P16 Vivo‐Morpholino Treatment Attenuates Hepatic Senescence and Non‐Alcoholic Fatty Liver Disease Phenotypes in a Diet‐Induced Rodent Model

Abstract

Non-alcoholic fatty liver disease (NAFLD), which affects approximately 100 million Americans, is characterized by macro-vesicular steatosis, hepatic inflammation and fibrosis. NAFLD patients have increased biliary expression of p16 and p21 (key markers of cellular senescence), and p16 is critical in the progression of liver phenotypes the in a cholestasis mouse model of primary sclerosing cholangitis. Furthermore, inhibition of p16 in a cholestatic mouse model ameliorates hepatic damage, biliary senescence and fibrosis. Thus, we aimed to determine the role of p16 inhibition on ductular reaction, biliary senescence, steatosis, inflammation and liver fibrosis in a model of NAFLD. Methods: Wild-type (WT, C57BL6) male mice at 4 weeks of age were either fed control diet (CD) or HFD (high fat, trans-fat and 0.2% cholesterol) along with a high fructose corn syrup equivalent supplemented in drinking water for 16 weeks. Both the CD and HFD fed mice received two doses of either p16 Vivo-Morpholino (to inhibit hepatic p16 expression) or mismatch Morpholino by tail vein injection (TVI) twice a week at 16 wks of feeding before they were harvested for tissues and serum. Hepatic p16 expression post morpholino treatment was evaluated by co-immunofluorescence staining of p16 and cytokeratin-19 (CK-19) in cholangiocytes and p16 and HNF4α in hepatocytes. We performed hematoxylin and eosin (H&E) staining to determine hepatic damage and steatosis was measured by Oil-Red-O staining. Liver fibrosis and hepatic stellate cell activation (HSC) was evaluated by Sirius Red staining and co-immunofluorescence (co-IF) for desmin and CK-19 (biliary marker). Hepatic inflammation was measured by F4/80 (Kupffer cell marker) staining. Results p16 immunoreactivity increased in HFD fed WT mice compared to CD, and when mice were treated with p16 Vivo Morpholino, p16 expression was decreased in both cholangiocytes and hepatocytes. HFD feeding in WT mice increased (i) ductular reaction, hepatic damage and steatosis;(ii) HSC activation and hepatic fibrosis; and (iii) inflammation compared to WT mice fed CD; however, knockdown of p16 reduced these parameters in HFD fed mice. Conclusion Biliary and hepatocyte senescence promotes NAFLD phenotypes and progression. Attenuation of hepatic p16 expression may be a potential therapeutic approach in improving NAFLD phenotypes and subsequent progression to NASH.