Amniotic Fluid Reduces Liver Fibrosis By Attenuating Hepatic Stellate Cell Activation
Background & Aims: Persistent activation of hepatic stellate cells (HSCs) drives liver fibrosis, marked by myofibroblast activation (MFA) and epithelial-mesenchymal transition (EMT). Strategies to prevent or reverse this will be critical to treat liver fibrosis successfully. We previously showed that full-term, cell-free human amniotic fluid (cfAF) inhibits MFA and EMT in fibroblasts in vitro. We hypothesize that cfAF treatment can attenuate HSC activation and limit liver fibrosis.  Methods: HSC activation was assessed using murine models of acute (DMN) and chronic (CCl4) liver damage, three-dimensional hepatic spheroids, and HSC cultures. EMT and MFA were induced in vitro using ethanol (spheroids) or TGFß (HSCs) and evaluated via scratch assays, multi-omics approaches, and RNA/protein analyses.  Results: cfAF treatment prevented weight loss in mice with chronic liver damage without adverse events. Histological analysis revealed a modest reduction in fibrosis with preserved liver architecture in both chronic and acute murine liver damage models. Gene expression profiling and immunostaining indicated cfAF administration to CCl4-treated mice led to reduced EMT- and MFA-related biomarkers along with changes in transcripts associated with liver metabolism, immune regulatory pathways, and cell signaling. In hepatic spheroids exposed to ethanol cfAF treatment lowered COL1A1 protein levels and smooth muscle actin (SMA) RNA abundance in a dose-dependent manner. Treating primary or LX2 hepatic stellate cells with cfAF strongly represses EMT, and multi-omics analyses revealed that it also attenuates TGFß-induced MFA and the inflammatory phenotype. Thus cfAF treatment prevents liver fibrosis by safeguarding hepatic stellate cell activation.  Conclusions: cfAF treatment limits liver fibrosis by repressing HSC activation in liver fibrosis models. These findings suggest cfAF may be a safe and effective therapy for reducing liver fibrosis and preventing the development of cirrhosis and/or hepatocellular carcinoma. Overall design: RNA-seq to measure transcript abundance changes in human LX2 hepatic stellate cells, either cultured with or without cell-free human amniotic fluid (cfAF) and in the presence of absence of TGF-beta (and/or with TGF-beta blocker A-83-01)