Ghrelin protects against experimental sepsis by inhibiting high-mobility group box 1 release and by killing bacteria

Sepsis, a life-threatening complication of infections and the most common cause of death in intensive care units, is characterized by a hyperactive and out-of-balance network of endogenous proinflammatory cytokines. None of the current therapies are entirely effective, illustrating the need for novel therapeutic approaches. Ghrelin (GHR) is an orexigenic peptide that has emerged as a potential endogenous anti-inflammatory factor. In this study, we show that the delayed administration of GHR protects against the mortality in various models of established endotoxemia and sepsis. The therapeutic effect of GHR is mainly mediated by decreasing the secretion of the high mobility box 1 (HMGB1), a DNA-binding factor that acts as a late inflammatory factor critical for sepsis progression. Macrophages seem to be the major cell targets in the inhibition of HMGB1 secretion, in which GHR blocked its cytoplasmic translocation. Interestingly, we also report that GHR shows a potent antibacterial activity in septic mice and in vitro. Remarkably, GHR also reduces the severity of experimental arthritis and the release of HMGB1 to serum. Therefore, by regulating crucial processes of sepsis, such as the production of early and late inflammatory mediators by macrophages and the microbial load, GHR represents a feasible therapeutic agent for this disease and other inflammatory disorders.     

Inhibition of high-mobility group box 1 protein (HMGB1) enhances bacterial clearance and protects against Pseudomonas Aeruginosa pneumonia in cystic fibrosis

Pulmonary infection with Pseudomonas aeruginosa and neutrophilic lung inflammation significantly contribute to morbidity and mortality in cystic fibrosis (CF). High-mobility group box 1 protein (HMGB1), a ubiquitous DNA binding protein that promotes inflammatory tissue injury, is significantly elevated in CF sputum. However, its mechanistic and potential therapeutic implications in CF were previously unknown. We found that HMGB1 levels were significantly elevated in bronchoalveolar lavage fluids (BALs) of CF patients and cystic fibrosis transmembrane conductance regulator (CFTR )(-/-) mice. Neutralizing anti-HMGB1 monoclonal antibody (mAb) conferred significant protection against P. aeruginosa-induced neutrophil recruitment, lung injury and bacterial infection in both CFTR(-/-) and wild-type mice. Alveolar macrophages isolated from mice treated with anti-HMGB1 mAb had improved phagocytic activity, which was suppressed by direct exposure to HMGB1. In addition, BAL from CF patients significantly impaired macrophage phagocytotic function, and this impairment was attenuated by HMGB1-neutralizing antibodies. The HMGB1-mediated suppression of bacterial phagocytosis was attenuated in macrophages lacking toll-like receptor (TLR)-4, suggesting a critical role for TLR4 in signaling HMGB1-mediated macrophage dysfunction. These studies demonstrate that the elevated levels of HMGB1 in CF airways are critical for neutrophil recruitment and persistent presence of P. aeruginosa in the lung. Thus, HMGB1 may provide a therapeutic target for reducing bacterial infection and lung inflammation in CF.     

Alpha-melanocyte-stimulating hormone protects against mesenteric ischemia-reperfusion injury

Mesenteric ischemia-reperfusion (I/R) injury to the intestine is a common and often devastating clinical occurrence for which there are few therapeutic options. alpha-Melanocyte-stimulating hormone (alpha-MSH) is a tridecapeptide released by the pituitary gland and immunocompetent cells that exerts anti-inflammatory actions and abrogates postischemic injury to the kidneys and brainstem of rodents. To test the hypothesis that alpha-MSH would afford similar protection in the postischemic small intestine, we analyzed the effects of this peptide on intestinal transit, histology, myeloperoxidase activity, and nuclear factor-kappaB (NF-kappaB) activation after 45 min of superior mesenteric artery occlusion and 相似文献   

Macrophage endocytosis of high-mobility group box 1 triggers pyroptosis

Macrophages can be activated and regulated by high-mobility group box 1 (HMGB1), a highly conserved nuclear protein. Inflammatory functions of HMGB1 are mediated by binding to cell surface receptors, including the receptor for advanced glycation end products (RAGE), Toll-like receptor (TLR)2, TLR4, and TLR9. Pyroptosis is a caspase-1-dependent programmed cell death, which features rapid plasma membrane rupture, DNA fragmentation, and release of proinflammatory intracellular contents. Pyroptosis can be triggered by various stimuli, however, the mechanism underlying pyroptosis remains unclear. In this study, we identify a novel pathway of HMGB1-induced macrophage pyroptosis. We demonstrate that HMGB1, acting through RAGE and dynamin-dependent signaling, initiates HMGB1endocytosis, which in turn induces cell pyroptosis. The endocytosis of HMGB1 triggers a cascade of molecular events, including cathepsin B release from ruptured lysosomes followed by pyroptosome formation and caspase-1 activation. We further confirm that HMGB1-induced macrophage pyroptosis also occurs in vivo during endotoxemia, suggesting a pathophysiological significance for this form of pyroptosis in the development of inflammation. These findings shed light on the regulatory role of ligand-receptor internalization in directing cell fate, which may have an important role in the progress of inflammation following infection and injury.Infection and injury, the most challenging factors to the survival of species throughout evolution, are still major causes of human death worldwide. Infection and severe trauma share many overlapping features in the mechanism of activation of the innate immune system through pathogen-associated molecular pattern molecules derived from microbes and damage-associated molecular pattern (DAMP) molecules released by damaged tissues.^{1, 2} High-mobility group box 1 (HMGB1), a highly conserved ubiquitous protein present in the nucleus and cytoplasm of nearly all cell types, is the prototypic DAMP molecule.³ During infection and sterile tissue injury, HMGB1 is released from cells and serves as a necessary and sufficient mediator of inflammation to induce a variety of cellular responses including cell chemotaxis and release of pro-inflammatory cytokines.^{4, 5} Inflammatory functions of HMGB1 are mediated by binding to the cell surface receptors, including the receptor for advanced glycation end products (RAGEs), Toll-like receptor (TLR)2, TLR4, and TLR9.^{6, 7}RAGE is a type I transmembrane protein and a member of the immunoglobulin superfamily expressed in many cell populations, including endothelial cells, vascular smooth muscle cells, neurons, neutrophils, and macrophages/monocytes.⁸ RAGE has been implicated as a receptor mediating the chemotaxis and cytokine activity of HMGB1 in macrophages and tumor cells.^{7, 9, 10} RAGE engagement by multiple ligands is linked to a range of signaling pathways including activation of NF-κB,^{11, 12} PI3K/Akt,¹³ MAPKs,^{14, 15} Jak/STAT,¹⁶ and Rho GTPases,¹⁷ although how RAGE transduces the signaling is not fully addressed.Pyroptosis is a caspase-1-dependent programmed cell death, which is morphologically and mechanistically distinct from other forms of cell death such as apoptosis and necrosis.¹⁸ Previous observations suggested that pyroptosis features rapid plasma membrane rupture and release of proinflammatory intracellular contents, contrasting with the characteristic of apoptosis, which packs cellular contents and induces non-inflammatory phagocytic uptake of the apoptotic bodies by macrophages.¹⁹ Pyroptosis can be triggered by various pathological stimuli, such as microbial infection, stroke, heart attack, or cancer;¹⁸ however, the mechanism that underlies pyroptosis formation in response to inflammatory mediators remains unclear. In this study, we identify a novel pathway of HMGB1-induced pyroptosis. We demonstrate that HMGB1 acting through RAGE on macrophages triggers dynamin-dependent endocytosis of HMGB1, which in turn induces cell pyroptosis. The endocytosis of HMGB1 initiates a cascade of molecular events, including cathepsin B (CatB) activation and release from ruptured lysosomes, followed by pyroptosome formation and caspase-1 activation. We further confirm that this in vitro observation of HMGB1-induced macrophage pyroptosis also occurs in vivo during endotoxemia, suggesting a pathophysiological significance for pyroptosis in the development of inflammation.     

Glucagon-like peptide-1 receptor agonist protects against hyperglycemia-induced cardiocytes injury by inhibiting high mobility group box 1 expression

Glucagon-like peptide-1 (GLP-1), a gut incretin hormone secreted from L cells, and a GLP-1 receptor agonist, exendin-4 (Ex-4) has been shown to be cardioprotective and could exert beneficial effects through its anti-inflammatory property. However, the mechanism remains unclear. The purpose of this study was to investigate whether Ex-4 could ameliorate myocardial cell injury by inhibiting high mobility group box 1 (HMGB1) expression under high glucose condition. Neonatal rat ventricular myocytes were prepared and then cultured with high glucose and different concentration of Ex-4. Lactate dehydrogenase (LDH), creatine kinase (CK), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), malondialdehyde (MDA) and superoxide dismutase (SOD) were measured. HMGB1 expression was assessed by western blotting. Ex-4 significantly inhibited the increase in LDH, CK, TNF-α, IL-1β and MDA levels induced by high glucose, especially at the 1 and 10?nM concentrations as well as suppressed the decrease in SOD level. Meanwhile, HMGB1 expression was markedly increased after 12?h of hyperglycaemia (P?<?0.05), which was significantly inhibited by Ex-4, especially at the 1 and 10?nM concentrations (P?<?0.05). The present study suggested that Ex-4 could reduce high glucose-induced cardiocytes injury, which may be associated with the inhibition of HMGB1 expression.     

Dexmedetomidine protects against high mobility group box 1‐induced cellular injury by inhibiting pyroptosis

Dexmedetomidine (DEX) is a widely used clinical anesthetic with proven anti‐inflammatory effects. Both high mobility group box 1 (HMGB1) and pyroptosis play an important role in the inflammatory response to infection and trauma. Thus far, there have been no studies published addressing the effect of DEX on HMGB1 and pyroptosis. In order to fill this gap in the literature, bone marrow‐derived macrophages (BMDMs) were exposed to HMGB1 (4 µg/mL) with or without DEX (50 μM) pretreatment. The production of pro‐inflammatory cytokines [such as tumor necrosis factor α (TNF‐α), interleukin 1β (IL‐1β), and IL‐18], phosphorylation of extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2) and P38, and the activation of caspase‐1 were measured by enzyme immunosorbent assay, western blot analysis, confocal microscope, and flow cytometry, respectively. We found that DEX protected against HMGB1‐induced cell death of BMDMs. In addition, DEX suppressed the generation of TNF‐α, IL‐1β, and IL‐18 as well as the phosphorylation of ERK1/2 and P38. Moreover, DEX inhibited caspase‐1 activation and decreased pyroptosis. Taken together, these findings demonstrate the protective effect of DEX in mediating HMGB1‐induced cellular injury, thus indicating that DEX may be a potential therapeutic candidate for the management of infection and trauma‐derived inflammation.     

Cardiac mTOR protects the heart against ischemia-reperfusion injury

Cardiac mammalian target of rapamycin (mTOR) is necessary and sufficient to prevent cardiac dysfunction in pathological hypertrophy. However, the role of cardiac mTOR in heart failure after ischemic injury remains undefined. To address this question, we used transgenic (Tg) mice with cardiac-specific overexpression of mTOR (mTOR-Tg mice) to study ischemia-reperfusion (I/R) injury in two animal models: 1) in vivo I/R injury with transient coronary artery ligation and 2) ex vivo I/R injury in Langendorff-perfused hearts with transient global ischemia. At 28 days after I/R, mortality was lower in mTOR-Tg mice than littermate control mice [wild-type (WT) mice]. Echocardiography and MRI demonstrated that global cardiac function in mTOR-Tg mice was preserved, whereas WT mice exhibited significant cardiac dysfunction. Masson's trichrome staining showed that 28 days after I/R, the area of interstitial fibrosis was smaller in mTOR-Tg mice compared with WT mice, suggesting that adverse left ventricular remodeling is inhibited in mTOR-Tg mice. In the ex vivo I/R model, mTOR-Tg hearts demonstrated improved functional recovery compared with WT hearts. Perfusion with Evans blue after ex vivo I/R yielded less staining in mTOR-Tg hearts than WT hearts, indicating that mTOR overexpression inhibited necrosis during I/R injury. Expression of proinflammatory cytokines, including IL-6 and TNF-α, in mTOR-Tg hearts was lower than in WT hearts. Consistent with this, IL-6 in the effluent post-I/R injury was lower in mTOR-Tg hearts than in WT hearts. These findings suggest that cardiac mTOR overexpression in the heart is sufficient to provide substantial cardioprotection against I/R injury and suppress the inflammatory response.     

SCD1 deficiency protects mice against ethanol-induced liver injury

Stearoyl-CoA desaturase 1 (SCD1) is a delta-9 fatty acid desaturase that catalyzes the synthesis of mono-unsaturated fatty acids (MUFA). SCD1 is a critical control point regulating hepatic lipid synthesis and β-oxidation. Scd1 KO mice are resistant to the development of diet-induced non-alcoholic fatty liver disease (NAFLD). Using a chronic-binge protocol of ethanol-mediated liver injury, we aimed to determine if these KO mice are also resistant to the development of alcoholic fatty liver disease (AFLD).Mice fed a low-fat diet (especially low in MUFA) containing 5% ethanol for 10 days, followed by a single ethanol (5 g/kg) gavage, developed severe liver injury manifesting as hepatic steatosis. This was associated with an increase in de novo lipogenesis and inflammation. Using this model, we show that Scd1 KO mice are resistant to the development of AFLD. Scd1 KO mice do not show accumulation of hepatic triglycerides, activation of de novo lipogenesis nor elevation of cytokines or other pro-inflammatory markers. Incubating HepG2 cells with a SCD1 inhibitor induced a similar resistance to the effect of ethanol, confirming a role for SCD1 activity in mediating ethanol-induced hepatic injury.Taken together, our study shows that SCD1 is a key player in the development of AFLD and associated deleterious effects, and suggests SCD1 inhibition as a therapeutic option for the treatment of this hepatic disease.     

SCUBE1-enhanced bone morphogenetic protein signaling protects against renal ischemia-reperfusion injury

We previously reported that the membrane-bound SCUBE1 (signal peptide-CUB-epithelial growth factor domain-containing protein 1) forms a complex with bone morphogenetic protein 2 (BMP2) ligand and its receptors, thus acting as a BMP co-receptor to augment BMP signal activity. However, whether SCUBE1 can bind to and facilitate signaling activity of BMP7, a renal protective molecule for ischemia-reperfusion (I/R) insult, and contribute to the proliferation and repair of renal tubular cells after I/R remains largely unknown. In this study, we first showed that I/R-induced SCUBE1 was expressed in proximal tubular cells, which coincided with the expression of renoprotective BMP7. Molecular and biochemical analyses revealed that SCUBE1 directly binds to BMP7 and its receptors, functioning as a BMP co-receptor to promote BMP7 signaling. Furthermore, we used a new Scube1 deletion (Δ2) mouse strain to further elucidate the renal pathophysiological function of SCUBE1 after I/R injury. As compared with wild-type littermates, Δ2 mice showed severe renal histopathologic features (extensive loss of brush border, tubular necrosis, and tubular dilation) and increased inflammation (neutrophil infiltrate and induction of monocyte chemoattractant protein-1, tumor necrosis factor-α and interleukin-6) during the resolution of I/R damage. They also showed reduced BMP signaling (phosphorylated Smad1/5/8) along with decreased proliferation and increased apoptosis of renal tubular cells. Importantly, lentivirus-mediated overexpression of SCUBE1 enhanced BMP signaling and conferred a concomitant survival outcome for Δ2 proximal tubular epithelial cells after hypoxia–reoxygenation treatment. The protective BMP7 signaling may be facilitated by stress-inducible SCUBE1 after renal I/R, which suggests potential targeted approaches for acute kidney injury.     

2-APB protects against liver ischemia-reperfusion injury by reducing cellular and mitochondrial calcium uptake

Ischemia-reperfusion (I/R) injury is a commonly encountered clinical problem in liver surgery and transplantation. The pathogenesis of I/R injury is multifactorial, but mitochondrial Ca(2+) overload plays a central role. We have previously defined a novel pathway for mitochondrial Ca(2+) handling and now further characterize this pathway and investigate a novel Ca(2+)-channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB), for preventing hepatic I/R injury. The effect of 2-APB on cellular and mitochondrial Ca(2+) uptake was evaluated in vitro by using (45)Ca(2+). Subsequently, 2-APB (2 mg/kg) or vehicle was injected into the portal vein of anesthetized rats either before or following 1 h of inflow occlusion to 70% of the liver. After 3 h of reperfusion, liver injury was assessed enzymatically and histologically. Hep G2 cells transfected with green fluorescent protein-tagged cytochrome c were used to evaluate mitochondrial permeability. 2-APB dose-dependently blocked Ca(2+) uptake in isolated liver mitochondria and reduced cellular Ca(2+) accumulation in Hep G2 cells. In vivo I/R increased liver enzymes 10-fold, and 2-APB prevented this when administered pre- or postischemia. 2-APB significantly reduced cellular damage determined by hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining of liver tissue. In vitro I/R caused a dissociation between cytochrome c and mitochondria in Hep G2 cells that was prevented by administration of 2-APB. These data further establish the role of cellular Ca(2+) uptake and subsequent mitochondrial Ca(2+) overload in I/R injury and identify 2-APB as a novel pharmacological inhibitor of liver I/R injury even when administered following a prolonged ischemic insult.     

Mycobacterial infection induces the secretion of high-mobility group box 1 protein

High-mobility group box protein 1 (HMGB1) is a non-histone nuclear protein that acts as a pro-inflammatory cytokine and is released by monocytes and macrophages. Necrotic cells also release HMGB1 at the site of tissue damage which induces a variety of cellular responses, including the expression of pro-inflammatory mediators. This study investigated the secretion of HMGB1 in mycobacterial infection by macrophages in vitro and in the lungs of infected guinea pigs. We observed that infection by mycobacterium effectively induced HMGB1 release in both macrophage and monocytic cell cultures. Culture filtrate proteins from Mycobacterium tuberculosis induced maximum release of HMGB1 compared with different subcellular fractions of mycobacterium. We demonstrated that HMGB1 is released in lungs during infection of M. tuberculosis in guinea pigs and increased HMGB1 secretion in lungs of guinea pigs was delayed by prior vaccination with Mycobacterium bovis BCG. The secretion of cytokines like tumour necrosis factor alpha (TNF-alpha) and Interleukin-1beta was significantly increased when M. bovis BCG-infected cultures of J774A.1 cells were incubated with HMGB1. Among different mycobacterial toll-like receptor ligands, heat-shock protein 65 (HSP65) was found to be more potent in inducing HMGB1 secretion in RAW 264.7 cells. Pharmacological suppression of p38 or extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases with specific inhibitors failed to inhibit HSP65-induced HMGB1 release, but inhibition of c-Jun NH(2)-terminal kinase activation attenuated HMGB1 release. Inhibition of the inducible NO synthase and neutralizing antibodies against TNF-alpha also reduced HMGB1 release stimulated by HSP65. We conclude that HMGB1 is secreted by macrophages during tuberculosis and it may act as a signal of tissue or cellular injury and enhances immune response.     

A1 adenosine receptor allosteric enhancer PD-81723 protects against renal ischemia-reperfusion injury

Activation of A(1) adenosine receptors (ARs) protects against renal ischemia-reperfusion (I/R) injury by reducing necrosis, apoptosis, and inflammation. However, extrarenal side effects (bradycardia, hypotension, and sedation) may limit A(1)AR agonist therapy for ischemic acute kidney injury. Here, we hypothesized that an allosteric enhancer for A(1)AR (PD-81723) protects against renal I/R injury without the undesirable side effects of systemic A(1)AR activation by potentiating the cytoprotective effects of renal adenosine generated locally by ischemia. Pretreatment with PD-81723 produced dose-dependent protection against renal I/R injury in A(1)AR wild-type mice but not in A(1)AR-deficient mice. Significant reductions in renal tubular necrosis, neutrophil infiltration, and inflammation as well as tubular apoptosis were observed in A(1)AR wild-type mice treated with PD-81723. Furthermore, PD-81723 decreased apoptotic cell death in human proximal tubule (HK-2) cells in culture, which was attenuated by a specific A(1)AR antagonist (8-cyclopentyl-1,3-dipropylxanthine). Mechanistically, PD-81723 induced sphingosine kinase (SK)1 mRNA and protein expression in HK-2 cells and in the mouse kidney. Supporting a critical role of SK1 in A(1)AR allosteric enhancer-mediated renal protection against renal I/R injury, PD-81723 failed to protect SK1-deficient mice against renal I/R injury. Finally, proximal tubule sphingosine-1-phosphate type 1 receptors (S1P(1)Rs) are critical for PD-81723-induced renal protection, as mice selectively deficient in renal proximal tubule S1P(1)Rs (S1P(1)R(flox/flox) PEPCK(Cre/-) mice) were not protected against renal I/R injury with PD-81723 treatment. Taken together, our experiments demonstrate potent renal protection with PD-81723 against I/R injury by reducing necrosis, inflammation, and apoptosis through the induction of renal tubular SK1 and activation of proximal tubule S1P(1)Rs. Our findings imply that selectively enhancing A(1)AR activation by locally produced renal adenosine may be a clinically useful therapeutic option to attenuate ischemic acute kidney injury without systemic side effects.     

内质网应激预处理条件下大鼠肝脏缺血再灌注损伤模型的建立

目的:通过建立了内质网应激预处理条件下的大鼠肝脏缺血再灌注损伤模型,探讨内质网应激预处理在体内的应用.方法:以衣霉素为内质网应激诱导剂,采用大鼠肝脏70%缺血再灌注损伤模型.按照不同给药剂量分为50μ g/kg组、100μ g,kg组、200μ g/kg组、对照组,观察不同给药剂量条件下,血清转氨酶水平的变化.结果:给药100μ g/kg体重、诱导5天条件下大鼠术后转氨酶水平显著低于对照组.其它组与对照组无统计学差异.肝组织病理切片证实该预处理条件对肝脏缺血再灌注损伤有显著保护作用.结论:在特定的给药剂量条件下,衣霉素诱导的内质网应激预处理对大鼠肝脏缺血再灌注损伤有显著的保护作用.     

Cutting edge: C1q protects against the development of glomerulonephritis independently of C3 activation

C1q-deficient (C1qa-/-) mice develop antinuclear Abs and glomerulonephritis (GN) characterized by multiple apoptotic bodies. To explore the contribution of C3 activation to the induction of spontaneous GN, C1qa-/- mice were crossed with factor B- and C2-deficient (H2-Bf/C2-/-) mice. GN was present in 64% of the 45 C1qa/H2-Bf/C2-/- mice compared with 8% of the 65 H2-Bf/C2-/- mice and none of the 24 wild-type controls. IgG was detected in the glomeruli of diseased C1qa/H2-Bf/C2-/- kidneys. However, glomerular staining for C3 was absent. Increased numbers of glomerular apoptotic bodies were detected in undiseased C1qa/H2-Bf/C2-/- kidneys. These findings support the hypothesis that C1q may play a role in the clearance of apoptotic cells without the necessity for C3 activation and demonstrate that the activation of C3 is not essential for the development of GN in this spontaneous model of lupus-like disease.     

Bax ablation protects against myocardial ischemia-reperfusion injury in transgenic mice

The role of the proapototic Bax gene in ischemia-reperfusion (I/R) injury was studied in three groups of mice: homozygotic knockout mice lacking the Bax gene (Bax(-/-)), heterozygotic mice (Bax(+/-)), and wild-type mice (Bax(+/+)). Isolated hearts were subjected to ischemia (30 min, 37 degrees C) and then to 120 min of reperfusion. The left ventricular developed force of Bax-deficient vs. Bax(+/+) hearts at stabilization and at 120 min of reperfusion was 1,411 +/- 177 vs. 1,161 +/- 137 mg and 485 +/- 69 vs. 306 +/- 68 mg, respectively. Superior cardiac function of Bax(-/-) hearts after I/R was accompanied by a decrease in creatine kinase release, caspase 3 activity, irreversible ischemic injury, and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cardiomyocytes. Electron microscopic evaluation revealed reduced damage to mitochondria and the nuclear chromatin structure in Bax-deficient mice. In the Bax(+/-) hearts, the damage markers were moderate. The superior tolerance of Bax knockout hearts to I/R injury recommends this gene as a potential target for therapeutic intervention in patients with severe and intractable myocardial ischemia.