The Repair Mechanism Of Exosome Derived From Human Umbilical Cord Mesenchymal Stem Cell In Renal Interstitial Fibrosis

Objective Previous research results indicated that human umbilical cord mesenchy stem cell(huc MSC)and huc MSC-exosome could alleviate cisplatin-induced rat renal tubular epithelial cells injury and micro RNA(mi RNA)play an important role in this process.Basing on these we establish unilateral ureteral obstruction(UUO)induced renal interstitial fibrosis(RIF)model in vivo and transforming growth factor1(TGF-?1)induced NRK-52 E cells in vitro.Through different experiments we explore repair effect and mechanism of huc MSC-exosome on renal interstitial fibrosis in vivo and vitro.Methods In vivo Sprague-Dawley rats(SD rats)were divided into three groups.Sham-operation group:SD rats' left abdomen were opened and their skin were sutured after finding the ureter.UUO group:SD rats which performed UUO operation.Huc MSC-exosome intervention group: Huc MSC-exosome were injected via caudal vein 24 h after UUO operation.We used vivo imaging technology to observe the location and distribution of huc MSC-exosome in tissues.SD rats' serum of each group were collected at different time points after the operation(12h,24 h,48h,72 h,7d,14d)and tested the level of creatinine(Cr)and urea nitrogen(BUN).The rats were sacrificed at 14 days after UUO surgery and the renal tissues were collected for the next experiments.Hematoxylin and eosin(HE)staining was used to assess the renal pathogeny structure and masson staining was applied to evaluate the range of collagen deposition in differen groups.We applied Immunohistochemistry,immuneofluore-scence and Western blot to detect the location and level of epithelia-mesenchymal transition(EMT)associated proteins(E-cadherin,N-cadherin,LOXL2)and fibrosis associated proteins(?-SMA,TGF-?1,COL1A1).In vitro,NRK-52 E cells were cultured in 6-well plates.NRK-52 E cells without any treatment was control group.In the induced group NRK-52 E cells were cultured with 40 ng TGF-?1 for 24 h.Intervention group was that induced cells were co-cultured with huc MSC-exosome for 48 h.Inverted microscope was used to observe the cellular morphology and cell density of NRK-52 E cells in different groups.We also detected the expression of EMT associated proteins(E-cadherin,N-cadherin)and fibrosis associated proteins(?-SMA ?TGF-?1?COL1A1)through Western blot.We used RT-PCR to evaluate the level of mi RNAs(mi R-199 a,mi R-146b)in different groups.Results Imaging in vivo result showed that fluorescence labeled huc MSC-exosome injected via vein intravenous reached the UUO renal.Right renal,spleen,lung had slightly fluorescence expression.In sham operation group there is no fluorescence expression in organs.Huc MSC-exosome has ability of chemotaxis towards injured organ.The level of BUN and Cr was significantly decreased in huc MSC-exosome intervention group compared with UUO group.HE staining displayed that abnormal dilatation of the tubules,massive accumulation of inflammatory cells and fibrotic changes happened in UUO group.Huc MSC-exosome could reverse these pathological changes and resotred the structure integrity of renal tubular.Collagen deposition in the interstitial area of the renal cortex could be effectively alleviated by huc MSC-exosome.Comparing with UUO group the expression of E-cadherin increased and N-cadherin decresed in huc MSC-exosome interverntion group which indicated that huc MSC-exosome could ameliate EMT progress.Fibrosis associated proteins(?-SMA,TGF-?1,COL1A1)decreased in intervention group which demonstrated renal fibrosis can be suppressed by huc MSC-exosome.Immunohistochemistry,immunofluorescence and Western blot displayed that the expression of LOXL2 that play an important role in EMT increase in UUO group and decrease in huc MSC-exosome intervention group.In Vitro results showed when we used TGF-?1 treating NRK-52 E cells the cellular morphology transformed from smooth-ovoid shape to irregular-spindle shape and cells' density decreased remarkably.Western blot results demonstrated that the expression of E-cadherin decreased and N-cadherin increased in the induced group comparing with the control group.Huc MSC-exosome treatment could increase the cells' density and recover cells' morphology.The expression of E-cadherin restored and N-cadherin,?-SMA,TGF-?1,COL1A1 decreased in the huc MSC-exosome intervention group.In addition,q RT-PCR proved the level of mi R-199 a downregulated induced by TGF-?1 and upregulated after co-cultured with huc MSC-exosome.(P<0.05)Conclusion Experiments in vivo and in vitro revealed that huc MSC-exosome could repair renal interstitial fibrosis.The mechanism of the therapeutic effect maybe associate with LOXL2 and micro RNA parceled in huc MSC-exosome.