| Non-alcoholic fatty liver disease(NAFLD)is the most common form of chronic liver disease in the world.It is characterized by steatosis and insulin resistance(IR),which can cause a variety of complications and related deaths.The common cause of NAFLD is hepatitis B virus(HBV)infection,which induces liver steatosis and IR through x protein(HBx).The occurrence of IR is accompanied by increased liver glycogen phosphorylase(PYGL)activity to promote glycogenolysis.Moreover,IR is closely related to endoplasmic reticulum(ER)stress and its autophagy.ER stress is an important pathological mechanism of NAFLD caused by HBV.It inhibits insulin receptor substrate 1(IRS-1)signaling by activating transcription factor 4 to promote IR.Reticulophagy regulator 1(RETREG1,also known as FAM134B),as an ER resident protein,maintains ER homeostasis by participating in lysosome(Lyso)-mediated catabolism of stress/damaging ER.ER is an important membrane source of extracellular vesicles(EVs),Lyso degradation can inhibit the biological occurrence of EVs.Autophagy disorder promotes the release of EVs from hepatocytes to activate hepatic stellate cells(HSCs)and mediate liver fibrosis.In summary,whether ER-phagy mediates hepatocyte insulin sensitivity and activation of hepatic stellate cells by hepatic glycogen metabolism and EVs release,and participates in the phenomenon and regulation mechanism of HBV/HBx-induced NAFLD injury remains to be elucidated.Objective:In order to explore FAM134B-dependent ER-phagy regulation and molecular mechanism in HBV/HBx-related NAFLD.By establishing an in vivo and in vitro experimental model of HBx expression inducing human hepatocyte insulin sensitivity decrease and activation of HSCs,to prove mechanism of FAM134B regulates PYGL in HBx-induced glycogen metabolism disorders for causing insulin sensitivity decrease,to prove the function of FAM134B mediating ER-phagy to participate in the CD63-related EVs-Lyso degradation pathway for regulating EVs release,and to explore the function of FAM134B regulating RBPs mediating miR-122 and other sorting into EVs and inducing HSCs activation via EVs Function.Finally clarifying that FAM134B-mediated ER autophagosome communicating with Lyso plays the role on HBx-induced hepatocyte releases EVs and intercellular communication,to provide clues for screening intervention targets in HBx-induced liver insulin resistance and HSCs activation-induced NAFLD.Methods:(1)GEO analysis:The GEO database was used to analyze the relative transcription levels and correlations of autophagy-related genes(FAM134B,SQSTM1,LC3B)and EVs marker protein-related TSG101 genes in liver tissue samples from HBV-infected people.(2)In vitro:Give human liver cancer HepG2 cells pcDNA3.1-HBV,-HBx,-HBV-xNULL or control plasmid transient(1.0 μg)transfection for 8 hours,HepG2-Tet-ON-HBx cells with Tet-ON switch(expressed by DOX)were treated with DOX(0.25,0.5,1.0,2.0,4.0 μg/mL)for 24 h to establish an HBx expression model.Insulin INS(0.2 IU/mL,0.5h)was administered to establish an insulin induction model.Palm Acid PA(120μmol/L,24 h)was administered to establish insulin resistance positive cell model.HepG2.2.15 cells were used to transfect HBx monoclonal antibody(anti-HBx)expression plasmid to interfere with HBx expression;LX2 cells were used as HSCs activation model.Autophagy inhibitor CQ(10μmol/L,24 h),autophagy agonist CCCP(15 μmol/L,24 h),RAPA(10 μg/L,24 h),ATF4 small interfering RNA(siATF4),FAM134B small interfering RNA(siFAM134B)intervention model was validated;CRISPR/Cas9 technology was used to construct HepG2-Cas9-FAM134B with stable and low expression of FAM134B and its control cells.①Cell triglyceride enzymatic method to measure intracellular triglyceride levels.②Cell glycogen content detection kit to measure intracellular glycogen content levels,Cell glycogen PAS staining to observe intracellular glycogen distribution,Cell glycogen synthase(GCS)activity detection kit to measure the level of glycogen synthesis in hepatocytes,glycogen phosphorylase a(GPa)activity detection kit to measure the level of glycogen decomposition in hepatocytes,cell glucose content detection kit to measure the level of intracellular glucose content,ATP detection kit to measure the energy supply level in hepatocytes.③Real-time fluorescent quantitative PCR(qRT-PCR)to measure the mRNA levels of ER stress-related proteins PERK,ATF4,ATF6,HBx,and the mRNA levels of EVs marker proteins TSG101,CD63.④Western blotting(WB)to measure intracellular insulin downstream proteins ADIPOQ,IRS-1,endoplasmic reticulum stress proteins PERK,IRE1,EIFα,ER-phagy-related proteins FAM134B,LC3B,p62,LAMP2,mTOR,p-mTOR,EVs marker proteins TSG101,CD63,MLKL,LX2-activating-protein α-SMA and other expression levels.⑤Immunofluorescence(IF)experiment and laser confocal microscope to observe ER(ER-Tracker label)and Lyso(Lyso-Tracker label)and related proteins FAM134B,LC3B,PYGL,TSG101,CD63 co-localization,and mTagRFP-mWasabi-LC3B and p62 were used to analyze the state of autophagic flux.⑥Transmission electron microscope(TEM)to observes the ER morphology and ER-phagy structure,and to identify EVs morphology and purity.⑦Proximal ligation experiment(PLA)to measure the spatial proximity level of hepatocytes FAM134B and CD63.⑧Co-immunoprecipitation(Co-IP)to measure the interaction of endoplasmic reticulum protein FAM134B with PYGL and CD63.⑨Ultracentrifugation and kit method to extract EVs components,Dynamic light scattering(DLS)particle size technology to identify the morphology and purity of EVs.⑩CD63 enzyme-linked immunosorbent assay(ELISA)test to measure the release levels of extracellular vesicles,scratch test to measure Migration ability of LX2 cells.(3)In vivo:10-12 weeks old HBx transgenic(HBx-Tg)mice and wild-type(WT)C57BL/6 mice were fed with choline-deficient high-fat diet(CD),and siFAM134B tail vein injection was used for intervention.From the 11th week of rearing,the mice were injected with 8 nmol every three days for a total of 5 times.Mouse serum samples and liver tissue samples were tested as follows:①Intraperitoneal glucose tolerance test(IPGTT)to measure the sensitivity of insulin to glucose in each group of mice.②Insulin tolerance test(ITT)to measure the insulin sensitivity of each group of mice.③Tissue triglyceride enzymatic method was used to measure the hepatocyte triglyceride level of mice in each group.④Tissue glycogen content test kit to measure the hepatocyte glycogen level of each group of mice.⑤Tissue ATP test kit to measure energy supply level of each group of mice.⑥qRT-PCR test to measure the levels of mouse hepatocyte ER-phagy related genes(Retregl,Atg5,Sqstm1,Map1lc3b),hepatic stellate cells activation related genes(Col-1,Acta2)and extracellular Vesicle miRNAs(miR21a,miR-122,miR-155,miR-203,miR-205,miR-233).⑦Animal in vivo ultrasound imaging(US)test to measure images of liver fibrosis in mice.⑧immunohistochemistry(IHC)test to measure the expression and distribution of α-SMA,autophagy-related protein(FAM134B,LAMP2)and EVs-related protein CD63 in mouse liver tissue.⑨CD63-ELISA test to measure the release levels of extracellular vesicles.⑩Ultracentrifugation method Isolation,TEM and DLS to identify EVs morphology and purity.Results:(1)GEO analysis:The analysis of GEO database(GSE83148)showed that compared with normal controls(n=6),the mRNA levels of SQSTM1 and LC3B genes increased in liver tissue samples of HBV-infected persons(n=122),suggesting that there is autophagic flow in liver tissues infected by HBV;Moreover,the mRNA level of FAM134B gene decreased and that of TSG101 increased significantly.It showed a significant negative correlation(P<0.05),suggesting a potential correlation between FAM134B and EVs biogenesis in liver tissues infected by HBV.(2)HBx expression induces hepatocyte endoplasmic reticulum-related insulin resistance:①Compared with the blank control,in insulin-acting cells,the level of glycogen,GCS activity were increased,the area of glycogen PAS staining was increased,and the level of glucose,GPa activity were significantly decreased,ATP level was significantly increased,all above had P<0.05,suggesting that hepatocytes insulin induction Model establishment.②Compared with insulinacting cells,in HBx-expressing HepG2 cells,glycogen levels were significantly reduced(P<0.05),glycogen PAS staining area decreased,consistent with the positive model PA treatment,GPa activity increased(P<0.05),ATP level was significantly reduced(P<0.05),glucose levels significantly reduced after siATF4 intervention(P<0.05),suggesting that HBx expression promotes glycogen decomposition and reduces insulin sensitivity,and endoplasmic reticulum stress is involved in hepatocyte insulin resistance.Compared with the control group,HBxexpressing HepG2 cells:③The triglyceride level was significantly increased(P<0.05),consistent with the positive model PA treatment,suggesting that lipid accumulation in HBxexpressing hepatocytes.④The protein levels of ADIPOQ and IRS-1 decreased,consistent with the results of CQ treatment,the expression levels of mRNA such as PERK and ATF4 increased,and the levels of UPR protein such as EIFα,PERK,IRE1 increased,suggesting that HBx expression induces hepatocyte ER stress and insulin resistance,Autophagy may be a potential intervention pathway.⑤IF showed that the number of ER(ie.the green fluorescent focus)and the level of PYGL protein(ie.the red fluorescent focus)increased,and the co-localization level of the two(ie.the yellow fluorescent focus)increased,indicating that HBx-expressing hepatocytes promote PYGL produce and accumulate in ER.⑥siFAM134B intervention caused a significant increase in protein levels such as PERK and IRE1,suggesting that inhibition of ER-phagy promotes ER stress.⑦Compared with insulin-acting cells,glycogen levels were significantly reduced after siFAM134B intervention(P<0.05),in HepG2-Cas9-FAM134B cells,triglyceride levels increased(P<0.05),and glycogen levels were significantly reduced(P<0.05),suggesting that ER-phagy inhibition causes cellular lipid accumulation,which is related to insulin resistance.⑧Compared with the control group,Co-IP in HBx expressing HepG2 cells showed FAM134B binding with PYGL,suggesting that FAM134B interacted with PYGL in HBx-expressing hepatocytes.(3)Endoplasmic reticulum FAM134B participates in ER-phagy-mediated endosomeLyso organelle communication:Compared with the control group,in HBx-expressing hepatocytes:①IF shows that Lyso and ER colocalization(ie.yellow fluorescent focus)is reduced(P<0.05),TEM showed that the endoplasmic reticulum expanded and the lysosomal phagocytosis of the endoplasmic reticulum was reduced,inconsistent with the results of RAPA treatment,suggesting that HBx expression inhibits hepatocyte ER-phagy.②IF showed that FAM134B and LC3B colocalization(ie.yellow fluorescent focus)increased,p62 protein level(ie.green fluorescent focus)increased significantly,mTagRFP-mWasabi-LC3B showed yellow fluorescent focus.WB showed FAM134B,p62,LC3B and other autophagy related protein levels increased in a dose-dependent manner with the induced expression of HBx,consistent with the results of CQ treatment,suggesting that HBx expression promoted the formation of hepatocyte ER autophagosomes but inhibited the degradation of autophagic cargo proteins,thereby inhibiting the FAM134B-mediated ER-phagy.③The expression of LAMP2 decreased in a dose-dependent manner with HBx induction,while the endosome-related protein MLKL increased in a dose-dependent manner.suggesting that HBx expression may inhibit the formation of lysosomes and promote endosome production in hepatocytes.(4)HBV/HBx expression promoted the release of EVs and mediated HSCs activation:Compared with the control group,in HBx expressing HepG2 cells:①The mRNA levels of TSG101,and CD63 increased in a dose-dependent manner with the induced expression of HBx,the protein levels of p-mTOR,mTOR,FAM134B,TSG101,and CD63 increased,and the protein level of LAMP2 decreased,consistent with the results of CQ treatment,suggesting that HBx-expressing liver cells inhibitted ER-phagy and promoted the production of extracellular vesicle-related proteins.②IF showed that the number of ER(ie.green fluorescent focus)and TSG101 protein level(ie.red fluorescent focus)increased,the co-localization level both(i.e.yellow fluorescent focus)increased,Lyso and TSG101 co-localization(ie.yellow fluorescent focus)was significant decrease(P<0.05),suggesting that HBx expression promoted the production of TSG101 in hepatocytes,and the ability of lysosomes to degrade extracellular vesicle-related proteins was weakened.③After siFAM134B intervention,the levels of TSG101,CD63 and other proteins increased,and the level of CD63 protein increased significantly in HepG2-Cas9-FAM134B cells,suggesting that FAM134B participates in extracellular vesicle formation in HBx-expressing hepatocytes.④IF showed that the co-localization level of FAM134B and CD63(ie.yellow fluorescent focus)increased significantly,PLA showed that the volume and number of red fluorescent focus adjacent to the spatial position between FAM134B and CD63 increased significantly,and Co-IP showed that the enhanced degree of binding between FAM134B and CD63,suggests that FAM134B and CD63 directly interacted each other in HBx-expressing hepatocytes.⑤Compared with the control group,in HBV expressing HepG2.2.15 cells,the CD63 release level was significantly increased(P<0.05),consistent with siFAM134B intervention,anti-HBx ntervention caused a significant decreased level of CD63(P<0.05),and The release level of CD63 was significantly increased(P<0.05)in HBx-expressing HepG2 cells,suggesting that HBx induces the release of extracellular vesicles in HBV-expressing hepatocytes,and HBx inhibits FAM134B-mediated ER-phagy played a regulatory role.⑥TEM and DLS identified goblet-shaped vesicles with a diameter of about 100nm,the proteins levels such as TSG101,CD63 and FAM134B increased in a dose-dependent manner with HBx in EVs,suggesting that HBx expression promoted the release of endoplasmic reticulum-derived extracellular vesicles in hepatocytes.⑦Compared with LX2 cells co-cultured with HepG2 cells,LX2 cells co-cultured with HepG2.2.15 cells have higher α-SMA protein expression and enhanced migration ability,suggesting that HBV expression promote hepatic stellate cells activation.⑧Compared with LX2 cells treated with Ctrl-EVs,the levels of activation-related genes such as TGFB1,ACTA2,COL1A1,etc.,were significantly increased(P<0.05)in LX2 cells treated with HBV-EVs,the protein level of α-SMA was increased,and the migration ability was enhanced,suggesting that HBV expressing hepatocytes release extracellular vesicles to promote hepatic stellate cell activation.(5)In vivo experiment to evaluate HBx expression-induced insulin resistance and liver injury:Compared with control WT mice,in HBx-Tg mice:①IPGTT showed that the blood glucose curve was significantly increased,and the AUC was significantly increased(P<0.05).②Insulin resistance index(HOMA-IR)was significantly increased(P<0.05).③Hepatic triglyceride levels were significantly increased(P<0.05).④Hepatic glycogen levels were significantly decreased(P<0.05);The hepatic ATP level was significantly reduced(P<0.05).it suggested that HBx-Tg mice had liver steatosis and insulin resistance.⑤The level of autophagic protein ATG5 decreased,and the protein levels of TSG101 and CD63 increased significantly,suggesting that HBx-Tg mice promote the production of extracellular vesiclesrelated proteins.In HBx-Tg mice with CD diet and HBx-Tg mice with siFA M134B intervention:the above experimental results have enhanced effects(P<0.05),suggesting that CD diet and siFAM134B intervention can significantly increase the obesity rate of mice,promote liver steatosis,insulin Resistance and release of extracellular vesicles.⑥US showed that the hepatomegaly and echogenic foci increased in HBx-Tg mice.⑦IHC showed increased vacuolation of liver tissue,increased protein levels of FAM134B,α-SMA,and CD63,while LAMP2 protein level decreased.⑧CD63 release levels increased significantly(P<0.05).⑨TEM and DLS showed an increase in cup-shaped vesicles with a diameter of about 100 nm.⑩The expression levels of miR-122,miR-155,and miR-203 increased significantly in EVs(P<0.05).It is suggested that endoplasmic reticulum-derived extracellular vesicles release miRNAs to promote liver fibrosis in HBx-Tg mice.In HBx-Tg mice with CD diet and HBx-Tg mice with siFAM134B intervention:the above experimental results have enhanced effects(P<0.05),and the levels of autophagic genes such as Sqstm1 and Map1lc3b were significantly increased(P<0.05),The mRNA level of Col-1was significantly increased(P<0.05),suggesting that the CD diet and siFAM134B intervention can promote the formation of autophagosomes and cause the accumulation of autophagic cargo proteins,and significantly enhance the level of extracellular vesicle-related liver fibrosis in mice.Conclusions:The study clarified that the molecular mechanism of FAM134B-dependent ERphagy is involved in CD63-related EVs-Lyso degradation pathway to promote the release of EVs in HBx-expressing hepatocytes,through RBPs-mediated miR-122 and other sorting into EVs to mediate HSCs activation.At the same time,it clarified FAM134B-dependent ER-phagy-lysosomal pathway regulates PYGL-related Gn metabolism in ER stress-related insulin sensitivity decrease of HBxexpressing hepatocytes.The result aim to provide theoretical basis for proving new targets in the EVsLyso degradation pathway,early symbol of HSCs activation and targeted interventions.Then revealing the mechanism of ER homeostasis and autophagy-mediated insulin resistance-related glycogen metabolism disorders in the occurrence and development of NAFLD... |
PDF Full Text Request