| Angiogenesis and vascular dysfunction play important roles in the occurrence and development of Crohn’s disease(CD),but relevant mechanistic research is lacking.This study aims to clarify the mechanism of vascular abnormalities in CD by using exosomal technology and epigenetics,and to explore the application of exosome micro RNA(mi RNA)in CD,so as to provide theoretical basis and basis for the subsequent treatment of CD by exosomal loading targeted drugs.Chapter1.Screening and etiology of plasma exosomes inCrohn’s diseaseBackground: The incidence of CD is increasing year by year,which seriously affects the quality of life of patients in the long term and causes a huge economic burden to patients and public health.The pathogenesis of CD has not been fully understood.Vascular dysfunction and angiogenesis are important pathogenesis of CD.Exosomal mediated transfer of circulating mi RNAs is believed to better promote cell-to-cell communication,but there are few studies on exosomal mi RNAs in CD,and the mechanism of mi RNAs’ involvement in CD is unclear.Objective: To screen specific or differential mi RNAs in plasma exosomes of patients with CD,and to investigate the effect of plasma exosomes derived from CD on vascular endothelial cells.Methods: Blood samples from CD patients(n=5)and healthy controls(n=5)were collected and plasma was isolated.Circulating exosomes were extracted from plasma by ultra-high speed centrifugation method.The quality control and identification of exosomes were carried out by electron microscopy,particle diameter detection technology and western blot detection.High-throughput sequencing was used to compare the mi RNA profiles of plasma derived exosomes from healthy individuals and patients with CD,and multiple genetic software and databases were used to predict the targets and signaling pathways of specific or highly differentially expressed mi RNAs.The exosomes in the CD group and the healthy control group were incubated with human umbilical vein endothelial cells(HUVECs),and cell growth(CCK-8 cell viability and Edu proliferation assay),apoptosis(flow cytometry),and cell migration(scratch healing and Transwell migration assay)were detected.And malondialdehyde(MDA)content,antioxidant enzyme glutathione peroxidase(GSH-PX),antioxidant enzyme superoxide dismutase(SOD),inducible nitric oxide synthase(i NOS)activity and other oxidative stress indicators were detected.Results: Compared to the control group,There were 18 Up-regulated mi RNAs in plasma exosome of CD group,which were mi R-21-5p,mi R-22-3p,mi R-548d-5p,mi R-190a-5p,mi R-3613-5p,mi R-144-3p,mi R-144-5p,mi R-101-3p,mi R-15a-5p,mi R-194-5p,mi R-25-3p,mi R-182-5p,mi R-486-5p,mi R-106b-5p,mi R-215-5p,mi R-20b-5p,mi R-192-5p,mi R-451 a and 6 Down-regulated mi RNAs were mi R-24-3p,mi R-744-5p,mi R-505-5p,mi R-768-5p,mi R-204-5p,mi R-483-3p.The main differential functions were cell growth,cell adhesion and antioxidant function.In the cell experiment,we used plasma exosomes of the CD group and the healthy group to intervene in HUVECs respectively,and found that the exosomes could enter cells autonomously within half an hour,among which the healthy group cells were in good condition.Compared with the healthy group,the growth and cell adhesion migration function of HUVECs cells treated by plasma exosomes in the CD group were significantly different,MDA content of lipid oxidation product was increased,GSH-Px activity was significantly decreased.The results of apoptosis flow analysis showed that there was no apoptosis of HUVECs in CD group after exosome intervention.Conclusions: There were 18 up-regulated mi RNAs and 6 downregulated mi RNAs in plasma exosomes in CD group.These differential mi RNAs are associated with cell growth,cell adhesion,and antioxidant function.Chapter2.Effect of plasma exosomal mi R-21 on vascularendothelium and its role in the pathological process of Crohn’sdiseaseBackground: Micro RNA-21(mi R-21)is one of the differential mi RNAs screened by sequencing of plasma derived exosome mi RNAs in the CD group and the healthy group.Exosome mi R-21 targets the phosphatase and tensin homolog(PTEN)gene on chromosome 10 to activate the PI3K/ AKT signaling pathway in malignant tumors.Exosome mi R-21 can regulate the proliferation of a variety of cells through PTEN.mi R-21 plays an important regulatory role in angiogenesis of a variety of diseases,but the role of exosome mi R-21 in CD has not been reported.Objective: To elucidate the effect and mechanism of mi R-21 on vascular endothelium,and to investigate the effect of circulating mi R-21 and mi R-21 antagonist on CD colitis mice and colonic vascular hyperplasia in mice.Methods:(1)Clinical validation: Quantitative reverse transcription polymerase chain reaction(q PCR)was used to verify the level of mi R-21 in plasma exosome of patients with CD(n=30 in active CD group,n=30 in healthy group);(2)Cell experiments: Plasma exosomes of patients with active CD and healthy subjects,mi R-21 mimic(in vitro mi R-21 mimic)and mimic control were added into HUVECs medium.The migration and tubulogenesis of plasma exosomes and mi R-21 to HUVECs in CD patients were evaluated by scratch healing assay,Transwell invasion assay and tubulogenesis assay,and the expression level of intracellular mechanism pathway proteins was determined by western blotting.Then mi R-21 inhibitor(in vitro mi R-21inhibitor)and inhibitor control were added into HUVECs medium of plasma exosomes pre-incubated in patients with active CD.The antagonistic effect of mi R-21 inhibitor on promoting HUVECs migration and tubulogenesis of plasma exosomes in CD patients was evaluated and the protein expression level of intracellular mechanism pathway was verified.(3)Animal experiments: Forty-eight C57 wild-type mice were randomly divided into 6 groups(n=8 for each group),control group 1,2,4,6-trinitrobenzenesulfonic acid(TNBS)group 5.Among them,4 TNBS mice were injected with agomir-21(in vivo mi R-21 mimics),Agomir control,Antagomir-21(in vivo mi R-21 antagonist)and Antagomir control via tail vein,respectively.Body weight and disease activity index(DAI)scores were recorded daily to determine the status of mice.The colon was taken for macroscopic(naked eye)and microscopic(histological HE staining)colon lesions.Serum interleukin-1 β(IL-1β)and tumor necrosis factor α(TNF-α)were detected by enzyme-linked immunosorbent assay(ELISA)to evaluate systemic inflammation.Immunohistochemical CD31 staining showed the distribution and expression of colonic blood vessels in mice.The expression of PTEN/ PI3K/Serine-threonine kinase(AKT)/vascular endothelial growth factor(VEGF)axin was detected by WB.Results:(1)Clinical verification: The level of mi R-21 in plasma exosomes of patients with CD was significantly increased.(2)Cell experiments: Plasma exosomes and mi R-21 mimic in CD patients significantly promoted the migration and tubulogenesis of human umbilical vein endothelial cells(HUVECs);mi R-21 inhibitor blocked the migration promoting and tubulogenic effects of CD exosomes on HUVECs.WB analysis of cell proteins showed that plasma exosomes and mi R-21 mimic in HUVECs of CD patients could inhibit PTEN,activate PI3K/AKT axis,and induce the expression of hypoxia-inducible factor 1α(HIF-1α)and VEGF.mi R-21 inhibitor antagonizes the inhibition of plasma exosomes on the PTEN/PI3K/AKT axis in PATIENTS with CD.(3)Animal experiment: TNBS enema mice showed colitis symptoms,DAI increased,serum inflammatory molecules TNF-α,IL-1β increased;Colitis in agomir-21 + TNBS mice was significantly aggravated,and the colonic lesion score in naked eye and histological pathology was increased,serum TNF-α and IL-1β were significantly increased,and CD31 fluorescence signal was increased.Colitis in antagomir-21 + TNBS mice was remission,the colonic lesion score in naked eye and histology was decreased,serum TNF-α and IL-1β were decreased,and CD31 fluorescence signal was decreased.WB results showed that Agomir-21 significantly inhibited TNBS-induced PTEN expression and activated PI3K/AKT/VEGF pathway,while Antagomir-21 antagonized this effect.Conclusion:(1)The level of plasma exosome mi R-21 increased in patients with CD;(2)Plasma exosome mi R-21 in PATIENTS with CD inhibits the expression of PTEN,activates PI3K/AKT signaling pathway,and promotes the migration and tubulogenesis of vascular endothelial cells;(3)TNBS induced increased colonic angiogenesis in CD colitis mice;(4)Caudal vein injection of mi R-21 can aggravate the symptoms of colitis and increase of colonic angiogenesis in TNBS-induced CD mice;caudal vein injection of mi R-21 antagonist can relieve the symptoms of colitis in TNBS mice and inhibit the proliferation of colonic vessels.Chapter3.Effect and mechanism of plasma exosomal mi R-106b-5p on vascular endothelium cellsBackground: Mi R-106b-5p is one of the differential mi RNAs screened by sequencing of plasma derived exosome mi RNAs in the CD group and the healthy group.mir-106b-5p also plays an important role in many vascular diseases.Mi RNAs can be loaded into exosomes,whose lipid envelope ensures that mi RNAs are not hydrolyzed by RNase in the extracellular environment and contributes to their stability.In addition,exosomes are also promising as drug delivery vectors due to their low immunogenicity and intrinsic interaction with target cells,which further highlights their potential in clinical therapy.Objective: To explore the method of exosome loading mi RNA,and clarify the effect and mechanism of exosome mi R-106b-5p on vascular endothelial.Methods:(1)Clinical validation: Quantitative reverse transcription polymerase chain reaction(q PCR)was used to detect the level of mi R-106b-5p in plasma exosome of patients with CD and healthy controls(n=30 in the CD group and n=30 in the healthy group).(2)Cell experiments: Mi R-106b-5p mimic,mimic control,mi R-106b-5p inhibitor and inhibitor control were transferred into healthy group exosomes by electric transfection,and incubated with HUVECs to simulate the intervention of exosomes loaded by foreign source.Si RNA was transfected into HUVECs by liposome transfection to construct STK11 silent cell model.STK11 overexpressed escherichia coli plasmid was transfected into HUVECs by plasmid transfection to construct STK11 overexpressed cell model.The double luciferase reporter gene verified the targeted inhibition of STK11,and the intracellular ATP content was detected to determine the energy metabolism level.Cell viability and growth cycle were evaluated by CCK-8 cell viability assay,Edu proliferation assay and cell cycle flow cytometry.The levels of intracellular mechanism pathway proteins were detected by western blot.Fluorescence in situ hybridization was used to evaluate the binding of mi R-106b-5p to target m RNA in colon tissue.The expression of CD31 and STK11 in colon tissue was evaluated by immunohistochemistry and immunodual fluorescence.Results:(1)Clinical verification: Mi R-106b-5p is highly enriched in plasma exosomes of patients with CD.(2)Cell experiments: CCK-8 cell viability and Edu proliferation test results showed that exosome mi R-106b-5p inhibited the proliferation of HUVECs.Flow cytometry further suggested that exosome mi R-106b-5p delayed the G1/G2 phase of HUVEC growth cycle.WB analysis showed that the expression of G1 cyclin D1 was decreased.Exogenous addition of exosome inhibitor CPZ and exosome mi R-106b-5p can counteract or alleviate the above symptoms.The double luciferin reporter gene assay suggested that mi R-106b-5p could bind to the 3’UTR end of STK11 m RNA,and the post-transcriptional inhibition of mi R-106b-5p on STK11 was verified by q PCR and WB analysis.In addition,the results of HUVECs after STK11 silencing were similar to those of HUVECs after exosomal mi R-106b-5p intervention in CCK-8,Edu and flow cytometry,while the results of HUVECs treated with STK11 overexpression were opposite.ATP assay confirmed that mi R-106b-5p and silencing of STK11 decreased ATP content in HUVECs cells.Overexpression of STK11 increased intracellular ATP content in HUVECs.In the mechanism studies,both exosomal mi R-106b-5p and STK11 silencing could inhibit the activation of Wnt/GSK3β/β-catenin pathway,inhibit the deposition of β-catenin,and down-regulate the expression of cyclin cyclin D1 downstream of the pathway.Conclusion:(1)Increased levels of circulating exosome mi R-106b-5p in patients with CD;(2)mi R-106b-5p can target STK11 m RNA to inhibit its posttranscriptional translation process,and down-regulate ATP level in endothelial cells by inhibiting STK11.(3)mi R-106b-5p mimic and mi R-106b-5p inhibitor can be loaded into exosomes and enter cells with exosomes;(4)Electrotransfected exosomes loaded with mi R-106b-5p can downregulate the expression of cyclin cyclin D1 and delay the growth cycle of endothelial cells by inhibiting STK11/Wnt/GSK3β/β-catenin axis. |
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