Animals ; Disease Progression ; Fatty Liver ; metabolism ; pathology ; Humans ; Inflammation ; Kupffer Cells ; metabolism ; Non-alcoholic Fatty Liver Disease

OBJECTIVETo investigate HMGB-1 expression and its extracellular release of cultured primary hepatic parenchymal cells (HC) and Kupffer cells (KC) that were induced by lipopolysaccharides (LPS).

METHODSPrimary hepatic parenchymal cells and Kupffer cells were cultured in flasks, and some cells were treated with 500 microg/L LPS for 24 hours (induced group) and some were not treated with LPS and served as controls. All of the cells were repeatedly frozen-thawed, and the expression levels of HMGB1-mRNA and HMGB1 proteins were detected by semi-quantitative RT-PCR and Western blot respectively. Then HC and KC were subcultured in 24-well culture plates for 6 h, 12 h, 24 h and 48 h, and the HMGB1 protein in culture fluids was detected by Western blot at each time point.

RESULTSCompared with the cells in the control group, the expression levels of HMGB1-mRNA in the induced group were significantly increased in both HC and KC at 24 h (t=31.32 and 45.90, P<0.05) and the protein levels of HMGB1 showed the same results (t=46.19 and 38.44, P<0.05). There was a small quantity of HMGB1 protein in the culture fluids of two control groups and the induced group of HC. However the HMGB1 protein in the induced group of KC were obviously increased with prolonged culture time (F=42.74, P<0.05). Compared with the control group, the level of HMGB1 protein in the induced group of KC was not increased at 6 h (t=9.57, P>0.05) but was significantly increased at 12 h, 24 h and 48 h (t=21.95, 32.39, 44.16, respectively P<0.05).

CONCLUSIONLPS could increase HMGB1 expression of HC and KC and HMGB1 release from KC, but not from HC. The results suggest that KC play an important role in triggering inflammation and liver injury.

Animals ; Cells, Cultured ; Female ; HMGB1 Protein ; metabolism ; Hepatocytes ; metabolism ; Kupffer Cells ; metabolism ; Lipopolysaccharides ; Liver ; cytology ; metabolism ; pathology ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar

The effects of estriol on oxygen uptake, glucose release, lactate and pyruvate production, beta-hydroxybutyrate and acetoacetate production in perfused rat liver as well as, carbon uptake in rat liver and intracellular calcium in isolated Kupffer cells were investigated. Basal oxygen consumption of perfused liver increased significantly in estriol or ethanol-treated rats. But these increased effects were blocked by gadolinium chloride pretreatment. In a metabolic study, pretreatment with estriol resulted in a decrease in glucose production and in glycolysis while an increase in ketogenesis. A more oxidized redox state of the mitochondria was indicated by increased ratios of perfusate [lactate]/[pyruvate] and decreased ratios of perfusate [beta-hydroxybutyrate]/[acetoacetate]. Carbon uptake of Kupffer-cell increased significantly in estriol-treated rats. But these increased uptake were not shown in rats pre-treated by gadolinium chloride blocking phagocytosis. In isolated Kupffer cells from estriol-treated rats, intracellular calcium was more significantly increased after addition of lipopolysaccharide (LPS) than in controls. These findings suggest that the metabolic effects of estriol (two mg per 100 mg body wt) can be summarized to be highly toxic in rat liver, and these findings suggest that oral administration of estrogens may induce hepatic dysfunctions and play a role in the development of liver disease.
3-Hydroxybutyric Acid/metabolism ; Acetoacetates/metabolism ; Animal ; Calcium/metabolism ; Carbohydrates/metabolism ; Carbon/metabolism ; Cells, Cultured ; Colloids/metabolism ; Estriol/pharmacology* ; Estriol/metabolism ; Ethanol/pharmacology ; Female ; Gadolinium/pharmacology ; Glucose/biosynthesis ; Intracellular Fluid/metabolism ; Kupffer Cells/metabolism ; Kupffer Cells/cytology ; Lactates/metabolism ; Lipids/metabolism ; Liver/metabolism ; Liver/drug effects* ; Oxygen Consumption ; Phagocytosis ; Pyruvic Acid/metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of angiotensin II type-1 (AT-1) alpha receptor gene silencing on nuclear factor-kappaB (NF-kappaB) activity in hepatic Kupffer cells.

METHODSThe expression of AT-1 alpha receptors in primary isolated cultured hepatic Kupffer cells was detected by immunohistochemistry. pSilencer/AT-1 alpha receptor siRNA plasmids were transfected into Kupffer cells, which were subsequently exposed to 10(-6) mol/L angiotensin II (Ang II) for 60 min. The changes in the DNA binding activity of NF-kappaB in the cells was assessed using electrophoretic gel mobility shift assay (EMSA).

RESULTSAT-1 alpha receptor expression was detected in Kupffer cells. NF-kappaB DNA binding activity was markedly increased in Kupffer cells after Ang II stimulation, and obviously inhibited by transfectiom with pSilencer/AT-1 alpha receptor siRNA plasmid.

CONCLUSIONAng II stimulation of Kupffer cell results in increased activation of NF-kappaB via AT-1 alpha receptor.

Cells, Cultured ; Humans ; Kupffer Cells ; cytology ; NF-kappa B ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptor, Angiotensin, Type 1 ; genetics ; metabolism

OBJECTIVETo investigate the mechanism underlying the inhibitory effect of tacrolimus (FK506) against acute liver graft rejection.

METHODSRat models of orthotopic liver transplantation were divided into 3 groups, namely the tolerance group with Brown Norway (BN) rats as the donors and Lewis rats as the recipients, rejection group with Lewis rats as donors and BN rats as recipients, and FK506 group with the same donor-recipient pair as in the rejection group and FK506 treatment. The recipients were sacrificed 7 days after the transplantation, and the hepatic histology, cytokine levels, and glucocorticoid-induced tumor necrosis factor-related protein ligand (GITRL) expression in the liver and Kupffer cells were observed and detected.

RESULTSCompared with the tolerance group, the rejection group showed increased GITRL expressions in the liver and Kupffer cells (P<0.05), which was significantly lowered by FK506 treatment (P<0.05). Acute liver graft rejection caused significantly elevated interferon-γ (IFN-γ) levels and decreased interleukin-10 (IL-10) levels in the plasma and Kupffer cells (P<0.05), and these changes were obviously attenuated by FK506 treatment (P<0.05).

CONCLUSIONThe effect of FK506 in suppressing acute liver graft rejection is probably associated with down-regulated GITRL expression in the liver and Kupffer cells.

Animals ; Carrier Proteins ; metabolism ; Graft Rejection ; prevention & control ; Kupffer Cells ; metabolism ; Liver ; metabolism ; Liver Transplantation ; Male ; Rats ; Rats, Inbred BN ; Rats, Inbred Lew ; Tacrolimus ; pharmacology

The inducible form of nitric oxide synthase (iNOS) is expressed in hepatic cells in pathological conditions. Its induction is involved in the development of liver fibrosis, and thus iNOS could be a therapeutic target for liver fibrosis. This review summarizes the role of iNOS in liver fibrosis, focusing on 1) iNOS biology, 2) iNOS-expressing liver cells, 3) iNOS-related therapeutic strategies, and 4) future directions.
Endothelial Cells/metabolism ; Hepatic Stellate Cells/metabolism ; Humans ; Kupffer Cells/metabolism ; Liver Cirrhosis/metabolism/*pathology ; Nitric Oxide/*metabolism ; Nitric Oxide Synthase Type II/antagonists & inhibitors/genetics/*metabolism ; Polymorphism, Single Nucleotide ; RNA, Untranslated/metabolism

OBJECTIVETo explore the mechanism of endotoxin tolerance (ET) through observing the expression of interleukin 1 receptor associated kinase-4 (IRAK-4) during endotoxin tolerance development in Kupffer cells (KCs).

METHODSIsolated KCs of Balb/c mouse were divided into two groups: the non-endotoxin tolerance (NET) group and the endotoxin tolerance (ET) group, which were pretreated with 10 ng/ml lipopolysaccharide (LPS) for 24 h. Then, the two groups were treated with 100 ng/ml LPS. The expressions of IRAK-4 gene and protein level were determined by RT-PCR and Western blot. The activities of NF-kappaB of KCs and the TNFalpha level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h after LPS stimulation.

RESULTSThe ultimate level of IRAK-4, the activities of NF-kappaB and the TNFalpha level were evidently lower in the ET group than those in the NET group (t = 12.4, 17.4 and 138.9 respectively, P<0.01).

CONCLUSIONSPretreatment with LPS on KCs could induce endotoxin tolerance of KCs and inhibition of IRAK-4 expression may be one of the reasons for its development.

Animals ; Cells, Cultured ; Endotoxins ; immunology ; Immune Tolerance ; Interleukin-1 Receptor-Associated Kinases ; biosynthesis ; genetics ; Kupffer Cells ; cytology ; immunology ; metabolism ; Lipopolysaccharides ; immunology ; Male ; Mice ; Mice, Inbred BALB C

OBJECTIVETo explore the inhibitory effects on the activation of endotoxin-induced Kupffer cells (KCs) through short hairpin RNA (shRNA) targeting interleukin-1 receptor associated kinase-4 (IRAK-4) gene.

METHODSTwo effective transfection shRNA plasmid (pSIIRAK-4-A, pSIIRAK-4-B) and one invalidated plasmids (pSIIRAK-4-C) targeting IRAK-4 gene were constructed. The isolated mouse KCs were divided into three groups: the normal control group, the RNAi control group (pSIIRAK-4-C) and the RNAi effective group (pSIIRAK-4-A, pSIIRAK-4-B). Then KCs were stimulated with 0.1 microg/ml lipopolysaccharide (LPS) after 24 h transfection with the constructed plasmid. The expression of IRAK-4 gene and protein level were determined by RT-PCR and Western blot at 6 h after LPS stimulation, and the activities of NF-kappaB in KCs and the TNFalpha level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h.

RESULTSThe level of IRAK-4, the activities of NF-kappaB and the TNF-alpha level in the RNAi effective group were evidently lower than those in normal and RNAi control groups (P < 0.01) at 1 h, 3 h, and 6 h. Especially, the pSIIRAK-4-A group in which the changes of the above indices were of no difference (P > 0.05), had better inhibited effects than that of the pSIIRAK-4-B group (P < 0.01).

CONCLUSIONThe shRNA targeting IRAK-4 gene could effectively inhibit the activation of endotoxin-induced KCs.

Animals ; Endotoxins ; Interleukin-1 Receptor-Associated Kinases ; genetics ; metabolism ; Kupffer Cells ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; RNA Interference ; RNA, Small Interfering ; genetics ; Signal Transduction ; physiology

OBJECTIVETo study changes of TLR2 signaling pathway expression in Kupffer cells during the process of hepatic ischemia/reperfusion in a mice model and the mechanism of TLR2 signaling pathway participating in hepatic ischemia/reperfusion injury.

METHODSBALB/c mice were divided into 3 groups: sham operation (SH), ischemia/reperfusion (I/R) and GdCl3 treatment (Gd) groups. After 4 h of reperfusion, the expression of TLR2 mRNA and membrane TLR2 protein were analyzed in ischemic lobes of the livers, and in Kupffer cells isolated from ischemic lobes. The expression of NF-kappaB in ischemic lobes was also examined. Levels of endotoxin, ALT and TNFalpha were measured at the same time point.

RESULTSThe expressions of TLR2 mRNA and protein in both ischemic hepatic lobes and Kupffer cells isolated from ischemic lobes were increased in the I/R group compared to those in the SH group, as well as the expression of NF-kappaB in ischemic lobes, which was down regulated by intravenous GdCl3 treatment. Levels of ALT and TNFalpha in the portal vein were higher in the I/R group than in the SH group, which also were decreased with treatment of GdCl3. The level of endotoxin in the three groups remained constant.

CONCLUSIONTLR2 signaling pathway in Kupffer cells is activated during the process of hepatic ischemic/reperfusion injury. The activation of TLR2 signaling pathway in Kupffer cells may play a role in this process.

Animals ; Kupffer Cells ; metabolism ; Liver ; blood supply ; Male ; Mice ; Mice, Inbred BALB C ; Reperfusion Injury ; metabolism ; Signal Transduction ; Toll-Like Receptor 2 ; biosynthesis ; genetics

OBJECTIVES: This study aimed to explore the functions and potential regulatory targets of local macrophages in nonalcoholic fatty liver combined with Porphyromonas gingivalis (P. gingivalis)infection. METHODS: Single-cell RNA sequencing was used to analyze the phenotypes and functional changes in various cells in the liver tissue of nonalcoholic steatohepatitis (NASH) mice fed with P. gingivalis. Real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay, and immunofluorescence staining were applied to observe the inflammation and expression levels of macrophage antigen presenting functional markers in the NASH liver. Oil red staining was performed to observe the accumulation of local adipose tissue in the NASH liver. Results were verified through RT-PCRand RNA sequencing using P. gingivalis-lipopolysaccharide treated mouse peritoneal macrophages. RESULTS: In comparison with healthy livers with Kupffer cells, the NASH liver combined with P. gingivalis infection-related macrophages showed significant heterogeneity. C1qb, C1qc, Mafb, Apoe, and Cd14 were highly expressed, but Cd209a, H2-Aa, H2-Ab1, and H2-DMb1, which are related to the antigen presentation function, were weakly expressed. Further in vivo and in vitro investigations indicated that the activation and infiltration of these macrophages may be due to local P. gingivalis-lipopolysaccharide accumulation. CONCLUSIONS P. gingivalis-lipopolysaccharide induces a local macrophage immunotolerance phenotype in nonalcoholic fatty liver, which may be the key mechanism of periodontitis pathogen infection that promotes NASH inflammation and pathogenesis. This study further clarifies the dysfunction and regulatory mechanisms of macrophages in the pathogenesis of P. gingivalis-infected NASH, thereby providing potential therapeutic targets for its clinical treatment.
Mice ; Animals ; Non-alcoholic Fatty Liver Disease/pathology* ; Kupffer Cells/pathology* ; Porphyromonas gingivalis ; Lipopolysaccharides/metabolism* ; Inflammation/pathology* ; Macrophages/metabolism* ; Mice, Inbred C57BL