Mechanism Of HucMSC-Ex Regulating NLRP3 Inflammasome In Macrophage To Repair Inflammatory Bowel Disease

Objective:Human umbilical cord mesenchymal stem cell-derived exosomes（hucMSC-Ex）repairing dextran sulfate sodium salt（DSS）induced mouse inflammatory bowel disease model was established in this study.The role of macrophage NLRP3inflammasome in the allevation of mice IBD by hucMSC-Ex administration was discussed.The mechanism of hucMSC-Ex regulating macrophage pyroptosis was further studied,which provides an experimental basis on clinical application of hucMSC-Ex relieving IBD.Methods:1)Extraction and identification of hucMSC-Ex:The supernatants of human umbilical cord mesenchymal stem cells（hucMSC）cultured in ultracentrifugal fetal calf serum were collected.HucMSC-Ex was isolated by ultracentrifugation and identified by electron microscopy,Nano Sight nanoparticle tracking analyzer and the detection of surface protein markers by western blot.2)IBD model was established by feeding water containing 3%DSS.BALB/c male mice were assigned to three groups:NEG group,IBD group and hucMSC-Ex group.Mice in IBD group and hucMSC-Ex group were given 3%DSS water,while 1mg hucMSC-Ex was injected intravenously to mice in hucMSC-Ex group.The body weight of mice was recorded and the disease activity index（DAI）was analyzed everyday.All mice were sacrificed on the 11^thday and histological score of colons was performed.Colonic mucosal protein and total RNA were extracted.H&E staining was used to analyze the microstructure of mice colon.QRT-PCR was used to detect the expression of inflammatory factors and the expression of NLRP3 inflammasome related molecules in colon tissues.The NLRP3 inflammasome protein in colonic tissues was detected by western blot and immunohistochemistry（IHC）.3)THP-1 cells and murine peritoneal macrophages were used for confirmatory experiments in vitro.Cells were divided into three groups:Ctrl group,LPS+NIG group and hucMSC-Ex group.Cells in LPS+NIG group and hucMSC-Ex group were stimulated with LPS and Nigericin to induce NLRP3 inflammasome,while 200μg/m L hucMSC-Ex was added to cells in hucMSC-Ex group to alleviate inflammation.The release of IL-1βin supernatant was determined by ELISA.The expression of NLRP3 inflammasome related molecules in cells were analyzed by q RT-PCR and western blot.The formation of ASC speck-like protein in cells was detected by immunofluorescence（IF）.4)To clarify whether hucMSC-Ex regulated macrophages pyroptosis,the expression of Caspase-1 p45 and Caspase-1 p20 in the colonic tissues of mice were detected by IHC,while the cleavage of GSDMD was detected by western blot.In vitro,CCK8 assay was used to detect cell viability.LDH activity in cell supernatant was analyzed by lactate dehydrogenase（LDH）kit.The cleavage of protein GSDMD was determined by western blot.The proportion of PI positive cells in THP-1 cells was analyzed by flow cytometry.Morphological changes of murine peritoneal macrophages were observed by microscope.5)In order to explore which key molecule in hucMSC-Ex regulated NLRP3inflammasome in macrophages,we set NLRP3 as the target molecule.The miRNAs predicted by the database that can target the 3’UTR of NLRP3 m RNA was cross-aligned with the hucMSC-Ex miRNA sequencing results to screen the coincident miRNAs.QRT-PCR detected the expression of the screened miRNAs and miR-378a-5p was identified as the key molecule.miR-378a-5p mimics,mimics NC,inhibitor and inhibitor NC were transfected into THP-1 cells.Protein and miRNA were extracted from transfected cells.QRT-PCR detected the expression of miR-378a-5p and western blot analyzed the expression of NLRP3 in cells.Results:1)HucMSC-Ex was successfully extracted.The analysis of transmission electron microscope and Nano Sight nanoparticle tracking analyzer showed that the diameter of hucMSC-Ex was 110 nm.Western blot analysis of the protein markers on the surface of exosomes showed positive expressions of CD9,CD63,CD81,HSP70and Alix in hucMSC-Ex,while negative expression of Calnexin.2)Compared with mice in NEG group,mice in IBD group decreased weight on the 8^thday and the DAI score also started to rise on the 8^thday,while the loss of mice body weight in hucMSC-Ex group was relieved and the DAI score was lower than that in IBD group.The colon of mice in IBD group was obviously shortened with disorganized intestinal gland structure,while the colon of mice in hucMSC-Ex group recovered and partial normal intestinal gland tissues were visible.The expression of NLRP3 inflammasome related molecules increased in the colonic tissues of mice in IBD group,but obviously decreased in the colonic tissues of mice in hucMSC-Ex group.3)Immunofluoresence staining of clinical colon tissue specimens showed that compared with healthy controls,the number of NLRP3 and ASC double positive cells in the colon of patients with inflammatory bowel disease was remarkably increased with the hyperactivation of NLRP3 inflammasome.4)NLRP3 inflammasome was induced by LPS and Nigericin in THP-1 cells and murine peritoneal macrophages in vitro.Compared with cells in the positive group,the expression of NLRP3 inflammasome related molecules and the extracellular release of pro-inflammatory factor IL-1βin hucMSC-Ex group cells were decreased.Meanwhile,hucMSC-Ex reduced the formation of ASC speck-like protein in THP-1 cells.5)The expression of Caspase-1 p45,Caspase-1 p20 and the cleavage of GSDMD in colonic tissues of mice in IBD group were remarkably increased,which were decreased in the colon of mice with hucMSC-Ex administration.HucMSC-Ex postponed cell pyroptosis in the colon of mice.In vitro,hucMSC-Ex increased cell vitality of macrophages under inflammatory enviroment,reduced LDH activity and the cleavage of GSDMD.6)NLRP3 inflammasome was induced after transfection with miR-378a-5p mimics,mimics NC,inhibitor,inhibitor NC.Compared with positive group,NLRP3protein in mimics group was significantly decreased,while NLRP3 protein expression in inhibitor group was not obviously changed.Conclusions:HucMSC-Ex protests against DSS-induced mice IBD via regulating macrophage pyroptosis by miR-378a-5p/NLRP3 axis.