Cyclooxygenase 2 Regulates Cholesterol Transport Of Hepatocytes Expressed Hepatitis B Virus X And Induces Inflammatory Activation Of Macrophages

The X protein(HBx)encoded by the Hepatitis B virus(HBV)genome X gene is essential in the replication and spread of HBV.It is involved in regulating the host cell genome stability,signal transduction and other processes.However,the relationship between lipid accumulation and inflammation-associated liver injury caused by HBx remains to be elucidated.Chronic HBV infection is closely related to diseases such as non-alcoholic steatohepatitis(NASH)in the population.As a key regulator,HBx can mediate lipid accumulation in liver.Among lipid,the increase of free cholesterol(FC)caused by the dysfunction of cholesterol transport between organelles is related to HBx-induced lipid accumulation.Caveolin 1(CAV1)is abundantly present in the membrane structure that is coupled between mitochondria and endoplasmic reticulum named mitochondrial-associated endoplasmic reticulum membrane(MAM),and is involved in cholesterol transport.Our previous research showed that the xenobiotics SPIONNPs can induce the translocation of cyclooxygenase 2(COX-2)to MAM and increased hepatocyte apoptosis.On the other hand,COX-2 is closely related hepatocyte lipid accumulation induced with HBx,but whether the molecular mechanism of MAM component regulating cholesterol transport involves the regulation of CAV1 by COX-2 remains to be explored.Most of protein translocation and activity levels are regulated by post-translational modifications such as phosphorylation modification,and COX-2 has phosphorylation sites.Therefore,it is speculated whether the translocation and activation of COX-2 is due to its phosphorylation/dephosphorylation modification.Protein phosphatase 2A(PP2A),which is abundant in mammals,plays an important role in the process of protein dephosphorylation modification and the B subunit has specific substrate recognition function.Therefore,in the dephosphorylation modification of COX-2,PP2A-B subunit has attracted our attention.The potential phosphorylation of COX-2 by the PP2A-B subunit and the translocation of MAM are the focus of our study.In addition,the liver has a unique regional immune structure and microenvironment,and contains a variety of immune cell subgroups and functional molecules.Among them,macrophages recognize damage-associated molecular patterns(DAMPs)via pattern recognition receptors(PRRs)to regulate inflammatory(such as NLRP3)transcription and activation to induce macrophage polarization and cause immunoinflammatory damage in the liver area.As a DAMPs,prostaglandin E2(PGE2)catalyzed by COX-2 activated NLRP3 to induce macrophage polarization may play an important role,but the specific mechanism remains to be elucidated.Objectives:To establish a model of COX-2 overexpression with HBx that resulting in lipid accumulation in hepatocytes and inflammatory activation in macrophages,finding out COX-2 expression and its MAM translocation are involved in PP2A-B56y and hepatocytes lipid accumulation mechanisms about HBx-induced COX-2-CAV1 related free Cholesterol(FC).Exploring liver regional immune regulation mechanisms accompanied by DAMPs(PGE2)that release to activate the immune cascade effect.These results are used to provide clues and theoretical basis in HBx-induced NAFLD/NASH caused by lipid accumulation and inflammatory injury.Methods:(1)GEO analysis:Using the GEO database(GSE89632),the mRNA levels of CAV1 and key genes for lipid synthesis(SREBF1,SREBF2)and their correlations in liver tissues of NASH patients and normal controls were analyzed.GEO database(GDS4387)was used to analyze the mRNA levels of PP2A-B subunit-related genes in liver tissues of patients with HBV-related liver injury and normal controls and use the GEO database(GSE83148)to analyze inflammation-related genes(PTGER1-PTGER4,NLRP3,NOS2,and CD68)mRNA levels,and the correlation between PPP2R5C and the aforementioned inflammation-related genes.(2)In vitro:HepG2.2.15 cell was transfected with HBx monoclonal antibody(anti-HBx)expression plasmid that was used to interfere with HBx expression,HepG2 cells were transfected with pcDNA3.1-HBx(pc3.1-HBx)plasmid 6-8 h,HepG2-Tet-ON-HBx cells were treated with DOX(1 μg/mL)for 48 h to induce HBx expression to establish a HBx expression model and detect related indicators.①WB detected the protein level of COX-2 and CAV1 with HBV/HBx.Under transfection of pcDNA3.1(pc3.1)or pc3.1-HBx,immunofluorescence(IF)experiment was used to observe COX-2/CAV1(blue fluorescent dot)fluorescence signal enhanced,and COX-2/CAV1 co-localized with MitoTracker-labeled mitochondria(red fluorescent dot)and ER-Tracker-labeled ER(green fluorescent dot),which was yellow fluorescent dots that formed to characterize MAM increased with a laser confocal microscope(confocal),showing changes in the colocalization distribution(white fluorescent dots)of COX-2 or CAV1/ER/mitochondria;MAM components of HepG2-Tet-ON-HBx cells were extracted by differential ultracentrifugation.The level of COX-2 and CAV1 protein were measured by WB,and the structure of MAM was observed by transmission electron microscope(TEM).② qRTPCR was used to detect the mRNA level of lipid-related genes in total RNA of HepG2-TetON-HBx cells;the kit was used to detect the contents of TC and FC.③HepG2 cells were used to construct HepG2-CAV1 cell line stably and highly expressing CAV1.The mRNA level of CAV1 was detected by qRT-PCR and WB was used to detect the protein level to verify the establishment of the model.After transiently transfect pc3.1 or pc3.1-HBx plasmid,confocal observed the change of FC(characterized by filipin)on MAM and observed the distribution of lipid droplets(characterized by BODIPY 493/503)and the kit detects the content of TC and FC.④HepG2-PTGS2 cells that constructed in our laboratory previous were used to transiently transfect pc3.1 or pc3.1-HBx plasmids;HepG2-Tet-ON-HBx cells were used to perform PTGS2 siRNA(siPTGS2)treatment to establish high COX-2 expression and gene intervention models,and qRT-PCR was used to detect the mRNA level of PTGS2,and WB was used to detect the COX-2 protein level to verify the establishment of the COX-2 intervention model.WB was used to detect the COX-2 and CAV1 levels in the COX-2 high-low expression model induced by HBx.confocal observed COX-2,FC and lipid droplet distribution on MAM;kit detected TC and FC content.⑤DOX dose points were used to treat HepG2-Tet-ON-HBx cells.WB was used to detect the expression of PP2A-B subunit protein and screen functional subunits under the action of HBx.⑥ HepG2.2.15 cells were transiently transfected with HBx monoclonal antibody(anti-HBx)expression plasmid to interfere with HBx expression,and WB was used to detect PP2A-B56γ protein levels.⑦HepG2-Tet-ON-HBx cells were used to observe the close proximity between COX-2 and PP2A-B56y with proximity ligation assay(PLA),and the interaction between proteins was detected by immunocoprecipitation(IP).⑧HepG2-Tet-ON-HBx cells were used for PPP2R5C siRNA(siPPP2R5C)treatment to establish a PP2A-B56y gene intervention model.WB was used to detect changes in protein levels of COX-2 and CAV1 in the PP2A-B56y gene intervention model induced by DOX.Confocal observed the changes of COX-2 on MAM;the kit detected the contents of TC and FC.⑨Using human liver L02 cells in the early stage of the laboratory,targeting serine(S)582,583,584,586 or 601 at the C-terminus of COX-2 to construct stable and high expression mimicking COX-2 hyperphosphorylation(D)or Site-directed mutant cell lines with low phosphorylation(A),WB detected the expression levels of COX-2 and CAV1 proteins in each mutant cell line,and screened the functional site of COX-2 to regulate CAV1 was COX-2Ser584.Confocal observed the distribution of CAV1 on MAM;the kit detected the content of TC and FC.⑩Coculture of HBx/HBV-expressing hepatocytes and human mononuclear macrophage cell line THP-1 cells in a transwell chamber to establish a liver regional immune exposure model;siPTGS2 treatment in liver cells,and EP4 antagonist ONO-AE3-208 in macrophages or NLRP3 small molecule inhibitor CY-09 treated PMA-induced THP-1 cells as an intervention group.WB detects COX-2 protein levels in hepatocytes.ELISA kit detected hepatocyte layer PGE2 and macrophage layer IL-1β release levels in co-culture systems;qRT-PCR detects PGE2 receptor family(EP1-EP4)genes(PTGER1-PTGER4)mRNA level,WB detected NLRP3 inflammatory related molecules,iNOS,CD206 protein levels in cells and cytoplasm and nuclear components.(3)In vivo:8-10 weeks HBx transgenic C57BL/6 mice(HBx-Tg mice)were used to establish HBx exposure model and wild type(WT)C57BL/6 mice were used as controls,and they are performing COX-2 selective intervention agent celecoxib(CELE,30 mg/kg)to establish an intervention model.And a methionine-choline deficiency diet(MCD)diet induced a positive model of NAFLD/NASH was used.①WB was used to detect the protein levels of HBx,PP2A-B56γ,COX-2 and CAV1,NLRP3 inflammatory related factors(NLRP3,ASC,IL1β)in liver tissue ②TEM was used to observe MAM structural changes and lipid droplet levels.Mouse serum samples and liver tissue samples were used.①to detect liver function indicators such as AST and ALT.②TC and TG content were detected by kits.③FC content were detected by kit.④enzyme-linked immunosorbent assay(ELISA)was used to detect the release levels of inflammatory factors PGE2 and IL-1β.Mouse liver tissue sections were used for ①Oil Red O staining to observe changes in lipid droplet levels in frozen sections;②Immunohistochemical experiment(IHC)detection to observe changes in various molecules in liver tissue;③Sirius red experiment to observe collagen deposition;④Hematoxylin-eosin(HE)stained sections were used to observe the inflammatory foci of liver tissue.Results:(1)GEO analysis:According to database analysis,compared with the healthy control,mRNA levels of PPP2R5C in PP2A-B subunits in patients with HBV-related liver injury were significantly increased.In HBV-infected patients,inflammation-related genes(PTGER1-PTGER4,NLRP3,NOS2,CD68)mRNA levels were significantly increased,and both were significantly positively correlated with PPP2R5C.(2)In vitro:①Compared with HepG2 cells,the expression of HBx in HepG2.2.15 cells carrying the HBV genome and transfection with anti-HBx plasmid can significantly inhibit the expression of HBx in the cells.Accordingly,compared with HepG2 cells,the COX-2 protein level increased and the CAV1 protein level decreased in HepG2.2.15 cells,while the COX-2 protein level decreased and the CAV1 protein level increased in the anti-HBx treatment group.Compared with the control group,DOX-treated HepG2-Tet-ON-HBx COX-2 protein levels increased,CAV1 decreased,COX-2 on MAM components increased,CAV1 decreased,and the cell MAM spacing distance increased significantly,approximately three times that of control cells(P<0.05).Compared with pc3.1 plasmid-transfected group,the COX-2(blue fluorescent dot)fluorescence signal in the pc3.1-HBx plasmid-transfected HepG2 cells was enhanced,which was expressed as a colocalized distribution of COX-2/ER/mitochondria(white fluorescent dots)increased.At the same time,a decrease in CAV1 fluorescence signal and a decrease in colocalization distribution with MAM were also observed.②Compared with the control group,genes that involved in intracellular lipid metabolism(SREBP1,PPARG,PPARA,CPT1A)in the DOX treatment group,the CAV1 mRNA level was significantly reduced(P<0.05),and the contents of TC and FC were significantly increased(P<0.05).③Compared with pc3.1-HBx-transfected HepG2-pB group,the distribution of FC on MAM and the number of LD in pc3.1-HBx-transfected HepG2-CAV1 cells decreased significantly(P<0.05),TC and FC levels were significantly reduced(P<0.05).④In the model of high and low expression of COX-2,compared with the control group,the CAV1 protein level decreases when COX-2 is highly expressed,and the CAV1 protein level increased when COX-2 knock down;distribution of FC and LD was increased in cells of over-expression COX-2.While the distribution of FC in MAM decreased and the distribution of LD decreased when COX-2 was low-expressed;the content of TC and FC increased when COX-2 was highly expressed.⑤Compared with the control group,the protein level of PP2A-B56y increased significantly with DOX treatment.⑥Compared with control HepG2 cells,the level of PP2A-B56y protein in HepG2.2.15 cells increased,and decreased with anti-HBx treatment.⑦Compared with the control group,the proximity effect between COX-2 and PP2AB56γ was increased in the DOX-treated group(P<0.05),and the interaction between COX-2 and PP2A-B56γ protein was enhanced by IP.⑧Compared with the control group,COX-2 protein level upregulated,CAV1 protein level downregulated,COX-2 distribution in MAM decreased,and TC and FC contents decreased in the siPPP2R5C combined DOX treatment group.⑨Compared with L02-pBabe and other high and low phosphorylation site mutation cell lines,CAV1 protein level is reduced in L02-COX-2S584A cells(low phosphorylation),and CAV1 is increased in L02-COX-2S584D(high phosphorylation);Moreover,compared with L02-COX-2S584D cells,the distribution of CAV1 in MAM,the number of LDs,TC and FC levels in L02-COX-2S584A cells were significantly increased.⑩Compared with control cells,the release of PGE2 was increased in HBx/HBV-expressing hepatocytes cultured in a single layer.Compared with the control-treated hepatocyte-THP-1 coculture,PGE2 and IL-1β increased in co-cultured with DOX-treated hepatocyte-THP-1,and NFκB(p65)decreased in the cytoplasm and increased in the nucleus.In this model,compared with control,NLRP3 and other inflammatory associated molecules were reversed by ONO-AE3-208 and CY-09.(3)In vivo:Compared with WT mice,HBx-Tg mice liver tissue:①increased lipid droplet content;②increased TC,TG and FC content;TEM observed increased MAM distance and increased lipid droplets;③increased number of inflammation foci;④increased collagen deposition;⑤increased COX-2,NLRP3 inflammatory and M1 polarization-related proteins;⑥increased COX-2,PP2A-B56y positive staining and decreased CAV1 positive staining;⑦PGE2 and IL-1 secretion increase.Compared with the HBx-Tg mouse group,the above indexes were rescued in the mice combined with the CELE intervention of HBx-Tg group;Compared with the MCD-diet WT mice group,there are more serious vacuolization in liver tissue sections of MCD-diet HBx-Tg group except above indicators increased.Conclusions:This study clarified that HBx promotes the increase of COX-2 expression and the translocation of MAM to cause cholesterol metabolism disorder and lipid accumulation.At the same time,the expression of COX-2 usually catalyzes the synthesis of PGE2 to mediate intercellular communication,triggering local immune-dependent liver injury,provide a theoretical basis for exploring the regulation mechanism and intervention target of NAFLD caused by HBx hepatotoxicity.