Abstract: Objective To explore the changes in Yes-associated protein (YAP) activity at the early stage of nonalcoholic steatohepatitis (NASH) and the spatiotemporal relationship between YAP and ductular reaction (DR). Methods Male C57BL/6J mouse models of NASH were established by feeding with a methionine- and choline-deficient (MCD) diet or a thioacetamide (TAA) diet for 12 weeks. At different time points during the feeding, liver histology of the mice was observed with HE and Masson trichrome staining. The mRNA expressions of YAP and its target genes (Ctgf, Cyr61, Acta2) were determined by qPCR, and the total protein expression level of YAP was measured with immunoblotting. The expression and distribution of YAP and the markers of DR (K19 and Sox9) were observed with immunohistochemical staining. Results At the early stage of NASH induced by MCD diet (1 to 4 weeks), the mRNA expression of YAP and its target genes and the total protein expression of YAP increased significantly (P<0.01). The number of YAP-positive hepatocytes reached the peak level of 90.8 (cells per ×400 field of view) at week 2 and then decreased to 30.8 at week 4 (P<0.001); YAP-positive ductular cells appeared near the portal area, where DR began to occur. From 8 to 12 weeks, numerous K19/Sox9-positive DR cells were observed in the hepatic lobules around the central vein (P<0.01), while only a few YAP-positive hepatocytes were present in the liver tissue (P>0.05), and the number of YAP-positive ductular cells gradually increased with time (P<0.001). At the early stage of NASH induced by TAA diet (3 days to 2 weeks), the mRNA expression of YAP and its target genes and the total protein expression of YAP increased significantly (P<0.05), and the number of YAP-positive hepatocytes reached the peak of 69.2 at week 2 and then decreased to 55.2 at week 4 (P<0.001); YAP-positive ductular cells first appeared at the initial location of DR near the central vein. From 6 to 12 weeks, numerous K19/Sox9- positive DR cells occurred in the hepatic lobules around the central vein (P<0.01). While the number of YAP-positive hepatocytes decreased (P<0.001), the number of YAP-positive ductular cells continued to increase (P< 0.001). Conclusion During the development of NASH, YAP activation occurs earlier than DR but they are spatiotemporally correlated. YAP activation in hepatocytes may participate in DR by promoting hepatocyte dedifferentiation.

Key words: YAP; nonalcoholic steatohepatitis; ductular reaction; spatiotemporal relationship