Imbalance Between α1‐Antitrypsin and Neutrophil Elastase in Simple Steatosis Promotes Inflammation and Fibrosis Through Regulation of Adenosine a3 Receptor Signaling in Non‐alcoholic Steatohepatitis

Study objectives Non‐alcoholic fatty liver disease (NAFLD) has been considered leading cause of developing liver failure. However, the underlying mechanisms for transition from benign steatosis to clinically significant non‐alcoholic steatohepatitis (NASH) and non‐alcoholic steatofibrosis (NASF) have not been fully understood. Method In mouse model, NAFLD was induced by fast food diet (FFD) with a high glucose‐fructose solution for 24 weeks. For secretome proteomics, we used primary hepatocytes (HPs) isolated from normal chow diet (NCD) or FFD‐fed liver. The biased agonist of A3 adenosine receptor (A3AR), FM101 was utilized for the study of A3AR signaling. Results In secretome proteomics, we found α1‐antitrypsin (A1AT) was decreased 0.2‐fold in FFD hepatocytes compared with NCD hepatocytes. Consistently, serum levels of A1AT from mice and human patients with NAFLD were significantly decreased compared to normal group. Conversely, the mRNA and protein level of Neutrophil Elastase (NE) were increased in FFD‐fed liver compared to NCD‐fed liver. We next confirmed inflammatory role of NE in Kupffer cells (KCs) as various cytokines (CCL2, TNF‐α, IL‐1β, iNOS and IL‐6) were increased in KCs with treatment of NE. Of note, NE has been reported to exert proteolytic activity that can prevent activation of G‐protein‐coupled receptors (GPCRs). We observed similar proteolysis that A3AR, a member of the adenosine receptor group of GPCRs, was cleaved in hepatic stellate cells (HSCs) after treatment of NE, indicating that A1AT‐NE system may regulate the A3AR signaling. Interestingly, A3AR was predominantly expressed in KCs and HSCs, but not in HPs. In vitro activation of A3AR signaling with FM101 significantly inhibited activation of KCs and HSCs as demonstrated by inflammatory cytokines (TNF‐α, IL‐6 and IL‐1β), and profibrogenic genes (Col1a1, Timp1 Acta2) expression, respectively. Moreover, in vivo activation of A3AR with FM101, lowered liver weight, liver to body ratio, and serum level of ALT and cholesterol induced by FFD. Furthermore, FM101 effectively improved steatofibrosis as assessed by quantification of collagen deposition. Mechanistically, FM101 treatment strongly blocked mitochondrial oxidative phosphorylation. Using pMito‐Timer and Mt‐Keima system, we observed FM101‐induced mitochondria vulnerability as demonstrated by increased accumulation of damaged mitochondria and subsequence mitophagic degradation. Consequently, CCCP‐induced cell death in macrophage was further enhanced by FM101 treatment. Conclusions The imbalance between A1AT and NE in simple steatosis mediates the progression of NASH and NASF through proteolytic inactivation of A3AR in KCs and HSCs. Thus, A3AR agonism with FM101 may have therapeutic potentials for treatment of NAFLD. Support or Funding Information National Research Foundation of Korea (NRF) ‐ 2019R1A2C1090178 FM101 ameliorates FFD‐induced steatohepatitis and stetofibrosis Figure 1 Adenosine A3 receptor signaling contributes to mitochondria vulnerability Figure 2

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