Characteristics Of Hepatitis B Virus (HBV)-Associated Acute-On-Chronic Liver Failure (ACLF) Identified By Liver Gene Expression Profiling And Pathogenic Mechanism Of Aclf-Hypothesis “PINRO”

Part One Characteristics of hepatitis B virus(HBV)-associated acute-on-chronic liver failure(ACLF) identified by liver gene expression profilingBackgroud and aims: A recent study demonstrated that submassive hepatic necrosis(SMHN) is the critical histological feature of hepatitis B virus(HBV)-associated acute-on-chronic liver failure(ACLF).This study aimed to investigate the genetic alterations regarding metabolism,immune response and regeneration after the occurrence of SMHN.Methods: From 2008 to 2011,we screened and enrolled 33 patients undergoing liver transplantation(LT) due to HBV-associated cirrhosis with acute decompensation(AD) in Renji Hospital affiliated to School of Mecicine Shanghai Jiao Tong University. Two groups were distinguished by the presence or absence of SMHN(defined as necrosis of 15-90% of the entire liver on explant). By the use of bioinformatics BRB-Array Tools, liver gene expression profiles relating to hepatic metabolism,local immune response and proliferation were compared between these groups.Results: SMHN was identified in 16 of 33 patients proven to have cirrhosis by histological means. Significance analysis of microarray(SAM) revealed that 2933 genes were significantly different between SMHN+/-,including 1868 up-regulated and 1065 down-regulated genes in SMHN+ group.The different expression genes mainly distributed in categories of substance and energy metabolism,cell cycle and proliferation,cell component,response to stimulation,coagulation system and immune response based on gene ontology analysis.According to the analysis of different expression genes based on the public database KEGG and Bio Carta pathways,the 4 up-regulated pathways in SMHN+ group were ECM-receptor interaction, lysosome, leukocyte transendothelial migration and regulation of actin cytoskeleton. In the 10 most down-regulated pathways,8(80%) were related to metabolism, including carbohydrate, fat, amino acid,drug and so on.Conclusion: According to the hepatic gene expression pattern, we could distinguish the HBV cirrhotics with AD into two groups,almost in accordance with the classification based on the presence of pathological SMHN or not.After the occurrence of SMHN, HBV-associated ACLF patients had liver metabolism dysfunction,focal immune system and active cell proliferation.Part Two Pathogenic mechanism of hepatitis B virus(HBV)-associated acute-on-chronic liver failure(ACLF)- hypothesis “PINRO”Backgroud and aims: “PIRO” is the current hypothesis for the mechanism of ACLF. After our group finding SMHN was the major pathological feature of HBV related ACLF patients, we hypothesis “PIN(necrosis)RO” is the mechanism for HBV related ACLF patients.Methods: From 2008 to 2011,we screened and enrolled 33 patients undergoing liver transplantation(LT) due to HBV-associated cirrhosis with acute decompensation(AD) in Renji Hospital affiliated to School of Mecicine Shanghai Jiao Tong University. 40%(69/174) had histological evidence of SMHN. Clinical features about Inflammatory response and multiple organ failure(MOF) were compared between SMHN positive and negative group whenever acute insults exiting or not.Results: Acute insults were identified in 58.6%(102/174) enrolled patients. There was no difference in either Inflammatory(white cell count,WCC) or MOF parameter(CLIF-SOFA) between insults identified and unidentified group(p>0.05 for all). However, In acute insults identified group, SMHN positive patients(n=44) had a significantly higher WCC(7.2±3.9*109/L vs 4.3±3.6*109/L), CLIF-SOFA(8(6,10) vs 4(2,6)) and MELD(32(25.3,36.5) vs 13.5(10,20.5)) than SMHN negative patients(n=58)( p<0.001 for all). In the meantime, SMHN positive patients(n=25) also had both significant more severe inflammation(WCC, 7.22±3.84*109/L vs 3.75±1.61*109/L) and MOF( CLIF-SOFA, 8(7.5,10) vs 4(4,6)) than SMHN negative patients(n=47) in acute insults un-identified group.Conclusion: Combined with the gene expression data in part one,our data showed the submassive hepatic necrosis(SMHN) determined the progression of first(the damage of hepatic metabolism) and second(immune) response as well as further developing to MOF. Thus "PINRO" concept which include predisposition(cirrhosis)-> injury-> submassive hepatic necrosis(SMHN)-> deterioration of liver function(first response) /deranged systemic inflammatory response(second response)-> MOF might be better to understand the mechanism of HBV related ACLF.Part Three Analysis of gene expression signature related to bile duct construction of hepatitis B virus(HBV)-associated acute-on-chronic liver failure(ACLF)Backgroud and aims: After the occurrence of SMHN in HBV-associated ACLF patients,hepatocytes and cholangiocytes were impared,resulting in cholestasis and calling hepatic progenitor cell(HPC) into action which was in response to injury within ductular reactions(DR). This study aimed to illustrate the alteration of gene expression signature related to bile duct construction after the occurrence of SMHN.Methods: From 2008 to 2011,we screened and enrolled 33 patients undergoing liver transplantation(LT) due to HBV-associated cirrhosis with acute decompensation(AD) in Renji Hospital affiliated to School of Mecicine Shanghai Jiao Tong University. and gene expression profilings were performed on liver specimens on explant.Referring to the literatures about bile duct construction pathways, comparison of the expression of these related genes between SMHN+/- group were conducted by the use of bioinformatics BRB-Array Tools.Results: The expression of critical genes regulating the HPC to differentiate into cholangiocyte,including in the 4 pathways,TGF ?pathway(TGFB1,TGFB2, TGFBR2)?Notch pathway(JAG1,RBPJ)?WNT pathway(DVL2, DVL3, AXIN1, AXIN2, APC, LEF1, TCF7, NUMB, JAG1)?FGF pathway(FGF2, FGF7)and transcription factors HNF1 B are up-regulated in SMHN+ group,so were the genes(TGFB1?TGFB2?SOX9?HNF1B) promoting the ductal plate to form primitive ductal structures.However, genes(HES1?FOXA1?FOXA2?ONECUT1) promoting the further development into mature bile duct were down-regulated in SMHN+ group, so were the transcription factors(TBX3?CEBPA?HNF4A) promoting the differentiation of HPC into hepatocytes. The expression of critical genes(FOXA2,?HNF4A? ONECUT1) in the transcriptional networks controlling maturation of hepatocytes were down-regulated in SMHN+ group,while HNF1 A and NR5A2 showed no differences between SMHN+/- groups.Conclusion: According to the hepatic gene expression pattern, bipotential HPC( differentiate into hepatocyte or cholangiocyte) in HBV-associated ACLF patients with the presence of SMHN could more likely give rise to cholangiocyte,but no mature bile duct was formed. Meanwhile, transcription factors controlling maturation of hepatocytes were deficient.This might be the cause of cholestasis and liver metabolism dysfunction in ACLF patients. Results need further validation.