Study On The Mechanism Of Exosomal MiR-103a-3p Isolated From Creeping Fat Derived-ASCs Regulating Intestinal Fibrosis In Crohn’s Disease

Background & Aims:Creeping fat(CF),also known as fat wrapping,is essentially hypertrophy of mesenteric adipose tissue,which is a hallmark of Crohn’s disease(CD).CF plays a dual role in the course of CD due to its pro-inflammatory and pro-fibrotic effects and its limiting effect on intestinal transmural inflammation.However,there are few relevant studies at present,and the effect and mechanism of CF on the intestine are not completely clear.It is reported that adipose-derived stem cells(ASCs)from patients with CD show a distinctive DNA methylation pattern compared with ASCs from healthy donors,and CF-ASCs presented a more proliferative,inflammatory,invasive,and phagocytic phenotype than equivalent cells from healthy donors,irrespective of the clinical stage.Considering the close relationship between CF and CD intestinal fibrosis,this study aims to explore the impact of CF-ASCs on intestinal fibrosis.Methods:First of all,CF of the diseased bowel(CF)and mesenteric adipose tissue of the surgical margin,which were macroscopically normal(Ctrl),were obtained during the surgery from patients with CD,to isolate the ASCs(CF-ASCs and Ctrl-ASCs).Macroscopically normal ileum serosa of the specimen margin was collected during surgery from patients with right colon cancer to isolate the primary intestinal fibroblasts(PIFs).The supernatant of ASCs culture solution was collected and then were divided into 3 components: complete supernatant(CM),supernatant without exosomes(CMNone Exos)and exosomes(Exos).The three components from the above two sources were co-cultured with PIFs respectively,and CCK8,Ed U,wound-healing assay,and western blot were used to detect the activation of PIFs(proliferation,migration and transformation to myofibroblasts).Transmission electron microscope,nanoparticle tracking analysis,and western blot were used to detect the isolated exosomes.Chronic colitis models were induced by dextran sulfate sodium(DSS),tail-vein injection was used to verify the effect of two exosomes on intestinal fibrosis in mice with chronic enteritis,and histopathological staining,q RT-PCR,and tissue immunofluorescence were used to evaluate the intestines.Then we detected the differential expression of mi RNAs in the two kinds of exosomes by conducting exosomal mi RNA sequencing,and determined the expression of mi R-103a-3p.q RT-PCR,western blotting,and luciferase reporter assay were used to confirm that mi R-103a-3p can directly target TGFBR3.We purchased inhibitor of mi R-103a-3p and lentiviral vectors(sh TGFBR3),and verified the regulatory relationship and mechanism of mi R-103a-3p and TGFBR3 on fibroblasts through rescue experiments.Finally,Intestinal samples from the diseased segments(D,wrapped by the CF)or at the resection margin(R),and the corresponding mesenteric samples(CF and RMAT)were collected from patients with CD.Intestinal samples at the resection margin(C)and the corresponding mesenteric samples(CMAT)were collected from patients with colon cancer.The degree of CF and intestinal fibrosis were evaluated by histopathological staining,while the expression of mi R-103a-3p in tissues was detected and its correlation with CF wrapping and fibrosis was explored.Results:Part Ⅰ: CF-Exos can significantly promote the proliferation,migration and transformation of fibroblasts to myofibroblasts compared with Ctrl-Exos in intro.Further analysis showed that the secretion of exosomes was enhanced in CF-ASCs compared with Ctrl-ASCs,while the characteristics of the two kinds of exosomes have not changed significantly and both of them can be internalized by fibroblasts.The results showed that the tail-vein injected exosomes can reach the intestine and be internalized by fibroblasts in the colon.In experiments in vivo,the results indicated that CF-Exos promoted intestinal fibrosis by activating fibroblasts in a dose-dependent manner.They continuously promoted progression of intestinal fibrosis even after DSS withdrawal.Part Ⅱ: Exosomal mi R-103a-3p was enriched in CF-Exos and mi R-103a-3p can be transferred from CF-ASCs to fibroblasts in the form of exosome.Mechanistically,mi R-103a-3p can directly target the 3’-UTR of TGFBR3 and mediate the its expression.CFASCs released exosomal mi R-103a-3p and promoted fibroblast activation by targeting TGFBR3 and promoting Smad2/3 phosphorylation.Part Ⅲ: The degree of CF significantly increased adjacent to the diseased intestine and was positively correlated with the degree of intestinal fibrosis.The expression of mi R-103a-3p was upregulated in CF and diseased bowel,and its expression level was correlated with the degree of CF encapsulation and intestinal fibrosis.Conclusion:Our findings showed that the expression level of mi R-103a-3p was positively correlated with the degree of CF and intestinal fibrosis.Exosomal mi R-103a-3p from CF-ASCs promoted intestinal fibrosis by targeting TGFBR3 and activating fibroblasts.Downregulation of mi R-103a-3p can significantly inhibit the intestinal fibrosis promoting effect of CF-Exos.All these results suggested that CF-ASCs would be a potential therapeutic target for CD intestinal fibrosis.