A Triple-Hit Alcoholic Liver Disease Model Linking High-Fat Diet, Endotoxins, and Neo-Antigen Formation

BACKGROUNDExisting rodent models of alcoholic liver disease (ALD) often fail to replicate its full clinical progression--from steatosis to alcoholic hepatitis and ultimately cirrhosis--and overlook key metabolic contributors. Clinically, high-fat diets and gut-derived endotoxins (from enteric dysbiosis) act as additional "hits" that exacerbate alcohol-induced liver injury. Moreover, alcohol metabolism generates oxidative stress and modifies self-proteins into neo-antigens, potentially triggering immune responses. A model that incorporates these elements is critical for studying ALD pathogenesis and its immunological aspects. METHODSMale Wistar rats were randomized into four groups. The AL group received alcohol (36% caloric equivalent) and a single LPS dose (1 {micro}g/kg) 24 hours before sacrifice. The AF group was fed a high-fat diet (35% caloric equivalent) for 15 days, then maintained on alcohol and fat. The ALF group was similarly primed with fat, maintained on alcohol and fat, and given LPS prior to sacrifice. The ICC group served as isocaloric controls. Liver injury was assessed via histology, biochemical markers (AST, ALT, MDA), and serum endotoxin levels. Antibody titers against liver proteins were analyzed by ELISA; neo-antigens were identified using proteomics. RESULTSAll treated groups showed elevated liver enzymes, MDA, and endotoxin levels, indicating progressive liver damage. The ALF group exhibited the most severe pathology within six weeks, progressing from steatosis to steatohepatitis and fibrosis. In contrast, the AF group showed steatosis alone, and the AL group displayed moderate inflammation. Neo-antigens were most abundant in the ALF group. CONCLUSIONWe present a novel, rapid-onset triple-hit model of ALD that mimics the diseases full spectrum. This model enables mechanistic studies on how diet and gut-derived endotoxins contribute to ALD and highlights the immunogenic potential of alcohol-induced neo-antigens.