The Preliminary Study Of Aspartyl-(Asparaginyl)-?-Hydroxylase In Fibrosis And Its Mechanism

?Objective? In this study, we investigated a central role of Aspartyl/Asparaginyl ?-ydroxylase(ASPH) in hepatic fibrosis and demonstrated the mechanism in regulating the pro-fibrotic action of ASPH in this progression.?Methods? To investigate ASPH action in vivo,we first assessed the progression of liver fibrosis in wild-type mice by bile duct ligation(BDL) treatment or chronic administration of carbon tetrachloride(CCl4). Here, we established a mice strain in which the genomic DNA encoding the enzymatic domain of ASPH was selectively mutated(ASPHdel) through CRISPR/Cas9, causing a lost of hydroxylase activity. We then assessed the progression of liver fibrosis induced by bile duct ligation(BDL) treatment in ASPHdel and wild-type mice.Finally, the in vivo antifibrotic efficacy of small molecule inhibitors(MO-I-1100) which was selected for reducing ASPH enzymatic activity was assessed in mice with liver fibrosis induced by CCl4 and bile duct ligation.?Results? We found a dramatic up-regulation of plasma ASPH in mice with BDL treatment.Carbon tetrachloride administration caused a robust increase in ASPH m RNA expression in the liver parenchyma as assessed by PCR and a slight decrease in Jagged-2 protein expression as assessed by Western blot. Limited PCR performed on tail DNA showed that the intensity of the ASPH PCR product was absent from ASPHdel mice. Additionally little amounts of immunoreactive ASPH in ASPHdel mice compared to wild-type mice were demonstrated by Western blot of li ver l ysates. T hen, we unexpectedly fo und t he reduced expression of C ollagen ?I,MMP2,TIMP1,a-SMA,TGF-?1,IL-1? and TNF-? in ASPHdel mice as well as the elevated expression of Jagged-2 in comparison with those in wild-type mice by bile duct ligation, and Sirius red staining showed that collagen deposition was even more reduced in ASPHdel mice. More importantly, we found more severe hepatonecrosis in the ASPHdel mice upon BDL treatment than that in wild-type mice.Eventually,We observed reduced m RNA expression of Collagen ??,TIMP-1,MMP-2,Jagged-2,Notch-3,IL-1? and TNF-? in the MO-I-1100-treated CCl4 livers.?Conclusions? Collectively, our preliminary data suggested the hydroxylase activity of ASPH might play an important role to promote the progression of liver fibrosis in down-regulating Jagged-2 expression to accelerate the synthesis of inflammatory factors by enhacing activation of Hepatic Stellate Cells and secretion of extracellular matrix.