Aspartyl Beta-hydroxylase Mediated Activation Of Hepatic Stellate Cells To Promote Hepatic Fibrosis And Its Underlying Mechanism

Background and aims:Hepatic fibrosis is a common pathological state caused by various chronic liver diseases and is typically characterised by excessive deposition of extracellular matrix during repeated liver injury and repair.Although significant progress has been made in previous studies on the mechanisms of hepatic fibrosis,there is a lack of clinically proven anti-fibrotic drugs.Previous inhibitors targeting key cytokines in the hepatic fibrosis process,such as TGFβ,ultimately failed to enter clinical use due to severe adverse effects.Screening and discovering new targets for liver fibrosis treatment,and then developing new therapeutic strategies,has long been a hot and difficult issue in this field.Hepatic stellate cells（HSC）play a key role in the development and evolution of liver fibrosis.Normal HSC are transformed into myofibroblasts by the action of various cytokines under the stimulation of various injuries,and the activated HSC are able to secrete extracellular matrix components,which are deposited in the liver and promote the formation of hepatic fibrosis.Aspartyl-beta hydroxylase（aspartyl/asparaginyl beta-hydroxylase,ASPH）,a 2-OG-dependent oxygenase,is mainly involved in the hydroxylation of aspartic acid and asparagine residues in proteins,and this type of protein modification is essential for cellular adhesion,migration and signal transduction.ASPH has been reported to play an important role in calcium regulation,oxygen sensing,tumor invasion and metastasis.However,there is still a lack of in-depth and systematic research on the specific mechanism of ASPH’s role in liver fibrosis.Our group found that the expression level of ASPH in paraneoplastic tissues of hepatocellular carcinoma was correlated with the degree of hepatic fibrosis,suggesting that the expression of ASPH may affect the occurrence and development of hepatic fibrosis,but the related mechanism has not been reported.In this study,we investigated the role of ASPH in the pathological progression of hepatic fibrosis by establishing different etiology-induced liver fibrosis models in mice using in vivo and in vitro experiments,and screened the ASPH inhibitors using the ASPH-specific enzyme activity inhibitor screening platform constructed by our group to explore the role of ASPH inhibitors on hepatic fibrosis in mice,which will provide new targets for understanding the pathological mechanisms of hepatic fibrosis and the prevention and treatment of hepatic fibrosis.The results will provide new targets for the understanding of the pathological mechanism of liver fibrosis and the prevention and treatment of liver fibrosis.Methods:1.Research on ASPH expression in fibrotic liver tissues:Establishment of CCl₄ and DDC diet-induced mouse liver fibrosis animal models;WB and RT-q PCR were used to detect ASPH expression in the liver tissues of experimental model mice;Sirius staining was used to detect the degree of fibrosis of mouse livers,and immunohistochemical staining was used to detect ASPH in liver tissues of hepatic fibrosis model mice,α-SMA,an activation marker of HSCs;to detect the distribution of ASPH expression and co-expression of other cellular characteristic molecules in the fibrotic regions of the liver of CCl₄ and DDC induced fibrosis model mice by multiplex immunofluorescence;Extract primary HSCs from mice,and the expression of ASPH in HSCs was detected in different activation modes by immunofluorescence and WB methods;Establish human HSCs（LX-2 cell line）activation model by TGF-β1,and the expression levels of ASPH andα-SMA were detected by WB and RT-q PCR in the activation process of LX-2 cells;ASPH knockdown and overexpression LX-2 cells were constructed by using interfering lentivirus,and the effects of knockdown and overexpression of ASPH on the activation indexes of HSCs（α-SMA and COL1A1）.2.TGF-βaffects the process of liver fibrosis by regulating the expression of ASPH in HSCs:TGF-β1 and TGFβpathway inhibitor SB431542 were co-applied in LX-2 cells,and the expression levels of ASPH andα-SMA were detected by WB and RT-q PCR;bioinformatic prediction of the binding of transcription factors in the TGFβsignal pathway to the promoter of ASPH was carried out;and the binding of transcription factors in the TGFβsignal pathway to the ASPH promoter was predicted by bioinformatics;the regulation of ASPH expression by SMAD2 and SMAD3 was detected by dual luciferase reporter gene assay in 293T cells,;the specific binding sites of SMAD2 and SMAD3 transcription factors to the ASPH promoter were detected by CUT&Tag assay in LX-2 cells;the effect of ASPH on the TGF-β/SMAD pathway was detected by WB and RT-q PCR signal transduction process and SMAD2/3 phosphorylation level;Co IP assay to verify the existence of inter-protein binding between ASPH and LTBP1;ELISA to detect the TGF-β1 expression level after interfering with the expression of ASPH in LX-2 cells;and HDOCK and Py MOL to predict the protein binding site of ASPH and LTBP1.3.The role of ASPH inhibitors in ameliorating hepatic fibrosis:an ASPH enzyme activity assay system was established to detect the effects of the ASPH enzyme activity inhibitors（Cepharanthine and Compound 6）on ASPH enzyme activity.The expression levels of ASPH andα-SMA were detected by WB and RT-q PCR;a CCl₄-induced drug treatment model of hepatic fibrosis in mice was established,and the degree of hepatic fibrosis lesions in mice was evaluated by HE,PSR and Masson staining;the levels of liver function（ALT and AST）were detected by enzyme assay in serum,and the expression of fibrosis-related molecules in liver tissues of mice was detected by RT-q PCR.Results:1.Expression study of ASPH in fibrotic liver tissues:The results of immunohistochemical staining,PSR,Western Blot and RT-q PCR showed that the expression level of ASPH was significantly correlated with the expression level ofα-SMA in liver tissues of mouse liver fibrosis models induced by CCl₄ and DDC diets,and that the expression of ASPH increased with the severity of liver fibrosis;the expression of ASPH in the liver tissues of CCl₄ and DDC mouse multiplex immunofluorescence staining of fibrotic liver tissues revealed that there was a significant co-expression of ASPH withα-SMA,a marker of activated HSCs;in vitro experiments,the expression level of ASPH was significantly up-regulated in primary HSCs of mice activated in different ways,and HSCs were activated in LX-2 cells using TGF-β1,and the expression level of ASPH and hepatic fibrosis was significantly correlated with that of COL1A1,a correlate of hepatic fibrosis.The expression of COL1A1 andα-SMA,which are related indicators of liver fibrosis,was up-regulated and showed a concentration correlation with the concentration of TGF-β1;ASPH overexpression and interference lentivirus were obtained,and lentiviral were transfected in LX-2 cells,and the transfection efficacy of lentiviral was verified by Western-Blot and RT-q PCR.The expression ofα-SMA and COL1A1 was significantly up-regulated by overexpression of ASPH in LX-2 compared with that of control;the expression ofα-SMA and COL1A1 was inhibited by interfering with ASPH.2.TGF-βaffects the process of liver fibrosis by regulating the expression of ASPH in HSCsCombined application of TGF-β1 and TGF-β1 inhibitors in HSCs revealed that ASPH expression was regulated by TGF-β;bioinformatics analyses showed that SMAD2/3 in the TGF-βsignal pathway could bind to the ASPH promoter region and was involved in the regulation of ASPH expression;the dual-luciferase reporter gene indicated that the SMAD2/3 transcription factors binding and up-regulating ASPH expression;Cut&Tag assay verified that SMAD2/3 transcription factor binds to the ASPH promoter region;Knockdown of ASPH in LX-2 cells did not significantly affect the expression level of SMAD2/3,but significantly decreased the expression level of p SMAD2/3,suggesting that ASPH can affect the TGFβsignal pathway in the SMAD2/3 phosphorylation;Co IP showed that ASPH could directly bind to LTBP1,and ELISA assay revealed that the expression level of activated TGF-β1 was significantly reduced after knocking down ASPH in LX-2 cells,suggesting that ASPH could bind to LTBP1 to regulate the activation of TGF-β1.3.Role of ASPH inhibitors in ameliorating liver fibrosisASPH enzyme activity assay Cepharanthine and compound 6 can exert the inhibition of ASPH enzyme activity at 0.1μM;CCK-8 assay indicated Cepharanthine does not affect the proliferation of cells below 500 n M in LX-2 cells,Cepharanthine at 100 n M 500 n M inhibited the ASPH andαSMA expression;HE,PSR,Masson staining,mouse serum ALT,AST,and mouse liver RT-q PCR assays showed that administration of low and high doses of Cepharanthine reduced liver fibrosis in these mouse models either when initiated immediately after injury or when initiated after fibrosis established.CCK-8 assay indicated compound 6 did not affect cell proliferation below 50μM in LX-2 cells,compound 6 at 10μM 50μM inhibited the ASPH andαSMA expression;HE,PSR,Masson staining,mouse serum ALT,AST,and mouse liver RT-q PCR assays showed that administration of low and high doses of compound 6 reduced liver fibrosis in these mouse models either when initiated immediately after injury or when initiated after fibrosis established.Conclusions:ASPH expression was up-regulated in mouse hepatic fibrotic liver tissues and positively correlated with the severity of hepatic fibrosis,and its expression changes were mainly concentrated in HSCs.The expression of ASPH was induced and regulated by TGF-β.ASPH could bind to LTBP1 to regulate the activation of TGF-β1 and thus affect the activation of the TGF-βsignal pathway.The ASPH inhibitors（Cepharanthine and Compound 6）ameliorated CCl₄-induced liver fibrosis in mice.Those data suggested that ASPH can be a potential therapeutic target for the treatment of liver fibrosis.