| Objective Renal interstitial fibrosis is a common pathological injury process in the development of all chronic kidney disease to the end stage.Previous research studies have found that human umbilical cord mesenchymal stem cell derived exosomes?hucMSC-Ex?can alleviate renal interstitial fibrosis in rats,but the specific mechanism is still unclear.Exosomes-derived microRNAs?miRNAs?mediate intercellular communication behavior,participate in many cellular processes,and play an important role in the fibrosis process.Therefore,this study will explore the role and specific mechanism of hucMSC-Ex carrying miRNAs in relieving renal interstitial fibrosis in vitro and in vivo.Methods hucMSCs were cultured and exosomes?hucMSC-Ex?were isolated from culture supernatants.Morphological size and surface markers were performed for hucMSC-Ex.A rat modelof unilateral ureteral ligation?UUO?renal interstitial fibrosis was established.First,the tail vein was injected with DIR-labeled hucMSC-Ex,In vivo imaging of small animals examined the localization of hucMSC-Ex.The UUO rats were injected hucMSC-Ex though tail vein,with PBS or HFL1-Ex as control.HE and Sirius red staining for kidney tissue structure and collagen deposition,immunohistochemistry and Western blot detection for fibrosis-related genes to evaluate the effect of hucMSC-Ex.Human tubular epithelial cells?HK-2?were induced by TGF-?1 recombinant protein and co-cultured with hucMSC-Ex or HFL1-Ex.Cell morphology was observed under microscope.QRT-PCR,Western blot and immunofluorescence staining were performed to detect fibrosis-related genes.The miRNAs were sequenced an screened for differentially expressed miRNA NC000019.1013474?miR-13474?by hucMSC-Ex and HFL1-Ex and verified by qRT-PCR.QRT-PCR detected the expression of miR-13474 in tissues and cells.The downstream target genes were predicted and verified by luciferase reporter gene to be a disintegrin and metalloproteinase domain 17?ADAM17?,and its expression in tissues and cells was analyzed.QRT-PCR,Western blot and immunofluorescence staining examined the expression of ADAM17 in tissues and cells.Next,the effect of miR-13474 on fibrosis was observed using miR-13474 mimics and inhibitor.QRT-PCR,Western blot and immunofluorescence staining were performed to detect ADAM17,TGF-? and ?-SMA expression levels.The miR-13474 mimics were imported into hucMSC-Ex by ultrasound to treat cells.QRT-PCR,Western blot and immunofluorescence staining were used to detect the relevant gene expression,respectively.Finally,plate cloning and cell apoptosis test were performed in HK-2 cells treated with miR-13474 mimics to explore the biological function of miR-13474.Results The isolated cultured hucMSCs exhibited osteogenic and adipogenic differentiation ability.Both hucMSC-Ex and HFL1-Ex accorded with exosomes characteristics.hucMSC-Ex was able to direct migration to damaged kidneys to alleviate renal interstitial fibrosis in rats.Compared with the UUO group,the renal tubule dilatation was slowed,the inflammatory cell infiltration was reduced,the interstitial collagen deposition was decreased,and the fibroblast hyperplasia was not obvious;besides the expression of ?-SMA,TGF-?1 and collagen were all decreased in the hucMSC-Ex intervention group.In vitro experiments,hucMSC-Ex intervention reversed cellular fibroid alterations induced by TGF-?1,with downregulation of ?-SMA,TGF-? and FAP.hucMSC-Ex overexpressed miR-13474 and in bote tissues and cells,hucMSC-Ex intervention group contained high levels of miR-13474,which was able to play a role in alleviating fibrosis by binding specifically to the ADAM17 3'-UTR side.QTR-PCR,Western blot and immunofluorescence staining revealed that ADAM17 was significantly down-regulated and the fluorescence intensity in the cytoplasm decreased in hucMSC-Ex treatment group.After overexpression of miR-13474,the expression of ADAM17,TNF-?,TGF-? and ?-SMA were all down-regulated,while knocking down miR-13474,these genes were all up-regulated.The miR-13474 content of hucMSC-Ex increased after ultrasound by qRT-PCR detection,and hucMSC-Ex-miR-13474 significantly promoted the accumulation of intracellular miR-13474.In addition,the expression of ADAM17,TNF-?,TGF-? and ?-SMA was downregulated compared with the hucMSC-Ex treatment group.Plate cloning and apoptosis experiments found that miR-13474 could inhibit cell proliferation but had no effect on apoptosis.Conclusion hucMSC-Ex can home to injury site of kidney to play a therapeutic effect,and high expression of miR-13474;hucMSC-Ex carrying miR-13474 regulated the expression of ADAM17,inhibited the expression of TGF-? and ?-SMA,and relieved renal fibrosis in vivo and in vitro.Ultrasound could make hucMSC-Ex overexpression miR-13474 and play a better therapeutic effect.Suggesting that hucMSC-Ex transport miRNAs in the treatment of renal interstitial fibrosis is expected to be a new treatment. |
PDF Full Text Request