Decoction and the western medicine group(p<0.05);compared with the Compared with the blank group,the water content of rats in the model group was significantly reduced,and the difference was statistically significant(p<0.01);after 4 w of drug intervention,the water content of each treatment group was significantly increased compared with that of the model group(p<0.01);compared with the western medicine group,the water content of rats in the low and medium dose groups of Huayu Tongbian Decoction was significantly increased(p<0.01);compared with the western medicine group,the water content of rats in the low and medium dose groups of Huayu Tongbian Decoction was significantly reduced(p<0.01).Compared with the western medicine group,the water content in the low and middle dose groups of Huayu Tongbian Decoction was lower(p<0.05),while the difference between the high dose group and the western medicine group was not significant and not statistically different(p>0.05);(6)Transmission electron microscopy revealed that the proximal colonic ICCs of the rats in the blank control group were more numerous,with better morphology,abundant organelles,dense cytoplasm and more intercellular connections.In the model control group,ICCs were damaged,with degenerative necrosis of organelles,mild lysis of cytoplasm,significantly reduced intercellular connections and broken connection structures.The morphology of ICCs in the colon of rats in the low and medium dose groups was basically normal and the damage was mild,with slightly blurred intercellular connections and fewer organelles.(7)Comparison of c-kit expression counts:the damage of Cajal mesenchymal cells in each intervention group was improved and repaired,and the number of c-kit positive cells was significantly increased(p<0.01),and the improvement and repair of Cajal mesenchymal cells in the high-dose group of Huayu Tongbian Decoction and western medicine control group were obvious,and the trend of increasing the number of c-kit positive cells was significant;(8)The expression levels of c-kit,HCN1,and HCN2 proteins in the treatment groups increased to different degrees(p<0.05),and the trend of increasing c-kit,HCN1,and HCN2 protein expression levels in the high-dose group and western medicine control group was significant.4 Based on PI3K/AKT/mTOR signaling pathway,the effect of Huayu Tongbian Decoction on colon cell autophagy in STC rats was investigated(1)Compared with the blank control group,the expression of autophagy-related proteins Beclin-1,LC3B and Atg5 were up-regulated and P62 expression was down-regulated in the model group(p<0.01);compared with the model group,the low,medium and high doses of Huayu Tongbian Decoction groups all had different degrees of down-regulation of Beclin-1,LC3B and Atg5 protein expression and up-regulation of P62 expression,among which the difference between the high dose group and the model The difference between the high-dose group and the model group was statistically significant(p<0.01);the difference in the expression of autophagy-related proteins between the high-dose and blank control groups was not significant(p>0.05).(2)The trend of change of Beclin-1,LC3B,Atg5 and P62 gene ratio by RT-PCR was similar to the trend of Western Blot results,which again strengthened the validation of the results.(3)Compared with the blank control group,the relative gray values of p-mTOR,p-AKT,and p-PI3K were significantly higher in the model control group(p<0.01),indicating that the expression of p-mTOR,p-AKT,and p-PI3K was increased in the model control group;compared with the model group,there were different degrees of decrease in the relative gray values of p-mTOR,p-AKT,and p-PI3K in each treatment group(p<0.01),among which the decrease in the integral ratio between the high-dose group of Huayu Tongbian Decoction and the western drug control group was large,indicating that the drugs in each treatment group could reduce the expression of p-mTOR,p-AKT and p-PI3K to different degrees,and the effect of the high-dose group of Huayu Tongbian Decoction and the western drug control group was obvious.Compared with the western medicine control group,there was no difference in the relative grayscale values of p-mTOR,p-AKT and p-PI3K in the high-dose group of Huayu Tongbian Decoction(p>0.05),and there were differences in the relative grayscale values of p-mTOR,p-AKT and p-PI3K in the middle and low-dose groups of Huayu Tongbian Decoction(p<0.05),indicating that the high-dose group of Huayu Tongbian Decoction and the western medicine control group reduced the expression of p-mTOR,p-AKT and p-PI3K.AKT and p-PI3K expression in the high-dose group and the control group,and the therapeutic effects were better than those in the low and medium-dose groups.(4)Compared with the blank control group,the expression of mTOR mRNA in the model group increased(p<0.01),and the expression of mTOR mRNA in each treatment group decreased to different degrees(p<0.01),among which the integral ratio of the high-dose group of Huayu Tongbian Decoction and the western control group decreased to a greater extent,indicating that the drugs in each treatment group could reduce the expression of mTOR mRNA to different degrees,and the expression of mTOR mRNA in the high-dose group of Huayu Tongbian Decoction and the western control group decreased to a greater extent.The results of mRNA detection were consistent with the trend of Western Blot detection.Conclusion:1 The study of network pharmacology technology found that Huayu Tongbian Decoction has multiple pathways of action with slow transmission constipation(STC),including PI3K/AKT/mTOR signaling pathway and autophagy related biological processes.2 compound diphenoxate 50mg/(Kg d)has good modeling stability,high safety and low mortality;The histopathological examination of colon showed obvious changes,the damage of Cajal interstitial cells and the changes of autophagy signal proteins were obvious.It is the optimal dose for STC rat modeling.3 The morphology of colon ICCs in STC model was impaired and the number of ICCs decreased,which may be related to the intensification of autophagy in model group.4 Huayu Tongbian Decoction can accelerate the recovery of the growth rate of body mass in rats with slow transit constipation,improve and repair Caj al interstitial cells,significantly increase the number of Cajal interstitial cells,repair the morphology and function of ICCs,thus affecting the intestinal transport function and treating STC.Among them,Huayu Tongbian Decoction high-dose group had the most obvious improvement effect.5 Huayu Tongbian Decoction can down-regulate the expression of p-mTOR,p-AKT and p-PI3K,and can treat STC by regulating the signal pathway PI3K/AKT/mTOR6 Huayu Tongbian Decoction can down-regulate Beclin-1,LC3B,Atg5 expression and inhibit colon cell autophagy,up-regulate P62 expression and promote colon cell repair,thus promoting intestinal peristalsis and rapid excretion of feces;7 Huayu Tongbian Decoction can regulate the autophagy mediated by PI3K/AKT/mTOR signaling pathway to treat the colon ICCs of STC rats;8 The efficacy of Huayu Tongbian Decoction in treating STC is related to dosage,and the high-dose Huayu Tongbian Decoction in this experiment has the best effect in treating STC. |
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