YAP Activity Changes At The Early Stage Of NASH And Its Possible Involvement In The Development Of Ductular Reaction

Background:Nonalcoholic steatohepatitis（NASH）is the progressive subtype of nonalcoholic fatty liver disease（NAFLD）.Ductular reaction（DR）was closely associated with NASH progression and hepatic fibrosis stage.Yes-associated protein（YAP）activation plays an important role in the initiation and progression of NASH-related liver fibrosis,and participates in the development of DR in cholestatic liver injury.However,the activation of YAP at the early stage of NASH remains unclear and there are few data on the effects of YAP activation on the development of DR.Aim:We aimed to characterize the change of YAP activation and identify its possible involvement in the development of DR at the early stage of steatohepatitis.Methods:Male C57BL/6 mice fed with methionine choline-deficient（MCD）,thioacetamide（TAA）,western diet（WD）and normal control（NC）diet for 12 weeks were used to represent the disease progression in NASH with different pathogenic mechanisms.Primary mouse hepatocytes in culture treated with palmitic acid（PA）for 24h were used as a cellular model.In vitro and in vivo,the mRNA levels of YAP and its target genes（Ctgf,Cyr61,Acta2 and Ankrd1）,Notch pathway target genes（Hes1,Jag1 and Hey1）,a cell proliferation marker Ki67 and several markers of liver injury（Il1b,Tgfb,116,Tnfa and Rela）were measured by qPCR,and the protein levels of YAP and Phospho-YAP（Ser127）,ductular-specific markers（K19 and Sox9）and a marker for Notch pathway activation NICD were measured by immune quantification,and the spatial distributions of YAP,K19 and Sox9 were examined by immunostaining.Results:1.Both the transcription levels of YAP and its target genes and total YAP protein level in liver tissue were significantly increased（YAP 2wk/4wk:2.34,6.77 fold increase;Ctgf 1wk/2wk/4wk:1.30,3.53,4.61 fold increase;Cyr612wk/4wk:3.70,4.85 fold increase;Acta2 2wk/4wk:1.45,1.41 fold increase;total YAP protein 1wk/2wk/4wk:13.9,61.3,35.9 fold increase;vs NC,p<0.05）at the early stage of steatohepatitis in MCD-induced NASH model.After 1wk,the mRNA levels of injury-related markers were significantly increased（116:0.98 fold increase,Tnfa:5.50 fold increase,Rela:1.75 fold increase;vs NC lwk,p<0.001）,and about 34.0 YAP-positive hepatocytes were observed per 40×field.The number of YAP-positive hepatocytes reached a peak of 90.8 at wk2 and then decreased to 30.8 at wk4 while YAP-positive ductular cells appeared near YAP-positive hepatocytes.During 8-12wk,a large number of K19-positive/Sox9-positive cells were observed while the number of YAP-positive hepatocytes continued to decline from 11.0 to 5.8,and the number of YAP-positive ductular cells increased from 97.2 to 120.2.2.Both the transcription levels of YAP and its target genes and total YAP protein level in liver tissue were significantly increased（YAP 2wk:1.88 fold increase;Ctgf 2wk:1.52 fold increase;Cyr61 1wk/2wk:0.33,0.80 fold increase;Acta2 1wk/2wk:0.69,1.67 fold increase;total YAP protein 3d/lwk/2wk:28.4,34.0,53.8 fold increase;vs NC,p<0.05）at the early stage of steatohepatitis in TAA-induced NASH model.After 3d,mRNA levels of injury-related markers were significantly increased（116:14.35 fold increase,Tnfa:5.03 fold increase,Rela:1.96 fold increase;vs NC 3d,p<0.001）,and about 43.7 YAP-positive hepatocytes close to central vein were observed per 40×field.The number of YAP-positive hepatocytes reached a peak of 69.2 at wk2 and then decreased to 55.2 at wk4 while YAP-positive ductular cells appeared near YAP-positive hepatocytes.A large number of K19-positive/Sox9-positive cells were observed during 6-12wk,while the number of YAP-positive hepatocytes continued to decline from 48.0 to 25.6,and the number of YAP-positive ductular cells increased from 85.8 to 105.1.3.In PA-induced hepatocytes injury,both the mRNA levels of Il1b,Rela and Tgfb and the activity of YAP were significantly increased（including the increased mRNA expressions of target genes Ankrdl,Cyr61,Ctgf and Acta2,the increased protein level of total YAP protein,the decreased ratio of pYAP to YAP,and increased nuclear expression of YAP）.Following the increased YAP activity,the expression levels of K19 and Sox9 and the activity of Notch pathway were increased（including the increased mRNA expressions of target genes Hes1,Jag1 and Hey1 and the increased protein level of NICD）.When the transcriptional activity of YAP was inhibited by VP,the mRNA levels of YAP target genes Ankrdl and Acta2 were decreased with the concomitant decrease of K19 and Sox9 expression levels and Notch pathway activity.When the activity of Notch pathway was inhibited by DAPT,YAP activity was not influenced（no changes of the mRNA levels of target genes Ctgf and Acta2,protein levels of total YAP protein,and the ratio of pYAP to YAP）,but the expression levels of K19 and Sox9 were decreased.4.Both the transcription levels of YAP and its target genes and total YAP protein level in liver tissue were significantly increased（YAP 4wk/8wk/12wk:5.70,3.63,3.19 fold increase;Ctgf 8wk/12wk:2.18,3.09 fold increase;Cyr61 4wk/8wk/12wk:2.05,4.42,2.81 fold increase;Acta2 4wk/8wk/12wk:2.41,3.69,4.42 fold increase;total YAP protein 4wk/8wk/12wk:10.50,33.3,37.0 fold increase;vs NC,p<0.05）at the early stage of steatohepatitis in WD-induced NASH model.After 4wk,mRNA levels of injury-related markers were significantly increased（116:0.19 fold increase,Tnfa:0.66 fold increase,Rela:0.26 fold increase;vs NC 3d,p<0.001）,and the number of YAP-positive ductular cells and the mRNA level of Ki67 increased gradually along with the time（the number of YAP-positive ductular cells 4wk/8wk/12wk:12.8,22.0,30.4;Ki67 4wk/8wk/12wk:4.13,4.77,11.71 fold increase）,and the number of K19-positive/Sox9-positive cells were significantly increased at wk12.Conclusions:Hepatic YAP activation was earlier than the development of DR in NASH models.YAP activation could participate in the development of DR through different mechanism at the early stage of steatohepatitis.Liver injury was much severe and hepatocyte YAP activation may involve in the development of DR through hepato-biliary transdifferentiation mediated by notch at the early stage of steatohepatitis in MCD/TAA-induced NASH model.Liver injury was relatively mild and YAP activation could participate in the development of DR by promoting ductular cells proliferation at the early stage of steatohepatitis in WD-induced NASH model.The extent of liver injury influenced YAP expression in liver epithelial cells.The different changes of YAP activation among the different diet-induced NASH models suggested that the pathophysiological characteristics of non-obese NASH patients mimicked by MCD/TAA was different from that of obese NASH patients mimicked by WD,and may provide valuable insights into diagnosis and management of NASH patients with different pathogenic mechanisms.