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Characterization of cellular senescence mechanisms in alcoholic liver injury
Author(s) -
Meng Fanyin,
Lorenzo Sugeily Ramos,
Francis Heather,
Glaser Shan,
Alpini Gianfranco
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.804.3
Subject(s) - senescence , viability assay , biology , liver injury , gene silencing , microbiology and biotechnology , chemistry , andrology , cell , endocrinology , biochemistry , medicine , gene
Background Cellular senescence is a stress‐responsive program limiting the proliferation of damaged cells and leading to stable cell‐cycle arrest. Accumulation of senescent hepatocytes may contribute to loss of functional hepatic mass and lead to liver decompensation and fibrosis. The current study aimed to characterize the functional role of cellular senescence during alcoholic‐induced hepatitis. Methods Senescence related gene expression was assessed using a Cellular Senescence PCR Array and/or real‐time PCR analysis in ethanol‐ and LPS‐treated normal human hepatocytes (N‐Heps) and cholangiocytes (HiBEC), as well as in liver specimen from mice fed alcohol for 5 weeks relative to control liver tissue. Cellular senescence and viability were measured by SA‐β‐gal Activity and MTS assay. The upstream modulators of senescence were defined in ethanol/LPS treated hepatocytes and cholangiocytes in vitro, and in TLR‐4 knockout mice in vivo. Results We identified that 5 week of ethanol feeding significantly increased total liver histopathology score and hepatocellular senescence by PCR array and SA‐β‐gal assay. The up‐regulation of hepatic senescence initiators, PAI‐1 and EGR1, were further verified by real‐time PCR assay. Treatment of N‐Heps and HiBECs with ethanol (86 mM) and LPS (20 ng/ml) for 72 hr significantly increased PAI‐1 and EGR1 expression, along with the enhanced SA‐β‐gal activity and reduced cellular viability detected by MTS assay. Silencing of PAI‐1 decreased ethanol and LPS‐ induced senescence in both N‐Heps and HiBECs, whereas inhibition of EGR1 only reduced the senescence and increased viability in N‐Hep cells. Interestingly, silencing of PAI‐1 and EGR1 also reduced the pro‐fibrotic markers, α‐SMA and MMP‐9 in N‐Hep cells, whereas silencing of LPS receptor TLR4 decreased these markers in the same group of N‐Hep cells, suggesting LPS‐mediated cellular senescence during alcohol‐induced liver fibrosis. Furthermore, the expression of TLR4 and the verified LPS related senescence markers, including E2F1, ID1 and IGFBP3 were significantly altered in ethanol‐fed mouse liver specimens compared to controls. TLR4 knockout mice displayed less sensitivity to alcoholic injury, along with reduced PAI‐1 and EGR1 levels and recovered expressions of α‐SMA and MMP‐9. Summary and Conclusion Our results show that PAI‐1 and EGR1 are the critical regulator of LPS induced cellular senescence and alcoholic hepatitis. The findings provide new insight into the function of LPS regulated cellular senescence and increase opportunities for the development of novel treatment paradigms for the management of alcoholic liver diseases. Support or Funding Information VA Merit Review Awards and NIH/NIDDK R01 Grants