Mechanism Of Cyclooxygenase-2 On The Regulated Necroptosis And Lipid Accumulation In Hepatocytes Induced By The Combined Exposure Of Aflatoxin B1 And Hepatitis B Virus X Protein

Aflatoxin B1(AFB1),hepatitis B virus(HBV)infection,high-fat diet,intestinal microbiota,and genetic factors all lead to lipid accumulation in hepatocytes.AFB1 intake can cause disturbance of lipid metabolism in liver cells in human and animals.Hepatitis B virus x protein(HBx)is one of the four constituent proteins of HBV which can cause liver lipid accumulation.Studies have shown that AFB1 or HBx exposure can cause hepatic lipid accumulation in human and animals,but the combination of the two exposure induced mitochondrial COX-2 dependent death patterns and regulatory mechanisms have not yet been clarified.Necroptosis is a form of programmed cell death,and the formation of necrosomes is a signal of its initiation.Necroptosis plays an important role in the process of liver injury and disease.Inhibition of necroptosis can reduce alcohol-induced hepatic lipid accumulation.Mitochondrial dynamics is one of the important molecular events that affect lipid accumulation in hepatocytes.The necrosomes formed by receptor-interacting protein kinase(RIP)1 and RIP3 localizes to mitochondria,and RIP3 interacts with phosphoglycerate mutase family protein 5(PGAM5),resulting in increased mitochondrial fission and reduced fusion.Mitochondrial fusion protein 2(Mfn2)promotes mitochondrial? oxidation,accelerates fatty acid metabolism,and slows down lipid accumulation.Fatty acids enter the mitochondria via carnitine palmitoyltransferase 1A(CPT1A)to oxidize fatty acids and provide energy to cells.A decrease in CPT1A leads to accumulation of lipids in hepatocytes.In the process of HBx expression combined with AFB1 exposure induced lipid accumulation in hepatocytes,the mode of mitochondrial dynamic changes and the regulation of mitochondria-associated target molecules on lipid accumulation remain to be elucidated.Cyclooxygenase 2(COX-2)is not expressed under most physiological conditions and can be induced by external environmental stimuli such as common environmental pollutants and viral infections.It is a protein that can regulate hepatic lipid accumulation.Our group has found that HBx expression combined with AFB1 exposure can induce elevated COX-2 expression and mitochondrial translocation in liver cells,and participate in mitochondrial quality control(MQC)and dynamic changes;suggesting the relationship between mitochondrial COX-2,hepatotoxicity and lipid accumulation and its mediating liver toxicity damage,and the regulatory mechanisms involved in necroptosis remain to be further studied.Objectives:This study aims to investigate the combined toxic effects of HBx expression and AFB1 exposure via hepatocyte lipid accumulation induced by necroptosis,and to explore the interaction between COX-2-induced expression of RIP3 and its mitochondrial translocation,their interactions with mitochondria,oxidative mediation of lipid metabolism and molecular mechanisms involved in necroptosis regulation pathway.The results will provide a basis for potential targets and strategies for the prevention and treatment of hepatic mitochondria-dependent deaths in liver.Methods:(1)In vitro:The three different groups of HBx-expressing hepatocytes used in this study include human hepatocyte HepG2 cell line,HepG2.2.15 cells with stable HBV genome replication(including HBx expression);HepG2-Tet-ON-HBx cells with Tet-ON that are switchable(inducible by DOX expression);human hepatic progenitor cell line HepaRG cells with metabolic enzyme activity,and differentiated cells induced by DMSO.The above cells were grouped and tested as follows:(DHepG2 and HepG2.2.15 cells were treated with solvent(DMSO)and AFB1(1?mol/L)for 48 h or 72 h;the HepG2(Ctrl)and HepG2.2.15 control group(HBV expression group containing HBx expression)were used as the control group of AFB1 exposure group,and AFB1 exposure group of HepG2.2.15(HBV+AFB1 group)respectively.?HepG2-Tet-ON-HBx cells were treated with DOX(1?g/mL)to induce HBx expression,and then treated with solvent(DMSO)and AFB1(1?mol/L)for 48 h or 72 h and divided into four groups:(Ctrl),AFB1 exposure group,HBx expression group(DOX+DMSO)and HBx expression combined with AFB1 exposure group(HBx+AFB1).?HepaRG cells were induced to differentiate in 1.8%DMSO for 28 days.Real-time quantitative PCR(qRT-PCR)was used to detect mRNA levels related to liver function(HNF4A,ALB),metabolism-related genes(NR112,RXR,CYP1A2,CYP3A4,GSTM1)in HepaRG cells.HepaRG cells were then transfected with pcDNA3.1-HBx or control plasmid(1?g/mL)for 12 h and treated with AFB1(0.5?mol/L)for another 48 h or 72 h;divided into control group(Ctrl),AFB1 exposure group,HBx expression group(pHBx+DMSO)and HBx expression combining with AFB1 exposure group(pHBx+AFB1)?The above groups of hepatocytes were treated for 48 h,and the mRNA levels of PPARA,PPARG,CPT1A,PTGS2,and ACTB were detected by qRT-PCR.The necroptosis-related proteins(RIP1,RIP3,p-MLKL),mitochondrial dynamic related protein(Drp1,p-Drp1ser616,Mfn2),and COX-2 expression levels were detected by Western blot(WB).?The above groups of hepatocytes were treated with oil O red for 72 h,lipid droplet distribution in the cells was observed and semi-quantitative analysis was performed.The total cholesterol(TC)content in cells was detected by TC enzyme method,and the total triglyceride(TG)content of cells was detected by TG enzyme method.?The control group(Ctrl)and HBx expression combining with AFB1 exposure group(HBx+AFB1)above were pretreated with RIP1 inhibitor Nec-1(15?mol/L)for 12 h,respectively,and detect cell viability using MTT colorimetric method.?Necroptotic intervention model using NS A(1 mmol/L)pretreated HepaRG cells in the control group(Ctrl),HBx expression combining with AFB1 exposure group(pHBx+AFB1)for 12 h,WB was used to detect mitochondrial lipid metabolism-related proteins(CPT1A,StAR,PPAR?,PPAR?),and the contents of TC and TG in cells were detected.Oil red O staining was used to observe the intracellular lipid droplet distribution and semi-quantitative analysis.?Gene database was used to analyze the expression levels and correlation between PTGS2,MLKL and DNM1L in liver tissue of chronically infected HBV patients.Furthermore,in the Drpl intervention model,HepG2-Tet-ON-HBx cells were treated with Drpl siRNA(siDNM1L).WB was used to detect necroptosis and mitochondrial dynamic related protein expression.Immunofluorescence(IF)was used to observe the mitochondrial morphology and detect the intracellular distribution of p-Drp1Ser616 protein.The content of TC and TG in the cells was detected.Oil red O staining and semi-quantitative analysis was used to observe the lipid droplet distribution in the cells.?COX-2 intervention model was used in HepG2-Tet-ON-HBx cells with COX-2 small interfering RNA(siPTGS2).WB was used to detect necroptosis and mitochondrial dynamic related protein expression.IF was used to detect COX-2 and mitochondrial outer membrane marker protein(TOM20).Co-localization distribution,orbital ligation assay(PLA)detects the spatial proximity of COX-2 and RIP3;Co-IP detects the interaction between COX-2 and RIP3;detection of TC and TG content in cells,oil red O staining was used to observe intracellular lipid droplet distribution and semi-quantitative analysis.?HepG2-Cas9-PTGS2 and control cells HepG2-Cas9-NC were constructed using CRISPR/Cas9 technology;12 h after transfection with pcDNA3.1-HBx plasmid,AFB1(1?mol/L)was treated for 48 h or 72 h as HBx+AFB1 exposed group;pcDNA3.1 plasmid were transfected and treated with equal volume of DMSO as control group;necroptosis and mitochondrial dynamic-related protein were detected,mitochondrial components were also extracted to detect RIP3 and COX-2 proteins' translocation to mitochondrial;the content of TC,oil red O staining and semi-quantitative analysis were used to observe the intracellular lipid droplet distribution.(2)Ex vivo:Human liver chimeric(HLC)mice were used to construct HBV infected(including HBx expression)model by injecting HepaAD38 cells(induced with tetracycline)supernatant which contains HBV virus particles;HLC mice injected with HepaAD38 cells without HBV virus particles(induction with tetracycline)were used as controls.The ALB or HNF4? were marked to sort out the regenerated human primary hepatocytes(PHHs)by flow cytometry,and then AFB1(0.5?mol/L)was treated for 48 h or 72 h.The volume of DMSO was used as a solvent control.Control group(Ctrl),AFB1 exposure group,HBV infection(HBx expression)group,and HBV infection combined with AFB1 exposure group(HBV+AFB1).The cell morphology was observed and the following experiments were performed:?qRT-PCR was performed to detect mRNA levels of specific human liver genes(HNF4A,ALB)and metabolism-related genes(CYP1A2,CYP2B6,CYP3A4).?WB was used to detect the expression of necroptosis-related proteins(RIP 1,RIP3,p-MLKL),mitochondrial dynamic-related proteins(Drp1,p-Drp1Ser616 Mfn2),mitochondrial?-oxidation-related protein CPT1A and COX-2.?TC enzyme method and TG enzyme method were used to detect the content of TC and TG in the cells.Oil red O staining was used to observe the distribution of lipid droplets in the cells and semi-quantitative analysis was performed.(3)In vivo:First,12-month-old HBx transgenic(HBx-Tg)mice and wild-type(WT)C57BL/6J mice were sacrificed to detect?FABP1 PPARA,PPARG,CPT1A,PTGS2,and ACTB mRNA level by qRT-PCR.?WB detection of liver HBx,COX-2,mitochondrial dynamics and lipid anabolism related proteins in mice.?The contents of TC and TG in liver tissue were detected.After frozen section of liver tissue,oil red O staining was used to observe lipid droplet distribution and semi-quantitative analysis in liver tissue.HE staining was used to observe the pathological changes of the liver.Next,6-month-old HBV-transgenic(HBV-Tg)mice and wild-type(WT)mice were further treated with AFB1(5 mg/kg.bw)by intraperitoneal injection as HBV(containing HBx expression)+AFB1 group and AFB1 exposure groups,and equal volume of solvent(peanut oil)were used as HBV(containing HBx)expression group and control group(Ctrl),the injections were performed once every two days,and injected three times within a week to construct an in vivo AFB1 exposure model.?After the last exposure of 24 hours,the blood was taken by removalling eyeballs.The whole blood and liver tissue were used for experiments.?The whole blood was still.standing at 37? for 15 min and then centrifuged at 3000 rpm for 15 min.The upper serum was used to measure the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST).WB detected liver HBx expression in mice,as well as COX-2,necroptosis,mitochondrial dynamics-related proteins,CPT1A and so on.?HE staining was used to observe the pathological changes of the liver.?Immunohistochemistry(IHC)assay detected the distribution and expression of RIP3,COX-2,p-Drp1Ser616,HBx and other proteins in liver tissue.?TC and TG content were detected in liver tissue;oil O red staining was perfprmed to observe intracellular lipid droplet distribution in frozen section of liver tissue.Results:(1)In vitro:Firstly,comparing with the control groups,HepG2.2.15 and HepG2 cells,HepG2-Tet-ON-HBx cells,HepaRG cells,?the level of PPARG mRNA level was slightly increased in HepG2 cells exposed to AFB1,and the mRNA level of PPARA and mitochondrial ?-oxidation rate-limiting enzyme CPTIA increased slightly(P>0.05);The mRNA levels of PPARG,PPARA and CPT1A in neither AFB1 or HBV/HBx group changed(P>0.05);the PPARG mRNA level in HBV+AFB1 group was significantly increased 1-2 folds(P<0.05),and the mRNA levels of PPARA and CPT1A were slightly increased(P<0.05,P>0.05).The above results were similarly verified in HBx and AFB1treated HepG2-Tet-ON-HBx cells.In HepaRG cells treated with HBx+AFB1,mRNA levels of liver function-related genes(HNF4A,ALB)and metabolism-related genes(NR1I2,RXR,CYP1A2,CYP3A4,GSTM1)were significantly increased(P<0.05),suggesting those are related to HepaRG's metabolic enzymes activation.The PPARG mRNA level in HepaRG cells exposed to AFB1 or HBx expression group was significantly increased by 5 folds(P<0.05),PPARA mRNA level was decreased to 50%(P<0.05),and CPT1A mRNA level was increased by 2 folds(P<0.05);The level of PPARG mRNA in HepaRG cells in AFB1 group was significantly increased by 16 folds(P<0.05),PPARA mRNA level was decreased to 50%(P<0.05),and CPT1A mRNA level was increased by 5 folds(P<0.05).?Three different kinds of hepatocytes were treated for 48 h.In HepG2.2.15 cells and HepG2 cells,necroptosis,mitochondrial dynamic-related proteins,and COX-2 expression levels were changed in HepG2 cells exposed to AFB1;In HepG2.2.15 cells(HBV protein expression group),RIP1,RIP3 and p-MLKL proteins,Drp1 and p-Drp1Ser616,and COX-2 protein levels increased,Mfn2 expression decreased;necroptosis-related proteins,mitochondria dynamic-related proteins in HepG2.2.15 cells.The expression levels of dynamic related protein and COX-2 were significantly increased compared with the single treatment group,and the expression level of Mfn2 was significantly decreased in HBV+AFB1 group.The above results were also verified in HepG2-Tet-ON-HBx cells and HepaRG cells.?After three different groups of hepatocytes treatment for 72 h,the levels of TC and TG in HepG2.2.15 and HepG2 cells remained unchanged in the AFB1 exposed group and HBV protein expression group except TG level increased in HepaRG transfected with pcDNA3.1-HBx,and the TC levels in HBV+AFB1 group increased significantly(P<0.05).The above results were also verified in HepG2-Tet-ON-HBx cells and HepaRG cells.In HepG2.2.15 cells and HepG2 cells,Oil Lip O staining revealed a slight increase in lipid droplets in the average single cells in the AFB1 exposed group and the HBV protein expression group(P>0.05).The distribution of lipid droplets in the HBV+AFB1 group cells was increased and averaged individually.The lipid droplets in the cells were significantly increased(P<0.05);the above results were also confirmed in HepG2-Tet-ON-HBx cells and HepaRG cells.?Three different levels of hepatocytes,MTT results showed that in HepG2.2.15 cells and HepG2 cells,the cell viability of AFB1 exposed group and HBV protein expression group was decreased,and the survival rate of HBV+AFB1 group cells was significantly decreased(P<0.05);The survival rate of HBV+AFB1 cells was increased by Nec-1 treatment(P<0.05).The above results were also confirmed in HepG2-Tet-ON-HBx cells and HepaRG cells.Secondly,?comparing with the control group(Ctrl),the expression of mitochondrial division-related protein in HepaRG cells in pHBx+AFB1 group increased,the expression of Mfn2 and CPT1A decreased;after pretreatment in HepaRG cells with NS A in pHBx+AFB1 group,the expression of mitochondrial division-associated protein was decreased to the level of control group,the expression of fusion-associated protein and CPT1A increased to the level of the control group;?comparing with the control group(Ctrl),the TC content in pHBx+AFB1 treated HepaRG cells was significantly increased(P<0.05).The distribution of lipid droplets in oil red O stained cells increased;the TC content in HepaRG cells treated with Nec-1 or NSA was lower than that in pHBx+AFB1 group(P<0.05),but there's no significant difference in control group(P>0.05).Thirdly,?from GSE83148 database,GEO analysis found that the DNM1L,PTGS2,and MLKL mRNA levels were significantly higher in chronic HBV infected individuals(n=122)than in normal population(n=6),and PTGS2 and DNMIL(r=0.5116).(P<0.05)and MLKL(r=0.5816),(P<0.05)were both significantly correlated.Furthermore,in HepG2-Tet-ON-HBx cells exposed to HBx+AFB1,siDNM1L reduced Drpl,p-MLKL,and RIP3,restored mitochondrial morphology,decreased the intracellular distribution of p-Drp1Ser616 protein,and reduced the expression of p-Drp1Ser616,Mfn2 expression increased,TC content decreased,intracellular lipid droplets stained by oil red O decreased.Fourthly,?the expression of COX-2,necroptosis-related protein and mitochondrial division related protein p-Drp1ser616 was decreased in HepG2-Tet-ON-HBx cells exposed to siPTGS2 in the HBx+AFB1 exposure group,and Mfn2 expression partially rescued and recovered.COX-2 co-localization with TOM20 decreased,the number of PLA fluorescence foci of COX-2 and RIP3 decreased,the Co-IP expression level of COX-2 and RIP3 decreased,TC content decreased,and content of intracellular lipid droplets decreased by oil red O staining.Fifthly,?by recombinant plasmid sequencing,qRT and WB cell identification of transfected cells,the results proved that HepG2-Cas9-PTGS2 cells were successfully constructed,the knocked down efficiency of COX-2 in cells was 50.95%±1.82%;comparing with control in HepG2-Cas9-NC cells,the expression of necroptosis-related protein(RIP1,RIP3,p-MLKL),mitochondrial dynamic-related protein(Drp1,p-Drp1Ser616)decreased,and the expression of Mfn2 and CPT1A increased in the combined exposure group of HBx+AFB1.The distribution of RIP3 and COX-2 in the mitochondrial fraction decreased;the level of TC in the cells decreased(P<0.05),and lipid droplet distribution of oil red O staining per unit area decreased(P<0.05).There was no significant difference in the above phenomena in HepG2-Cas9-PTGS2 cells.(2)Ex vivo:?PHHs were cultured after perfusion,the cell morphology was round,oval or polygonal,like mint egg-like,with more binucleated and multinucleated cells.?Compared with mouse hepatocytes,HNF4A and ALB mRNA levels were highly expressed in PHHs.IHC staining also detected ALB expression in HLC mouse livers,suggesting those are human primary hepatocytes;compared with immortalized HepG2 cells,CYP1A2,CYP2B6,CYP3A4 mRNA levels in PHHs were significantly increased(P<0.05),suggesting that PHHs have good potential metabolic activity.?Compared with PHHs from uninfected HLC mice,the expression of HBx protein in PHHs from HBV-infected mice were significantly higher than those of uninfected HLC mice,indicating that HBx was expressed in HBV-infected PHHs.?At the same time,compared with the control group,the expression levels of CYP1A2,CYP2B6,and CYP3A4 mRNA in HBV infection(HBx expression)group and AFB1 exposure group were significantly higher(P<0.05);HBV+AFB1 was compared with HBV infection group and AFB1 exposure group.The expression levels of CYP1A2,CYP2B6,and CYP3A4 mRNA in the exposed group were further increased(P<0.05);there was no significant change in the cell morphology in each group.?Compared with the control group,the expression levels of RIP1,RIP3 and p-MLKL,Drp1 and p-Drp1Ser616,CPT1A,and COX-2 in HBV infection(HBx expression)group and AFB1 exposure group cells were not significantly changed;and HBV infection(HBx expression)The levels of RIP1,RIP3 and p-MLKL,Drp1 and p-Drp1Ser616 in the HBV+AFB1 group were higher than those in the AFB1 group,and the level of CPT1A was decreased,suggesting changes in mitochondrial dynamics and lipid oxidation.?Compared with the control group,the TC and TG contents in the HBV-infected(HBx-expressing)and AFB1-exposed groups were increased,and the distribution of lipid droplets in the oil O red-stained cells increased;compared with the HBV-infected(HBx-expressing)group and the AFB1 exposed group,HBV and AFB1 group TC and TG levels were significantly higher,the distribution of lipid droplets increased(P<0.05).Results Validation of HBV infection in humans(HBx expression)hepatocytes combined with AFB1 exposure can induce necroptosis,mitochondrial dynamic changes,and lipid accumulation in PHHs(3)In vivo:Firstly,?in the liver tissue of HBx-Tg mice,the lipid synthesis-related FABP1 and PPARG genes and PTGS2 mRNA levels were significantly increased in the liver tissue of HBx-Tg mice,and the fatty acid metabolism-related gene PPARA in liver cells was observed.CPT1A mRNA levels decreased.?HBx,COX-2,PPARy protein expression levels increased,PPARa and CPT1A protein expression decreased.?The level of TC and TG increased;the lipid droplet distribution of oil O red staining increased in liver tissue(P<0.05).?HE staining liver cells in HBx-Tg mice were observed,suggesting that the liver may have lipid accumulationSecondly,compared with WT mice,?HBV protein(including HBx)expression group,AFB1 exposure group,and HBV(including HBx expression)+AFB1 group were smaller in HBV-Tg mice than in WT control group in each treatment group.The levels of ALT and AST increased in the serum of mice(P<0.05),but there was no significant difference between the HBV(including HBx)+AFB1 group and the AFB1 alone or HBV alone(including HBx)groups.?HBx expression in hepatic tissue of HBV-Tg mice,liver tissue expression of HBV protein(including HBx)and AFB1 exposed mice liver tissue necroptosis and mitochondrial dynamic related protein,and COX-2 protein expression slightly increased;HBV(including Expression of RIP 1,RIP3,p-MLKL,Drp1,p-Drp1ser616,and COX-2 in liver tissue of HBx+AFB1 group was significantly increased,and CPT1A expression was significantly decreased.?Liver pathological examination revealed that hepatocytes in HBV(containing HBx expression)+AFB1 group had vacuolization of hepatocytes,deep staining of nuclei,rupture of hepatic cords,and inflammatory infiltration.?The proportions and expression levels of positive RIP3,COX-2,and p-Drp1ser616 proteins in hepatocytes in HBV protein(including HBx)-expressing group and AFB 1-exposed group were increased,and HBV(containing HBx expression)+The expression levels of HBx,RIP3,COX-2,and p-Drp1Ser616 proteins in the liver of AFB1 mice were increased at the same site of liver cells in serial sections,suggesting that COX-2 was expressed in HB V infection(HBx expression)combining with AFB1-exposed liver tissue(hepatocytes),and mitochondrial dynamics,and necroptosis associated with and potential regulatory effects.?The levels of TC and TG in the liver of HBV protein(including HBx)-expressing group and AFB1-exposed group were slightly increased,and the distribution of lipid droplets in oil red O-stained liver cells was slightly increased;TC in the liver of mice infected with HBV(containing HBx)+AFB1 group The level of TG was significantly increased,and lipid droplet distribution of oil O red staining in hepatocytes was significantly increased(P<0.05).It is suggested that HBV infection(HBx expression)combining with AFB1 exposure mediates intracellular lipid accumulation in liver tissue.Conclusions:Establishment of in vitro,ex vivo and in vivo models of HBx-expressing hepatocytes and HBV-infected liver(hepatocytes)combined with AFB1 exposure can induce necroptosis of hepatocytes and mediate lipid accumulation in hepatocytes,and induce hepatocyte COX-2 expression The interaction between mitochondrial translocation and RIP3 mediates mitochondrial dynamic and lipid accumulation.Targeted intervention of COX-2 expression can regulate HBx expression and AFB1 co-exposure-induced hepatocyte necroptosis and lipid accumulation.The results provide a basis for further exploring the regulatory mechanisms of hepatocyte toxicity induced by combined exposure of HBx and AFB1 and exploring potential targets for prevention and control of liver injury and related diseases.