*Purpose: Effective interventions for hepatic inflammation and fibrosis remain a dire unmet need in transplant medicine. Cell-free amniotic fluid (AF) represents a potential alternative to current therapies due to its anti-inflammatory properties. We hypothesize that amniotic fluid will reduce liver inflammation and fibrosis by diminishing stellate cell activation and promoting hepatocyte homeostasis.

*Methods: We modeled stellate cell activation and liver inflammation/fibrosis in vitro and in vivo and measured epithelial-to-mesenchymal transition (EMT), pro-inflammatory signaling, and fibrosis. We used the scratch test assay and measured Vimentin mRNA levels by RT-qPCR in LX2 human hepatic stellate cells with and without ethanol and AF supplementation to measure EMT. We measured total and phosphorylated (p-) STAT3 by western blotting to determine if AF could abrogate pro-inflammatory signaling in ethanol-treated LX2 cells. Hepatic spheroids consisting of LX2 cells and immortalized human hepatocyte cells (PH5CH2) were also treated with ethanol and cultured with or without AF, then stained for type I collagen to measure fibrosis by confocal microscopy or RNA was extracted and we performed RT-qPCR. We measured fibrosis and liver homeostasis in vivo by treating mice with the liver toxin dimethylnitrosamine (DMN), with or without AF, and performed histological analyses.

*Results: The scratch assays indicate that the addition of AF is sufficient to reduce cell migration. Vimentin levels were also significantly reduced compared to controls, upon the addition of AF. Strikingly, LX2 cells treated with ethanol show strongly reduced p-STAT3 levels, but the addition of AF is able to restore the p-STAT3 levels to those observed in untreated cells. Hepatic spheroids treated with AF displayed markedly reduced type 1 collagen levels, and a dose-dependent reduction in smooth muscle actin RNA levels. Lastly, H&E and Mason’s trichrome staining from mice treated with DMN+AF displayed a significant reduction in necrotic cells or liver fibrosis (respectively), compared to mice treated with DMN alone.

*Conclusions: Together, the data suggest that AF can reduce liver inflammation and fibrosis. This is mediated by inhibiting EMT and pro-inflammatory signaling. Therefore we conclude that cell-free AF may be an effective treatment for liver inflammation and fibrosis. Further studies will determine the mechanisms through which these effects are executed.

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