The Mechanisms Of Macrophage-derived Exosomes In The Pathogenesis Of Non-alcoholic Steatohepatitis

Aims: Non-alcoholic steatohepatitis(NASH)is a progressive stage of non-alcoholic fatty liver disease(NAFLD)and is closely associated with poor prognosis such as cirrhosis and hepatocellular carcinoma.Exosomes contain a variety of biologically active substances that mediate intercellular communication and are involved in the regulation of many cellular functions.Several studies have shown that exosomes play an important role in the development and progression of NASH.Inflammation is the hallmark transition process in the progression of NAFL to NASH,and the macrophages are the main immune cells in NASH liver inflammation.But the role and mechanisms associated with macrophage-derived exosomes in NASH have not been explored.The aim of this study was to clarify the role of macrophage-derived exosomes in the development of NASH,to investigate the mechanisms involved in the increased secretion of macrophage-derived exosomes,and to provide a theoretical basis for the search of possible therapeutics for NASH.Methods:(1)To clarify the role of hepatic macrophage-derived exosomes in the development of NASH.Firstly,a high fat and high cholesterol diet(HFHC)-induced NASH mouse model was constructed to detect the changes in the number of total exosomes and macrophage-derived exosomes in serum.Then,liver macrophages were isolated from NASH mice to clarify the changes in the number of exosomes secreted by macrophages in vitro.Secondly,in vivo transfusion of macrophage-derived exosomes was performed to investigate the effect of macrophage-derived exosomes on HFHC-induced liver injury,steatosis,inflammation and fibrosis was assessed by detection of liver function indicators and pathology-related indicators.Thirdly,macrophagederived exosomes were labelled with fluorescent dyes for in vivo tracing,and were used to identify the main target cells for endocytosis of exosomes.The effect of macrophage-derived exosomes on target cells was also explored in vitro.(2)To investigate the mechanism of increased secretion of macrophage-derived exosomes in NASH.First,the expression levels of the exosomal secretion-related protein RAB27 A on NASH liver tissue and on liver macrophages were clarified using HFHC-induced NASH mice and NASH patients confirmed by liver biopsy.In addition,RAB27 A inhibitors were used to act on bone marrowderived macrophages to clarify the role of RAB27 A in macrophage exosome secretion.Secondly,mass spectrometry was used to identify E3 ubiquitin ligases that interact with RAB27 A protein.Further analysis was used to screen for candidate molecules with differential expression on liver macrophages in NASH.Plasmid overexpression on macrophage cell lines was used to identify E3 ubiquitin ligases that regulate RAB27 A protein expression levels and exosome secretion.Thirdly,the interaction between the E3 ubiquitin ligase STUB1 and RAB27 A was determined by immunoprecipitation assay and immunofluorescence staining.The mechanism of STUB1 on RAB27 A was analyzed using ubiquitination assays and protein degradation assays.By stimulating bone marrowderived macrophages with fatty acids and lipopolysaccharides,the protein expression levels of STUB1 and RAB27 A under pathological stimulation and the changes in the number of exosomes secreted by macrophages were clarified.The role of STUB1 and RAB27 A in macrophage exosome secretion was then verified by knocking down Stub1 on bone marrowderived macrophages.(3)To explore therapeutic agents that target the mechanism of blocking the increased abnormal secretion of exosomes from macrophage.First,to predict and design peptide with blocking effects by analyzing the α-helical peptide sequence in the protein interaction structural domain of the E3 ubiquitin ligase STUB1 and RAB27 A.Second,peptides with the ability to block the interaction between STUB1 and RAB27 A were screened in HEK293 T cells using an immunoprecipitation assay,and their blocking effect on macrophage exocytosis was verified in an in vitro bone marrow-derived macrophage model.Thirdly,HFHC-induced NASH mice were treated with peptides to assess the effects of peptide on HFHCinduced liver injury,steatosis,inflammation and fibrosis.Results:(1)Macrophages-derived exosomes promoted lipid accumulation and inflammatory response in hepatocytes,leading to the development of NASH.In HFHC-induced NASH mice,serum levels of total exosomes and macrophage-derived exosomes were significantly increased.And secretion of exosomes from liver macrophages was increased.Macrophage-derived exosomes were found to exacerbate HFHC-induced impaired glucose tolerance as well as insulin sensitivity,liver damage,liver steatosis,inflammation and fibrosis by in vivo reinfusion experiments.In vivo transfusion of fluorescently labelled exosomes showed that macrophagederived exosomes were predominantly endocytosed by hepatocytes.Macrophage-derived exosomes,when applied to primary hepatocytes in vitro,promoted lipid accumulation and inflammatory responses in hepatocytes.(2)STUB1-RAB27 A mediates the secretion of hepatic macrophage exosomes in NASHRAB27A expression was upregulated in liver tissues of NASH mice and NASH patients,and was highly expressed on liver macrophages of NASH mice.And inhibition of RAB27 A protein function in vitro reduced exosome secretion from bone marrow-derived macrophages.STUB1 and TRIM21 were identified by mass spectrometry and experimentally validated as differentially expressed E3 ubiquitin ligases in liver macrophages from NASH,which also interacted with RAB27 A.Upregulation of STUB1 was found to cause high expression of RAB27 A protein and increased secretion of macrophage exosomes on macrophage cell lines.Mechanistic studies revealed that STUB1 could upregulate the protein stability of RAB27 A by interacting with RAB27 A and ubiquitinating RAB27 A,resulting in increased expression of RAB27 A protein levels.In vitro,STUB1 and RAB27 A protein and exosome secretion levels in bone marrow-derived macrophages increased with the duration of pathological stimulation,whereas inhibition of STUB1 gene expression blocked the increase in RAB27 A protein and exosome secretion.(3)Peptide Pep Rab2 ameliorated NASH by blocking STUB1-RAB27AThree peptide precursors were designed and synthesized based on the protein-protein interaction structural domain of STUB1-RAB27 A,of which peptide Pep Rab2 blocked the STUB1-RAB27 A interaction and reduced exosome secretion from bone marrow-derived macrophages in response to pathological stimuli.Treatment of HFHC diet-induced NASH mice with peptide Pep Rab2 attenuated HFHC-induced glucose tolerance as well as impaired insulin sensitivity,liver damage,liver steatosis,inflammation and fibrosis.Conclusions: Exosome secretion from macrophages was increased in NASH and was involved in promoting the development of NASH by promoting lipid synthesis and accumulation and inflammatory response of hepatocytes.Highly expressed STUB1 on liver macrophages could increase RAB27 A protein stability,upregulate RAB27 A protein expression level and increase RAB27A-mediated exosome secretion through ubiquitination modification of RAB27 A.Blockade of STUB1-RAB27 A using the peptide Pep Rab2 had a good therapeutic effect on NASH and might be one of the potential therapeutics for NASH.