The Role Of Puromycin-sensitive Aminopeptidase In Nonalcoholic Fatty Liver Disease

BackgroundNonalcoholic fatty liver disease(NAFLD)is a metabolic stress liver injury characterized by triglyceride(TG)accumulation in the liver.It is closely related to metabolic risk factors such as insulin resistance,diabetes and obesity,and has become the most common chronic liver disease in the world.Puromycin-sensitive aminopeptidase(PSA)is an important zinc metallopeptidase,and is widely expressed in many tissues.Recently,PSA has been reported to be related to adipocyte dysfunction,but its role in lipid metabolism is still unclear.This is the first subject to investigate and discuss the role and mechanism of PSA in liver lipid metabolism.ObjectiveThis study aims to explore the regulatory role and potential mechanisms of PSA in liver lipid metabolism,and provides new target for clinical treatment of NAFLD.Methods1.The protein and mRNA levels of PSA expression in fatty liver from NAFLD patients and mice(HFD、 ob/ob and db/db)were examined by western blot(WB),immuno histochemistry(IHC)and quantitative real time polymerase chain reaction(qRT-PCR).2.Huh7 hepatocytes were transfected with si RNA or plasmid for PSA inhibition or overexpression,and then treated with or without free fatty acid(FFA)for 24 h.The oil red O staining and the triglyceride(TG)content was determined enzymatically.The protein and mRNA levels of lipid metabolism target genes were detected by WB and qRT-PCR.Mito Tracker staining,mt DNA content,Seahorse OCR and protein levels of the OXPHOS complexes were determined.3.Huh7 hepatocytes were transfected with si RNA or plasmid for PSA inhibition or overexpression,and then treated with FFA for 24 h.ROS generation were determined by Mito SOX staining and flow cytometry assay,and GSH/GSSG ratio was measured.The mRNA and protein levels of indicated genes related to NRF2 signaling pathway were determined by qRTPCR and WB.PSA attenuates lipid accumulation through inducing the NRF2 antioxidant response.Huh7 hepatocytes co-transfected with PSA plasmid and NRF2 si RNA were treated by FFA for 24 h.The mRNA and protein levels of indicated genes related to NRF2 signaling pathway were determined.The oil red O staining and the TG content was determined enzymatically.The protein and mRNA levels of lipid metabolism target genes were detected by WB and qRT-PCR.Mito Tracker staining,mt DNA content,Seahorse OCR and protein levels of the OXPHOS complexes were determined.The ubiquitination and the half-life of NRF2 were detected by immunoblotting and immunoprecipitation analysis.4.8-week-old ob/ob mice were injected with AAV8-PSA or AAV8-GFP as control via tail vein,and the mice were examined 4 weeks later.Body weight(BW)and the ratio of liver weight to BW(LW/BW)were determined.Liver TG,TC and NEFA were determined enzymatically.The serum lipid profiles(TG,TC,LDL,HDL and NEFA)、 enzyme activity and β-hydroxybutyric acid were tested enzymatically.H&E,Oil red O and Mito SOX staining of liver sections were determined.The protein levels of indicated genes related to NRF2 signaling pathway were determined by WB.The mRNA levels of indicated genes related to lipid metabolism,inflammatory response and fibrosis were determined by qRT-PCR.The protein levels of indicated genes related to liver fibrosis were determined by WB.F4/80 IHC and Sirius Red staining of liver sections were determined.5.Human liver tissue microarrays with 78 cases were used to detect protein expression of PSA by IHC.Representative images of liver from normal(n=16),hepatic steatosis with inflammation(n=7),cirrhosis(n=28)and liver cancer(n=27)were stained for PSA.All stained tissue sections were semi-quantitatively scored.Results1.Both the mRNA and protein levels of PSA were decreased in the liver of NAFLD patients and mice compared with the control groups.2.TG accumulation were enhanced by PSA-si RNA,and attenuated when PSA was overexpressed under FFA treatment but not under basal conditions.PSA regulated hepatic TG metabolism by modulating lipogenesis and fatty acid β-oxidation.Further findings demonstrated that PSA affected mitochondrial biogenesis and metabolism.3.PSA overexpression activated antioxidative response by up-regulating NRF2 signaling pathway.NRF2 interference inhibited PSA-induced reduction of ROS and decreased lipid accumulation.PSA activated NRF2 by decreasing the ubiquitination of NRF2,leading to an increase in NRF2 stability.4.AAV8-PSA treatment led to PSA overexpression exclusively in the liver and it increased the NRF2 antioxidative pathway.AAV8-PSA significantly reduced the ratio of LW/BW,hepatic TG content,serum LDL and the liver enzyme activities in ob/ob mice,and increased serum β-hydroxybutyrate levels.Hepatic steatosis,lipid droplet accumulation,ROS levels,inflammation and fibrogenesis were markedly attenuated in AAV8-PSA-treated ob/ob mice.5.Human tissue microarray results showed that compared with normal human liver tissues,the expression of PSA was down-regulated in the liver of patients with fatty liver,cirrhosis and hepatocellular carcinoma.Conclusion1.PSA expression is downregulated in the liver of NAFLD patients and mice.2.Knockdown of PSA exacerbates TG accumulation by regulating both fatty acid synthesis and β-oxidation.3.PSA enhances the antioxidant effect in hepatic cells by activating the NRF2 pathway.4.Rescue of PSA alleviates hepatic steatosis in genetically induced ob/ob mice.5.PSA is a potential marker during the progression of NAFLD in human liver samples.