| Objective1.To explore the effect of acupuncture and herb-partitioned moxibustion on Crohn’s disease and its effect on autophagy.2.To clarify the role of chromatin remodeling in Crohn’s disease and the regulatory role of acupuncture and herb-partitioned moxibustion on chromatin remodeling mediated autophagy in Crohn’s disease.MethodsPart 1: 60 male SD rats were randomly divided into normal group,model group,acupuncture group,herb-partitioned moxibustion group and herb-partitioned moxibustion + acupuncture group.The rats of CD model were prepared by 2,4,6-trinitrobenzenesulfonic acid(TNBS).In the acupuncture group,"Zusanli" and "Shangjuxu" of CD rats were treated with acupuncture,"Tianshu" and "Qihai" separately treated with herb-partitioned moxibustion,and in the herb-partitioned moxibustion + acupuncture group,separately treated with herb-partitioned moxibustion and acupuncture.The model group and the normal group only received the same fixation without any treatment.The effect of acupuncture and herb-partitioned moxibustion on CD was observed by observing the general condition of rats,disease activity degree,macro damage score of colon tissue and pathological score of colon tissue.The levels of autophagy related factors Atg16L1,IRGM and IL-18 in peripheral blood were detected by ELISA,Western blot and RT-q PCR were used to detect the protein and m RNA expressions of autophagy related factors Atg16L1,IRGM,IL-18 in colon tissues of rats in each group.We tested the above indicators to observed the effect of acupuncture and herb-partitioned moxibustion on CD autophagy.Part 2: 60 male SD rats were randomly divided into normal group,model group,acupuncture group,herb-partitioned moxibustion group and herb-partitioned moxibustion + acupuncture group.The modeling and treatment methods were the same as in Part I.RNA-seq sequencing and bioinformatics were used to analyze the different genes and pathways in each group,and the genes related to autophagy,immunity and inflammation as well as the pathways closely related to CD were screened.We tested protein and m RNA expressions of chromatin remodeling related enzymes Brg1,LSD1,HDAC3,HDAC9 and h MOF in colon tissues of rats in each group were detected by Western blot and RT-q PCR to observe the effect of acupuncture and herb-partitioned moxibustion on chromatin remodelase.Ch IP-seq was used to analyze the different genes and the enrichment level of H3K27me3,H3K4me2 and H3K27 ac in autophagy-related genes Atg16L1,IRGM and IL-18’s promoter in colon tissues of rats in each group to observe the effect of acupuncture and herb-partitioned moxibustion on autophagy of chromatin remodeling.ResultsPart 1: 1.Comparison of diarrhea scores of rats in each group: compared with the normal group,diarrhea score of model group was significantly increased(P<0.01);Compared with model group,the diarrhea score of acupuncture group,herb-partitioned moxibustion group and herb-partitioned moxibustion + acupuncture group were all decreased(Pall<0.05).Comparison of occlusive blood scores in each group: compared with normal group,the occlusive blood score in model group was significantly increased(P<0.01).Compared with model group,the occlusive blood score in acupuncture group,herb-partitioned moxibustion group and herb-partitioned moxibustion + acupuncture group were all decreased(P<0.05,P<0.05,P<0.01).Comparison of macroscopical tissue damage scores in each group: compared with normal group,model group’s macroscopical tissue damage score was significantly increased(P<0.01),compared with model group,acupuncture group,herb-partitioned moxibustion,herb-partitioned moxibustion + acupuncture group’s macroscopical tissue damage score were all lower(Pall<0.05).Comparison of scores of CMDI in each group: compared with normal group,the CMDI score in model group was significantly increased(P<0.01);Compared with model group,the CMDI score of colon in acupuncture group,herb-partitioned moxibustion group and herb-partitioned moxibustion + acupuncture group were significantly decreased(Pall<0.01).Compared with acupuncture group,the CMDI score of colon in herb-partitioned moxibustion group and herb-partitioned moxibustion + acupuncture group were significantly decreased(P<0.01).Compared with herb-partitioned moxibustion group,the CMDI score of colon in herb-partitioned moxibustion + acupuncture group was significantly decreased(P<0.01).2.The levels of autophagy related factors Atg16L1,IRGM and IL-18 in peripheral blood serum of rats in each group: compared with normal group,the levels of autophagy related factors Atg16L1,IRGM and IL-18 in peripheral blood serum of rats in model group were significantly increased(Pall<0.01);Compared with model group,the levels of ATG16L1,IRGM and IL-18 in peripheral blood of rats in acupuncture group,herb-partitioned moxibustion group and herb-partitioned moxibustion + acupuncture group were significantly decreased(Pall<0.01).3.The protein and m RNA expressions of autophagy related factors Atg16L1,IRGM,IL-18 in colon tissue of rats in each group: compared with normal group,the protein and m RNA expressions of Atg16L1,IRGM,IL-18 in colon epithelial tissue of rats in model group were significantly increased(Pall<0.01);Compared with model group,the protein and m RNA expressions of Atg16L1,IRGM,IL-18 in the colon epithelial tissue of rats in herb-partitioned moxibustion + acupuncture group were significantly decreased(Pall<0.01).Part 2: 1.Observation of different genes and pathways in colon tissues of rats in each group: RNA-seq: the expressions of Atg16L1 and IL-18 in herb-partitioned moxibustion + acupuncture group were decreased compared with model group;Ch IP-seq: The expressions of ATG16L1 and IRGM in the H3K27me3 of herb-partitioned moxibustion + acupuncture group were higher than model group;The expressions of ATG16L1 and IRGM in the H3K4me2 of herb-partitioned moxibustion+ acupuncture group were lower than model group.The expressions of ATG16L1 and IRGM in the H3K27 ac of herb-partitioned moxibustion + acupuncture group were lower than model group.The genes of higher enrichment after treatment were response to hypoxia and response to decreased oxygen levels.Different pathway after treatment was mainly PPAR pathway.2.The protein and m RNA expressions of chromatin remodeling related enzymes Brg1,LSD1,HDAC3,HDAC9 and h MOF in colon tissue of rats in each group:compared with normal group,protein and m RNA expressions of LSD1,HDAC3,HDAC9 and h MOF in colon epithelial tissue of rats in model group were significantly increased(Pall<0.01),while protein and m RNA expressions of Brg1 were significantly decreased(Pboth<0.01).Compared with model group,the protein and m RNA expressions of LSD1,HDAC3,HDAC9 and h MOF in the colon epithelial tissue of rats in herb-partitioned moxibustion + acupuncture group were significantly decreased(Pall<0.01),while the protein and m RNA expressions of Brg1 were significantly increased(Pboth<0.01).3.The enrichment of ATG16L1,IRGM and IL-18’s gene promoter peaks in H3K27me3,H3k4me2 and H3K27 ac of each group: The enrichment of ATG16L1,IRGM,IL-18’s gene promoter peaks in H3K27me3: there was almost no enrichment of model group;there was a small amount of enrichment in acupuncture group and herb-partitioned moxibustion group;the enrichment was more in normal group and herb-partitioned moxibustion + acupuncture group.The enrichment of ATG16L1,IRGM and IL-18’s gene promoter peaks in H3K4me2: the enrichment was more in model group;there were a small amount of enrichment in acupuncture group and herb-partitioned moxibustion group;there was less enrichment in normal group and herb-partitioned moxibustion + acupuncture group.The enrichment of ATG16L1,IRGM and IL-18’s gene promoter peaks in H3K27ac: the enrichment was more in model group;there were a small amount of enrichment in acupuncture group and herb-partitioned moxibustion group;there was less enrichment in normal group and herb-partitioned moxibustion + acupuncture group.Conclusions1.Acupuncture and herb-partitioned moxibustion can adjust the expression of ATG16L1,IRGM and IL-18.2.Acupuncture and herb-partitioned moxibustion can regulate the abnormal expression of chromatin remodeling related enzymes Brg1,LSD1,HDAC3,HDAC9 and h MOF.3.Acupuncture and herb-partitioned moxibustion can affect the enrichment of H3K27me3,H3K4me2 and H3K27 ac in the autophagy-related factors Atg16L1,IRGM and IL-18 promoter regions of CD to regulate the transcription and expression of Atg16L1,IRGM and IL-18.4.Acupuncture and herb-partitioned moxibustion may regulate autophagy by adjusting the abnormal expression of chromatin remodeling LSD1,HDAC3,HDAC9 and h MOF,which in turn affect enrichment of ATG16L1,IRGM and IL-18’s gene promoter peaks in H3K27me3,H3K4me2 and H3K27 ac to regulate the transcription and expression of ATG16L1,IRGM and IL-18. |
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