| Objective: Non-alcoholic fatty liver disease(NAFLD)is characterized by fatty degeneration and fat accumulation in liver parenchymal cells,and is one of the most important public health issues in the 21 st century.Despite extensive research,there is no currently approved strategy for the treatment of NAFLD.In previous studies,we found that human umbilical cord mesenchymal stem cell-derived exosomes(huc MSC-Ex)carry active molecules such as proteins and nucleic acids from originated cells were alleviated acute liver injury and liver fibrosis.However,whether it is invovled in the pathogenesis of NAFLD remains unknown.The present study is aimed at examing the effect of huc MSC-Ex in inhibiting lipid accmulation and alleviating the progression of NAFLD,and exploring the fatty acid regulation mechanism of CAMKK1 carried by huc MSC-Ex to provide a basis for the application of huc MSC-Ex in NAFLD.Methods:(1)huc MSC were isolated and then huc MSC-Ex was extracted.The morphology of exosomes was analyzed using transmission electron microscope(TEM)and atomic force microscope(AFM).The size and relative intensity of exosomes were observed by nanoparticle tracking analysis(NTA).The CD9,CD63,and CD81 molecules which frequently located on the surface of exosomes were analyzed using Western blot,and the membrane surface markers CD9,CD63 and CD81 were detected by imaging flow cytometry.(2)human hepatocytes LO2 steatosis was induced by free fatty acids and huc MSC-Ex was added.The lipid deposition was observed by Nile Red staining,the fatty acid oxidation rate was detected by kit,and the fatty acid metabolites were detected by non-targeted lipidomics.(3)The NAFLD mice model induced by high fat diet(HFD)were established,and huc MSC-Ex was injected into the tail vein.The positive effect of huc MSC-Ex was evaluated by HE staining,oil red "O" staining,liver index,liver tissues and serum lipid,liver function indexes(TG,TC,AST,ALT).(4)Total RNA of LO2 cells before and after huc MSC-Ex treatment was collected,and the fatty acid metabolism pathway regulated by huc MSC-Ex was analyzed by transcriptome microarray,and verified by Western blot,q PCR,and immunofluorescence.(5)Proteins related to fatty acid metabolism regulation in huc MSC-Ex were analyzed by mass spectrometry,and verified in liver tissues and cells by Western blot and immunofluorescence.(6)The regulatory protein was overexpressed in LO2 and 293 T cells,and the regulation of lipid metabolism and its signal pathway were observed by Western blot,q PCR,immunofluorescence and Nile Red staining.Regulatory proteins knock down huc MSC-Ex were constructed,and the effects of regulatory proteins in huc MSC-Ex on inhibition of fatty acid metabolism were analyzed.Results:(1)The huc MSC-Ex obtained by ultracentrifugation expressesed exosomal marker proteins CD9,CD63,Alix and TSG101,which are biconcave disc-shaped vesicles with a diameter of about 110 nm and has typical characteristics of exosomes.(2)huc MSC-Ex treatment were reduced the lipid deposition of LO2 cells in a concentration-dependent manner,increased the fatty acid oxidation rate and inhibited the production of fatty acid metabolites.(3)Further,we found that huc MSC-Ex treatment were alleviated fatty vacuole degeneration and lipid deposition in the liver of mice,reduced the weight of mice,increased liver index and improved liver function in NAFLD mice model induced by high-fat diet.(4)Calcium/calmodulin-dependent protein kinase kinase alpha(Ca MKK1)promoted fatty acid oxidation and inhibited fatty acid production by regulating AMPK/PPARa and AMPK/SREBP-1C/FASn pathways.(5)Importantly,knockdown of CAMKK1 in huc MSC abolished the anti-hepatic steatosis abilities of huc MSC-Ex,and reduced the hepato-protective effects of huc MSC-Ex in vitro and in vivo.Conclusion: huc MSC-Ex may promote fatty acid oxidation,inhibit fatty acid synthesis and attenuate the progression of NAFLD through the transport of CAMKK1,which is a potential treatment for NAFLD. |
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