Exosomes Derived From Human Umbilical Cord Mesenchvmal Stem Cells Alleviate Acute Liver Failure By Reducing The Activity Of The NLRP3 Inflammasome In Macrophages

Part 1:Observation of the effect of hUCMSCs-Exosomes on inflammasome NLRP3 activation in RAW264.7 macrophageObject:To obverse whether hUCMSCs-Exosomes can directly inhibit the activation of inflammasome NLRP3 and effect the expression level of inflammatory mediator in LPS-induced RAW264.7 cell from mice mononuclear phagocyte systemMethods:Exosomes were collected from supernatant of hUCMSCs cell c?lture medium.Western blot was used to analyze exosome characteristic proteins.Exosomes concentration and particle size were detected by nanoparticle tracking analysis.Average exosomes diameter was obtained through flow cytometry analysis.To observe exosome characteristic properties,transmission electron microscope(TEM)was used.For exosomes uptake experiment,exosomes were labeled with PKH 67 to observe the uptake of exosomes by macrophage.Macrophage was planted in 6-well plates at 2×106 cells per well and randomly divided into 4 groups:the macrophage control group,the macrophage+LPS group,the macrophage+LPS+hUCMSCs-Exosomes group and hUCMSCs-Exosomes group,for each group was made in triplicate.Culture medium supernatant and cell protein were collected and NLRP3,caspase-1 and IL-1? expression level were detected by western blotting.IL-1?and IL-6 expression level in c?lture medium supernatant were tested by ELISA.Result:Western blotting and ELISA res?lts demonstrated that NLRP3,Caspase-1,IL-6 and IL-1? expression level significantly increased in macrophage+LPS group,the activation of NLRP3,caspase-1,IL-6 and IL-1? decreased in macrophage+LPS+hUCMSCs-Exosomes group when compared to macrophage+LPS group(P<0.05).Conclusion:hUCMSCs-Exosomes can decrease the activation of inflammasome NLRP3 in LPS induced macrophage.Part 2:The protective effect of hUCMSCs-Exosomes in mice ALF modelObject:To further confirm hUCMSCs-Exosomes can directly inhibit ALF mice liver inflammasome NLRP3 activation and influence inflammatory mediator expression level in serum in vivo.Elucidate the significance and potential mechanisms of hUCMSCs-Exosomes in alleviate acute liver injury.Methods:24 male C57BL/6 mice were randomly divided into 4 groups:the control group,the LPS(5mg/kg)+D-GalN(150mg/kg)model group,the model+hUCMSCs-Exosomes group and the hUCMSCs-Exosomes group.The mice in control group were intraperitoneally and tail-intravenously injected with PBS,the animals in model+ hUCMSCs-Exosomes group and the hUCMSCs-Exosomes group were intraperitoneally injected with LPS(5mg/kg)+D-GalN(150 mg/kg).One hour after the injection,PBS and exosomes(100?g/250?1)were intraperitoneally injected respectively.The hUCMSCs-Exosomes group was intraperitoneally injected with PBS and tail-intravenously injected with exosomes(100?g/250?l).The liver tissues were collected and stained with hematoxylin and eosin to evaluate liver damage and inflammation level.NLRP3 and Caspase-1 expression level in hepatocyte were examined by western blotting.ALT,AST in liver tissues and IL-1? IL-6 level in serum were tested by ELISA.Results:1.The control group had no change;inflammatory cells,bleeding and necrocytosis increased in the model group.Tissue damage and inflammatory cells decreased in the model+hUCMSCs-Exosomes group.Necrosis was not observed in hUCMSCs-Exosomes group.2.The expression of NLRP3 and Caspase-1 protein in hepatocytes was the highest in the model group and was significantly decreased in model+hUCMSCs-Exosomes group(P<0.05),the difference is statistically significant,while there was no obvious change between hUCMSCs-Exosomes group and control group(P>0.05).3.The expression of ALT and AST in hepatocytes were the highest in the model group,ALT and AST levels were decreased in model+hUCMSCs-Exosomes group(P<0.05),the difference is statistically significant,while there was no obvious change between hUCMSCs-Exosomes group and control group(P>0.05).Conclusion:Tail intravenous injection of hUCMSCs-Exosomes can alleviate the acute liver damage that induced by intraperitoneally injected LPS(5mg/kg)+D-GalN(150 mg/kg)and inflammasome NLRP3 expression level in mice liver was reduced.