The Role And Mechanism Of DRAM In Promoting Hepatocyte Exosome Secretion In The Development And Progression Of NAFLD

Objective: Non-alcoholic fatty liver disease(NAFLD)characterized by excessive fat deposition in liver cells refers to a clinical pathological syndrome,which becomes currently one of the most common chronic liver diseases.Although liver biopsy is the gold standard for the diagnosis of NAFLD,it is an invasive examination with low acceptance by patients.Therefore,there is an urgent need to develop a non-invasive examination method with high sensitivity and specificity.The amount and contents of exosomes change in different disease states,making them gradually become biomarkers for disease diagnosis.Previous studies have shown that exosomes are all significantly increased in patients with non-alcoholic steatohepatitis,mice treated with high-fat diet(HFD),and hepatocytes treated with fatty acids(FAs).However,the biogenetic and diagnostic value of exosomes in NAFLD is still unclear.The purpose of this study is to explore the mechanism of exosome biogenesis and its diagnostic value during the occurrence and development of NAFLD.Methods:(1)We used three methods,including nanoparticle tracking analysis(NTA),bicinchoninic acid(BCA)assays after exosome isolation using ultracentrifugation and phosphatidylserine Enzyme-linked immunosorbent assay(ELISA)to evaluate the level of plasma exosomes in patients with NAFLD,and the efficacy of the three methods was compared.(2)Genes related to exosome secretion in NAFLD were screened by GEO database and Gene Set Enrichment Analysis.(3)DRAM knockout mice were constructed using CRISPR/Cas9 technology,and the level of plasma exosomes in mice was determined by BCA,NTA,ELISA and Western Blot.(4)After knockdown or overexpression of DRAM in Hep G2 cells,three methods including Western Blot,BCA after exosome isolation and ELISA,which detected exosomes’ level directly,were used to detect the level of exosomes.(5)Lysosomal LGALS3/galectin puncta assay and detection of cathepsin B level in the cytoplasm and lysosome after lysosome extraction were used to determine lysosomal membrane permeability,and mice were injected with bafilomycin A1(a lysosomal inhibitor)to explore whether inhibition of lysosomal function can reverse the inhibitory effect of DRAM knockdown on exosome secretion.(6)Co-Immunoprecipitation experiment and Immunofluorescence assay were conducted to explore the interaction between DRAM and STOM.Results:(1)We found that NTA,BCA and ELISA have similar detection capability in detecting the number of exosomes,and the exosome level in NAFLD patients was significantly higher than that in healthy people.(2)By analyzing the GEO database and gene enrichment analysis,it is confirmed that DRAM was highly expressed in nonalcoholic fatty liver population,and that the expression was positively correlated with indicators related to liver injury and steatosis.And genes related to exosome secretion were enriched in the DRAM high expression group,which confirmed that DRAM might be one of the genes related to exosome secretion in NAFLD.(3)The results showed loss or knockdown of DRAM could down-regulate exosome secretion from hepatic cells using a knock-out mouse model and a knock-down cell model.(4)HFD and FAs can induce the expression of DRAM and increase the exosome secretion of hepatic cells.Knockdown of DRAM could reverse the increase of exosomes induced by FAs.(5)Overexpression of DRAM could promote the lysosomal membrane permeability of hepatocytes,and silencing DRAM could reverse FAs-induced lysosomal membrane permeabilization(LMP)and exosome secretion in hepatic cells.Inhibition of lysosomal function by baflomycin A1 could reverse the decrease of exosome secretion in DRAM knockout mice.(6)DRAM could interact with STOM,promote its lysosomal localization.Meanwhile,knocking down of STOM could inhibit DRAM-induced exosome secretion.Conclusions&Significance: The study illustrated that lipid-induced DRAM could interact with STOM,promote its lysosomal localization and induce LMP to further promote exosome secretion of hepatic cells in NAFLD.We not only revealed DRAM-mediated mechanism for exosome secretion from hepatic cells during the occurrence and development of NAFLD,but also provided a theoretical foundation for exosomes as a new biomarker for patients with NAFLD.