Strategy for mass production of lytic Staphylococcus aureus bacteriophage pSa-3: contribution of multiplicity of infection and response surface methodology.

BACKGROUND: Antibiotic-resistant bacteria have emerged as a serious problem; bacteriophages have, therefore, been proposed as a therapeutic alternative to antibiotics. Several authorities, such as pharmacopeia, FDA, have confirmed their safety, and some bacteriophages are commercially available worldwide. The demand for bacteriophages is expected to increase exponentially in the future; hence, there is an urgent need to mass-produce bacteriophages economically. Unlike the replication of non-lytic bacteriophages, lytic bacteriophages are replicated by lysing host bacteria, which leads to the termination of phage production; hence, strategies that can prolong the lysis of host bacteria in bacteria-bacteriophage co-cultures, are required. RESULTS: In the current study, we manipulated the inoculum concentrations of Staphylococcus aureus and phage pSa-3 (multiplicity of infection, MOI), and their energy sources to delay the bactericidal effect while optimizing phage production. We examined an increasing range of bacterial inoculum concentration (2 × 108 to 2 × 109 CFU/mL) to decrease the lag phase, in combination with a decreasing range of phage inoculum (from MOI 0.01 to 0.00000001) to delay the lysis of the host. Bacterial concentration of 2 × 108 CFU/mL and phage MOI of 0.0001 showed the maximum final phage production rate (1.68 × 1010 plaque forming unit (PFU)/mL). With this combination of phage-bacteria inoculum, we selected glycerol, glycine, and calcium as carbon, nitrogen, and divalent ion sources, respectively, for phage production. After optimization using response surface methodology, the final concentration of the lytic Staphylococcus phage was 8.63 × 1010 ± 9.71 × 109 PFU/mL (5.13-fold increase). CONCLUSIONS: Therefore, Staphylococcus phage pSa-3 production can be maximized by increasing the bacterial inoculum and reducing the seeding phage MOI, and this combinatorial strategy could decrease the phage production time. Further, we suggest that response surface methodology has the potential for optimizing the mass production of lytic bacteriophages.

Myeloid Cell Expression of LACC1 Is Required for Bacterial Clearance and Control of Intestinal Inflammation.

BACKGROUND & AIMS: Loss-of-function variants in the laccase domain containing 1 (LACC1) gene are associated with immune-mediated diseases, including inflammatory bowel disease. It is not clear how LACC1 balances defenses against intestinal bacteria vs intestinal inflammation or what cells are responsible for this balance in humans or mice. METHODS: Lacc1-/- mice and mice with myeloid-specific disruption of Lacc1 (Lacc1Δmye) were given oral Salmonella Typhimurium or dextran sodium sulfate. CD45RBhiCD4+T cells were transferred to Lacc1-/-Rag2-/- mice to induce colitis. Organs were collected and analyzed by histology and protein expression. Bone marrow-derived macrophages and dendritic cells, lamina propria macrophages, and mesenteric lymph node dendritic cells were examined. We performed assays to measure intestinal permeability, cell subsets, bacterial uptake and clearance, reactive oxygen species, nitrite production, autophagy, signaling, messenger RNA, and cytokine levels. RESULTS: Lacc1-/- mice developed more severe T-cell transfer colitis than wild-type mice and had an increased burden of bacteria in intestinal lymphoid organs, which expressed lower levels of T helper (Th) 1 and Th17 cytokines and higher levels of Th2 cytokines. Intestinal lymphoid organs from mice with deletion of LACC1 had an increased burden of bacteria after oral administration of S Typhimurium and after administration of dextran sodium sulfate compared with wild-type mice. In macrophages, expression of LACC1 was required for toll-like receptor-induced uptake of bacteria, which required PDK1, and of mitogen-activated protein kinase (MAPK)- and nuclear factor κB-dependent induction of reactive oxygen species, reactive nitrogen species, and autophagy. Expression of LACC1 by dendritic cells was required for increasing expression of Th1 and Th17 cytokines and reducing expression of Th2 cytokines upon coculture with CD4+ T cells. Mice with LACC1-deficient myeloid cells had an increased burden of bacteria and altered T-cell cytokines in intestinal lymphoid organs, similar to Lacc1-/- mice. Complementation of cytokines produced by myeloid cells to cocultures of LACC1-deficient myeloid cells and wild-type CD4+ T cells restored T-cell cytokine regulation. When S Typhimurium-infected Lacc1Δmye mice were injected with these myeloid cell-derived cytokines, intestinal tissues increased production of Th1 and Th17 cytokines, and bacteria were reduced. CONCLUSIONS: Disruption of Lacc1 in mice increases the burden of bacteria in intestinal lymphoid organs and intestinal inflammation after induction of chronic colitis. LACC1 expression by myeloid cells in mice is required to clear bacteria and to regulate adaptive T-cell responses against microbes.

Microstructural and Very High Cycle Fatigue (VHCF) Behavior of Ti6Al4V-A Comparative Study.

In this study, an investigation is carried out to evaluate and compare the material and physical properties of Grade 5 Titanium alloy (Ti6Al4V G5) samples of three different impeller manufacturers. The study aims to identify the efficient impeller core material from different Ti6Al4V G5 manufacturers. Ultrasonic fatigue test for Ti6Al4V samples of 100 horsepower (hp) centrifugal compressor impeller parts is performed before and after heat treatment. The effect of microstructure on Very High Cycle Fatigue (VHCF) behavior of Ti6Al4V is also analyzed and discussed in detail. Optical Microscopy (OM) and Scanning Electron Microscopy (SEM) observation are carried out to investigate the microstructure of different Ti6Al4V material samples. The dynamic elastic properties are measured by the Impulse Excitation Technique (IET) at room temperature. The fracture behavior of the tensile specimens is analyzed by SEM. Post-heat-treatment analysis of Ti6Al4V is also carried out and presented which affects the grain size of the material sample and thus considerable effect in the mechanical properties. Chemical composition investigation of Ti6Al4V using SEM and Energy Dispersive X-ray Spectroscopy (EDS) also included in this study.

Genomic characterization of bacteriophage pEt-SU, a novel phiKZ-related virus infecting Edwardsiella tarda.

A bacteriophage infecting Edwardsiella tarda (named pEt-SU) was isolated from freshwater collected in Chung-ju, South Korea. The whole genome of pEt-SU was 276,734 bp in length, representing the first giant phage infecting Edwardsiella reported to date. A total of 284 putative open reading frames were predicted and annotated. Morphology and genome analyses verified that pEt-SU may be distantly related to the phiKZ-like phages, a well-known giant myovirus. The findings in this study provide new insights into the phages infecting E. tarda ads well as fundamental data for the study of giant phages.

Enhanced bath immersion vaccination through microbubble treatment in the cyprinid loach.

Immunization by bath immersion is likely the simplest method of fish vaccination. Although the route of immunogenicity has not been fully identified, immersion vaccination is clearly a useful labor-saving technique. In this study, microbubble (MB) treatment was assessed for its ability to improve the efficacy of bath immersion vaccination in the cyprinid loach. MBs are commonly defined as minute particles of gas with a diameter of less than 100 µm, which generated free radicals. Here, the efficacy of MB treatment for vaccination enhancement in the cyprinid loach was assessed in direct challenge experiments using the virulent Aeromonas hydrophila JUNAH strain; assessments comprised agglutination titer assay and non-specific parameter analysis. Agglutination titers were high in loaches that were immunized via injection with inactivated cells (FKC group); however, non-specific immune activation parameters (e.g., lysozyme, superoxide dismutase, and phagocytic activity) were more increased in loaches that were immunized via bath immersion with MB treatment. Moreover, MB-treated loaches showed comparable survival rates, relative to loaches immunized via injection with formalin inactivated cells. Thus, higher levels of non-specific immune parameters suggest increased efficacy of this vaccine approach. Improving the effectiveness of bath immersion vaccine will increase its affordability and ease of application in aquaculture.

Resolvin D1 activates the sphingosine-1-phosphate signaling pathway in murine livers with ischemia/reperfusion injury.

Resolvins (Rvs) are endogenous lipid mediators that promote resolution of inflammation and return to homeostasis. We previously reported that RvD1 both facilitates M2 macrophage polarization of Kupffer cells (KCs) and efferocytosis and modulates thioredoxin 2-mediated mitochondrial quality control in liver ischemia/reperfusion (IR) injury. However, the specific cellular or molecular targets of RvD1 remain poorly understood. Sphingosine-1-phosphate (S1P), the natural sphingolipid ligand for a family of G protein-coupled receptors (S1P1-S1P5), regulates lymphocyte circulation and various immune responses. Here we investigated the role of RvD1 in IR-induced hepatocellular damage with a focus on S1P signaling. Male C57BL/6 mice were subjected to partial hepatic ischemia for 60 min, followed by reperfusion. Mice were pretreated with RvD1 (15 µg/kg, i.p.) 1 h prior to ischemia and immediately before reperfusion. To deplete KCs, liposome clodronate was administered (100 µL/mice, i.v.) 24 h prior to ischemia. Mice were pretreated with VPC23019 (100 µg/kg, i.p.), an antagonist for S1P1/S1P3 10 min prior to initial RvD1 treatment. Exogenous RvD1 attenuated IR-induced hepatocellular damage as evidenced by serum HMGB1 release. RvD1 attenuated the decrease in hepatic S1P concentration induced by IR. KC depletion by liposome clodronate did not alter the effect of RvD1 on sphingosine kinases (SKs) and S1P receptors, suggesting independency of KCs. Moreover, in purified hepatocytes of mice exposed to IR, mRNA expression of SK1, SK2, S1P1, and S1P3 decreased significantly, and this was attenuated by RvD1. Finally, VPC23019 pretreatment abolished the hepatoprotective effects of RvD1 in serum HMGB1 release. Our findings suggest that RvD1 protects the liver against IR injury by activating S1P signaling.

Immunostimulation of Cyprinus carpio using phage lysate of Aeromonas hydrophila.

Over the last 50 years, various approaches have been established for the development of antigens for immunostimulation. We used phage lysate (PL), composed of inactivated antigens by the lytic bacteriophage pAh 6-c for Aeromonas hydrophila JUNAH strain to develop a vaccine for the prevention of A. hydrophila infection in Cyprinus carpio (common carp). We also assessed the poly D,L lactide-co-glycolic acid (PLGA) microparticles encapsulation method to increase the efficiency of the vaccine. Six groups of vaccines involving encapsulated by PLGA, formalin killed cells, or phage lysate at low or high concentration were prepared for intraperitoneal injection in C. carpio. Blood specimens and head kidney samples were collected at various time points for bacterial agglutination assay and to assess relative expression of immune-related genes interleukin-1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), lysozyme C, and serum amyloid A (SAA). The vaccine groups using high dose phage lysate antigen showed significantly higher agglutination titers than all other groups at 4- and 6-weeks post vaccination (wpv), with the titer of the PLGA encapsulated vaccine group being highest from 10 wpv to the end of the experiment. The survival rate of fish immunized with the phage lysate vaccines were higher than that of fish immunized with the formailin killed cells vaccine in the challenge experiment conducted 6 wpv. Additionally, the PLGA-encapsulated high dose phage lysate antigen vaccinated groups showed the best protective efficacy in the challenge experiment 12 wpv. Vaccines using the phage lysate antigen also showed higher IL-1ß and lysozyme C gene expression at 7 days post vaccination (dpv) and 2 wpv, and higher TNF-α gene expression was seen at 7 dpv. Higher SAA gene expression was seen in these groups at 1 dpv. These results suggest that phage lysate antigen has the potential to induce robust immune responses than formalin killed cells-based vaccines, and could be more effective as a novel inactivated antigen in preventing A. hydrophila infection in C. carpio.

Resolvin D1 attenuates liver ischaemia/reperfusion injury through modulating thioredoxin 2-mediated mitochondrial quality control.

BACKGROUND AND PURPOSE: Liver ischaemia and reperfusion (IR) injury is a sterile inflammatory response involving production of ROS. Mitochondrial homeostasis is maintained by mitochondrial quality control (QC). Thioredoxin (TRX) 2 is a key mitochondrial redox-sensitive protein. Resolvin D1 (RvD1), a specialized pro-resolving lipid mediator, exerts anti-inflammatory and antioxidant activities. We investigated mechanisms of RvD1 protection against IR-induced oxidative damage to the liver, focusing on TRX2-mediated mitochondrial QC. EXPERIMENTAL APPROACH: Mice underwent partial warm IR. RvD1 was administered 1 h before ischaemia and immediately prior to reperfusion. Human liver carcinoma HepG2 cells were exposed to hypoxia/reoxygenation and transfected with TRX2 siRNA. Immunohistochemistry, Western blotting and enzyme assays were used to follow changes in mitochondrial structure and function. KEY RESULTS: RvD1 attenuated hepatocellular damage following IR, assessed by serum aminotransferase activities and histology. RvD1 reduced mitochondrial swelling, lipid peroxidation and glutamate dehydrogenase release. Impaired activities of mitochondrial complexes I and III were restored by RvD1. RvD1 enhanced expression of the mitophagy-related protein, Parkin and inhibited accumulation of PTEN-induced putative kinase 1. RvD1 restored levels of mitochondrial biogenesis proteins including PPARγ coactivator 1α, nuclear respiratory factor 1 and mitochondrial transcription factor A and mtDNA level. RvD1 attenuated the increase in levels of the mitochondrial fission-related protein, dynamin-related protein 1. IR reduced TRX2 levels while increasing TRX2 association with TRX-interacting protein. RvD1 attenuated these changes. The regulatory effects of RvD1 on mitochondrial QC were abolished by TRX2 knockdown. CONCLUSIONS AND IMPLICATIONS: We suggest that RvD1 ameliorated IR-induced hepatocellular damage by regulating TRX2-mediated mitochondrial QC.

Afzelin ameliorates D-galactosamine and lipopolysaccharide-induced fulminant hepatic failure by modulating mitochondrial quality control and dynamics.

BACKGROUND AND PURPOSE: Fulminant hepatic failure (FHF) is a fatal clinical syndrome that results in excessive inflammation and hepatocyte death. Mitochondrial dysfunction is considered to be a possible mechanism of FHF. Afzelin, a flavonol glycoside found in Houttuynia cordata Thunberg, has anti-inflammatory and antioxidant properties. The present study elucidated the cytoprotective mechanisms of afzelin against D-galactosamine (GalN)/LPS induced FHF, particularly focusing on mitochondrial quality control and dynamics. EXPERIMENTAL APPROACH: Mice were administered afzelin i.p. 1 h before receiving GalN (800 mg·kg-1 )/LPS (40 µg·kg-1 ), and they were then killed 5 h after GalN/LPS treatment. KEY RESULTS: Afzelin improved the survival rate and reduced the serum levels of alanine aminotransferase and pro-inflammatory cytokines in GalN/LPS-treated mice. Afzelin attenuated the mitochondrial damage, as indicated by diminished mitochondrial swelling and mitochondrial glutamate dehydrogenase activity in GalN/LPS-treated mice. Afzelin enhanced mitochondrial biogenesis, as indicated by increased levels of PPAR-γ coactivator 1α, nuclear respiratory factor 1 and mitochondrial transcription factor A. Afzelin also decreased the level of mitophagy-related proteins, parkin and PTEN-induced putative kinase 1. Furthermore, while GalN/LPS significantly increased the level of fission-related protein, dynamin-related protein 1, and decreased the level of fusion-related protein, mitofusin 2; these effects were attenuated by afzelin. CONCLUSIONS AND IMPLICATIONS: Our findings demonstrated that afzelin protects against GalN/LPS-induced liver injury by enhancing mitochondrial biogenesis, suppressing excessive mitophagy and balancing mitochondrial dynamics.

Resolvin D1 protects the liver from ischemia/reperfusion injury by enhancing M2 macrophage polarization and efferocytosis.

Resolution of inflammation is an active process involving a novel category of lipid factors known as specialized pro-resolving lipid mediators, which includes Resolvin D1 (RvD1). While accumulating evidence suggests that RvD1 counteracts proinflammatory signaling and promotes resolution, the specific cellular targets and mechanisms of action of RvD1 remain largely unknown. In the present study, we investigated the role and molecular mechanisms of RvD1 in ischemia/reperfusion (IR)-induced sterile liver inflammation. Male C57BL/6 mice underwent 70% hepatic ischemia for 60min, followed by reperfusion. RvD1 (5, 10, and 15µg/kg, i.p.) was administered to the mice 1h before ischemia and then immediately prior to reperfusion. RvD1 attenuated IR-induced hepatocellular damage and the proinflammatory response. In purified Kupffer cells (KCs) from mice exposed to IR, the levels of M1 marker genes (Nos2a and Cd40) increased, while those of M2 marker genes (Arg1, Cd206, and Mst1r) decreased, demonstrating a proinflammatory shift. RvD1 markedly attenuated these changes. Depletion of KCs by liposome clodronate abrogated the effects of RvD1 on proinflammatory mediators and macrophage polarization. In addition, RvD1 attenuated increases in myeloperoxidase activity and Cxcl1 and Cxcl2 mRNA expression. RvD1 markedly augmented the efferocytic activity of KCs, as indicated by increases in F4/80(+)Gr-1(+) cells in the liver. However, antagonist pretreatment or gene silencing of the RvD1 receptor, ALX/FPR2, abrogated the anti-inflammatory and pro-resolving actions of RvD1. These data indicate that RvD1 ameliorates IR-induced liver injury, and this protection is associated with enhancement of M2 polarization and efferocytosis via ALX/FPR2 activation.

Enhanced nitric oxide-mediated autophagy contributes to the hepatoprotective effects of ischemic preconditioning during ischemia and reperfusion.

Ischemic preconditioning (IPC) protects against liver ischemia/reperfusion (I/R) injury. Autophagy is an essential cytoprotective system that is rapidly activated by multiple stressors. Nitric oxide (NO) acts as an inducer of IPC. We examined the impact of autophagy in liver IPC and its regulation by NO. Male C57BL/6 mice were subjected to 60 min of hepatic ischemia followed by 6 h of reperfusion. IPC was achieved for 10 min of ischemia followed by 10 min of reperfusion prior to sustained ischemia. N(ω)-Nitro-l-arginine methyl ester (L-NAME, 15 mg/kg, i.v., all NOS inhibitor) and aminoguanidine (AG, 10 mg/kg, i.v., iNOS inhibitor) were injected 10 min before IPC. SB203580 (10 mg/kg, i.p., p38 inhibitor) was injected 30 min before IPC. I/R increased serum alanine aminotransferase activity. IPC attenuated this increase, which was abolished by L-NAME, but not AG. Microtubule-associated protein-1 light chain 3-II levels increased and p62 protein levels decreased after I/R; these changes were augmented by IPC and abolished by L-NAME. I/R increased liver protein expression of autophagy-related protein (Atg)12-Atg5 complex and lysosome-associated membrane protein-2. IPC augmented the expression of these proteins, which were abolished by L-NAME, but not AG. IPC also augmented the level of phosphorylated p38 MAPK induced by I/R and this phosphorylation was abolished by L-NAME. Our findings suggest that IPC-mediated NO protects against I/R-induced liver injury by enhancing autophagic flux.

Opuntia ficus-indica seed attenuates hepatic steatosis and promotes M2 macrophage polarization in high-fat diet-fed mice.

Opuntia ficus-indica (L.) is a popular edible plant that possesses considerable nutritional value and exhibits diverse biological actions including anti-inflammatory and antidiabetic activities. In this study, we hypothesized that DWJ504, an extract of O ficus-indica seed, would ameliorate hepatic steatosis and inflammation by regulating hepatic de novo lipogenesis and macrophage polarization against experimental nonalcoholic steatohepatitis. Mice were fed a normal diet or a high-fat diet (HFD) for 10 weeks. DWJ504 (250, 500, and 1000 mg/kg) or vehicle (0.5% carboxymethyl cellulose) were orally administered for the last 4 weeks of the 10-week HFD feeding period. DWJ504 treatment remarkably attenuated HFD-induced increases in hepatic lipid content and hepatocellular damage. DWJ504 attenuated increases in sterol regulatory element-binding protein 1 and carbohydrate-responsive element-binding protein expression and a decrease in carnitine palmitoyltransferase 1A. Although DWJ504 augmented peroxisome proliferator-activated receptor α protein expression, it attenuated peroxisome proliferator-activated receptor γ expression. Moreover, DWJ504 promoted hepatic M2 macrophage polarization as indicated by attenuation of the M1 marker genes and enhancement of M2 marker genes. Finally, DWJ504 attenuated expression of toll-like receptor 4, nuclear factor κB, tumor necrosis factor α, interleukin 6, TIR-domain-containing adapter-inducing interferon ß, and interferon ß levels. Our results demonstrate that DWJ504 prevented intrahepatic lipid accumulation, induced M2 macrophage polarization, and suppressed the toll-like receptor 4-mediated inflammatory signaling pathway. Thus, DWJ504 has therapeutic potential in the prevention of nonalcoholic fatty liver disease.

Melatonin enhances mitophagy and mitochondrial biogenesis in rats with carbon tetrachloride-induced liver fibrosis.

Liver fibrosis leads to liver cirrhosis and failure, and no effective treatment is currently available. Growing evidence supports a link between mitochondrial dysfunction and liver fibrogenesis and mitochondrial quality control-based therapy has emerged as a new therapeutic target. We investigated the protective mechanisms of melatonin against mitochondrial dysfunction-involved liver fibrosis, focusing on mitophagy and mitochondrial biogenesis. Rats were treated with carbon tetrachloride (CCl4) dissolved in olive oil (0.5 mL/kg, twice a week, i.p.) for 8 wk. Melatonin was administered orally at 2.5, 5, and 10 mg/kg once a day. Chronic CCl4 exposure induced collagen deposition, hepatocellular damage, and oxidative stress, and melatonin attenuated these increases. Increases in mRNA and protein expression levels of transforming growth factor ß1 and α-smooth muscle actin in response to CCl4 were attenuated by melatonin. Melatonin attenuated hallmarks of mitochondrial dysfunction, such as mitochondrial swelling and glutamate dehydrogenase release. Chronic CCl4 exposure impaired mitophagy and mitochondrial biogenesis, and melatonin attenuated this impairment, as indicated by increases in mitochondrial DNA and in protein levels of PTEN-induced putative kinase 1 (PINK1); Parkin; peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α); nuclear respiratory factor 1 (NRF1); and transcription factor A, mitochondrial (TFAM). CCl4-mediated decreases in mitochondrial fission- and fusion-related proteins, such as dynamin-related protein 1 (DRP1) and mitofusin 2, were also attenuated by melatonin. Moreover, melatonin induced AMP-activated protein kinase (AMPK) phosphorylation. These results suggest that melatonin protects against liver fibrosis via upregulation of mitophagy and mitochondrial biogenesis, and may be useful as an anti-fibrotic treatment.

The role of heme oxygenase-1 in drug metabolizing dysfunction in the alcoholic fatty liver exposed to ischemic injury.

This study was designed to investigate the role of heme oxygenase-1 (HO-1) in hepatic drug metabolizing dysfunction after ischemia/reperfusion (IR) in alcoholic fatty liver (AFL). Rats were fed a Lieber-DeCarli diet for five weeks to allow for development of AFL and were then subjected to 90min of hepatic ischemia and 5h of reperfusion. Rats were pretreated with hemin (HO-1 inducer) or ZnPP (HO-1 inhibitor) for 16h and 3h before hepatic ischemia. After hepatic IR, ethanol diet (ED)-fed rats had higher serum aminotransferase activities and more severe hepatic necrosis compared to the control diet (CD)-fed rats. These changes were attenuated by hemin and exacerbated by ZnPP. The activity and gene expression of HO-1 and its transcription factor (Nrf2) level increased significantly after 5h of reperfusion in CD-fed rats but not in ED-fed rats. After reperfusion, cytochrome P450 (CYP) 1A1, 1A2, and 2B1 activities were reduced to levels lower than those observed in sham group, whereas CYP2E1 activity increased. The decrease in CYP2B1 activity and the increase in CYP2E1 activity were augmented after hepatic IR in ED-fed animals. These changes were significantly attenuated by hemin but aggravated by ZnPP. Finally, CHOP expression and PERK phosphorylation, microsomal lipid peroxidation, and levels of proinflammatory mediators increased in ED-fed rats compared to CD-fed rats after reperfusion. These increases were attenuated by hemin. Our results suggest that AFL exacerbates hepatic drug metabolizing dysfunction during hepatic IR via endoplasmic reticulum stress and lipid peroxidation and this is associated with impaired HO-1 induction.

Melatonin attenuates carbon tetrachloride-induced liver fibrosis via inhibition of necroptosis.

We investigated the protective mechanisms of melatonin (MLT) associated with necroptosis signaling and damage-associated molecular patterns, which are mediated by the activation of pattern recognition receptors in liver fibrosis. Rats were given an intraperitoneal injection of carbon tetrachloride (CCl4) dissolved in olive oil (1:3, vol/vol) twice a week (0.5 mL/kg) for 8 weeks. During this period, MLT was administered orally at 2.5, 5, and 10 mg/kg once a day. Chronic CCl4 administration increased hepatic hydroxyproline content and hepatocellular damage. MLT attenuated these increases. The expression levels of transforming growth factor ß1 and α-smooth muscle actin that were increased by chronic CCl4 exposure were attenuated by MLT. CCl4 significantly increased receptor-interacting protein 1 (RIP1) expression, the formation of the RIP1 and RIP3 necrosome complex, and the level of mixed lineage kinase domain-like protein in liver tissue, which were attenuated by MLT. MLT also attenuated CCl4-induced increases in serum high-mobility group box 1 (HMGB1) and interleukin 1α, as well as the interaction between HMGB1 receptors for advanced glycation end products (RAGE). The increases in toll-like receptor 4 expression, p38, c-Jun N-terminal kinases phosphorylation, and nuclear factor κB translocation were suppressed by MLT. MLT attenuated the overexpression of RAGE, increased level of early growth response protein 1, and increased messenger RNA level of macrophage inflammatory protein 2. Our findings suggest MLT may prevent liver fibrosis by inhibiting necroptosis-associated inflammatory signaling.

DAMPs activating innate immune responses in sepsis.

Sepsis refers to the deleterious and non-resolving systemic inflammatory response of the host to microbial infection and is the leading cause of death in intensive care units. The pathogenesis of sepsis is highly complex. It is principally attributable to dysregulation of the innate immune system. Damage-associated molecular patterns (DAMPs) are actively secreted by innate immune cells and/or released passively by injured or damaged cells in response to infection or injury. In the present review, we highlight emerging evidence that supports the notion that extracellular DAMPs act as crucial proinflammatory danger signals. Furthermore, we discuss the potential of a wide array of DAMPs as therapeutic targets in sepsis.

Role of Kupffer cells in ischemic injury in alcoholic fatty liver.

BACKGROUND: This study was designed to evaluate the role of Kupffer cells (KCs) in hepatic drug metabolizing dysfunction after hepatic ischemia-reperfusion (IR) in alcoholic fatty liver. MATERIALS AND METHODS: Rats were fed the Lieber-DeCarli diet for 5 wk to develop alcoholic fatty liver, then were subjected to 90 min of hepatic ischemia and 5 h of reperfusion. For ablation of KCs, rats were pretreated with gadolinium chloride (GdCl3) 48 and 24 h before the IR procedure. RESULTS: After the IR procedure, ethanol diet (ED)-fed rats had higher serum aminotransferase activity compared with the control diet-fed rats. These changes were attenuated by GdCl3. The ED-fed rats exhibited increased hepatic microsomal total cytochrome P450 (CYP) content and nicotinamide adenine dinucleotide phosphate-CYP reductase and CYP1A1, 1A2, 2B1, and 2E1 isozyme activity. After hepatic IR, these increases were reduced to lower levels than observed in the sham group, except CYP2E1 activity. Increases in CYP2E1 activity and its expression were augmented after hepatic IR in ED-fed animals, but were attenuated by GdCl3. Finally, toll-like receptor 4 and myeloid differentiation primary response gene 88 protein expression, nuclear translocation of nuclear factor-κB and activator protein 1, and levels of proinflammatory mediators were further increased in ED-fed animals compared with control diet-fed animals after IR. These increases were attenuated by GdCl3. CONCLUSIONS: We suggest that KCs contribute to hepatic drug metabolizing dysfunction during hepatic IR in alcoholic fatty liver via the toll-like receptors 4-mediated inflammatory response.

Protective Effects of Chlorogenic Acid against Experimental Reflux Esophagitis in Rats.

Esophageal reflux of gastric contents causes esophageal mucosal damage and inflammation. Recent studies show that oxygen-derived free radicals mediate mucosal damage in reflux esophagitis (RE). Chlorogenic acid (CGA), an ester of caffeic acid and quinic acid, is one of the most abundant polyphenols in the human diet and possesses anti-inflammatory, antibacterial and anti-oxidant activities. In this context, we investigated the effects of CGA against experimental RE in rats. RE was produced by ligating the transitional region between the forestomach and the glandular portion and covering the duodenum near the pylorus ring with a small piece of catheter. CGA (10, 30 and 100 mg/kg) and omeprazole (positive control, 10 mg/kg) were administered orally 48 h after the RE operation for 12 days. CGA reduced the severity of esophageal lesions, and this beneficial effect was confirmed by histopathological observations. CGA reduced esophageal lipid peroxidation and increased the reduced glutathione/oxidized glutathione ratio. CGA attenuated increases in the serum level of tumor necrosis factor-α, and expressions of inducible nitric oxide synthase and cyclooxygenase-2 protein. CGA alleviates RE-induced mucosal injury, and this protection is associated with reduced oxidative stress and the anti-inflammatory properties of CGA.

Impaired expression of caveolin-1 contributes to hepatic ischemia and reperfusion injury.

Caveolae are membrane structures enriched in glycosphingolipids and cholesterol, and caveolin-1 (Cav-1) has been recognized to be pivotal in ischemic tolerance. Sphingosine-1-phosphate (S1P), one of the sphingolipid metabolites, is well known for its anti-apoptotic properties, counteracting ischemia and reperfusion (IR) injury. Here, we investigated the cytoprotective mechanism of Cav-1 against IR injury. Male C57BL/6 mice underwent 70% hepatic ischemia for 60 min, followed by reperfusion. Mice were pretreated with methyl-beta-cyclodextrin (MßCD, 10, 25 and 50mg/kg, i.p.), a caveolae disruptor, or saline 48 and 24h before ischemia. Serum and liver tissues were collected at the end of ischemia, at 0, 1, 4 and 24h of reperfusion. Decreases in the expression of Cav-1 protein and in the number of caveolae of the liver ultrastructure were observed during IR, which were augmented by pretreatment with MßCD. MßCD also augmented the IR-induced increases in serum alanine aminotransferase and tumor necrosis factor-α levels. IR decreased the levels of sphingosine kinase 2 (SK2) and S1P receptor 2 (S1P2) mRNA expressions, while MßCD also augmented these decreases. Moreover, IR resulted in increases of mitochondrial cytochrome c release, caspase 3, 8 activities and Bax/Bcl-xL ratio, and MßCD augmented all of these apoptotic parameters. MßCD also increased p38 MAPK and JNK phosphorylation, but did not affect ERK and PI3K/Akt. Our findings demonstrate that downregulation of Cav-1 mediates IR-induced liver damage by inhibiting SK2/S1P2 signaling and enhancing the apoptotic pathway.

Melatonin inhibits mTOR-dependent autophagy during liver ischemia/reperfusion.

BACKGROUND: Autophagy is a self-digestion system responsible for maintaining cellular homeostasis and interacts with reactive oxygen species produced during ischemia/reperfusion (I/R). Melatonin (MLT) is a potent and endogenous anti-oxidant that has beneficial effects in liver I/R injury. In this study, we examined the cytoprotective mechanisms of MLT in liver I/R, focusing on autophagic flux and associated signaling pathways. METHODS: Male C57BL/6 mice were subjected to 70% liver ischemia for 60 min followed by reperfusion. MLT (10 mg/kg, i.p.) was injected 15 min prior to ischemia and again immediately before reperfusion. Rapamycin (Rapa, 1 mg/kg, i.p.), which induces autophagy, was injected 1.5 h before ischemia. RESULTS: Liver I/R increased autophagic flux as indicated by the accumulation of LC3-II and degradation of sequestosome1/p62. This increase was attenuated by MLT. Likewise, electron microscopic analysis showed that autophagic vacuoles were increased in livers of mice exposed to I/R, which was attenuated by MLT. I/R decreased phosphorylation of mammalian target of rapamycin (mTOR) and 4E-BP1 and 70S6K, downstream molecules of the mTOR pathway, but increased expression of calpain 1 and calpain 2. MLT attenuated the decrease in mTOR, 4E-BP1 and 70S6K phosphorylation. Pretreatment of Rapa reversed the effect of MLT on autophagic flux as well as mTOR pathway. CONCLUSION: Our findings suggest that MLT downregulates autophagy via activation of mTOR signaling, which may in turn contribute to its protective effects in liver I/R injury.