Pretreatment Of Exosomes Derived From HUCMSCs With TNF-? Ameliorates Acute Liver Failure By Inhibiting The Activation Of NLRP3 In Macrophage

Part 1:The effect of TNF-? pretreatment of hUCMSCs exosomes hsa-miR-299-3p on activation of NLRP3 inflammasome on macrophage Golgi apparatusObjective:In this study,we first determined that whether TNF-? pretreated human umbilical cord mesenchymal stem cells(hUCMSCs)-derived exosomes(T-Exo)have better inhibition of LPS-induced over-activation of NLRP3 inflammatory corpuscle complex and over-expression of downstream inflammatory factors in macrophage in mice than untreated hUCMSCs-derived exosomes(Exo),and preliminary exploration of the molecular mechanism of its action,provides new potential ideas for the treatment of acute liver failure.Methods:1.Collect the culture supernatant of hUCMSCs after TNF-? pretreatment for 3 days and extract the exosomes,analyze the characteristic protein of exosomes by Western blot and flow cytometry,and detect the concentration and diameter of exosomes by nanoparticle tracing analysis.The typical shape of exosomes was observed with a transmission electron microscope,and hUCMSCs exosomes without TNF-? pretreatment were used as a control.2.Label exosomes with PKH 67 fluorescent dye,observe the uptake of exosomes by macrophages;and determine the effect of Exo and T-Exo on the proliferation of macrophages by CCK-8 experiment.3.LPS stimulates macrophages to construct an in vitro inflammation model,which is treated with Exo and T-Exo,collects the cell culture supernatant and total cell protein,detects the concentration of proinflammatory cytokines in the cell culture supernatant by ELISA,and the expression of NLRP3 pathway-related proteins in macrophages was detected by Western blotting.4.Carry out preliminary mechanism investigation and further observe the expression of anti-inflammatory related miRNA in T-Exo through q-PCR and speculate its effect on the activation of NLRP3 pathway.Results:1.Exo and T-Exo treatment did not affect the proliferation of macrophages;in macrophages+LPS group,NLRP3,Caspase-1 and ASC protein expression levels were significantly increased,proinflammatory cytokines IL-1?,IL-6 and IL-18 secretion is increased;the expression of NLRP3,Caspase-1 and ASC in macrophages+LPS group is reduced compared to macrophages+LPS+T-Exo group,The secretion of IL-1?,IL-6 and IL-18 is reduced;the macrophage+LPS+T-Exo group has more significant anti-inflammatory effect.2.q-PCR experiments showed that compared with untreated hUCMSCs exosomes,related anti-inflammatory hsa-miR-299-3p was significantly up-regulated in TNF-?-stimulated umbilical cord mesenchymal stem cell exosomes,the difference was statistically significant,P<0.05;and partially transferred to macrophages after T-Exo was incubated with macrophages3.Co-localization between T-Exo,NLRP3 protein and macrophage trans-Golgi network structure(TGN):the untreated control group has a complete TGN structure and NLRP3 is not activated;under LPS stimulation,NLRP3 is activated,TGN is decomposed into a decentralized trans-Golgi network structure(dTGN),and the co-localization of NLRP3 and dTGN is observed on the Merge diagram;while the structure of TGN in the T-Exo treatment group is almost complete.It indicates that TNF-? pretreatment of exosomes can significantly inhibit the recruitment and activation of NLRP3 on TGNConclusion:TNF-? pretreatment of hUCMSCs exosomes can reduce LPS-induced over-activation of the NLRP3 inflammatory corpuscle complex in macrophages and the expression level of proinflammatory cytokines in the cell supernatant.Part 2:Hepatic protection of hUCMSCs exosomes pretreated by TNF-? in a mouse model of acute liver failureObjective:In vivo experiments further verified that T-Exo can inhibit the activation of NLRP3 inflammatory corpuscle complex in the liver of acute liver failure mice and the expression of inflammatory factors in peripheral blood serum.To elucidate the significance and potential mechanism of TNF-? pretreatment of exosomes derived from hUCMSCs in relieving acute liver failureMethods:1.Construction of acute liver failure mouse model:24 male C57BL/6 mice were randomly divided into 4 groups:control group(CON);model group(ALF):LPS(5mg/kg)+D-GalN(150mg/kg);Model+Exo control treatment group;Model+T-Exo treatment group.The mice in the control group were intraperitoneally injected with equal volume of saline and the tail vein with equal volume of PBS buffer;the model group,model+Exo control treatment group and model+T-Exo treatment group were all given intraperitoneal injection of LPS(5mg/kg)+D-GalN(150 mg/kg)was given the same volume of PBS buffer,Exo suspension(100?g/250ul)and T-Exo suspension(100?g/250ul)after 1 hour.2.Collect the liver of the mouse,weigh it,observe the HW/BW coefficient,and use HE staining to assess the degree of liver damage and inflammation.3.ELISA and coupled enzyme assays were used to determine the expression of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)respectively.4.Western blot was used to detect the expression levels of NLRP3,Caspase-1 and ASC protein in liver tissue;immunohistochemistry was used to detect the expression levels of NLRP3 and Caspase-1 protein in liver tissue again.5.Collect serum from the peripheral blood of mice and determine the expression level of proinflammatory cytokines in the serum by ELISA.Results:1.Pathological changes in liver tissue:no change in the control group;inflammatory cell exudation and hepatocyte necrosis in the model group;liver damage in the model+Exo control treatment group and model+T-Exo treatment group is reduced,and inflammatory exudation is reduced;Compared with the model+Exo group,the+T-Exo group had a more obvious therapeutic effect.2.The expression levels of NLRP3,Caspase-1 and ASC protein in liver tissue were the highest in the model group,and the model+T-Exo group was significantly lower than that of the model group and model+Exo group.3.The expression levels of AST and ALT in liver tissue are the highest in the model group,and the model+T-Exo group is significantly lower than the model group and model+Exo group;the expression levels of IL-1?,IL-6 and IL-18 in serum are the highest in the model group,the model+T-Exo group and the model+Exo group were significantly reduced,compared with the model+Exo group,the model+T-Exo group had a more obvious treatment effect.The differences were statistically significant(P<0.05)Conclusion:Pretreatment of hUCMSCs exosomes with TNF-? in the tail vein can inhibit the expression of the NLRP3 inflammatory corpuscle complex in the mouse liver,thereby alleviating acute liver failure induced by LPS+D-GalN in mice.