Mechanism Of Exosomes Secreted By Hepatic Stellate Cells Affecting The Metabolism Of Hepatic Non-parenchymal Cells

Objective:Liver fibrosis is a reversible wound-healing process.The activation of hepatic stellate cells is the main source of extracellular matrix,which acts as the key pathologic lesion in liver fibrosis.Exosomes are membranous vesicles secreted by cells and play an important role in cell communication.We hypothesized that exosomes released by activated hepatic stellate cells participate in the repair process of damaged liver.The purpose of this work is to explore the mechanism of exosomes released by hepatic stellate cells in activation and metabolism of HSC and other liver nonparenchymal cells,thus further studying the mechanism of liver fibrosis,providing theoretical basis for the reversal of liver fibrosis.Methods:Electron microscopic imaging,Western blot and AchE activity assay were used to identify the exosomes extracted from culture supernatants of human hepatic stellate cell line LX-2.CoCl₂-treated or LPS-treated LX-2 cells were pretreated by 2ME?Hif-1 inhibitor?or GW4869?inhibitor of exosome release?and exosome secretion,expression of?-SMA and Hif-1,glucose uptake in LX-2 cells were detected.The expression of GLUT1 and PKM2 in CoCl₂-treated or LPS-treated LX-2 cells and exosomes were tested by Western blot or imunofluorescence staining.The exosomes secreted by activated hepatic stellate cells?aHSC?were incubated with quiescent hepatic stellate cells?qHSC?,mouse peritoneal macrophages and human umbilical vascular endothelial cells.The uptake of exosomes,the expression of?-SMA,GLUT1,PKM2 and VEGF were detected by imunofluorescence staining or Western blot.Results:Exosomes secreted by LX-2 cells have a typical structure of exosomes,expressed exosomes-specific molecules and possessed AchE activity.CoCl₂-or LPS-treatment increased the secretion of exosomes from LX-2 cells,which was inhibited by Hif-1chemical inhibitor 2ME.Inhibition of the exosomes release by GW4869 suppressed the expression of?-SMA and Hif-1 and the glucose uptake of LX-2.The expression of GLUT1 and PKM2 in CoCl₂-treated or LPS-treated LX-2 cells and in exosomes were increased.The expression of?-SMA,GLUT1,PKM2 and VEGF in qHSC were increased after the incubation with exosomes from aHSC.The expression of GLUT1 and PKM2 in macrophages and endothelial cells were increased after the incubation with exosomes from aHSC.Conclusion:Hepatic stellate cells can secrete exosomes and the secretion of exosomes is regulated by Hif-1.Exosome secretion might be involved in the activation and metabolism process of hepatic stellate cells.Exosomes extracted from activated hepatic stellate cells contained glycolysis regulatory proteins involved in Warburg effect,which might affect the metabolism of hepatic non-parenchymal cells such as liver residential macrophages and endotholial cells.