| Objective:To evaluate the clinical efficacy of the Changyun Formula under the guidance of the Yunpi Method on slow transit constipation with spleen deficiency and qi stagnation syndrome,to investigate the role of intestinal microbiota on the mechanism of slow transit constipation,and to explore the microbiological mechanism of the Yunpi Method in the treatment of slow transit constipation,to enrich the connotation of the Yunpi Method and provide new ideas for the prevention and treatment of constipation in Traditional Chinese Medicine.Methods:1.60 patients with the syndrome of spleen deficiency and qi stagnation in slow transit constipation were involved in the prospective randomized controlled study and were randomly assigned to the observation group and the control group.To evaluating treatment effectiveness,the number of weekly complete spontaneous bowel movements(CSBMs),the constipation main symptom scores,and the TCM syndrome scores were all recorded before and after treatment.2.Compound Diphenoxylate was used to create a mouse model of slow transit constipation.For 14 days,the Changyun Formula was gavaged to the high-dose(H-CYF),medium-dose(M-CYF),and low-dose(L-CYF)groups in the relevant dosages,lactulose to the control group(L),and equal volumes of normal saline to the normal group(N)and model group(M).The mice’s general health,the time of the first black feces,the amount of water in the feces,and the rate of intestinal ink propulsion were used to evaluate the modeling results and the intestinal transmission function.Colonic PGP9.5,C-Kit,and SCF protein and mRNA were tested using Western blot and Real-time PCR to evaluate the expression levels of neurons in the enteric nervous system(ENS)and interstitial cells of Cajal(ICC).3.Fecal microbiota transplants were performed on pseudo-germfree mice caused by the antibiotic cocktail to induce a slow transit constipation model,and then randomly divided into fecal microbiota transplant-normal group(N-FMT),fecal microbiota transplant-model group(M-FMT),and Changyun Formula group(CYF),with the N-FMT group receiving fecal microbiota transplants from group N mice and the M-FMT and CYF groups receiving fecal microbiota transplants from successfully modelled group M mice.Following successful modeling,the Changyun Formula was gavaged to the CYF group at a optimal dose for 14 days,whereas the normal saline was administered at an equal dose to the N-FMT and M-FMT groups.The modeling findings and the mice’s intestinal transmission function were assessed using the time of the first black feces,the rate of fecal water content,and the rate of intestinal ink propulsion.Western blot and Real-time PCR were used to detect the expression levels of colonic PGP9.5,C-Kit,SCF,Bcl-2,Bax,and Caspase-3 proteins and mRNA,and TUNEL was used to detect the apoptosis rate in colon cells,to explore the effect of intestinal microbiota on the apoptosis of neurons and ICC in ENS,and to explore the mechanism of action of the Yunpi Method.4.Mice in the N,M,N-FMT,M-FMT,and CYF groups had fresh feces collected.To confirm the transplantation of fecal microbiota and further look into how the Yunpi Method regulates intestinal microecology,16S rDNA was utilized to find intestinal microbiota and GC-MS to find short-chain fatty acids.Results:1.Clinical efficacy of the Yunpi Method in the treatment of slow transit constipation with spleen deficiency and qi stagnation syndrome.(1)After 4 weeks of therapy,the observation group’s overall effective rate was 89.29%while the control group’s overall effective rate was 70.37%(P<0.01).(2)CSBMs:Both groups were successful in enhancing the patients’ weekly CSBMs(P<0.01),however,the observation group performed better than the control group(P<0.01).(3)Constipation main symptom scores:The observation group improved the total and individual symptom scores of constipation(P<0.01).The total and individual symptom scores(frequency of defecation,duration of defecation,Incomplete defecation and anal distension,abdominal distension)of the observation group were higher than those of the control group(P<0.05/P<0.01).(4)The TCM syndrome scores:The observation group significantly reduced the total TCM syndrome scores as well as the single symptom scores for decreased defecation frequency,decreased awareness of defecation,dry stools,abdominal fullness,food less poor and appetite,fatigue,and sluggish speaking,and weakness after stools(P<0.01).Compared to the control group,the scores for total TCM syndrome as well as the single symptom for decreased defecation frequency,decreased awareness of defecation,abdominal fullness,food less poor and appetite,fatigue and sluggish speaking,and weakness after stools were improved(P<0.05/P<0.01).2.The impact of the Yunpi Method on gut motility in mice with slow transit constipation.(1)Intestinal transmission function:Compared with group N,group M mice had a longer time of the first black feces,lower rate of fecal water content and rate of intestinal ink propulsion(P<0.01),and the slow transit constipation model was successfully established and maintained with the Compound Diphenoxylate.Compared with the M group,the H-CYF,M-CYF,and L groups had a shorter time of the first black feces,a higher rate of fecal water content,and a higher rate of intestinal ink propulsion(P<0.05/P<0.01),mice in the L-CYF group had a shorter time of the first black feces,a higher rate of intestinal ink propulsion(P<0.01)but increasing trend with the rate of fecal water content(P>0.05).(2)Expression of ENS,ICC related proteins and mRNA:colonic PGP9.5,C-Kit,SCF protein,and mRNA expression were lower in group M compared to group N(P<0.01).Colonic PGP9.5,C-Kit,SCF protein,and mRNA expression were higher in the H-CYF group compared to the M group(P<0.05/P<0.01);colonic C-Kit,SCF protein,and mRNA expression were higher in the M-CYF group(P<0.05/P<0.01),while PGP9.5 protein and mRNA expression tended to be higher in the M-CYF group(P>0.05).Colonic C-Kit protein and mRNA expression were higher in the L-CYF group,as was SCF protein expression(P<0.01)and there was a tendency for enhanced SCF mRNA expression(P>0.05);colonic C-Kit and SCF protein,and mRNA expression were elevated(P<0.05/P<0.01),and PGP9.5 protein expression was elevated(P<0.05).PGP9.5 mRNA expression was found to be enhanced(P>0.05).3.The relationship between intestinal microbiota and the etiology of slow transit constipation.(1)Intestinal transmission function:Mice in the M-FMT group had a longer time of the first black feces and a lower rate of fecal water content and rate of intestinal ink propulsion(P<0.05/P<0.01)compared to the N-FMT group,and the fecal microbiota transplantation approach successfully caused a flora-related slow transit constipation model.Mice in the CYF group had a shorter time of the first black feces,a greater rate of fecal water content,and a higher rate of intestinal ink propulsion than mice in the M-FMT group(P<0.05).(2)Expression of ENS,ICC related proteins and mRNA:as compared to the N-FMT group,colonic PGP9.5,C-Kit,and SCF protein and mRNA expression in the M-FMT group were lower(P<0.05/P<0.01).When compared to the M-FMT group,colonic PGP9.5 mRNA expression was higher in the CYF group(P<0.05),PGP9.5 protein expression was higher(P>0.05),and the expression of protein and mRNA of c-kit and SCF increased(P<0.05/P<0.01).(3)Apoptosis-related protein and mRNA expression and apoptosis rate:compared to the N-FMT group,colonic Bcl-2 protein and mRNA expression was decreased in the M-FMT group(P<0.01),Bax and Caspase-3 protein and mRNA expression was increased(P<0.05/P<0.01),Bcl-2/Bax ratio was decreased(P<0.01),and the relative apoptosis rate In comparison to the M-FMT group,the CYF group demonstrated elevated colonic Bcl-2 protein and mRNA expression(P<0.05/P<0.01),decreased Bax and Caspase-3 protein and mRNA expression(P<0.05/P<0.01),raised Bcl-2/Bax ratio(P>0.05),and decreased relativeapoptosis rate(P<0.05).4.Effect of the Yunpi Method on intestinal microecology in mice with slow transit constipation.(1)Features of the intestinal microbiota:Following fecal microbiota transplantation,the N-FMT and M-FMT groups enhanced the number of OUT shared by their respective donor groups.Alpha diversity analysis revealed that there was no statistically significant difference between the two groups’ Chao1 and Shannon indexes and the corresponding donor groups(P>0.05).The 2 groups essentially mirrored the features of the intestinal microbiota of their respective donors,according to species relative abundance clustering analysis and beta diversity analysis,and the colonization of the flora was successful.Following the Changyun Formula intervention,there were more OUTs shared with the N-FMT group and fewer OUTs shared with the M-FMT group in the CYF group.The results of the alpha diversity analysis indicated that there was no significant change in the Chao1 index comparing the groups(P>0.05),a trend towards a decrease in the Shannon index comparing the M-FMT with the N-FMT group(P>0.05),and a significant decrease in the Shannon index comparing the M-FMT with the N group(P<0.05).The structure of the microbiota of the M-FMT and N-FMT groups was considerably distinct,and the features of the intestinal flora of the CYF group were similar to those of the N-FMT group,according to analyses of beta diversity and species relative abundance clustering.In the M-FMT group,as compared to the N-FMT group,the relative abundance of Firmicutes,Desulfobacterota,and Actinobacteriota declined,the relative abundance of Bacteroidota and Proteobacteria rose,and the Bacteroidota/Firmicutes ratio increased.The CYF group reduced the relative abundance of Muribaculaceae_Unclassified,Muribaculum,Bacteroides,increased the relative abundance of Lachnospiraceae_NK4A136_group,Desulfovibrio,A2,and reduced the Bacteroidota/Firmicutes ratio.LefSe analysis revealed that A2,Anaerovorax,Butyricicoccus,and Ruminococcaceae,all from Firmicutes,were the genera enriched in the CYF group that contributed to significant variations across groups.(2)Short-chain fatty acid content:Statistically,the N-FMT and M-FMT groups did not vary from the corresponding donor groups(P>0.05).The M-FMT group differed significantly from the N-FMT group in that it had higher levels of acetic acid(P<0.01),lower levels of butyric acid,valeric acid,isovaleric acid,and hexanoic acid(P<0.01),and no discernible change in propionic acid(P>0.05).Acetic acid decreased in the CYF group compared to the M-FMT group(P<0.01),whereas butyric acids,valeric acids,isovaleric acids,and hexanoic acids increased(P<0.05/P<0.01).Propionic acid did not alter significantly(P>0.05).Conclusions:1.When treating slow transit constipation with spleen deficiency and qi stagnation syndrome,the Changyun Formula used under the guidance of the Yunpi Method performed clinically better than the control group and was more effective in improving CSBMs,the main symptoms of constipation as well as the spleen deficiency and qi stagnation syndrome.2.The Compound Diphenoxylate method can successfully establish a mouse model of slow transit constipation.The Changyun Formula,under the guidance of the Yunpi Method,has been shown to significantly up-regulate the expression of PGP9.5,C-Kit and SCF,thus up-regulate the expression of ENS neurons and ICC.3.The slow transit constipation method using fecal microbiota transplantation was effective.By controlling the expression of Bcl-2,Bax,and Caspase-3 and by influencing the expression of ENS neurons and ICC,the intestinal microbiota contributes to the etiology of delayed transit constipation.The Yunpi Method and the Changyun Formula can suppress colon cell apoptosis and enhance the expression of ENS neurons and ICC.4.Using The Yunpi Method with the Changyun Formula,the intestinal micro-ecology of mice with slow-transit constipation converged to that of healthy mice by increasing the variety of intestinal microbiota and regulating the structure of intestinal microbiota and the amount of short-chain fatty acids.In summary,the clinical efficacy of the Yunpi Method in the treatment of slow transit constipation with spleen deficiency and qi stagnation syndrome is confirmed.The Yunpi Method may regulate intestinal microecology,improve excessive apoptosis of colonic cells brought on by dysbiosis,and restore the expression of ENS neurons and ICC to improve intestinal motility and treat slow transit constipation. |
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