| Research background and purpose:Liver is the largest and most important organ in the viscera of the human body.In the body,it can not only oxidize,store liver sugar,synthesize secretory protein,but also play a key role in the metabolism,detoxification and secretion of drugs and toxins.Acute liver failure(ALF)is a rare,life-threatening disease characterized by jaundice,coagulation disorders,and encephalopathy caused by a sudden decline in liver function,and affects many organ systems with a mortality rate of up to 90%.However,due to donor sources,liver transplantation indications,economic burden and rapid deterioration of ALF patients,orthotopic liver transplantation is still an effective method for the treatment of ALF,and many factors such as migration in the United States and patient acceptance limit the coverage rate of ALF patients receiving liver transplantation,making it unable to become a routine clinical treatment method in China.Therefore,it is urgent to find a new method to treat acute liver failure.Human umbilical cord mesenchymal stem cells(h UCMSCs)are a kind of stromal cells with the ability of self-renewal and multidirectional differentiation.Some scholars have found that mesenchymal stem cells can produce some cytokines with repair effect through paracrine mode,which can act on the liver and kidney to promote their injury repair.Exosomes is a kind of extracellular vesicles,which have shown great therapeutic potential in various diseases especially the liver diseases and own broad application prospects in regenerative medicine,disease model,drug screening,personalized medicine and other fields.Are exosomes derived from human umbilical cord mesenchymal stem cells(h UCMSC-Exo)a therapeutic option for acute liver failure? What is its specific mechanism of action and molecular mechanism? So far,there has been no report.The aim of this study was to explore the therapeutic effect and mechanism of h UCMSC-Exo on Acetaminophen(APAP)-induced acute liver failure,so as to provide experimental basis for finding new methods and new targets for the treatment of acute liver failure.Methods:1.Isolation,culture and identification of h UCMSCs and h UCMSC-Exo: 1)Isolation and culture of h UCMSCs from umbilical cord by trypsin;2)Molecular markers on the surface of h UCMSCs were detected by flow cytometry;3)The multidirectional differentiation potential of h UCMSCs in vitro was detected by osteogenic and adipogenic differentiation;4)h UCMSC-Exo was extracted by ultracentrifugation,and the morphology and particle size of h UCMSC-Exo were analyzed by TEM.5)Western blot was used to detect h UCMSC-Exo marker protein.2.Study on the potential of h UCMSC-Exo to inhibit hepatocyte necrosis and apoptosis in APAP-induced acute liver failure mice and its mechanism of action: 1)After the injection of APAP at a dose of 380 mg /kg,the survival rate of APAP-injured mice was evaluated after the injection of PBS or h UCMSC-Exo through the tail vein,and the site of action was determined by in vivo imaging after the injection of p KH26 labeled h UCMSC-Exo through the tail vein;2)The apoptosis and liver function of injured liver cells in different groups of mice were detected by HE staining,TUNEL apoptosis staining and liver function.3)A series of indexes related to oxidative stress in mice with acute liver failure,such as GSH,SOD,MDA,4-HNE,CYP2E1,were detected by immunofluorescence and enzyme plate analyzer.4)The expression of inflammatory cytokines in ALF mice was detected by q RT-PCR.3.h UCMSC-Exo inhibits oxidative stress and apoptosis of APAP-damaged LO2 cells by activating IGF-1R/PI3K/ Akt and ERK1/2 signaling pathways: 1)h UCMSC-Exo is easily absorbed by LO2 cells;2)h UCMSC-Exo can reduce the contents of ROS and MDA in APAP-injured LO2 cells;3)h UCMSC-Exo inhibits oxidative stress and apoptosis of APAP-injured LO2 cells by activating IGF-1R/PI3K/ Akt and ERK1/2 signaling pathways.Results:1.The characteristics of h UCMSCs and h UCMSC-Exo: 1)HUCMSCs with spindle-like cell morphology could be isolated and cultured from umbilical cord by trypsin digestion,and the morphology of h UCMSCs cells tended to whirlpool-shaped growth or parallel arrangement;2)Flow cytometry showed that h UCMSCs expressed mesenchymal stem cell marker genes CD29,CD90,CD73 and CD105,but did not express hematopoietic stem cell marker genes CD34 and CD45;3)h UCMSCs can differentiate into osteocytes and adipocytes in vitro;4)Transmission electron microscopy showed that h UCMSC-Exo was a vesicle with a diameter of 30-150nm;5)h UCMSC-Exo expressed exosome marker proteins CD9,CD63,CD81 and Rab5.2.In vivo h UCMSC-Exo transplantation can promote the repair of APAP-induced acute liver failure injury by inhibiting hepatocyte apoptosis and alleviating oxidative stress: 1)In vivo h UCMSC-Exo transplantation can significantly improve the survival rate of ALF mice,and in vivo imaging showed that most h UCMSC-Exo was enriched in liver;2)TUNEL apoptosis test showed that h UCMSC-Exo could inhibit the apoptosis of liver cells,HE staining showed that h UCMSC-Exo could significantly reduce APAP-induced central lobular necrosis of liver,and liver function index test showed that h UCMSC-Exo could significantly improve the liver function of ALF mice;3)h UCMSC-Exo can significantly restore the activities of GSH and SOD in ALF mice,and inhibit the increase of MDA content.Immunofluorescence staining showed that h UCMSC-Exo could significantly reverse the overexpression of 4-HNE and CYP2E1 in liver tissues of ALF mice;4)q RT-PCR assay showed that h UCMSC-Exo could significantly reduce the levels of pro-inflammatory cytokines IL-6,IL-1β and TNF-α in APAP-damaged liver tissues.3.h UCMSC-Exo inhibits oxidative stress and apoptosis of APAP-damaged LO2 cells by activating IGF-1R/PI3K/ Akt and ERK1/2 signaling pathways: 1)h UCMSC-Exo is easily absorbed by LO2 cells;2)h UCMSC-Exo can reduce the contents of ROS and MDA in APAP-injured LO2 cells;3)h UCMSC-Exo inhibits oxidative stress and apoptosis of APAP-injured LO2 cells by activating IGF-1R/PI3K/ Akt and ERK1/2 signaling pathways.Conclusion:1.h UCMSCs have strong proliferative ability,express the marker genes of mesenchymal stem cells,but do not express the marker genes of hematopoietic stem cells,and can be successfully induced to differentiate into adipocytes and bone cells with multidirectional differentiation potential.The derived exosomes were consistent in size and expressed exosomal marker proteins.2.In vivo transplantation of h UCMSC-Exo can promote the recovery of liver function in APAP-induced acute liver failure mice,the mechanism of which is related to the inhibition of necrosis and apoptosis of injured liver cells and the alleviation of oxidative stress;3.h UCMSC-Exo can be taken up by LO2 cells,and then activate APAP to injure the IGF-1R/PI3K/ Akt and ERK1/2 signaling pathways in LO2 cells,thereby inhibiting the oxidative stress and apoptosis of APAP-injured LO2 cells. |
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