Protective Effect And Mechanism Of HMBOX1 In LPS/D-GalN-induced Acute Liver Injury

Object:Acute liver injury(ALI)is a clinical syndrome characterized by sudden and severe impairment of liver function,with diffuse intrahepatic infiltration by inflammatory cells with massive multilobular necrosis.ALI is a severe liver injury accompanying hepatic encephalopathy and causes multi-organ failure with an extremely high mortality rate,even if intensive care is provided.The only proven therapy for ALI patients are unlikely to recover is liver transplantation.ALI has been considered to be caused by several hepatitis viruses,various drugs,toxins,and metabolic disorders.The model of hepatic injury induced by simultaneous injection of D-galactosamine(DGalN)and lipopolysaccharide(LPS)has been widely used to examine the mechanisms of ALI.D-GalN can deplete liver UTP,disrupt protein synthesis and sensitize hepatocytes to challenge agents such as LPS.In the model,kupffer cells were activated and secreted inflammatory factors,such as TNF-?,IL-6 and IL-1?,to induce secondary liver injury,which is the major mechanism of ALI induced by LPS/GalN.HMBOX1(homeobox containing 1)was isolated from human pancreatic cDNA library,and has been detected in several tissues,including pancreas,brain liver and kidney.More interestingly,HMBOX1 was expressed in hepatic carcinoma with a lower level compared with the adjacent tissues,indicating it might play a role in the process of HCC happening and development,but the biological function of HMBOX1 in HCC was basically unknown.Recently,several studies reported the biological function of HMBOX1,including the Eeffects on the differentiation of bone marrow stromal cells(BMSCs)to endothelial cells(ECs),cell senescence and cell autophagy.In our previous work,we found HMBOX1 was a negative regulator in natural killer(NK)cells and displayed a role of transcription repression on the transcription activity of interferon ?(IFN-?)promoter.Thus,HMBOX1 might play a role in the process of inflammatory regulation,but the mechanism was basically unknown.In this study,we demonstrated the protected role of HMBOX1 in D-GalN/LPS-induced ALI by inhibiting macrophage infiltration into injury liver through HMBOX1/NF-?B/CCL2 pathway.In additional,HMBOX1 overexpression in hepatocytes could increase the activity of coincubated macrophage.These results suggested that HMBOX1 might shed new light on ALI therapy,and might be a novel therapeutic target for human ALI.Methods1.HMBOX1 and the immune response in hepatocytes1.1 HMBOX1 and liver metabolism-related genesHepG2 cells were transfected with pEGFP-HMBOX1 plasmid or HMBOXl-siRNA.Microarray analysis method was used to evaluated the genes regulated withHMBOX1,and liver metabolism-related genes in HepG2 cells were detected with q-PCR assay.1.2 HMBOX1 and the expression of inflammatory factors in hepatocytesHepG2 cells were transfected with pEGFP-HMBOX1 plasmid,and microarray analysis was used to analyze the potential inflammatory factors regulated by HMBOX1.LPS/D-GalN or LPS was used to induce the inflammatory in BNL CL.2 cells,and HMBOX1 in hepatocytes was detected with q-PCR.In vivo,LPS/D-GalN was injected intraperitoneally to induce acute liver injury model,then the level of HMBOX1 in primary hepatocytes was detected with q-PCR and Western blot methods.1.3 HMBOX1 and cell autophagyHepG2 cells were transfected with pEGFP-HMBOX1 plasmid.LC3?/LC3 ?ratio were detected with Western blot assay and LC3B level was determined with immunofluorescence.Then mTOR and its upstream activators,including p38MAPK and AKT were also analyzed with Western blot assay.2.HMBOX1 and the immune response in macrophagesRAW264.7 cells were stimulated with LPS,then HMBOX1 and inflammatory factors were detected with q-PCR.The activation of RAW264.7 were analysed with flow cytometry.The concentrations of TNF-? and IL-6 in supernatant were determined with ELISA methods.Neutral red phagocytosis experiment was used to assess the phagocytic function of macrophages.3.Generation of HMBOX1 liver-specific knockout mice modelCre-Ioxp conditional knockout methods were used to generate HMBOX1 liver-specific knockout mice with hybridization of HMBOX1 floxed mice(Hmf/f mice)and Alb-cre mice.DNA genome was collected for identification,and mRNA and protein level of HMBOX1 in hepatocytes were analysed with q-PCR and Western blot,respectively.By comparing the character of the diet,activity,weight,liver tissue appearance and inflammatory level between Hm?hep mice and Hmf/f mice(littermate control),we evaluated the effect of HMBOX1 liver-specific knockout on the quality of life of the mice offspring.4.Protection role of HMBOX1 in ALI induced by LPS/D-GalNIntraperitoneal injection with 400 mg/kg D-galactosamine(GalN)and 5?g/kg LPS to induce ALI model,we evaluated the liver injury level with hepatic hyperemia and serum ALT level.Liver organizational integrity was assessed with HE-staining method,and hepatocyte apoptosis was evaluated with Western blot and TUNEL assay.5.Protection role of HMBOX1 in ALI induced by LPS/D-GaIN5.1 The influences of HMBOX1 on system inflammatoryAfter challenged with LPS/D-Ga1N,mRNA level of inflammatory factors in liver were determined with q-PCR,then cytokines in serum were detected with ELISA assay.In additional,the activation of NF-?B and MAPK signaling were analysed with Western blot method.5.2 HMBOX1 inhibites CDllb+Gr-1dimF4/80+ macrophages infiltration into liver through NF-?B/CCL2 pathwayMacrophages infiltrated into liver were analysed with flow cytometry assay;CCL2,CCL4 and CCL5 concentrations in liver tissue homogenate were determined with ELISA.To ensure the regulation function of HMBOX1 on CCL2,Hepal-6 cells were transfected with pcDNA3.1-HMBOX1 or vector plasmid,and CCL2 mRNA level was detected.Then Western blot methods and luciferase report assay were used to indentify the function of HMBOX1 on NF-?B pathway.Furthermore,a transwell migration assay was performed to confirm the function of HMBOX1 on macrophage migratory ability.To neutralize CCL2 chemokine,CCL2 neutralization antibody was injected into tain vein just followed by LPS/D-GalN administration.Then CD11b+Gr-ldimF4/80+macrophage level was analysed with flow cytometry assay and ALT in serum was detected.5.3 HMBOX1 in hepatocytes inhibites macrophage activiation.Hepal-6 cells were transfected with pcDNA3.1-HMBOX1 plasmid or blank vector and coincubated with RAW264.7 cells,TNF-?and IL-6 concentraion in supernatants were determined with ELISA.Transfected Hepal-6 cells were coincubated with peritoneal macrophages with or without LPS stimulation,the concentration of TNF-?and IL-6 in supernatants and the activated markers(MHCII and CD80)on macrophages were analysed with flow cytometry assay.5.4 HMBOX1 overexpression relieves liver injury induced by LPS/D-GalNTo further definite the role of HMBOX1 on inflammatory,Hm?hep mouse were hydrodynamic injected pcDNA3.1-HMBOX1 plasmid through vein venous to recovery HMBOX1 level and stimulated with LPS/D-GalN for 6 hours,then liver hyperemia,liver/weight ratio and the inflitration level of CD11b+Gr-1dimF4/80+macrophage were detected.Results1.HMBOX1 and the immune response in hepatocytes1.1 HMBOX1 and liver metabolism-related genesMicroarray analysis results showed that HMBOX1 overexpression in HepG2 cells could promote liver metabolism-related genes(APOA1BP,CYP2W1,CYP46A1,CYP26B1 and HPD)level.Q-PCR results also showed increased level of liver metabolism-related genes in HMBOX1-overexpressed HepG2 cells,and HMBOX1 silence inhibited their expression.1.2 HMBOX1 and the expression of inflammatory factors in hepatocytesWith the challenge of LPS/D-GalN,HMBOX1 level was decreased significantly in BNL CL.2 cells.In vivo,acute liver injury model was induced by LPS/D-GalN with peritoneal injection,and HMBOX1 level in hepatocytes was downregulated obviously.1.3 HMBOX1 and cell autophagyHMBOX1 could increase the ratio of LC3 ?/LC3 ? and LC3B expression in HepG2 cells.At the same time,the phosphorylation level of mTOR,AKT and p38 were inhibited apparently.All the results indicated that HMBOX1 has the ability to promote cell antophagy.2.HMBOX1 and the immune response in macrophagesIn LPS-induced RAW264.7 cells,HMBOX1 level decreased obviously.After transfected with pcDNA3.1-HMBOX1,the level of CD80,CD86 on cell surface and the concentrations of TNF-? and IL-6 in supernatants were downregulated significantly.In additionally,the phagocytic function of RAW264.7 cells was inhibited obviously for the overexpressed HMBOX1.3.Generation and identify of HMBOX1 liver-specific knockout mice)To generate liver-specific HMBOX1 deletion mice(Hm?hep mice),HMBOX1loxP/loxP(Hmf/f mice)mice were bred with Alb-Cre-recombinase transgenic mice.Mouse were genotyped by PCR amplification of sequences specific for the HMBOXI DNA from tail samples.Hepatocyte lysates from Hm?hep mice displayed a strong decrease in HMBOX1 mRNA and protein levels compared to the Hmf/f mice.Body weights,liver appearance,activity ability and mRNA levels of inflammatory cytokines were not significantly different between Hmf/f and Hm?hep mice.Primary hepatocytes obtained from Hmf/f and Hm?hep mice were cultured in vitro,and the ALT values in supernatants showed no significant difference.4.Protection role of HMBOX1 in ALI induced by LPS/D-GaINHm?hep and Hmf/f mouse were challenged with LPS/D-Ga1N,and higher level of hepatohemia,liver/body weight ratio and serum ALT were observed in Hm?hep mouse.Besidesly,tissue disorganization and apoptotic level in Hm?hep livers were increased significantly.5.Protection mechanism of HMBOX1 in ALI induced by LPS/D-GalN5.1 The influences of HMBOX1 on system inflammatoryAfter LPS challenge,TNF-? IL-6,IL-1,IL-17 and iNOS levels in HmAhep liver were much higher.Furthermore,TNF-a and IL-6 levels in the serum of Hm?hep mice were significantly higher in LPS/D-Ga1N-induced acute liver injury.However,no significant differences were observed in the levels of either IL-1?,IL-18,and iNOS,or the important negative regulatory cytokines(IL-10 and TGF-?).Hm?hep mice displayed higher NF-?B/MAPK activation in the liver accompanied with increased phosphorylation levels of NF-?B,ERK1/2,JNK,and c-Jun.5.2 HMBOXl inhibites CDllb+Gr-dimF4/80+ macrophages infiltration into liver through NF-?B/CCL signalingThe results showed that a significantly higher percentage and total number of CDllb+Gr-1dimF4/80+ cells were observed in Hm?hep liver tissues in response to LPS/D-Ga1N treatment.And CCL2 was significantly increased following the LPS/D-GalN challenge in Hm?hep livers compared to Hmf/f ones.Compared to vector-treated HepG2 cells,the activity of NF-?B in pEGFP-HMMBOX1 plasmid-transfected HepG2 cells was inhibited with or without LPS treatment.After transfected with pcDNA3.1-HMBOX1 plasmid,Hepal-6 cells expressed higher HMBOX1 and lower CCL2 compared with control.Then a transwell migration assay was performed and the results showed impaired migration ability of macrophage coincubated with HMBOX1-overexpressed Hepal-6 cells.5.3 HMBOX1 in hepatocytes inhibites macrophage activiation.Hepal-6 cells transfected with pcDNA3.1-HMBOX1 or a vector were co-incubated with intraperitoneal macrophages with or without LPS.The concentrations of TNF-a.and IL-6 in the supernatant were significantly lower than in the control.And both MHC ? and CD80 on peritoneal macrophages were significantly downregulated following co-incubation with Hepal-6 cells.5.4 HMBOX1 overexpression relieves liver injury induced by LPS/D-GalNAfter hydrodynamical injection with pcDNA3.1-HMBOXl plasmid,Hm?hep mice showed ameliorated liver hyperemia and decreased liver/body weight ratio.And the infiltration of CDllb+Gr-1dimF4/80+ cells was alleviated significantly by pcDNA3.1-HMBOX1 plasmid-treatment.Conclusion1.HMBOX1 has the function in inhibiting inflammatory level in hepatocyte.2.HMBOX 1 inhibites the activation and phagocytic function of macrophages.3.HMBOX 1 in hepatocytes promotes cell autophagy and inhibites inflammatory.4.Hepatic HMBOX1 can impede the infiltration of CDllb+Gr-ldmF4/80+macrophages into liver tissue by inhibiting NF-kB/CCL2 pathway.5.Hepatic HMBOX1 expression dampens macrophage activation.