Font Size: a A A

Effects Of Herb-Partitioned Moxibustion On SIRT1/FoxO1/ATG7 Signaling Pathway And Related MiRNAs In Rat With Crohn’s Disease Associated Intestinal Fibrosis

Posted on:2022-07-06Degree:DoctorType:Dissertation
Country:ChinaCandidate:M ZhaoFull Text:PDF
GTID:1524307295488724Subject:Acupuncture and Massage
Abstract/Summary:PDF Full Text Request
Objective: To explore the mechanism of herb-partitioned moxibustion in improving CD associated intestinal fibrosis induced by SIRT1/Fox O1/ ATG7 signaling pathway,and to predict the related mi RNAs target.Methods:Part I: Male Sprague-Dawley(SD)rats were randomly divided into normal group(NG),model group(MG),herb-partitioned moxibustion group(HM)and mesalazine group(MED).Intestinal fibrosis was established in rats with CD by repeated rectal administrations of 5% 2,4,6-trinitrobenzene sulfonic acid(TNBS)and 50% ethanol.In the HM group,Tianshu(ST25)and Qihai(CV6)acupoints were selected for treatment.The MED group approach to mesalazine by gavage.In this study,histological changes were examined by hematoxylin and eosin(HE)staining;and Masson’s trichrome staining was used to evaluate the degree of intestinal fibrosis;the morphology of autophagosome and autophagosolysosom were observed by transmission electron microscopy.The degree of intestinal fibrosis(E-cadherin,α-SMA,vimentin)and the activity of autophagy(LC3II/LC3 I,P62,ATG7,LAMP-1+LC3)were measured by immunohistochemistry,western blotting and the double immunofluorescent staining.Part II: Male SD rats were randomly divided into NG,MG,HM,M+EX and HM+EX groups.After model preparation completed,the M+EX group was intraperitoneally injected with EX527 solution,and the HM+EX group was treated with EX527 and herb-partitioned moxibustion.The colon injury and fibrosis were evaluated by gross observation,HE and Masson’s trichrome staining.Immunohistochemistry and western blotting were used to detect intestinal fibrosis indicators and the expressions of SIRT1,Ac-Fox O1 /Fox O1,ATG7,LC3II/LC3 I and P62.The interaction between SIRT1 and Fox O1,Ac-Fox O1 and ATG7 was detected by co-immunoprecipitation.Part III: The high-throughput sequencing was used to perform the bioinformatics analysis of micro RNA,screen significant difference micro RNAs intersection in NG,MG and HM groups,and focus on targets prediction and functional enrichment analysis.Thus it can predict mi RNA targets about HPM improving CD associated intestinal fibrosis,verified by the dual-luciferase reporter assay and real-time PCR.ResultsPart I: 1.Effect of herb-partitioned moxibustion on the gross morphology and histopathology of colon in rats with CD intestinal fibrosis.In NG group,the colonic tissue morphology was normal,with clear structure of each layer and orderly glands.In MG,intestinal lumen was significantly narrowed and dilated,intestinal wall was obviously thickened,and a large number of new small vessels and fibroblasts were observed.Compared with MG,the HM and MED group was relieved,accompanied by the infiltration of fibroblasts and repair under the microscope.Compared with NG,CMDI and TDI scores in MG group were significantly increased(P all<0.01).Compared with the MG group,the CMDI and TDI scores of HM groups were decreased(P>0.05,P<0.05);the CMDI and TDI scores of MED groups were decreased(P<0.01,P>0.05).2.Effect of herb-partitioned moxibustion on the degree of intestinal fibrosis.Masson staining showed that the expression of collagen fiber in MG group was significantly higher than that in NG group(P<0.01).Compared with the MG group,the expression of collagen fiber in HM and MED groups was significantly decreased(P all<0.01).Compared with NG group,the protein expression of α-SMA and vimentin in MG group was notably increased,while the expression of E-cadherin was obviously decreased(P<0.05).Compared with the MG group,the protein expressions of α-SMA and vimentin in HM and MED groups were decreased,while the protein expression of E-cadherin was increased(P<0.05).3.Effect of herb-partitioned moxibustion on autophagy activity in colon tissue of rats with CD associated intestinal fibrosis.The organelles in the NG group were normal,but the organelles in MG,HM and MED groups were deformed and swollen,with typical autophagosomes or autophagolysosomes.Compared with NG group,the levels of ATG7 protein and LC3II/LC3 I in MG group were significantly increased,while the levels of P62 protein were significantly decreased(P<0.05).Compared with the MG group,the levels of ATG7 protein and LC3II/LC3 I protein were downregulated,P62 were increased in HM and MED group(P<0.05).the expression of LAMP-1 and LC3 was stronger in the MG than NG group,and weaker in the HM and MED groups than MG group,with co-localization between them.Part II:1.Effect of herb-partitioned moxibustion on intestinal fibrosis in each group.The CMDI and TDI scores in MG and M+EX group were increased than NG(P all<0.01),which were higher than MG group in M+EX group(P<0.01);Herbpartitioned moxibustion decreased CMDI and TDI scores in rats with CD intestinal fibrosis(P<0.05,P>0.05),and those of HM+EX group were significantly higher than HM group(P<0.01,P<0.05).Herb-partitioned moxibustion significantly reduced collagen fiber deposition of model rats(P<0.01),and HM+EX group was significantly higher than that in HM group(P<0.05).In this study,HM down-regulated the expression of α-SMA and vimentin,and up-regulated the expression of E-cadherin(P<0.05).Compared with HM group,the expression of α-SMA and vimentin in HM+EX group were increased(P<0.05),while the expression of E-cadherin were decreased(P<0.05).2.Effects of herb-partitioned moxibustion on SIRT1/Fox O1/ATG7 signaling pathway in each group.Compared with the NG group,the expression of SIRT1 and P62 in MG and M+EX groups were decreased,while the expressions of ATG7,AcFox O1/Fox O1 and LC3II/LC3 I were increased.Compared with the MG group,the expression of SIRT1 and P62 in the HM group were up-regulated,while ATG7,AcFox O1/Fox O1 and LC3II/LC3 I were decreased.Compared with MG group,the expressions of SIRT1 and P62 in M+EX group were down-regulated,while ATG7,AcFox O1/Fox O1 and LC3II/LC3 I were up-regulated.Compared with HM group,the protein expressions of SIRT1 and P62 in HM+EX group were down-regulated,while the expressions of ATG7,Ac-Fox O1 /Fox O1 and LC3II/LC3 I were increased.The results about co-immunoprecipitation suggested that there were interactions between SIRT1/Fox O1,and ac-Fox O1/ATG7.Part III:1.Effect of herb-partitioned moxibustion on micro RNA expression in colon tissue of each group.There were 67 significantly differentially expressed mi RNAs in colon tissue between NG and MG group,and 81 significantly differentially expressed mi RNAs between MG and HM group,and the intersection results showed that there were common significantly differentially expressed mi RNAs among three groups.2.To predict the micro RNA target in colon tissue affected by herb-partitioned moxibustion.The prediction results showed that the target genes of rno-micro RNA-34a-5p included SIRT1,CD intestinal fibrosis susceptibility gene(ATG16L1),autophagy related genes(ATG14,ATG4 b,ATG9a),lysosomal associated membrane proteins(LAMP1,LAMP2),transforming growth factors(TGFα,TGFβ2),collagen protein(COL1a1,COL1a2,COL4a1),etc.Based on lots of literatures,it was suggested that rno-micro RNA-34a-5p might be related to the SIRT1/Fox O1/ATG7 signaling pathway.The dual-luciferase reporter assay indicated that SIRT1 was the target gene of rno-micro RNA-34a-5p,and the PCR results verified that the expression trend of rnomicro RNA-34a-5p and SIRT1 were consistent with the sequencing and those results in Part II.Conclusion:1.Herb-partitioned moxibustion could regulate the levels of LC3II/LC3 I,P62 and ATG7 in colon tissues,inhibit autophagy flux,and improve the degree of intestinal fibrosis.2.Herb-partitioned moxibustion could up-regulated the expression of SIRT1 protein,promoted Fox O1 deacetylation,inhibited the binding between ac-Fox O1 and ATG7,down-regulated the autophagy activity,and relieve CD intestinal fibrosis.3.The improvement of intestinal fibrosis by Herb-partitioned moxibustion is related to micro RNA.Among them,rno-micro RNA-34a-5p may have a certain target role,which may be related to the SIRT1/Fox O1/ATG7 autophagy pathway.
Keywords/Search Tags:Crohn’s disease, intestinal fibrosis, autophagy, SIRT1, small RNA sequencing, micro RNA, Herb-partitioned moxibustion
PDF Full Text Request
Related items