20 The results showed a significant inverse correlation between Doppler measurements and HVPG values. However, a correlation between the portal vein velocity and the HVPG was not confirmed in a recent study.21 Surprisingly, in these studies, neither hepatic artery resistance nor mesenteric artery resistance was correlated with the severity of portal hypertension. It should be noted that this method may not accurately characterize the portal blood flow because it measures only the peak velocity, whereas the flow is known

to be parabolic. Furthermore, see more this method is operator-dependent and has poor reproducibility in obese patients. Thus, further studies are needed to confirm these results, and studies should be performed in patients with asymptomatic cirrhosis to determine the portal vein velocity or flow values that correspond to the presence Napabucasin cell line of severe portal hypertension. Different factors contribute to the increased vascular resistance of the liver in patients with cirrhosis.22 One component is the hyperproduction of endogenous vasoconstrictors. For example, serum endothelin levels have

been shown to be significantly correlated with HVPG values in patients with cirrhosis.23 Thus, serum endothelin levels could be used to evaluate the degree of portal hypertension; however, further studies are needed to determine whether this dosage can be used in clinical practice. Recently, peripheral circulating cells associated with vascular injury were evaluated in patients with cirrhosis.24 The results showed

that the circulating endothelial cell count or the ratio of circulating endothelial cells to the platelet count is potentially a new biomarker of portal hypertension, but further clinical investigations are needed to confirm these results. Increased hepatic vascular resistance in patients with cirrhosis is also influenced by the presence and extent of fibrosis.4, 5 In one recent study, the area of liver collagen, which is 上海皓元 the major component of fibrous tissue, was measured by computer-assisted image analysis and was found to be significantly correlated with the HVPG in patients with cirrhosis.5 Accordingly, an evaluation of the extent of hepatic fibrosis may provide information about the presence and severity of portal hypertension. The noninvasive estimation of hepatic fibrosis has been a subject of extensive research in the last 10 years. However, only a few of these procedures have been evaluated for the noninvasive diagnosis of portal hypertension (Table 2). Only the methods that have evaluated the relationship between hepatic fibrosis and portal hypertension are reported in this review. Different markers of hepatic fibrosis have been studied to assess portal hypertension.

By multivariate analysis, BMS-777607 nmr the combination of 3D-CRT with HAIC was an independent contributing factor for OS (hazard ratio, 3.2; 95% confidence interval, 1.692–6.021; P < 0.001) among intrahepatic HCC non-responders to HAIC. 3D-CRT for PVTT combined with HAIC could

provide survival benefit to non-responder to HAIC. “
“Background and Aim:  Platelets provide many functions in the body, especially to the liver. The purpose of this study is to investigate the effect of thrombocytosis with acute hepatitis induced by anti-Fas antibody and its mechanism. Methods:  Acute hepatitis was induced by administration of anti-Fas antibody in normal and thrombocytotic C57BL6J mice. For thrombocytosis, thrombopoietin; PEG-rHuMGDF was injected 5 days before and just prior to administration of anti-Fas Panobinostat cost antibody. To investigate the mechanisms, hepatocyte cell line (AML12) and sinusoidal endothelial cell line (M1) were induced apoptosis by staurosporine. They were cultured with platelets or thrombopoietin. Examination items were as follows: platelet number, alanine aminotransferase (ALT), histological findings, TUNEL (TdT-mediated dUTP-biotin Nick

End Labeling) staining, and the expression of proteins associated with apoptosis in vivo and in vitro. Results:  Platelets were significantly increased in the thrombocytotic group (P < 0.01). Serum ALT levels were significantly reduced by thrombocytosis at 6, 24 and 72 h after the administration (P < 0.05). In histological findings, hemorrhagic necrosis was observed in the normal group, but not observed in the thrombocytotic group. TUNEL-positive hepatocytes were reduced and the expression of cleaved caspase-3 was significantly decreased 上海皓元医药股份有限公司 in the thrombocytotic

group. The phosphorylation of Akt, the increment of Bcl-xL and the decrease of cleaved caspase-3 were observed in AML12 cells cultured with platelets, but were not observed cultured with thrombopoietin. Platelets and thrombopoietin had no anti-apoptotic effect on M1 cells. Conclusion:  Increase of platelets has a preventative effect against acute hepatitis induced by the anti-Fas antibody. It is suggested that platelets have a direct protective effect against apoptosis of hepatocytes. “
“Goel GA, Deshpande A, Lopez R, Hall GS, van Duin D, Carey WD. Increased rate of spontaneous bacterial peritonitis among cirrhotic patients receiving pharmacologic acid suppression. Clin Gastroenterol Hepatol 2012;10:422-427. (Reprinted with permission.) BACKGROUND & AIMS: Patients with cirrhosis frequently receive proton pump inhibitor (PPI) or H2-receptor antagonist therapies. We investigated whether acid-suppressive therapy is associated with spontaneous bacterial peritonitis (SBP) in cirrhotic patients with ascites.

By multivariate analysis, PD-0332991 in vitro the combination of 3D-CRT with HAIC was an independent contributing factor for OS (hazard ratio, 3.2; 95% confidence interval, 1.692–6.021; P < 0.001) among intrahepatic HCC non-responders to HAIC. 3D-CRT for PVTT combined with HAIC could

provide survival benefit to non-responder to HAIC. “
“Background and Aim:  Platelets provide many functions in the body, especially to the liver. The purpose of this study is to investigate the effect of thrombocytosis with acute hepatitis induced by anti-Fas antibody and its mechanism. Methods:  Acute hepatitis was induced by administration of anti-Fas antibody in normal and thrombocytotic C57BL6J mice. For thrombocytosis, thrombopoietin; PEG-rHuMGDF was injected 5 days before and just prior to administration of anti-Fas selleck kinase inhibitor antibody. To investigate the mechanisms, hepatocyte cell line (AML12) and sinusoidal endothelial cell line (M1) were induced apoptosis by staurosporine. They were cultured with platelets or thrombopoietin. Examination items were as follows: platelet number, alanine aminotransferase (ALT), histological findings, TUNEL (TdT-mediated dUTP-biotin Nick

End Labeling) staining, and the expression of proteins associated with apoptosis in vivo and in vitro. Results:  Platelets were significantly increased in the thrombocytotic group (P < 0.01). Serum ALT levels were significantly reduced by thrombocytosis at 6, 24 and 72 h after the administration (P < 0.05). In histological findings, hemorrhagic necrosis was observed in the normal group, but not observed in the thrombocytotic group. TUNEL-positive hepatocytes were reduced and the expression of cleaved caspase-3 was significantly decreased medchemexpress in the thrombocytotic

group. The phosphorylation of Akt, the increment of Bcl-xL and the decrease of cleaved caspase-3 were observed in AML12 cells cultured with platelets, but were not observed cultured with thrombopoietin. Platelets and thrombopoietin had no anti-apoptotic effect on M1 cells. Conclusion:  Increase of platelets has a preventative effect against acute hepatitis induced by the anti-Fas antibody. It is suggested that platelets have a direct protective effect against apoptosis of hepatocytes. “
“Goel GA, Deshpande A, Lopez R, Hall GS, van Duin D, Carey WD. Increased rate of spontaneous bacterial peritonitis among cirrhotic patients receiving pharmacologic acid suppression. Clin Gastroenterol Hepatol 2012;10:422-427. (Reprinted with permission.) BACKGROUND & AIMS: Patients with cirrhosis frequently receive proton pump inhibitor (PPI) or H2-receptor antagonist therapies. We investigated whether acid-suppressive therapy is associated with spontaneous bacterial peritonitis (SBP) in cirrhotic patients with ascites.

Results: Complete data were available in 301 patients with cirrhosis (cryptogenic n = 94, non-cryptogenic n = 207). Patients with cryptogenic cirrhosis were older (mean SAHA HDAC in vivo age 66.4 vs. 60.7, p < 0.0001), had more females (43.6% vs. 26.6%, p = 0.003), less severe disease severity (Child Pugh C 8.5% vs 15.9%, p = 0.042) and a higher prevalence of the metabolic syndrome (83% vs. 51.2%, p < 0.0001)

compared with non-cryptogenic cirrhosis. During the 5-year study period, adults with cryptogenic cirrhosis had a longer total hospital admission stay compared to non-cryptogenic cirrhosis (median 10.5 vs 8 days, p = 0.08). Further analysis demonstrated a longer hospital admission duration for cryptogenic cirrhosis due to non-liver related morbidity (median 19.0 days vs. 13.0 days, p = 0.04), rather than liver related morbidity (median 14.0 days vs 11.0 days, p = 0.06). A higher proportion of stroke (6% vs 2%, p = 0.02) and cardiovascular disease (6% vs 3%, p = 0.05) were responsible for the increased hospitalization for non-liver related morbidity in

cryptogenic compared to non-cryptogenic cirrhotic patients. Kaplan-Meier survival analysis showed no significant difference in survival between both types of cirrhosis during the period of study (Log rank statistic 0.56). Conclusion: Cryptogenic GSI-IX molecular weight cirrhosis is associated with an increased morbidity, but not mortality, compared to non-cryptogenic cirrhosis. This difference is due to a greater burden of non-liver related complications in the 上海皓元 former. Key Word(s): Na Presenting Author: KALAIYARASI KALIYAPERUMAL Additional Authors: Na Corresponding Author: KALAIYARASI KALIYAPERUMAL Affiliations: Tan Tock Seng Hospital Objective: We present a 57 year old gentleman, who has liver cirrhosis from likely chronic systemic iron overload, haemolysis and iron deposition due to a rare form of non transfusion dependant thalassaemia. His medical problems include hypergonadotrophic hypogonadism, osteoporosis and subclinical hypothyroidism. He had never undergone any blood or blood product transfusions in the past. He is a teetotaller. On physical examination he had

short stature, bronze skin, scleral icterus and multiple stigmata of chronic liver disease with hepatosplenomegaly. He had biochemical evidence of hemolysis and iron overload in addition to raised aspartate aminotransferase and unconjugated hyperbilirubinemia. Investigations done to rule out other causes of liver cirrhosis was negative in particular HFE gene mutation analysis (C282Y and H63D mutations were not detected). An ultrasound of the liver showed coarsened liver echo texture and nodular surface outline. Fibroscan stiffness reading was 27.4 kPa. Oesophagogastroduodenoscopy (OGD) showed the presence of portal hypertensive gastropathy. DNA sequence analysis revealed a rare IVSInt1 mutation in his Beta globin gene, forming an extremely rare and unusual compound heterozygote for a Beta globin and an unknown HPFH thalassaemia mutation.

After treatment with TGF-β (10 ng/ml, 96 hours) the methylation of E-cadherin promoter was induced (35.5 ± 1.96% in PLC/PRF/5), which was abrogated by pre-treatment with a methylation inhibitor 5-aza-2′-deoxycytidine (5-Aza) indicating the involvement of DNA methylation in this process. Treatment of

HCC cells with TGF-β (10 ng/ml, 72 hours) increased protein expression of DNMT3B and DNMT1, which are reported as targets of miR-29a. After treatment of cells with TGF-β (10 ng/ml), expression of miR-29a decreased by 27.8% in PLC/RF/5 cells and by 26.7% in HepG2 cells by 72 hours. Transfection of precursor miR-29a in PLC/PRF/5 cells partially blocked the suppression of E-cadherin protein expression (control, 48%; miR-29a, 72%) and methylation level of the promoter CpG islands (control, 27.3 ± 9.15%; miR-29a, 13.7 ± 2.85%) induced by TGF-β. [Conclusion] We show that the involvement

PD0325901 of miR-29a in TGF-β-induced EMT via epigenetic regulation of E-cadherin in HCC cells. These observations identify miR-29a as a unique mechanism of the regulation of EMT in HCC. Disclosures: The following people have nothing to disclose: Takayuki Kogure, Yasuteru Kondo, Eiji Kakazu, Masashi Ninomiya, Osamu Kimura, Tomoaki Iwata, Tatsuki Morosawa, Yasuyuki Fujisaka, Tooru Shimosegawa Background: selleck screening library Protein kinases MST1 & MST2 are the core of the Hippo pathway, which inactivate the transcriptional co-activator YAP through LATS phosphorylation. Inhibition of MST1 & MST2 leads to the activation of YAP where it translocates to the nucleus and promotes the medchemexpress transcription of pro proliferative genes. We hypothesize that knockdown

(KD) of MST1 & MST2 will push hepatocytes into cell cycle through activation of YAP. Methods:We are exploring a gene therapy approach using siRNAs coupled with liposomes to target the Hippo pathway to promote hepatocyte proliferation in non-regenerating livers. Results: We identified siRNA sequences that lead to 92 and 89% KD of MST1 and MST2 in a mouse liver hepatoma cell line in vitro. siRNA:liposome complexes injected i.v. resulted in 80% KD of expression in the liver using FVII as a control gene target. Using siRNAs targeting MST1 & MST2 with liposomes reduced expression to 66 and 40%, respectively in liver after 72 hours. The KD was confirmed by RT-qPCR and immunoblot. KD of MST1 and MST2 in mouse liver resulted in an increase of nuclear Yap localization and hepatocyte proliferation measured by incorporation of EdU and Ki67 immunostaining. After MST1 and MST2 KD there was a 3 and 5-fold increase of BIRC5/survivin and Foxm 1, respectively -both YAP target genes normally up-regulated in a regenerating liver. Conclusion: The KD of MST1 & MST2 provokes nuclear YAP translocation and hepatocyte proliferation in wild-type mice.

After treatment with TGF-β (10 ng/ml, 96 hours) the methylation of E-cadherin promoter was induced (35.5 ± 1.96% in PLC/PRF/5), which was abrogated by pre-treatment with a methylation inhibitor 5-aza-2′-deoxycytidine (5-Aza) indicating the involvement of DNA methylation in this process. Treatment of

HCC cells with TGF-β (10 ng/ml, 72 hours) increased protein expression of DNMT3B and DNMT1, which are reported as targets of miR-29a. After treatment of cells with TGF-β (10 ng/ml), expression of miR-29a decreased by 27.8% in PLC/RF/5 cells and by 26.7% in HepG2 cells by 72 hours. Transfection of precursor miR-29a in PLC/PRF/5 cells partially blocked the suppression of E-cadherin protein expression (control, 48%; miR-29a, 72%) and methylation level of the promoter CpG islands (control, 27.3 ± 9.15%; miR-29a, 13.7 ± 2.85%) induced by TGF-β. [Conclusion] We show that the involvement

see more of miR-29a in TGF-β-induced EMT via epigenetic regulation of E-cadherin in HCC cells. These observations identify miR-29a as a unique mechanism of the regulation of EMT in HCC. Disclosures: The following people have nothing to disclose: Takayuki Kogure, Yasuteru Kondo, Eiji Kakazu, Masashi Ninomiya, Osamu Kimura, Tomoaki Iwata, Tatsuki Morosawa, Yasuyuki Fujisaka, Tooru Shimosegawa Background: BGB324 cell line Protein kinases MST1 & MST2 are the core of the Hippo pathway, which inactivate the transcriptional co-activator YAP through LATS phosphorylation. Inhibition of MST1 & MST2 leads to the activation of YAP where it translocates to the nucleus and promotes the MCE transcription of pro proliferative genes. We hypothesize that knockdown

(KD) of MST1 & MST2 will push hepatocytes into cell cycle through activation of YAP. Methods:We are exploring a gene therapy approach using siRNAs coupled with liposomes to target the Hippo pathway to promote hepatocyte proliferation in non-regenerating livers. Results: We identified siRNA sequences that lead to 92 and 89% KD of MST1 and MST2 in a mouse liver hepatoma cell line in vitro. siRNA:liposome complexes injected i.v. resulted in 80% KD of expression in the liver using FVII as a control gene target. Using siRNAs targeting MST1 & MST2 with liposomes reduced expression to 66 and 40%, respectively in liver after 72 hours. The KD was confirmed by RT-qPCR and immunoblot. KD of MST1 and MST2 in mouse liver resulted in an increase of nuclear Yap localization and hepatocyte proliferation measured by incorporation of EdU and Ki67 immunostaining. After MST1 and MST2 KD there was a 3 and 5-fold increase of BIRC5/survivin and Foxm 1, respectively -both YAP target genes normally up-regulated in a regenerating liver. Conclusion: The KD of MST1 & MST2 provokes nuclear YAP translocation and hepatocyte proliferation in wild-type mice.

The histological findings were evaluated by three blinded, experienced liver-specific pathologists and were validated by discussion. Predictive variables associated with selleck compound each stage of liver fibrosis were assessed using multivariate analyses. The diagnostic performances of the markers were expressed as diagnostic specificity, sensitivity, and area under the receiving operator characteristic (AUROC) curve. The AUROC curve values for predicting the stage of fibrosis of serum WFA+-M2BP were compared with five other indicators (i.e., platelets, hyaluronic acid, AST/ALT ratio, AST-to-platelet ratio index, and FIB-4 index). Results:

The serum WFA+-M2BP value in patients with stage 0 (n = 35), stage 1 (n =

113), stage 2 (n = 49), stage 3 (n = 41), and stage 4 (n = 51) fibrosis had cutoff indexes of 0.57, 0.70, 1.02, 1.57, and 2.96, respectively. All pairs of groups differed significantly from each other according to the Steel-Dwass test. Multivariate regression analysis showed that the serum WFA+-M2BP values predicted the stage of fibrosis (> stage 2). The AUROC curve, sensitivity, and specificity of ABT-263 supplier serum WFA+-M2BP were 0.876, 85.9%, and 74.6% for severe fibrosis, respectively, (> stage 3) and 0.879, 74.6%, and 87.0%, for cirrhosis, respectively. When compared with the other surrogate markers and scoring systems, serum WFA+-M2BP had the greatest AUROC curve for diagnosing severe fibrosis and cirrhosis. 上海皓元 Conclusions: The measurement of serum WFA+-M2BP values based on glycan-based immunoassay provides an accurate and reliable method for assessing the stage of liver fibrosis in patients with NAFLD. This method appears quite

promising as a means for evaluating the natural course of the disease, therapeutic effects, and the suitability of liver biopsy. Disclosures: Michiie Sakamoto – Grant/Research Support: MSD, Canon The following people have nothing to disclose: Masanori Abe, Teruki Miyake, Atsushi Kuno, Yasuharu Imai, Yoshiyuki Sawai, Keisuke Hino, Yuichi Hara, Shuhei Hige, Masaaki Korenaga, Yoichi Hiasa, Masashi Mizokami, Hisashi Narimatsu Background and Objectives: In our animal studies, we showed that bone marrow cells (BMCs) infused via a peripheral vein efficiently repopulate the cirrhotic liver and produce collage-nases, resulting in reduced liver fibrosis, elevated serum albumin levels, and a significant increase in survival. We also confirmed that frequent BMC infusion contributed to suppression of tumor initiation in hepatocarcinogenic cirrhotic mice. Based on these results, we started “Autologous bone marrow cell infusion therapy” for liver cirrhotic patients as liver regeneration therapy using non-cultured autologous whole BMCs, and subsequently reported its safety and efficacy.

The separation AZD4547 manufacturer of hepatic parenchymal and nonparenchymal cells was performed essentially as previously described.24 The procedure is described in detail in the supporting information. A rat hepatic SEC line (NP31)25 was cultured on type I collagen–coated dishes (Iwaki, Chiba, Japan) in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C with 5% CO2. A retrovirus vector (pMxIG)26 and a retrovirus packaging cell line (Plat-E)27 were used to

generate recombinant retroviruses. Hemagglutinin (HA)-tagged complementary DNAs (cDNAs) of full-length Cas (Cas FL) and a Cas mutant lacking the SH3 domain (Cas ΔSH3)28 were subcloned into pMxIG, and ecotropic retroviruses were produced by the transient transfection of Plat-E cells with viral vectors using FuGENE (Roche, Basel, Switzerland). Infection was performed in the presence of 8 μg/mL Polybrene (Sigma, St Louis, MO). After fixation selleck screening library in 4% paraformaldehyde in phosphate-buffered saline for 10 minutes, cells were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 5 minutes at room temperature and incubated with Alexa 594–conjugated phalloidin (1:40; Invitrogen, Carlsbad, CA) in 1% bovine serum albumin in phosphate-buffered saline for 30 minutes at 37°C. Cells were mounted

with Vectashield MCE公司 and observed on an Axioplan2 microscope with AxioCam MRm controlled by Axiovision software (Carl Zeiss, Germany). NP31 cells, cultured on glass cover

slips, were fixed in 2% glutaraldehyde buffered with a 1 M cacodylate buffer (pH 7.4) for 12 hours at 4°C and then with 1% osmium tetroxide in a cacodylate buffer (pH 7.4) for 1 hour at 4°C. After dehydration in a graded series of ethanol solutions, cells were dried to a critical point and sputter-coated with osmium. Cell surfaces were examined with an S-4300 scanning microscope (Hitachi, Tokyo, Japan) at a 30-kV accelerating voltage. To create a reduction-of-function Cas allele by gene targeting, we deleted exon 2 of the Cas gene, which encodes the entire SH3 and the N-terminal part of the SD domain containing one YLVP motif and four YQxP motifs. To this end, we constructed a targeting vector containing Cas exon 2 flanked by two locus of X-over P1 (loxP) sequences and followed by the Frt-flanked neomycin resistance (Neo) gene (Fig. 1A). When the floxed Cas exon 2 was correctly excised, exon 1 joined in frame to exon 3, and this resulted in a Cas transcript devoid of the exon 2–derived segment. Correctly targeted embryonic stem cells, identified by Southern blotting and genomic polymerase chain reaction (PCR; Fig. 1B, left and middle panels), were selected and used for the generation of heterozygous mice (Cas+/floxNeo).

The separation selleck chemicals of hepatic parenchymal and nonparenchymal cells was performed essentially as previously described.24 The procedure is described in detail in the supporting information. A rat hepatic SEC line (NP31)25 was cultured on type I collagen–coated dishes (Iwaki, Chiba, Japan) in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C with 5% CO2. A retrovirus vector (pMxIG)26 and a retrovirus packaging cell line (Plat-E)27 were used to

generate recombinant retroviruses. Hemagglutinin (HA)-tagged complementary DNAs (cDNAs) of full-length Cas (Cas FL) and a Cas mutant lacking the SH3 domain (Cas ΔSH3)28 were subcloned into pMxIG, and ecotropic retroviruses were produced by the transient transfection of Plat-E cells with viral vectors using FuGENE (Roche, Basel, Switzerland). Infection was performed in the presence of 8 μg/mL Polybrene (Sigma, St Louis, MO). After fixation see more in 4% paraformaldehyde in phosphate-buffered saline for 10 minutes, cells were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 5 minutes at room temperature and incubated with Alexa 594–conjugated phalloidin (1:40; Invitrogen, Carlsbad, CA) in 1% bovine serum albumin in phosphate-buffered saline for 30 minutes at 37°C. Cells were mounted

with Vectashield MCE公司 and observed on an Axioplan2 microscope with AxioCam MRm controlled by Axiovision software (Carl Zeiss, Germany). NP31 cells, cultured on glass cover

slips, were fixed in 2% glutaraldehyde buffered with a 1 M cacodylate buffer (pH 7.4) for 12 hours at 4°C and then with 1% osmium tetroxide in a cacodylate buffer (pH 7.4) for 1 hour at 4°C. After dehydration in a graded series of ethanol solutions, cells were dried to a critical point and sputter-coated with osmium. Cell surfaces were examined with an S-4300 scanning microscope (Hitachi, Tokyo, Japan) at a 30-kV accelerating voltage. To create a reduction-of-function Cas allele by gene targeting, we deleted exon 2 of the Cas gene, which encodes the entire SH3 and the N-terminal part of the SD domain containing one YLVP motif and four YQxP motifs. To this end, we constructed a targeting vector containing Cas exon 2 flanked by two locus of X-over P1 (loxP) sequences and followed by the Frt-flanked neomycin resistance (Neo) gene (Fig. 1A). When the floxed Cas exon 2 was correctly excised, exon 1 joined in frame to exon 3, and this resulted in a Cas transcript devoid of the exon 2–derived segment. Correctly targeted embryonic stem cells, identified by Southern blotting and genomic polymerase chain reaction (PCR; Fig. 1B, left and middle panels), were selected and used for the generation of heterozygous mice (Cas+/floxNeo).

33 We, see more therefore, investigated whether HO-1 might mediate CB2-induced anti-inflammatory effects in alcohol-fed mice and, first, characterized the impact of JWH-133 treatment on Kupffer-cell HO-1 protein expression by double immunohistochemistry combining antibodies to HO-1 and F4/80. Alcohol-fed mice treated with JWH-133 displayed a strong HO-1 protein increase in Kupffer cells, compared to alcohol-fed control animals (82% ± 2% versus 57% ± 3%, P < 0.05; Fig. 5A). In keeping with in vivo findings, JWH-133 induced HO-1 mRNA and protein expression in isolated Kupffer cells and in RAW264.7 macrophages, either

alone or in combination with LPS (Fig. 5B,C). We next investigated the impact of zinc protoporphyrin (ZnPP), a specific competitive inhibitor of HO-1 activity, on LPS-treated RAW264.7 macrophages exposed to JWH-133. Strikingly, ZnPP blunted the inhibitory effect of JWH-133 on LPS-induced nuclear factor-kappa B (NF-κB) translocation into the nucleus (Fig. 6A). In addition, ZnPP also prevented the inhibitory effect of JWH-133 on IL-6 and IL-1β expressions, whereas its effect on TNF-α did not reach statistical

significance (Fig. 6B). These data demonstrate that CB2 activation induces HO-1 in macrophages, thereby limiting NF-κB activation and the proinflammatory M1 response. The present study demonstrates that during alcoholic liver disease, activation of CB2 receptors expressed in Kupffer cells phosphatase inhibitor library reduces proinflammatory M1 response and favors M2 polarization, thereby eliciting antisteatogenic effects via paracrine interactions with hepatocytes (Fig. 7). Sustained inflammation constitutes the initial hepatic response to chronic alcohol consumption.8, 12 Experimental and clinical studies have shown that Kupffer cells play a pivotal role in this process. Thus, Kupffer cells undergo activation by gut-derived LPS and release several inflammatory mediators, such as TNF-α or IL-1β, suggesting that

they may adopt 上海皓元医药股份有限公司 a proinflammatory M1 profile.8, 11, 34 In the present study, we provide evidence for a mixed M1/M2 response of Kupffer cells in alcohol-fed animals. Indeed, alcohol triggers hepatic induction of proinflammatory mediators characteristic of a classical M1 profile, including cytokines, such as TNF-α and IL-6, or chemokines, such as CCL3 and CCL4. At the same time, alcohol feeding also enhances liver expression of alternative M2 markers, such as Arg1, Mrc2, and CD163. As yet, mechanisms regulating M1/M2 Kupffer-cell polarization remain largely unexplored. Recent studies in experimental models of obesity have shown that the transcription factor, peroxisome proliferator-activated receptor delta, promotes the transition of Kupffer cells to an M2 phenotype, thereby reducing liver inflammation and fatty liver.