| Objective Liver fibrosis is the inevitable intermediate link in the process of liver cirrhosis.The pathological process is characterized by activation of hepatic stellate cell(HSC)into myofibroblats,excessive deposition of extracellular matrix(ECM)and destruction of liver structure.Our previous studies have demonstrated that human umbilical cord mesenchymal stem cell(huc MSC)-derived exosomes(huc MSC-Ex)could ameliorate mouse liver fibrosis,but the underlying mechanism remains unclear.The present study aims to further discuss the mechanism of anti-fibrotic effects of huc MSC-Ex.Methods Huc MSC were isolated and incubated in vitro.Huc MSC-Ex were extracted using sucrose density gradient ultracentrifugation.The shape of huc MSC-Ex was measured by TEM and diameter distribution was detected by Nanoparticle Tracking Analysis.CD9 and CD63 expressed on the surface of huc MSC-Ex,were detected by Western blot.BALB/c mice model of liver fibrosis were established by injection CCl4 and followed by administration of huc MSC-Ex.Mice were divided into groups as follow: PBS group(n = 10),huc MSC-Ex group(n = 10).Maestro in Vivo Imaging System and immunohistochemistry were applied to observe the distribution of huc MSC-Ex in vivo.Tissue structure and collagen deposition of mice fibrotic livers were observed via optical images,H&E and Masson staining.Expression of lysyl oxidase-like 2(LOXL2)and HSC activation markers,Fibroblast Activation Protein(FAP)and ?-Smooth Muscle Actin(?-SMA),were detected by immunohistochemistry and Western blot.In vitro experiment,LX-2 cells were activated and followed by co-cultivation with huc MSC-Ex.RT-PCR,Western blot and Immunofluorescence were used to detected expression of LOXL2,collagen ?(COL1)and FAP.Yes-associated protein(YAP)expression in mouse livers suffered from liver fibrosis and in LX-2 cells were analyzed by immunohistochemistry and Western blot,respectively.Overexpression or knock-down of YAP in cell lines was administrated by adenovirus or plasmid and the expression of LOXL2 in these cells were detected by Western blot.Luciferase reporter system was applied to detect the transcriptional activity of YAP against LOXL2.14-3-3? levels in huc MSC-Ex were analyzed by Western blot.Exosomes derived from huc MSCs modified by adenovirus to overexpress 14-3-3?(Ad-14-3-3?-Ex)were extracted and analyzed.Ad-14-3-3?-Ex or Ad-GFP-Ex were appling to co-culture with activated LX-2 cells.Western blot and immunofluorescence were used to observe the relationship between 14-3-3? expression and YAP nuclear translocation.Results Huc MSC-Ex presented spherical,membrane-bound vesicles and expressed exosomal markers,CD9 and CD63.The diameter of huc MSC-Ex was approximately 30 ~ 100 nm.Through in vivo imaging,fluorescence dye-labelled huc MSC-Ex were observed to locate in liver.Livers with the administration of huc MSC-Ex were found to express exosomal marker,CD63.Compared to the PBS group,huc MSC-Ex attenuated liver fibrosis and in these livers,decreased liver necrosis,reduced collagen deposition and down-regulation of LOXL2 were found.Expression of HSC activation markers,FAP and ?-SMA also decreased.High expression of YAP and LOXL2 were detected in fibrotic livers and activated LX-2 cells,suggesting the relevance between YAP and LOXL2.To further prove this,YAP were overexpressed in 293 T cells,LX-2 cells and HL7702 cells,resulting in increased expression of LOXL2,while YAP knockdown led to LOXL2 expression decrease.Luciferase reporter system also showed the the transcriptional activity of YAP against LOXL2.Appling Ad-14-3-3?-Ex or Ad-GFP-Ex to activated LX-2 cells showed that Ad-14-3-3?-Ex owned much stronger effects on inhibition expression of LOXL2 and nuclear translocation of YAP.Conclusion Huc MSC-Ex could ameliorate mouse liver fibrosis by down-regulating LOXL2 expression and this mechanism may be attributed to effects of 14-3-3? derived from huc MSC-Ex on reduction of YAP nucleartranslocation in HSC. |
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