The Research On The Effect Of MiR-223 In Human Mast Cells-derived Exosomes On Gut Barrier Function

Background Inflammatory bowel disease(IBD)is a chronic nonspecific inflammatory disease of the intestinal tract with unknown etiology.The incidence of IBD has gradually increased in recent years.The patient's quality of life is seriously affected by IBD,and IBD increases the financial burden of the family and society.In order to find new drug targets and treatment methods,the research on the pathogenesis of IBD still needs to be further deepened.In recent years,people have paid more and more attention to the human mast cells(HMCs)in the occurrence and development of IBD,which is mainly to promote the inflammatory response and destroy the barrier function.The specific pathogenic mechanism of HMCs in IBD has not been fully elucidated.As an important carrier for intercellular material transmission and signal transduction,exosomes can be secreted by many types of cells.Exosomes derived from different cells have different biological functions.After the exosomes taken up by the recipient cells,the biologically active molecules contained in the exosomes can regulate the expression of genes or proteins in the recipient cells,such as mi RNA(Micro RNA),lnc RNA(Long non-coding RNA).Many studies showed that exosomes play an important role in immune regulation,they are closely related to the pathogenesis of many autoimmune diseases,such as systemic lupus erythematosus and rheumatoid arthritis.Multiple studies indicated that the expression of mi RNA derived from exosomes in serum of IBD patients is abnormal.The intestinal mucosal barrier is an important line of defense for the body against foreign harmful substances,which can prevent the occurrence of IBD and restore the intestinal function of IBD patients.The maturation of dendritic cells into antigenpresenting cells can be promoted by HMCs-derived exosomes,which assist the inflammatory response,too.However,there are few reports about the effect of HMCs on intestinal barrier function,and it is not clear whether HMCs can participate in the regulation of intestinal barrier function through exosomes and their internal mi RNAs.Objective This experiment intends to investigate whether exosomes derived from HMCs act on intestinal epithelial cells.The effect of mi RNA carried by HMCs-derived exosomes on the barrier function of intestinal epithelial cells was further explored.Methods(1)The exosomes were separated by differential ultracentrifugation,and the morphology and size of the exosomes were identified by transmission electron microscopy(TEM).Western blotting was used to detect whether the isolated exosomes contained labeled proteins CD63 and TSG101 and the negative control proteins GM130,Histone and Calnexin were detected.(2)The extracted exosomes labeled by fluorescent dye PKH67,and co-culture with Ca CO2,NCM460 and HT29 cells for 24 h.Then staining the cell nuclei with DAPI.The PKH67 which used to mark the fluorescent location of exosomes in three intestinal epithelial cells was observed under a fluorescent microscope.(3)Two intestinal epithelial cell barrier models,Ca CO2 and NCM460,were established to evaluate the effect of exosomes on the permeability of intestinal epithelial cells.The OD value of the liquid at the base of the Transwell cell base with or without exosomes was measuring by Multiscan Spectrum.(4)Immunofluorescence experiments were performed after co-cultivation of exosomes with Ca CO2 cells for 72 hours and used to observed the expression of tight junction proteins CLDN8,OCLN and ZO-1.After co-culturing of exosomes with Ca CO2 and NCM460 cells for 72 hours,the expression of tight junction proteins Claudin8,Occludin and ZO-1 were detected by Western blotting method.(5)In order to observe the mi RNA expression in the obtained exosomes,the HMCsderived exosomes was sent to professional biological company for mi RNA chip detection.(6)After co-cultivation of exosomes with HT29 cells for 72 h,RNA was extracted by Trizol method,and the expression level of mi RNA in HT29 cells co-cultured with exosomes was detected by fluorescence quantitative PCR method.(7)HMCs was infected with the lentivirus Lv-mi RNA-223 which containing mi R-223 sequence and lentivirus Lv-mi RNA-NC.The HMCs which stably overexpressing mi R-223 was constructed and selected.The exosomes from HMCs which overexpressing mi R-223 was extracted and co-cultured with HT29 and NCM460 cells for 72 h,and counterstained with DAPI for observing the distribution of green fluorescence(mi R-223).Flow cytometry was used to detect intestinal epithelial cells containing green fluorescence(mi R-223)after co-culture with exosomes.(8)After co-cultivation of exosomes with intestinal epithelial cells for 72 hours,the mi R-223 inhibitor was transfected into intestinal epithelial cells,and detected the level of CLDN8,a tight junction protein of intestinal epithelial cells.(9)Intestinal mucosa tissues of clinical IBD patients was collected,and detected the expression level of HMCs in intestinal mucosa tissues of healthy control group and IBD group by immunophenotype CD117(c-kit)immunohistochemistry.(10)The expression level of mi R-223 in intestinal mucosa tissues of healthy control group,UC group and CD group was detected by q PCR.Results(1)As we can observed by transmission electron microscope,the isolated exosomes have the typical morphological characteristics of exosomes,mostly round or oval,shaped like a "tea saucer",and the size is about 50-250 nm.The results of Western blotting showed that the extracted exosomes contained CD63 and TSG101 without GM130,Histone and Calnexin.(2)It can be observed that the cytoplasm of Ca CO2,NCM460 and HT29 contains a lot of green fluorescence by the fluorescence microscope,,which is aggregated,indicating that exosomes derived from HMCs can be taken up by intestinal epithelial cells.(3)Compared with the PBS group,the OD value of the liquid at the base of the Transwell chamber became larger after adding exosomes.Moreover,the trends of the two intestinal epithelial cell barrier models(Ca CO2 and NCM460),are consistent,which indicates that the ingestion of HMCs exosomes by intestinal epithelial cells increases the permeability of intestinal epithelial cells.(4)The results of immunofluorescence and Western blotting showed that compared with the control group,the expression levels of Claudin8,Occludin and ZO-1 in Ca CO2 cells decreased after exosome treatment.(5)The mi RNA chip test results show that a series of mi RNAs including mi R-223,mi R-21,mi R-16,etc.are highly expressed in exosomes derived from HMCs.(6)The results of q PCR indicated that exosomes may be used as a carrier for mi RNA transfer between HMCs and HT29 cells.(7)Exosomes with green fluorescence(mi R-223)can be observed in the cytoplasm of HT29 and NCM460 cells under a fluorescence microscope,which indicate that HMCs exosomes contain mi R-223 and the exosomes can be taken up by intestinal epithelial cells.Flow cytometry analysis showed that compare to the PBS group,intestinal epithelial cells took up exosomes with mi R-223 in the exosomes-treated group.(8)After transfecting with mi R-223 inhibitor,the inhibition of CLDN8 by exosomes can be alleviated.(9)Compared with the healthy control group,the number of HMCs in the intestinal mucosa of IBD patients increased.(10)Compared with the healthy control group,the expression of mi R-223 in the intestinal mucosa tissues of the UC group and the CD group increased.Conclusion Mi R-223 in HMCs-derived exosomes disrupts the intestinal barrier function by inhibiting the expression of the tight junction protein CLDN8 in intestinal epithelial cells.