Mechanism Of HucMSC-Ex Repairing IBD By Inhibiting Ferroptosis In Intestinal Epithelial Cells

Objective:To explore human umbilical cord mesenchymal stem cell-derived exosomes,hucMSC-Ex attenuates inflammatory bowel disease(IBD)induced by dextran sulfate sodium salt(DSS)in mice.Further investigation of the mechanisms of action of hucMSC-Ex in inhibiting lipid peroxidation in intestinal epithelial cells to reduce ferrocytosis and repair inflammatory bowel disease will help to provide a corresponding theoretical basis for the clinical application of hucMSC-Ex in the treatment of IBD.Methods:1)Isolation and identification of hucMSC-Ex: human umbilical cord mesenchymal stem cell(hucMSC)was obtained from umbilical cord tissue,digested and subcultured in the Petri dish,and cultured for 24-48 h in a complete medium prepared from fetal bovine serum with exosomes removed by ultracentrifugation,and the culture supernatant was collected for use in the next passage.HucMSC-Ex was extracted from culture supernatant by ultracentrifugation.The surface markers were identified by western blot,and the structure and particle size of hucMSC-Ex were analyzed by transmission electron microscope(TEM)and nanoparticle analysis(NTA).2)BALB/c mice were randomly divided into three groups: NC group,DSS group and hucMSC-Ex group.The mice in DSS group and hucMSC-Ex group were equipped with3% DSS,and the mice in DSS group and hucMSC-Ex group were freely used to establish the mouse IBD model.During this period,mice in the hucMSC-Ex group were injected with 1mg hucMSC-Ex every three days through the tail vein,and mice in the other two groups were injected with PBS solution at the same time.Every morning,the body weight and survival status of the mice were recorded,and the disease activity index(DAI)was calculated.On the 11 th day,the mice were sacrificed,and the colon and spleen of the mice were removed and measured.Scrape the colon mucosa of mice,extract total protein and RNA,weigh the tissue with the same mass and make it into tissue homogenate,keep part of the tissue embedded in paraffin,and slice it for later use.Hematoxylin and eosin staining(H&E)was used to analyze the integrity of colon and spleen nodules of mice.Real-time fluorescence quantification PCR(qRT-PCR)was used to detect the expression levels of inflammatory factors and ferroptosis related molecules ACSL4,DMT1,COX2,GPX4 in colon tissue.Western blot and immunohistochemistry(IHC)were used to detect the expression level and location of ferroptosis related proteins in colon tissues.Iron staining was performed on intestinal sections to detect the deposition of intestinal iron particles.The level of Total glutathione(T-GSH)in intestinal mucosa was measured to analyze the antioxidant level.3)Human normal colonic epithelial cell line HCoEpiC was selected to explore the effect of hucMSC-Ex on inflammatory bowel disease in mice in vitro.We used lipopolysaccharide(LPS)to induce inflammation of intestinal epithelial cells and established an IBD model in vitro.HCoEpiC was divided into three groups: NC group,LPS induction group and hucMSC-Ex group,LPS group and hucMSC-Ex group were treated with 2mg/m L LPS.In the hucMSC-Ex group,an additional 400μg/m L of hucMSC-Ex was added.QRT-PCR was used to detect the expression levels of inflammatory factors in HCoEpiC,and CCK-8was used to detect the survival rate of HCoEpiC.4)To verify whether hucMSC-Ex has a regulatory effect on ferroptosis in intestinal epithelial cells in vitro,ferroptosis related indicators ACSL4,DMT1,COX2 and GPX4 were detected in the cell model.Western blot and qRT-PCR were used to detect ferroptosis related indicators from the protein and mRNA expression levels,respectively.The level of T-GSH in cell homogenate was detected to analyze the effect of hucMSC-Ex on the antioxidant capacity of cells.5)In order to explore the specific mechanism of hucMSC-Ex regulating ferroptosis in intestinal epithelial cells,we obtained highly expressed miRNAs in hucMSC-Ex by small RNA sequencing,and predicted miRNAs targeting ACSL4 3 ’-UTR through the database,screened out miR-129-5p,and constructed mimics.mimics-NC,inhibitor and Inhibit-NC were transfected into HCoEpiC in vitro to verify their effects on ferroptosis of intestinal epithelial cells.Western blot and qRT-PCR were used to detect ferroptosis related indicators of HCoEpiC after transfection with miR-129-5p from the protein and mRNA expression levels,respectively.IF was used to detect the effect of miR-129-5p on ACSL4 and GPX4.The level of T-GSH in cell homogenate was detected,and the effect of miR-129-5p on the antioxidant capacity of cells was analyzed.6)To verify the effect of miR-129-5p on IBD in mice in vivo,miR-129-5p mimics,mimicsNC,inhibitor and inhibiter-NC were transfected into hucMSC-Ex.BALB/c mice were randomly divided into six groups: NC group,DSS group,mimics group,mimics-NC group,inhibitor group,inhibitor-NC group.NC group freely drinking sterilized tap water;DSS group,mimics group,mimics-NC group and inhibitor group were given 3% DSS.HucMSC-Ex transfected with miR-129-5p mimics,mimics-NC,inhibitor and inhibitor-NC was injected through tail vein.The body weight and survival status of mice were recorded every morning.The disease activity index(DAI)was calculated and the mice were sacrificed on day 10.The colon and spleen of the mice were removed and measured.The colonic mucosa of mice was scraped,total protein and RNA were extracted,and the same mass of tissue was weighed to make tissue homogenate,and part of the tissue was retained for paraffin embedding and sectioned for later use.The integrity of colon and spleen nodules was analyzed by hematoxylin and eosin staining.Western blot and qRT-PCR were used to detect the mRNA and protein expression levels of inflammatory factors and ferroptosis related molecules ACSL4,DMT1,COX2,GPX4 in colon tissues and the expression levels of inflammatory factors in colon tissues of mice.The expression level of ACSL4 in colon tissues was detected by IHC.Iron staining was performed on intestinal sections to detect the deposition of intestinal iron particles.The level of Total glutathione(T-GSH)in intestinal mucosa was detected to analyze the antioxidant level of hucMSC-Ex after miR-129-5p transfection.Results:1)We successfully isolated hucMSC from human umbilical cord for culture,and collected hucMSC-Ex by ultracentrifugation.The particle number and particle size of the hucMSCEx obtained by nanoparticle tracking analyzer met the requirements.Western Blot identified that the extracted hucMSC-Ex expressed exosome positive markers CD9,CD81,Alix,and did not express exosome negative marker Calnexin.2)The weight of DSS mice continued to decrease,and the weight loss was alleviated after injection of hucMSC-Ex.Mice in DSS group had severe diarrhea with severe bloody stool,and DAI score decreased after hucMSC-Ex injection.The spleen of DSS mice was enlarged,whereas the spleen of hucMSC-Ex injected mice was slightly larger than that of the normal group and smaller than that of the DSS group.The colon length was shortened in DSS mice and recovered in hucMSC-Ex mice.The intestinal gland structure of DSS mice was disordered and the splenic nodule was incomplete,while the intestinal gland structure of mice in the hucMSC-Ex group was restored and the splenic nodule was clear.The expression of inflammatory factors was significantly increased in the intestine of IBD mice,and hucMSC-Ex injection significantly reduced the level of inflammation.The expression of intestinal ferroptosis related molecules was up-regulated in IBD mice,and hucMSC-Ex injection reduced intestinal epithelial ferroptosis.3)HCoEpiC induced inflammation by LPS,the levels of pro-inflammatory factors increased,and the antioxidant capacity and survival rate decreased.After hucMSC-Ex treatment,the inflammation level was alleviated,and the antioxidant capacity and survival rate were improved.After LPS-induced inflammation of HCoEpiC,the levels of ferroptosis related molecules increased.hucMSC-Ex treatment reduced HCoEpiC ferroptosis.4)Database analysis showed that miR-129-5p could target ACSL4.We verified that miR-129-5p was highly expressed in hucMSC-Ex.Dual luciferase reporter assay verified that miR-129-5p had a binding site with the 3 ’-UTR of ACSL4.HucMSC-Ex may reduce lipid peroxidation in intestinal epithelial cells and alleviate IBD by inhibiting ACSL4.5)miR-129-5p mimics,mimics-NC,inhibitor,inhibitor-NC were transferred into HCoEpiC.Compared with LPS group,the transfection of mimics significantly reduced the protein expression level of ACSL4 and improved the antioxidant capacity.However,there were no significant changes in other ferroptosis related molecules,indicating that miR-129-5p could target ACSL4 to inhibit HCoEpiC ferroptosis.6)MiR-129-5p mimics,mimics-NC,inhibitor,inhibitors-NC were transferred into hucMSCEx to verify the effect of miR-129-5p in vivo.Compared with the DSS group,the weight of mice in the mimics group decreased slightly,the DAI score decreased,the colon length recovered,the glandular structure recovered clearly,the spleen nodule was intact,and the expression level of inflammatory factors recovered.7)The expression of ferroptosis related molecules was increased,the apoptosis rate was increased,the antioxidant capacity was decreased,iron particles were deposited,and the expression of ACSL4 was increased in the DSS group.In the mimics group,the expression level of ACSL4 in intestinal tissue of mice decreased,the apoptotic cells decreased,the antioxidant capacity increased,and the deposition of iron particles decreased.Conclusion:HucMSC-Ex targets ACSL4 through miR-129-5p,inhibits the lipid peroxidation process of intestinal epithelial cells,reduces cell ferroptosis,and alleviates DSS-induced IBD in mice.