| Objective:Functional constipation(FC)is a clinically recurrent refractory disease.Previous studies have shown that intestinal flora plays an important role in acupuncture treatment of FC,and mi RNA is a key regulatory substance mediating the dialogue between flora and host.This study aims to study the clinical efficacy of acupuncture,the relationship between intestinal flora and mi RNAs,explore the potential of intestinal flora and mi RNAs as intestinal markers for acupuncture treatment of FC,and explore the possible mechanism of acupuncture treatment of FC.Methods:1.This study was designed based on the methodology of clinical randomized controlled trials.From May 2021 to November 2022,patients with FC who were treated at the Department of Colorectal Surgery of Chengdu University of Traditional Chinese Medicine Affiliated Hospital were selected as the research subjects.At the same time,healthy volunteers of matching gender and age were recruited from the community as the normal control.A total of 65 FC patients and 30 healthy volunteers were enrolled,and FC patients were randomly assigned to the acupuncture group and sham acupuncture group.The acupuncture group received deep needling at the bilateral "Tianshu," "Fujie," and "Shangjuxu" acupoints,while the sham acupuncture group received non-acupoints between two meridians with no skin penetration for placebo treatment.Both groups were treated once a day,with five consecutive treatments per week for the first two weeks,followed by two days of rest,and three treatments per week for the next two weeks,with one day of rest in between,for a total of 16 treatments.The main outcome measure was the proportion of patients with an average of three complete spontaneous bowel movements(CSBMs)per week before and after treatment.The secondary outcome measures included changes in the average weekly Bristol stool form scale(BSFS),bowel movement difficulty,and constipation patient quality of life score compared to baseline.Healthy volunteers did not receive any treatment.2.A total of 60 fecal samples were collected,including 30 before and after treatment from two groups of patients,as well as 28 samples from healthy volunteers.The samples were subjected to high-throughput sequencing of the V3-V4 region of the 16 S r DNA gene,and species abundance annotation information was obtained.Diversity indices reflecting gut microbiota richness and diversity,and inter-group structural similarity were calculated using the QIIME2 microbiome analysis software.Based on these results,LEf Se and Venn analyses were performed to screen for candidate differential bacterial genera that may be affected by acupuncture.The Spearman method was then used to analyze the correlation between these candidate genera and changes in clinical efficacy indicators,ultimately identifying the key microbiota most closely related to acupuncture efficacy.Finally,changes in microbial functional genes and metabolic pathways were analyzed using PICRUSt.3.Six samples were selected from each group for mi RNA sequencing and eight samples were used for subsequent RT-PCR validation.High-throughput sequencing was conducted using small RNA transcriptome sequencing technology,with an FDR of 0.05 and Log2 FC of0.585 set as the threshold.Based on the principle of high expression abundance,differentially expressed mi RNAs in each group were selected.A Venn diagram of differential expression profiles of fecal mi RNAs before and after acupuncture treatment in FC patients and healthy controls,as well as in a sham acupuncture group,was generated to preliminarily screen key mi RNAs for acupuncture treatment of FC.The selected mi RNAs were further validated using RT-PCR to verify the accuracy of sequencing.Finally,the multimi R package in the R software was used to predict target genes of validated mi RNAs,and GO and KEGG analysis were performed to elucidate the relevant functions and signaling pathways of the target genes.4.After obtaining a specific gut bacteria genus and key mi RNAs involved in the action of acupuncture treatment for FC,the correlation coefficient matrix between the specific bacteria genus and key mi RNAs should be calculated using R software.Based on the filtered correlation coefficient values,a "gut microbiota-mi RNAs" interaction network for acupuncture treatment for FC should be constructed using Cytoscape software,with bacteria genus or mi RNA as nodes and the correlation coefficient values as edges.Finally,the diagnostic potential of these interacting bacteria genus and mi RNAs as gut biomarkers for acupuncture treatment for FC should be evaluated through a random forest model analysis.Results:1.Clinical efficacy of acupuncture for patients with FC:A total of 65 patients with functional constipation and 30 healthy volunteers were included in this study,with 32 in the acupuncture group and 33 in the sham acupuncture group.The proportion of patients in the acupuncture group who had an average of 3 CSBMs per week after treatment was 75.76%,while that in the sham acupuncture group was only 6.25%,and the difference between the two groups was significant(P < 0.01).Compared with baseline,both groups showed a significant increase in average weekly SBMs and a significant improvement in BSFS scores and difficulty of defecation(P < 0.01),leading to an improvement in the patients’ quality of life(P < 0.01).Compared with the sham acupuncture group,the acupuncture group showed a more significant increase in average weekly SBMs,improvement in difficulty of defecation,and improvement in quality of life of patients with functional constipation(P < 0.05).2.Acupuncture improves the gut microbiota of patients with FC:(1)Diversity analysis indicated that Chao1,observed OTUs,Pielou’s evenness,Shannon’s and Simpson’s indexes in FC patients were significantly higher than those in the healthy control group(P < 0.05).The scatter plot analysis demonstrated that the FC group and healthy group were separated by clusters(ADONIS,R2 = 0.97,P = 0.001).After acupuncture treatment,the diversities of intestinal microbiota in FC patients were slightly restored,but the difference was not significant(P > 0.05).However,the diversity of microbiota was significantly improved(ADONIS,R2 = 0.97,P = 0.017).On the other hand,in the sham acupuncture group,there were no significant changes in the diversity and richness of intestinal microbiota before and after intervention(P > 0.05).(2)Species difference analysis showed that at the genus level,the abundance of 20 microbial species including Intestinibacter,Coprobacter,and Fusobacterium was significantly increased(P < 0.05)in the intestinal microbiota of FC patients,while the abundance of Prevotellaceae＿UCG.001,Megamonas and ML635J-40 aquatic group was significantly decreased(P < 0.05).Acupuncture intervention significantly decreased the abundance of eight opportunistic pathogens such as Intestinibacter,Coprobacter,and Fusobacterium in the intestinal microbiota(P < 0.05).In contrast,sham acupuncture intervention only decreased the relative abundance of two intestinal microbiota species,Blautia and Clostridium＿sensu＿stricto＿1(P < 0.05).(3)The LEf Se analysis showed that at an LDA score of 2.5,42 microbial taxa,including Bacteroides,Escherichia-Shigella and Parabacteroides,were significantly enriched in the gut microbiota of patients with FC.After acupuncture treatment,seven gut microbes,including Lactobacillus,Prevotellaceae＿UCG.001,and Ralstonia,were significantly enriched in the gut microbiota of FC patients.In contrast,after sham acupuncture intervention,three gut microbes,including Erysipelotrichaceae＿UCG.003,Streptococcus,and Parasutterella,were more significantly enriched in the gut microbiota of patients.(4)Based on the analysis of species differences and LEf Se analysis,Wayne analysis was used to exclude species with the same direction of action in each group.After further exclusion of species that showed no significant correlation with clinical efficacy indicators through Spearman correlation analysis,a total of 24 specific gut microbes affected by acupuncture were identified.Among them,seven species,including Lactobacillus,Prevotellaceae＿UCG.001,and Butyrivibrio,were upregulated in abundance after treatment,while 17 species,including Coprobacter,Pseudomonas,and Erysipelotrichaceae＿UCG.003,were downregulated in abundance.(5)Functional prediction analysis using PICRUSt showed that acupuncture treatment for FC was mainly associated with multiple metabolic pathways,including cellular dynamic ion transmembrane transport injury and repair,carbohydrate metabolism,and amino acid metabolism(P < 0.05).In contrast,sham acupuncture treatment did not result in significantly different metabolic pathways(P < 0.05).3.Acupuncture modulates the expression of fecal mi RNAs in patients with FC:(1)Compared with healthy volunteers,there were 61 differentially expressed fecal mi RNAs in FC patients,with 33 upregulated and 28 downregulated;compared before and after acupuncture treatment,there were 37 differentially expressed fecal mi RNAs,with 22 upregulated and 15 downregulated;compared before and after sham acupuncture treatment,there were 82 differentially expressed fecal mi RNAs,with 68 upregulated and 14 downregulated.The Venn analysis of these three sets revealed that the downregulated mi RNAs in acupuncture treatment for FC were hsa-mi R-34c-5p and hsa-mi R-335-5p,and the upregulated mi RNA was hsa-mi R-205-5p.(2)The RT-PCR validation results showed that the expression of hsa-mi R-34c-5p was significantly increased in patients with functional constipation(FC)(P = 0.000),but was significantly reduced after acupuncture treatment(P = 0.000),while there was no significant change observed with sham acupuncture treatment(P = 0.222).The expression of hsa-mi R-335-3p was increased in FC patients(P = 0.328),and there was a slight increase after both acupuncture and sham acupuncture treatments,but these differences were not statistically significant(P = 0.779,P = 0.720).The expression of hsa-mi R-205-5p was significantly reduced in FC patients(P = 0.000),but was significantly increased after acupuncture treatment(P = 0.004),while there was no significant change observed with sham acupuncture treatment(P = 0.557).(3)The prediction of target genes for hsa-mi R-34c-5p and hsa-mi R-205-5p shows that the intersection of predicted target genes in various mapping datasets contains a total of 449 target genes.GO analysis indicates that these 449 target genes are mainly involved in regulating axon guidance,intestinal epithelial cell proliferation,and inhibition of intestinal motility.KEGG pathway analysis shows that the 449 target genes are involved in 69 enriched signaling pathways,including Hippo,autophagy,acetylcholine synaptic transmission,PI3 KAkt,and other signaling pathways that are associated with FC disease.4.Possible mechanism of acupuncture for patients with FC based on the gut microbiotami RNA interaction:(1)Co-occurrence network analysis showed a tightly interacting network of 2 mi RNAs and 8 bacterial genera in the acupuncture group.Topological parameter analysis revealed that the network had 10 nodes and 18 edges,with mi R-34c-5p having the highest degree of connectivity.mi R-34c-5p was significantly negatively correlated with Lactobacillus(weighted＿r =-0.0036)and Butyrivibrio(weighted＿r =-0.0045),while significantly positively correlated with Pseudomonas(weighted＿r = 0.0024),Coprobacter(weighted＿r = 0.0131),and Erysipelotrichaceae＿UCG.003(weighted＿r = 0.0015).Lactobacillus,Phascolarctobacterium,and Erysipelotrichaceae＿UCG.003 had the highest degree of connectivity among the bacterial genera.Lactobacillus was significantly positively correlated with Butyrivibrio(weighted＿r =0.0024)and significantly negatively correlated with Erysipelotrichaceae＿UCG.003(weighted＿r =-0.0028).Phascolarctobacterium was significantly negatively correlated with Butyrivibrio(weighted＿r =-0.0053)and significantly positively correlated with Pseudomonas(weighted＿r = 0.0186).Erysipelotrichaceae＿UCG.003 was significantly positively correlated with Coprobacter(weighted＿r = 0.0053).(2)In the sham acupuncture group,no significant correlations(FDR < 0.05)were found between the species that showed significant differences before and after intervention and mi RNAs,except for a positive correlation between mi R-34c-5p and mi R-205-5p(weight＿r =0.0054).All other nodes remained isolated and unconnected.This observation provides indirect evidence for the accuracy and reliability of the results from the acupuncture group study.(3)The analysis of a random forest model found that the combined diagnosis of eight gut bacterial genera and two mi RNAs under acupuncture treatment had an area under curve(AUC)of 1.000,indicating a high diagnostic efficacy and accurate ability to distinguish subjects before and after acupuncture treatment.The combined model performed better than the diagnosis results obtained from gut microbiota or mi RNAs alone(with AUCs of 0.771 and0.886,respectively).Conclusion:1.Acupuncture could effectively increase the frequency of complete spontaneous bowel movements and spontaneous bowel movements for patients with FC,while also softening the consistency of the feces and reducing the severity of difficulty in defecation,thus improving the quality of life of the patients.2.Acupuncture can regulate the species richness and diversity of intestinal microbiota in FC patients,and restore the disrupted microbial structure and metabolic pathways to exert therapeutic effects.Among them,7 probiotics with up-regulated abundance such as Lactobacillus,Prevotellaceae＿UCG.001,Butyrivibrio and 17 conditional pathogenic bacteria with down-regulated abundance such as Coprobacter,Fusobacterium,Erysipelotrichaceae＿UCG.003 may be specific gut microbiomes involved in the therapeutic effect of acupuncture for FC.3.Acupuncture could regulate the expression of mi RNAs in the feces of patients with FC,among which hsa-mi R-34c-5p and hsa-mi R-205-5p may be key mi RNAs involved in the therapeutic effect of acupuncture on FC.4.The interaction between hsa-mi R-34c-5p,hsa-mi R-205-5p,and gut microbiomes such as Lactobacillus,Phascolarctobacterium,Coprobacter,Erysipelotrichaceae＿UCG.003,Pseudomonas,Butyrivibrio,Bacteroides＿caccae,and Butyrivibrio＿crossotus under acupuncture can tightly regulate intestinal function.These intestinal biomarkers not only help to elucidate the mechanism of acupuncture treatment for functional constipation but also have the potential to develop as objective indicators for evaluating the gastrointestinal function of FC patients undergoing acupuncture,which deserves further validation in animal experiments. |
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