Rat Kupffer Cells Research Articles

Glycyrrhizin Prevents Liver Injury by Inhibition of High-Mobility Group Box 1 Production by Kupffer Cells after Ischemia-Reperfusion in Rats

High-mobility group box 1 (HMGB1) acts as an early mediator of inflammation and organ damage in hepatic ischemia-reperfusion (I/R) injury. Glycyrrhizin is a natural anti-inflammatory and antiviral triterpene in clinical use. The purpose of this study was to investigate the effect of glycyrrhizin on liver injury caused by I/R and production of HMGB1 by Kupffer cells in rats. In the first test period, rats were given saline or glycyrrhizin 20 min before segmental hepatic warm I/R. Serum alanine aminotransferase and HMGB1 levels and hepatic histopathological findings were evaluated after I/R. Furthermore, expression of HMGB1 in the liver was assessed by immunohistochemical staining after I/R. Kupffer cells were isolated by collagenase digestion and differential centrifugation, and production of HMGB1 was assessed. In another set of experiments, the effect of inhibition of Kupffer cells by injection of liposome-entrapped dichloromethylene diphosphonate (lipo-MDP) on liver injury and expression of HMGB1 were investigated after I/R. Liver injury was prevented in the glycyrrhizin group compared with the control group. Furthermore, serum HMGB1 levels were also significantly blunted in the glycyrrhizin group compared with the control group. Cells expressing HMGB1 were detected in the hepatic sinusoid by immunohistochemistry and recognized morphologically as Kupffer cells. Furthermore, the expression of HMGB1 was reduced in the glycyrrhizin group compared with the control group. Production of HMGB1 was reduced in Kupffer cells isolated from the glycyrrhizin group compared with the control group. It is noteworthy that treatment with lipo-MDP significantly blunted serum HMGB1 levels and prevented liver injury after I/R. These results suggest that glycyrrhizin has the therapeutic potential to prevent warm I/R-induced injury during hepato-biliary surgery.

TNF receptors in Kupffer cells

Introduction: Somatostatin is a mediator of immune functions and has been used as an antineoplastic agent in animal models and human neoplasias. We have demonstrated that Octreotide inhibits only LPS induced secretion of proinflammatory cytokines including TNFa by Kupffer cells (KC). We, therefore, tested the hypothesis that somatostatin modulates the expression of tumor necrosis factor alpha (TNFα) receptors and apoptosis of KC.Methods: Rat KC were isolated by centrifugal elutriation. TNFR1 and TNFR2 expression was studied by RT-PCR, quantitative PCR, Western Blot and immunofluorescence before and after Octreotide pre-incubation. Apoptosis was assessed by quantitative measurement of cytoplasmic histone-associated DNA fragments. TNFa mRNA expression was assessed by semiquantitative PCR and TNFa was measured in cell supernatants by ELISA.Results: TNFR1 and TNFR2 mRNA are constitutively expressed in KC. Octreotide incubation increased both receptors expression with a peak at 6 h and return to basal levels at 24 h. TNFR1 was mostly influenced. However, only increase in TNFR2 protein was identified, whereas a band at 90 kD was present instead of a band at 55 kD as expected for TNFR1. TNFα mRNA expression was inhibited by Octreotide and a significant inhibition was observed at 48 h. TNF had no effect on KC apoptosis, whereas Octreotide significantly increased their apoptosis, and this effect was not influenced by co-incubation with TNFa.Conclusion: TNFR1 and TNFR2 are constitutively expressed in KC and their expression is strongly increased by somatostatin. Moreover, somatostatin increases KC apoptosis. These findings may in part explain the antineoplasmatic effect of somatostatin.

Abstract 5252: Sequential biphasic changes in claudin1 and claudin4 expression correlate to colorectal cancer progression and liver metastasis

Abstract Terminal progression of colorectal cancer culminates in liver metastasis. To identify genes that are involved in the metastatic phenotype, cDNA microarrays were used to analyze mRNA expression profiles of CC531 rat colon adenocarcinoma cells for changes related to their homing into the liver. Briefly, CC531 cells were intraportally implanted into the liver of Wag-Rij rats and re-isolated after 3, 6, 9, 14 and 21 days. Compared to control CC531 cells, claudin1 and claudin4 were among the ≥8 fold initially down-regulated genes. The co-culture of tumor cells with isolated rat hepatocytes and Kupffer cells did not induce down-regulation of either claudin1 or 4. When the environment effective on circulating tumor cells was simulated by cell culture conditions favoring their adhesion, only claudin4 showed augmented expression. Knockdown of claudin1 and claudin4 mediated by siRNA caused significantly increased migration and decreased clonogenic growth of tumor cells (p<0.05), but had no effect on their proliferation. These experimental results were paralleled by increased claudin1 and claudin4 expression in human colorectal cancer samples in UICC stages l-lll, as evaluated by real-time PCR. Increased claudin4 levels were correlated with significantly reduced overall survival (log-rank test, p=0.018). Further, significantly (p<0.05) reduced expression of claudin1 and claudin4 was observed in stage IV and liver metastasis by immunohistochemistry. In conclusion, sequential biphasic changes in claudin1 and claudin4 expression occur during the homing of colorectal cancer cells to the liver. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5252. doi:10.1158/1538-7445.AM2011-5252

Somatostatin effect on TNFA receptors in Kupffer cells

<h3>Introduction</h3> Somatostatin is a mediator of immune functions and has been used as an antineoplastic agent in animal models and human neoplasias. We have demonstrated that octreotide-a somatostatin analogue- inhibits only LPS-induced secretion of pro-inflammatory cytokines including tumour necrosis factor (TNF) α by Kupffer cells.12 We therefore tested the hypothesis that somatostatin modulates the expression of TNFα receptors and apoptosis of Kupffer cells. <h3>Methods</h3> Rat Kupffer cells were isolated by centrifugal elutriation. TNFR1 and TNFR2 expression was studied by RT-PCR, quantitative PCR, Western Blot and immunofluorescence before and after octreotide pre-incubation. Apoptosis was assessed by quantitative measurement of cytoplasmic histone-associated DNA fragments (Roche). TNFα mRNa expression was assessed by a semiquantitative PCR and TNFα was measured in cell supernatants by ELISA. <h3>Results</h3> TNFR1 and TNFR2 m RNA are constitutively expressed in Kupffer cells. Octreotide incubation increased both receptors expression with a peak at 6 h and return to basal levels at 24 h. TNFR1 was mostly influenced. However only TNFR2 protein increase was identified by Western blot, while a band at 90 kD was present instead of a band at 55 kD as expected for TNFR1. TNFa mRNA expression and protein secretion was not influenced by octreotide at 24 h. However a significant inhibition was observed at 48 h. TNF had no effect on Kupffer cell apoptosis while octreotide significantly increased their apoptosis and this effect was not influenced by co-incubation with TNFα. <h3>Conclusion</h3> TNFR1 and TNFR2 are constitutively expressed in Kupffer cells and their expression is strongly increased by somatostatin. Moreover somatostatin increases Kupffer cell apoptosis. These findings may in part explain the antineoplasmatic effect of somatostatin.

Up-regulation of MicroRNA-155 in Macrophages Contributes to Increased Tumor Necrosis Factor α (TNFα) Production via Increased mRNA Half-life in Alcoholic Liver Disease

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) and Toll-Like Receptors 4 (TLR4)-LPS-mediated increase in TNFα production has a central role in the pathogenesis of alcoholic liver disease. Micro-RNA (miR)-125b, miR-146a, and miR-155 can regulate inflammatory responses to LPS. Here we evaluated the involvement of miRs in alcohol-induced macrophage activation. Chronic alcohol treatment in vitro resulted in a time-dependent increase in miR-155 but not miR-125b or miR-146a levels in RAW 264.7 macrophages. Furthermore, alcohol pretreatment augmented LPS-induced miR-155 expression in macrophages. We found a linear correlation between alcohol-induced increase in miR-155 and TNFα induction. In a mouse model of alcoholic liver disease, we found a significant increase in both miR-155 levels and TNFα production in isolated KCs when compared with pair-fed controls. The mechanistic role of miR-155 in TNFα regulation was indicated by decreased TNFα levels in alcohol-treated macrophages after inhibition of miR-155 and by increased TNFα production after miR-155 overexpression, respectively. We found that miR-155 affected TNFα mRNA stability because miR-155 inhibition decreased whereas miR-155 overexpression increased TNFα mRNA half-life. Using the NF-κB inhibitors, MG-132 or Bay11-7082, we demonstrated that NF-κB activation mediated the up-regulation of miR-155 by alcohol in KCs. In conclusion, our novel data demonstrate that chronic alcohol consumption increases miR-155 in macrophages via NF-κB and the increased miR-155 contributes to alcohol-induced elevation in TNFα production via increased mRNA stability.

Ethanol-induced HO-1 and NQO1 Are Differentially Regulated by HIF-1α and Nrf2 to Attenuate Inflammatory Cytokine Expression

Oxidative stress plays an important role in alcohol-induced inflammation and liver injury. Relatively less is known about how Kupffer cells respond to oxidative stress-induced expression of heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1) to blunt inflammation and liver injury. We showed that Kupffer cells from ethanol-fed rats and ethanol-treated rat Kupffer cells and THP-1 cells displayed increased mRNA expression of HO-1, NQO1, and hypoxia-inducible factor-1α (HIF-1α). Our studies showed that silencing with HIF-1α and JNK-1 siRNAs attenuated ethanol-mediated mRNA expression of HO-1, but not NQO1, whereas Nrf2 siRNA attenuated the mRNA expression of both HO-1 and NQO1. Additionally, JunD but not JunB formed an activator protein-1 (AP-1) oligomeric complex to augment HO-1 promoter activity. Ethanol-induced HO-1 transcription involved antioxidant response elements, hypoxia-response elements, and an AP-1 binding motif in its promoter, as demonstrated by mutation analysis of the promoter, EMSA, and ChIP. Furthermore, livers of ethanol-fed c-Jun(fl/fl) mice showed reduced levels of mRNA for HO-1 but not of NQO1 compared with ethanol-fed control rats, supporting the role of c-Jun or the AP-1 transcriptional complex in ethanol-induced HO-1 expression. Additionally, attenuation of HO-1 levels in ethanol-fed c-Jun(fl/fl) mice led to increased proinflammatory cytokine expression in the liver. These results for the first time show that ethanol regulates HO-1 and NQO1 transcription by different signaling pathways. Additionally, up-regulation of HO-1 protects the liver from excessive formation of inflammatory cytokines. These studies provide novel therapeutic targets to ameliorate alcohol induced inflammation and liver injury.

Adiponectin and Heme Oxygenase-1 Suppress TLR4/MyD88-Independent Signaling in Rat Kupffer Cells and in Mice after Chronic Ethanol Exposure

Alcoholic liver disease is mediated via activation of TLR4 signaling; MyD88-dependent and -independent signals are important contributors to injury in mouse models. Adiponectin, an anti-inflammatory adipokine, suppresses TLR4/MyD88-dependent responses via induction of heme oxygenase-1 (HO-1). Here we investigated the interactions between chronic ethanol, adiponectin, and HO-1 in regulation of TLR4/MyD88-independent signaling in macrophages and an in vivo mouse model. After chronic ethanol feeding, LPS-stimulated expression of IFN-β and CXCL10 mRNA was increased in primary cultures of Kupffer cells compared with pair-fed control mice. Treatment of Kupffer cells with globular adiponectin (gAcrp) normalized this response. LPS-stimulated IFN-β/CXCL10 mRNA and CXCL10 protein was also reduced in RAW 264.7 macrophages treated with gAcrp or full-length adiponectin. gAcrp and full-length adiponectin acted via adiponectin receptors 1 and 2, respectively. gAcrp decreased TLR4 expression in both Kupffer cells and RAW 264.7 macrophages. Small interfering RNA knockdown of HO-1 or inhibition of HO-1 activity with zinc protoporphyrin blocked these effects of gAcrp. C57BL/6 mice were exposed to chronic ethanol feeding, with or without treatment with cobalt protoporphyrin, to induce HO-1. After chronic ethanol feeding, mice were sensitized to in vivo challenge with LPS, expressing increased IFN-β/CXCL10 mRNA and CXCL10 protein in liver compared with control mice. Pretreatment with cobalt protoporphyrin 24 h before LPS challenge normalized this effect of ethanol. Adiponectin and induction of HO-1 potently suppressed TLR4-dependent/MyD88-independent cytokine expression in primary Kupffer cells from rats and in mouse liver after chronic ethanol exposure. These data suggest that induction of HO-1 may be a useful therapeutic strategy in alcoholic liver disease.

Differential Cell Sensitivity between OTA and LPS upon Releasing TNF-α

The release of tumor necrosis factor α (TNF-α) by ochratoxin A (OTA) was studied in various macrophage and non-macrophage cell lines and compared with E. coli lipopolysaccharide (LPS) as a standard TNF-α release agent. Cells were exposed either to 0, 2.5 or 12.5 µmol/L OTA, or to 0.1 µg/mL LPS, for up to 24 h. OTA at 2.5 µmol/L and LPS at 0.1 µg/mL were not toxic to the tested cells as indicated by viability markers. TNF-α was detected in the incubated cell medium of rat Kupffer cells, peritoneal rat macrophages, and the mouse monocyte macrophage cell line J774A.1: TNF-α concentrations were 1,000 pg/mL, 1,560 pg/mL, and 650 pg/mL, respectively, for 2.5 µmol/L OTA exposure and 3,000 pg/mL, 2,600 pg/mL, and 2,115 pg/mL, respectively, for LPS exposure. Rat liver sinusoidal endothelial cells, rat hepatocytes, human HepG2 cells, and mouse L929 cells lacked any cytokine response to OTA, but showed a significant release of TNF-α after LPS exposure, with the exception of HepG2 cells. In non-responsive cell lines, OTA lacked both any activation of NF-κB or the translocation of activated NF-κB to the cell nucleus, i.e., in mouse L929 cells. In J774A.1 cells, OTA mediated TNF-α release via the pRaf/MEK 1/2-NF-κB and p38-NF-κB pathways, whereas LPS used pRaf/MEK 1/2–NF-κB, but not p38-NF-κB pathways. In contrast, in L929 cells, LPS used other pathways to activate NF-κB. Our data indicate that only macrophages and macrophage derived cells respond to OTA and are considered as sources for TNF-α release upon OTA exposure.

CD36 Is a Novel Serum Amyloid A (SAA) Receptor Mediating SAA Binding and SAA-induced Signaling in Human and Rodent Cells

Serum amyloid A (SAA) is a major acute phase protein involved in multiple physiological and pathological processes. This study provides experimental evidence that CD36, a phagocyte class B scavenger receptor, functions as a novel SAA receptor mediating SAA proinflammatory activity. The uptake of Alexa Fluor 488 SAA as well as of other well established CD36 ligands was increased 5-10-fold in HeLa cells stably transfected with CD36 when compared with mock-transfected cells. Unlike other apolipoproteins that bind to CD36, only SAA induced a 10-50-fold increase of interleukin-8 secretion in CD36-overexpressing HEK293 cells when compared with control cells. SAA-mediated effects were thermolabile, inhibitable by anti-SAA antibody, and also neutralized by association with high density lipoprotein but not by association with bovine serum albumin. SAA-induced cell activation was inhibited by a CD36 peptide based on the CD36 hexarelin-binding site but not by a peptide based on the thrombospondin-1-binding site. A pronounced reduction (up to 60-75%) of SAA-induced pro-inflammatory cytokine secretion was observed in cd36(-/-) rat macrophages and Kupffer cells when compared with wild type rat cells. The results of the MAPK phosphorylation assay as well as of the studies with NF-kappaB and MAPK inhibitors revealed that two MAPKs, JNK and to a lesser extent ERK1/2, primarily contribute to elevated cytokine production in CD36-overexpressing HEK293 cells. In macrophages, four signaling pathways involving NF-kappaB and three MAPKs all appeared to contribute to SAA-induced cytokine release. These observations indicate that CD36 is a receptor mediating SAA binding and SAA-induced pro-inflammatory cytokine secretion predominantly through JNK- and ERK1/2-mediated signaling.

The anti‐inflammatory effects of adiponectin are mediated via a heme oxygenase‐1–dependent pathway in rat Kupffer cells†

Altered expression and activity of immunomodulatory cytokines plays a major role in the pathogenesis of alcoholic liver disease. Chronic ethanol feeding increases the sensitivity of Kupffer cells, the resident hepatic macrophage, to lipopolysaccharide (LPS), leading to increased tumor necrosis factor alpha (TNF-alpha) expression. This sensitization is normalized by treatment of primary cultures of Kupffer cells with adiponectin, an anti-inflammatory adipokine. Here we tested the hypothesis that adiponectin-mediated suppression of LPS signaling in Kupffer cells is mediated via an interleukin-10 (IL-10)/heme oxygenase-1 (HO-1) pathway after chronic ethanol feeding. Knockdown of IL-10 expression in primary cultures of Kupffer cells with small interfering RNA (siRNA) prevented the inhibitory effect of globular adiponectin (gAcrp) on LPS-stimulated TNF-alpha expression. gAcrp increased IL-10 mRNA and protein expression, as well as expression of the IL-10 inducible gene, HO-1; expression was higher in Kupffer cells from ethanol-fed rats compared with pair-fed controls. Although IL-10 receptor surface expression on Kupffer cells was not affected by ethanol feeding, IL-10-mediated phosphorylation of STAT3 and expression of HO-1 was higher in Kupffer cells after ethanol feeding. Inhibition of HO-1 activity, either by treatment with the HO-1 inhibitor zinc protoporphyrin or by siRNA knockdown of HO-1, prevented the inhibitory effect of gAcrp on LPS-stimulated TNF-alpha expression in Kupffer cells. LPS-stimulated TNF-alpha expression in liver was increased in mice after chronic ethanol exposure. When mice were treated with cobalt protoporphyrin to induce HO-1 expression, ethanol-induced sensitivity to LPS was ameliorated. gAcrp prevents LPS-stimulated TNF-alpha expression in Kupffer cells through the activation of the IL-10/STAT3/HO-1 pathway. Kupffer cells from ethanol-fed rats are highly sensitive to the anti-inflammatory effects of gAcrp; this sensitivity is associated with both increased expression and sensitivity to IL-10.

LIVER X RECEPTOR AGONIST GW3965 DOSE-DEPENDENTLY REGULATES LPS-MEDIATED LIVER INJURY AND MODULATES POSTTRANSCRIPTIONAL TNF-α PRODUCTION AND P38 MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION IN LIVER MACROPHAGES

Modulation of the host inflammatory response to infection may be a key approach to improve the outcome of patients with sepsis and organ injury. We previously reported that pretreatment of rats with the liver X receptor (LXR) agonist GW3965 reduced the liver injury associated with endotoxemia and attenuated the production of TNF-alpha by rat Kupffer cells. Here, we examine the dose-dependent effect of GW3965 on liver injury and cytokine production in a rat model of endotoxemia and explore the mechanisms underlying TNF-alpha attenuation in Kupffer cells. Low doses of GW3965 (0.1 or 0.3 mg/kg) administered 30 min before infusion of LPS and peptidoglycan significantly attenuated the increase in plasma levels of the liver injury markers alanine aminotransferase and bilirubin (6 h) as well as the inflammatory mediators TNF-alpha (1 h) and prostaglandin E2 (6 h) associated with endotoxemia. In contrast, pretreatment with a higher dose of GW3965 (1.0 mg/kg) had no such effect. Studies in primary cultures of rat Kupffer cells demonstrated that LXR agonist treatment attenuated both the secreted and cell-associated levels of TNF-alpha, whereas TNF-alpha mRNA levels were not altered. Phosphorylated p38 mitogen-activated protein kinase, which plays a major role in production of TNF-alpha at the posttranscriptional level, was attenuated by GW3965 treatment in Kupffer cells. Experiments in murine LXR-deficient Kupffer cells demonstrated enhanced production of TNF-alpha in Kupffer cells from LXR-alpha(-/-) mice when challenged with LPS compared with LXR-beta(-/-) and wild-type Kupffer cells. Taken together, these results argue in favor of a novel mechanism for LXR-mediated attenuation of liver injury by interfering with posttranscriptional regulation of TNF-alpha in Kupffer cells.

Cholera toxin‐sensitive GTP‐binding protein‐coupled activation of augmenter of liver regeneration (ALR) receptor and its function in rat kupffer cells

Mitogenic effect of augmenter of liver regeneration (ALR), a protein produced and released by hepatocytes, on hepatocytes in vivo but not in vitro suggests that the effect is mediated by nonparenchymal cells. Since mediators produced by Kupffer cells are implicated in hepatic regeneration, we investigated receptor for ALR and its functions in rat Kupffer cells. Kupffer cells were isolated from rat liver by enzymatic digestion and centrifugal elutriation. Radioligand ([(125)I] ALR) receptor binding, ALR-induced GTP/G-protein association, and nitric oxide (NO), tumor necrosis factor (TNF)-alpha, and interleukin-6 (IL-6) synthesis were determined. High-affinity receptor for ALR, belonging to the G-protein family, with K(d) of 1.25 +/- 0.18 nM and B(max) of 0.26 +/- 0.02 fmol/microg DNA was identified. ALR stimulated NO, TNF-alpha, and IL-6 synthesis via cholera toxin-sensitive G-protein, as well as p38-MAPK activity and nuclear translocation of NFkappaB. While inhibitor of NFkappaB (MG132) inhibited ALR-induced NO synthesis, MG132 and p38-MAPK inhibitor (SB203580) abrogated ALR-induced TNF-alpha and IL-6 synthesis. ALR also prevented the release of mediator(s) from Kupffer cells that cause inhibition of DNA synthesis in hepatocytes. Administration of ALR to 40% partially hepatectomized rats increased expression of TNF-alpha, IL-6, and inducible nitric oxide synthase (iNOS) and caused augmentation of hepatic regeneration. These results demonstrate specific G-protein coupled binding of ALR and its function in Kupffer cells and suggest that mediators produced by ALR-stimulated Kupffer cells may elicit physiologically important effects on hepatocytes.

Proteomics reveals acute pro‐inflammatory and protective responses in rat Kupffer cells and hepatocytes after chemical initiation of liver cancer and after LPS and IL‐6

Inflammation is a key event in the development of liver cancer. We studied early inflammatory responses of Kupffer cells (KCs) and hepatocyte (HC) after cancer initiation. The chemical carcinogen N-nitrosomorpholine (NNM) was used in a rat model. We applied a comprehensive analytical strategy including metabolic labeling, 2-D PAGE, LC-MS/MS-based spot identification and shotgun proteomics and thus determined the rates of synthesis of individual proteins, compared whole tissue with isolated constituent cells and performed in vivo to in vitro comparisons of NNM effects. NNM increased synthesis of overall and 138 individual proteins identified in HC and/or KC, indicating reprogramming of metabolism favoring protection, repair and replacement of cell constituents in HC and KC. Secretome analysis by 2-D PAGE and shotgun proteomics of HC revealed the induction of acute phase proteins, in case of KC of proteases, cytokines and chemokines, indicating inflammatory effects. All responses were induced rapidly, independently of signals from other cells, and closely mimicked the pro-inflammatory and protective effects of inflammation modulators LPS in KC and IL-6 in HC. In conclusion, the carcinogen NNM exerts pro-inflammatory effects in the liver, partially by direct activation of KC. The acute inflammation and its protective component will enhance formation, survival and proliferation of initiated cells and may therefore act synergistically with the genotoxic action of the carcinogen.

Demonstration of glucose-6-phosphate dehydrogenase in rat Kupffer cells by a newly-developed ultrastructural enzyme-cytochemistry.

Although various tissue macrophages possess high glucose-6-phosphate dehydrogenase (G6PD) activity, which is reported to be closely associated with their phagocytotic/bactericidal function, the fine subcellular localization of this enzyme in liver resident macrophages (Kupffer cells) has not been determined. We have investigated the subcellular localization of G6PD in Kupffer cells in rat liver, using a newly developed enzyme-cytochemical (copper-ferrocyanide) method. Electron-dense precipitates indicating G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of Kupffer cells. Cytochemical controls ensured specific detection of the enzymatic activity. Rat Kupffer cells abundantly possessed enzyme-cytochemically detectable G6PD activity. Kupffer cell G6PD may play a role in liver defense by delivering NADPH to NADPH-dependent enzymes. G6PD enzyme-cytochemistry may be a useful tool for the study of Kupffer cell functions.

Silibinin protects OTA‐mediated TNF‐α release from perfused rat livers and isolated rat Kupffer cells

We studied the inhibitory effect of silibinin on ochratoxin A (OTA) and LPS-mediated tumor necrosis factor alpha (TNF-alpha) release and the leakage of cytotoxic markers glutamate dehydrogenase (GLDH) and lactate dehydrogenase (LDH), from isolated blood-free perfused rat livers, and from isolated pure rat Kupffer cells. In the recirculation perfusion model at the end point 90 min, 2.5 micromol/L OTA released 2600 pg/mL TNF-alpha without effects on liver vitality. LPS at 0.1 microg/mL induced 3000 pg TNF-alpha/mL with slight leakage of GLDH and LDH. Under similar experimental conditions, the addition of silibinin 10 min prior to OTA and LPS showed dose-dependent protection against OTA or LPS-induced hepatic TNF-alpha release. High-dose of silibinin (12.5 microg/mL) also completely restored GLDH and LDH levels in the perfusate. Pretreatment of isolated Kupffer cells with 0.02, 0.1, 0.5, 2.5, and 12.5 microg silibinin/mL 30 min prior to OTA reduced OTA-induced TNF-alpha levels to 90, 70, 25, 25, and 25% at 4 h, respectively, and abrogated any TNF-alpha release at 24 h. Similarly, in the presence of silibinin LPS-induced TNF-alpha levels decreased at 4 h to 71, 57, 18, 22, and 18%, respectively. However, after 24 h of LPS exposition the protection by silibinin vanished and TNF-alpha partially recurred into the incubation medium under LPS. In summary, silibinin had hepatoprotective effects against OTA- or LPS-mediated TNF-alpha release and also reduced the cytotoxicity of both toxins. Isolated Kupffer cells were even more sensitive to the protective effect than perfused livers and responded to very low concentrations of silibinin with a strong inhibition of toxins-mediated TNF-alpha release.

18beta‐glycyrrhetinic acid prevents free fatty acid‐induced lipotoxicity by inhibiting ER stress and oxidative stress

The endoplasmic reticulum (ER) stress signal pathway has been linked to free fatty acid (FFA)‐induced inflammatory response and disease progression of nonalcoholic fatty liver disease in clinic. We have previously shown that 18?β‐glycyrrhetinic acid (GA) prevents FFA‐induced hepatic lipotoxicity by inhibiting cathepsin B expression and activity. But the underlying cellular mechanism remains unclear. This study determined if GA prevents FFA‐induced lipotoxicity by regulating ER stress signaling pathways.MethodsMouse macrophages, primary rat hepatocytes and Kupffer cells were used in this study. Cells were treated with palmitic acid (PA) and oleic acid (OA) in the presence or absence of GA. Activation of the UPR was determined by real time RT‐PCR and Western Blot analysis. The apoptosis was detected by fluorescence microscopy using Annexin V‐FITC/propidium iodide. Reactive oxygen species (ROS) was measured using dichlorofluorescein diacetate (DCFH‐DA).ResultsPA dose‐dependently induced UPR activation, ROS formation, and apoptosis both in macrophages and hepatocytes, while OA had no effect. GA not only inhibited PA‐induced UPR activation, but also inhibited PA‐induced ROS formation. PA‐induced ROS formation and cathepsin B expression were inhibited in CHOP knockout macrophages and hepatocytes.ConclusionGA prevents FFA‐induced lipotoxicity by inhibiting ER stress and oxidative stress.

Role of SIRT1 in regulation of LPS- or two ethanol metabolites-induced TNF-α production in cultured macrophage cell lines

Dysregulation of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of alcoholic liver injury. Sirtuin 1 (SIRT1) is an NAD(+)-dependent class III protein deacetylase that is known to be involved in regulating production of proinflammatory cytokines including TNF-alpha. In the present study, we examined the role of SIRT1 signaling in TNF-alpha generation stimulated by either lipopolysaccharide (LPS), acetaldehyde (AcH), or acetate (two major metabolites of ethanol) in two cultured macrophage cell lines. In both rat Kupffer cell line 1 (RKC1) and murine RAW 264.7 macrophages, treatment with either LPS, AcH, or acetate caused significant decreases in SIRT1 transcription, translation, and activation, which essentially demonstrated an inverse relationship with TNF-alpha levels. LPS, AcH, and acetate each provoked the release of TNF-alpha from RKC1 cells, whereas coincubation with resveratrol (a potent SIRT1 agonist) inhibited this effect. Conversely, addition of sirtinol (a known SIRT1 inhibitor) or knocking down SIRT1 by the small silencing SIRT1 plasmid (SIRT1shRNA) augmented TNF-alpha release, suggesting that impairment of SIRT1 may contribute to TNF-alpha secretion. Further mechanistic studies revealed that inhibition of SIRT1 by LPS, AcH, or acetate was associated with a marked increase in the acetylation of the RelA/p65 subunit of nuclear transcription factor (NF-kappaB) and promotion of NF-kappaB transcriptional activity. Taken together, our findings suggest that SIRT1-NF-kappaB signaling is involved in regulating LPS- and metabolites-of-ethanol-mediated TNF-alpha production in rat Kupffer cells and in murine macrophages. Our study provides new insights into understanding the molecular mechanisms underlying the development of alcoholic steatohepatitis.

Ferritin Expression in Rat Hepatocytes and Kupffer Cells after Lead Nitrate Treatment

Lead nitrate induces hepatocyte proliferation and subsequent apoptosis in rat livers. Iron is a constituent of heme and is also required for cell proliferation. In this study, the expression of ferritin light-chain (FTL), the major iron storage protein, was investigated in rat livers after a single intravenous injection of lead nitrate. Western blotting and immunohistochemistry revealed that FTL was increased in hepatocytes around the central veins and strongly expressed in nonparenchymal cells. Some FTL-positive nonparenchymal cells were identified as Kupffer cells that were positive for CD68. FTL-positive Kupffer cells occupied about 60% of CD68-positive cells in the periportal and perivenous areas. The relationships between FTL expression and apoptosis induction or the engulfment of apoptotic cells were examined. TUNEL-positive cells were increased in the treatment group, and enhanced expression of milk fat globule EGF-like 8 was demonstrated in some Kupffer cells and hepatocytes, indicating enhanced apoptosis induction and phagocytosis of apoptotic cells. FTL-positive Kupffer cells were not detected without lead nitrate treatment or in rat livers treated with clofibrate, which induces hepatocyte proliferation but not apoptosis. These results suggest that FTL expression in Kupffer cells after lead treatment is dependent on phagocytosis of apoptotic cells.

Effect of Poly(N-vinyl-pyrrolidone)-block-poly(d,l-lactide) as Coating Agent on the Opsonization, Phagocytosis, and Pharmacokinetics of Biodegradable Nanoparticles

The effect of the coating polymer poly(N-vinyl-pyrrolidone) (PVP) on the protein adsorption, phagocytosis, and pharmacokinetics of poly(D,L-lactide)-based nanoparticles was evaluated in vitro and in vivo. Control poly(ethylene glycol) (PEG)-coated nanoparticles were included for comparison. While no difference between PEG- and PVP-decorated nanoparticles in terms of amount of adsorbed protein was evident upon incubation in single protein solutions (BSA, IgG), incubation in serum revealed a protein adsorption pattern both quantitatively and qualitatively distinct. Larger amounts of complement components and immunoglobulins were found to adhere to PVP-coated particles, whereas PEG particles showed preferential adsorption of apolipoproteins. Furthermore, preopsonization in fresh rather than heat-inactivated serum enhanced uptake of both types of particles by murine RAW 264.7 macrophages. However, when isolated rat Kupffer cells were employed, activation of the complement system significantly enhanced the uptake of PVP-coated nanoparticles compared to PEG particles. Ultimately, PVP-coated nanoparticles exhibited considerably shorter circulation times compared to their PEG counterparts when administered intravenously to rats.

Role of JAK/STAT signaling pathway in pancreatic elastase-induced secretion of interleukin-18 within rat Kupffer cells

目的：探讨JAK／STAT信号通路在胰弹性蛋白酶（elastase）诱导Kupffer细胞分泌IL-18中的作用机制．方法：采用酶消化法及密度梯度离心法将提取的肝脏Kupffer细胞分为4组，A组：生理盐水组（正常对照组）；B组：脂多糖（LPS）处理组；C组：LPS和elastase处理组：D组：AG490预处理组．采用酶免疫吸附（ELISA）法检测Kupffer细胞上清液中IL-18含量；细胞免疫荧光染色技术和Western blot法检测细胞总蛋白中JAK2的表达．结果：与A组比较，B组在给予LPS刺激后，JAK2蛋白的表达以及上清液中IL-18含量均明显增加（IL-18：312．23±20．5ng／Lvs13．50±2．18ng／L，P〈0．01）；C组在同时给予LPS和elastase刺激后，JAK2的表达显著升高，上清液中IL-18含量与B组比较也显著增加（P〈0．01）；而D组在预先给予AG490处理后，JAK2表达明显下降，上清液中IL-18含量也有不同程度降低，与C组比较差异有统计学意义（317.31±25．24ng／L vs 438．86±21．32ng／L，P〈0．01），但与B组比较，各值仅有轻微变化，差异无统计学意义．结论：抑制JAK／STAT通路的活化可下调elastase诱导Kupffer细胞分泌促炎症因子IL-18的表达，这可能有助于减轻急性胰腺炎时炎症反应和肝损伤．