| Objective:To observe the effect of acupuncture and moxibustion on the species abundance and the diversity of the gut microbiota in rats with Crohn’s disease(CD);to elucidate that acupuncture and moxibustion can affect the expression of DNA methyltransferase in the colon tissue of CD rats under hypoxia environment,affect the abnormal DNA methylation level,and regulate the expression of immune-related molecules such as TNF-α,so as to achieve the purpose of acupuncture and moxibustion to inhibit colon inflammation;to reveal the genome-wide DNA methylation map of the colon of CD rats,and explore the effect of acupuncture and moxibustion on the DNA methylation of CD rat from the perspective of whole genomics,in order to provide experimental data and scientific basis for the clinical application of acupuncture and moxibustion treatment of CD.Methods:Male SD rats of clean grade were randomly divided into normal group,model group,herb-partitioned moxibustion group,electroacupuncture group and western medicine group.CD model rats were established by enema with 5 % TNBS.The rats in herb-partitioned moxibustion group were treated with moxibustion at “Tianshu”(bilateral)and “Qihai” acupoints,two moxa cones were used at each acupoint;The rats in electroacupuncture group were treated with electroacupuncture at “Tianshu”(bilateral)and “Qihai” acupoints,the needles were retained for 20 minutes each time;The rats in western medicine group were given sulfasalazine enteric-coated tablets by gavage,all treatment were performed once a day for 7 consecutive days.HE staining was used to observe the pathological changes of rat colon tissue;16S r DNA sequencing was used to observe the species structure,relative abundance and the diversity of the gut microbiota;ELISA was used to detect the protein content of CRP,IL-6,IL-1β,and TNF-α in serum.Western blot was used to detect the protein expression of DNMT1,DNMT3 a,DNMT3b,CEACAM6 in colon tissue;RT-q PCR was used to detect the m RNA expression of TNF-α and HIF-1α in colon tissue.Whole Genome Bisulfite Sequencing was used to observe the DNA methylation in colon tissue and construct a whole genome DNA methylation map,RT-q PCR was used to detect the m RNA expression of Hspa5,Cox6b1,Ctnnb1,Krt8 in colon tissue in normal group,model group,herb-partitioned moxibustion group and electroacupuncture group.Results:Part 1: Effect of acupuncture and moxibustion on the gut microbiota in CD rats1.Histopathological observation and scoring of the colon tissue: In the normal group,the colonic mucosa structure is intact,the intestinal mucosa epithelium is continuously covered,and the glands in the lamina propria of the mucosa are arranged neatly.There are no pathological changes such as hyperemia and ulcers.In the model group,the colonic mucosa,submucosa,and muscular layer were morphologically disordered,with fissuring ulcers,colonic mucosa congestion and edema,and a large number of inflammatory cell infiltrations.After intervention in the herb-partitioned moxibustion,electroacupuncture group and western medicine group,the above pathological changes were alleviated,and new mucosal epithelial repair,healing ulcers and the degree of inflammatory cell infiltration was lower than that of model group.Compared with the normal group,the colonic histopathology score of the model group was significantly increased(P<0.01),compared with the model group,the colonic histopathology score of the rats in the herb-partitioned moxibustion group,the electroacupuncture group and the western medicine group were significantly decreased(P<0.01).2.Effect of acupuncture and moxibustion on the relative abundance of species in the phyla,class,order,family,and genus levels of CD rats: Compared with the normal group,the relative abundance of Bacteroidetes,Actinobacteria,Bacteroidia,Bacteroidiales,Porphyromonadaceae,Prevotellaceae,Bacteroidaceae,Prevotella were increased,and Firmicutes,Proteobacteria,Clostridia,Clostridiales,Lachnospiraceae,Ruminococcaceae,Oscillibacter were decreased in the model group.After herb-partitioned moxibustion and electroacupuncture treatment,the relative abundances of most of the above-mentioned microorganisms are all improved to varying degrees,and the relative abundance of Lactobacillus could be increased by herb-partitioned moxibustion,Ruminococcaceae could be increased by electroacupuncture.3.Effect of acupuncture and moxibustion on the alpha diversity of the gut microbiota of CD rats: Compared with the normal group,the Chao1,Observed species,Shannon,and Simpson indexes were significantly decreased in the model group(P<0.05).Compared with the model group,the Chao1,Observed species,Shannon,and Simpson indexes were significantly increased in the herb-partitioned moxibustion group and electroacupuncture group(P<0.05).Part 2: Effect of acupuncture and moxibustion on the DNA methyltransferase in hypoxic environment of CD rats1.Effect of acupuncture and moxibustion on inflammatory cytokines in serum of CD rats: Compared with the normal group,the protein content of CRP,IL-6,IL-1β,and TNF-α in the serum of the model group increased significantly(P<0.01).Compared with the model group,the protein content of the above-mentioned inflammatory factors in the herb-partitioned moxibustion group,electroacupuncture group and western medicine group were significantly decreased(P<0.01).2.Effect of acupuncture and moxibustion on CEACAM6,TNF-α,HIF-1α in the colon tissue of CD rats: Compared with the normal group,the expression of CEACAM6 protein,TNF-α m RNA and HIF-1α m RNA in the colon tissue of the model group increased significantly(P<0.01).Compared with the model group,the expressions of CEACAM6 protein,TNF-α m RNA,and HIF-1α m RNA in the herb-partitioned moxibustion group and western medicine group were significantly decreased(P<0.05),the expression of CEACAM6 protein and HIF-1α m RNA were significantly decreased in the electroacupuncture group(P<0.05).3.Effect of acupuncture and moxibustion on DNA methyltransferase in the colon tissue of CD rats: Compared with the normal group,the protein expression of DNA methyltransferase DNMT1,DNMT3 a and DNMT3 b in the colon tissue of the model group increased significantly(P<0.01).Compared with the model group,the protein expression of the above-mentioned DNA methyltransferase in the herb-partitioned moxibustion group and the western medicine group were significantly decreased(P<0.01),and the expression of DNMT1 protein in the electroacupuncture group was significantly decreased(P<0.01).Part 3: Effect of acupuncture and moxibustion on the genome-wide DNA methylation map in CD rats1.Differentially methylated region(DMR)methylation level analysis: In the CG context,compared with the normal group,the number of hypermethylated DMR was2477 and hypomethylated DMR was 20610 in the model group;compared with the model group,the number of hypermethylated DMR was 5120 and hypomethylated DMR was 7982 in the herb-partitioned moxibustion group,the number of hypermethylated DMR was 7959 and hypomethylated DMR was 3161 in the electroacupuncture group.In the CHG context,compared with the normal group,the number of hypermethylated DMR was 11 and hypomethylated DMR was 192 in the model group;compared with the model group,the number of hypermethylated DMR was 1011 and hypomethylated DMR was 85 in the herb-partitioned moxibustion group,the number of hypermethylated DMR was 2565 and hypomethylated DMR was 118 in the electroacupuncture group.In the CHH context,compared with the normal group,the number of hypermethylated DMR was 180 and hypomethylated DMR was 864 in the model group;compared with the model group,the number of hypermethylated DMR was 3346 and hypomethylated DMR was 369 in the herb-partitioned moxibustion group,the number of hypermethylated DMR was 8059 and hypomethylated DMR was 382 in the electroacupuncture group.2.KEGG pathway enrichment analysis of genes in the promoter region that overlapped with the DMR: In the CG context,the KEGG pathways involved in the promoter regions in the differentially methylated regions of the normal group and the model group included PPAR signaling pathway,PI3K-Akt signaling pathway;the KEGG pathways involved in the promoter regions in the differentially methylated regions of the herb-partitioned moxibustion group and the model group included PI3K-Akt signaling pathway,intestinal immune network for Ig A production;the KEGG pathways involved in the promoter regions in the differentially methylated regions of the electroacupuncture group and the model group included ECM-receptor interaction.In the CHG context,the KEGG pathway involved in the promoter regions in the differentially methylated regions of the normal group and the model group included inflammatory bowel disease,colorectal cancer;the KEGG pathway involved in the promoter regions in the differentially methylated regions of the herb-partitioned moxibustion group and the model group included the ECM-receptor Interaction;the KEGG pathway involved in the promoter regions in the differentially methylated regions of the electroacupuncture group and the model group included leukocyte transendothelial migration,bacterial invasion of epithelial cells.In the CHH context,the KEGG pathway involved in the promoter regions in the differentially methylated regions of the normal group and the model group included colorectal cancer,bacterial invasion of epithelial cells;the KEGG pathway involved in the promoter regions in the differentially methylated regions of the herb-partitioned moxibustion group and the model group included m TOR signaling pathway,bacteria invasion of epithelial cells;the KEGG pathway involved in the promoter regions in the differentially methylated regions of the electroacupuncture group and model group included bacterial invasion of epithelial cells.3.Screening of genes in the promoter region that overlapped with the DMR: In the CG context,there are 41 differential methylated genes with intersection between model group vs.normal group,herb-partitioned moxibustion group vs.model group,and electroacupuncture group vs.model group,among them,Hspa5 and Cox6b1 were closely related to endoplasmic reticulum stress and energy metabolism.In the CHG context,there are 9 differential methylated genes with intersection between the above three different comparison combinations.In the CHH context,there are 60 differential methylated genes with intersection between the above three different comparison combinations,including oncogenes such as Ctnnb1 and Krt8.4.Effect of acupuncture and moxibustion on the genes in the promoter region that overlapped with the DMR: Compared with the normal group,the expressions of Hspa5,Cox6b1,Ctnnb1 and Krt8 m RNA in the colon tissue of the model group increased significantly(P<0.05).Compared with the model group,the expressions of the above genes m RNA in the herb-partitioned moxibustion group and electroacupuncture group were significantly decreased(P<0.05).This result was consistent with that the methylation levels of Hspa5,Cox6b1,Ctnnb1,and Krt8 in the model group are lower than the normal group,while the methylation levels of the herb-partitioned moxibustion group and electroacupuncture group were higher than the model group.Conclusion:1.Both herb-partitioned moxibustion and electroacupuncture can adjust the abundance and diversity of the gut microbiota in CD rats,and improve the intestinal microecology.The herb-partitioned moxibustion can also increase the relative abundance of probiotics such as Lactobacillus,and electroacupuncture can increase the relative abundance of probiotics such as Ruminococcaceae.2.Both herb-partitioned moxibustion and electroacupuncture can inhibit the expression of inflammatory factors such as CRP,IL-6,IL-1β,TNF-α,and HIF-1α,CEACAM6 and DNMT1 in CD rats.The herb-partitioned moxibustion can also inhibit the expression of DNMT3 a,and DNMT3 b,which may be one of the mechanisms of acupuncture to reduce CD intestinal inflammation.3.Both herb-partitioned moxibustion and electroacupuncture can regulate the genome-wide DNA methylation map of CD rats,and the main pathway involved are related to ECM-receptor interaction,bacterial invasion of epithelial cells,and biosynthesis of amino acid,and herb-partitioned moxibustion also involves intestinal immune network for Ig A production,m TOR signaling pathway,PI3K-Akt signaling pathway,while electroacupuncture also involves signaling pathway such as in leukocyte transendothelial migration and central carbon metabolism in cancer.4.Both herb-partitioned moxibustion and electroacupuncture can regulate the methylation and transcription levels of Hspa5 and Cox6b1,which were closely related to endoplasmic reticulum stress and energy metabolism,as well as oncogenes such as Ctnnb1 and Krt8 in colon tissue of CD rats.It may be one of the epigenetic regulation mechanisms of acupuncture in treating Crohn’s disease. |
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