Protective effects of cyclosporin A from endotoxin-induced myocardial dysfunction and apoptosis in rats

Myocardial depression can be demonstrated following administration of endotoxin. Proposed mechanisms of endotoxin-induced myocardial dysfunction include the release of proinflammatory mediators, focal myocardial ischemia, and the presence of activated leukocytes within the myocardium. Recently, myocardial caspase activation and mitochondria-related apoptotic events (i.e., release of cytochrome c) were demonstrated in the failing septic heart. Here, we tested the hypothesis that immunosuppressors, cyclosporin A and tacrolimus (FK 506), would improve inflammation, heart nuclear apoptosis, and myocardial dysfunction in endotoxin-treated rats. Myocardial contractility was assessed using an isolated heart preparation. Heart leukocyte infiltration was assessed by measurement of heart myeloperoxidase activity. Leukocyte activation was studied using the intravital microscopy of the mesenteric venule. Apoptosis was detected as myocardial DNA fragmentation, downstream caspase activation, and mitochondrial cytochrome c release. Both cyclosporin A and FK 506 reduced heart leukocyte sequestration and venular adhesion in endotoxin-treated rats. Cyclosporin A, which blocks mitochondrial cytochrome c release, was able to reduce endotoxin-induced myocardial end-stage nuclear apoptosis and heart dysfunction, whereas tacrolimus had no such effects. These effects could be related to the unique properties of cyclosporin A to act on mitochondria.     

Macrophage migration inhibitory factor is released from pituitary folliculo-stellate-like cells by endotoxin and dexamethasone and attenuates the steroid-induced inhibition of interleukin 6 release

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by peripheral immune cells and also by endocrine cells in the anterior pituitary gland. MIF exerts its proinflammatory actions in the host-defense system by blocking the inhibitory effects of glucocorticoids on the release of other proinflammatory cytokines (e.g. IL-1, IL-6, TNFalpha). Reports that pituitary folliculo-stellate (FS) cells share many characteristics with immune cells led us to propose that these cells may serve as an additional source of MIF in the pituitary and that pituitary-derived MIF may act in an autocrine or paracrine manner to modulate endotoxin-induced cytokine release from FS cells. In the present study we addressed this hypothesis by using 1) immunohistochemistry to localize MIF in primary pituitary tissue and 2) well-characterized FS (TtT/GF), corticotroph (AtT20), and macrophage/monocyte (RAW 264.7) cell lines to explore the effects of CRH, endotoxin, and dexamethasone on MIF release and to examine the effects of MIF on IL-6 release. Our immunohistochemical study showed that MIF is expressed in abundance in S100-positive FS cells and also in other pituitary cell types. All three cell lines expressed MIF protein and responded to endotoxin (10-1000 ng/ml, 24 h) and dexamethasone (100 pM to 10 nM, 24 h) with concentration-dependent increases in MIF release. CRH (10-100 nM) also stimulated MIF release from AtT20 cells but, unlike endotoxin and dexamethasone, it had no effect on MIF release from TtT/GF or RAW cells. Recombinant MIF did not affect the basal release of IL-6 from TtT/GF cells; however, it effectively reversed the inhibitory effects of dexamethasone (1 nM) on the endotoxin-induced release of IL-6 from these cells. The results suggest that the FS cells are both a source of and a target for MIF and raise the possibility that MIF serves as a paracrine/autocrine factor in the pituitary gland that contributes to the protective neuroendocrine response to endotoxin.     

Effects of the HIV-1 Protein Tat on Myocardial Function and Response to Endotoxin

HIV-1 infection has been associated with cardiomyopathy in a subset of patients. In order to determine whether HIV-1 alters myocardial function or the myocardial response to stress, transgenic mice that express the HIV-1 protein Tat were used. Heart function was assessed using the isolated working heart preparation. Response to infection was assessed by measuring heart function at various times after endotoxin administration. Since cytokines are implicated in myocardial dysfunction, plasma tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) and myocardial mRNA and protein levels of TNF-alpha and IL-6 were determined. Tat by itself did not cause myocardial dysfunction; however, 4 h after endotoxin, myocardial function was more severely compromised in the Tat mice than in control mice. Plasma TNF-alpha levels were elevated at 2 h and higher in the control group but myocardial levels were similar in the two groups. Plasma IL-6 was increased but myocardial levels were different only at 24 h at which time myocardial function was no longer depressed. Tat expression, by itself, did not impair intrinsic myocardial function but did increase myocardial injury induced by endotoxin. Although cytokines are associated with dysfunction, TNF-alpha and IL-6 were probably not responsible for the exaggerated dysfunction in Tat mice receiving endotoxin.     

Control of fibroblast-like synoviocyte proliferation by macrophage migration inhibitory factor

OBJECTIVE: The hyperplasia of fibroblast-like synoviocytes (FLS) is considered essential to the evolution of joint destruction in rheumatoid arthritis (RA), but the mechanisms underlying FLS proliferation remain poorly understood. Macrophage migration inhibitory factor (MIF) is a cytokine that has recently been shown to exert proinflammatory effects on RA FLS. This study sought to identify the mechanisms of activation of FLS by MIF, and to assess the effects of MIF on synovial cell proliferation. METHODS: Human RA FLS were treated with recombinant MIF, interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and/or anti-MIF monoclonal antibodies (mAb). Proliferation was measured with tritiated thymidine incorporation. Nuclear factor kappa B (NF-kappa B) and mitogen-activated protein (MAP) kinase activation were measured with immunohistochemistry and Western blotting, respectively. RESULTS: FLS proliferation was significantly increased by MIF. IL-1 beta and TNFalpha also induced proliferation, but these effects were prevented by neutralization with anti-MIF mAb. Activation of NF-kappa B was induced by IL-1 beta, but not by MIF. Anti-MIF mAb had no effect on IL-1 beta-induced NF-kappa B nuclear translocation. By contrast, MIF induced phosphorylation of extracellular signal-regulated kinase (ERK) MAP kinase. ERK antagonism, but not NF-kappa B antagonism, prevented the effect of MIF on FLS proliferation. CONCLUSION: These data suggest that MIF may regulate RA synovial hyperplasia by acting directly and via involvement in the effects of IL-1 beta and TNFalpha. In addition, the effects of MIF on FLS activation are independent of NF-kappa B, and dependent on ERK MAP kinase. These data suggest an important therapeutic potential for MIF antagonism in RA.     

Peroxynitrite decomposition catalysts prevent myocardial dysfunction and inflammation in endotoxemic rats

OBJECTIVES: The aim of this study was to test whether peroxynitrite neutralizers would reduce peroxynitrite accumulation and improve myocardial contractile dysfunction and inflammation in endotoxin-treated rats. BACKGROUND: Release of endogenous proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha in response to endotoxin is responsible for the production of large amounts of nitric oxide (NO), which may exert detrimental effects on the myocardium in animal models, isolated hearts, and isolated cardiac myocytes. Recent studies have indicated that many of the deleterious effects of NO are mediated by peroxynitrite, a powerful oxidant generated from a fast diffusion-limited reaction of NO and superoxide anion. METHODS: We studied the effects of peroxynitrite neutralizers, such as mercaptoethylguanidine (MEG) sodium succinate (10 mg/kg) and 5,10,15,20-tetrakis(4-sulfonatophenyl)-porphyrinato iron (III) (FeTPPS) (30 mg/kg) on peroxynitrite accumulation, in vivo endothelial cell-leukocyte activation on the mesenteric venule, and myocardial contractile dysfunction and inflammation in a model of sepsis induced by injection of endotoxin (10 mg/kg) in rats. RESULTS: Mercaptoethylguanidine sodium succinate and FeTPPS largely prevented the accumulation of peroxynitrite as measured by plasma rhodamine fluorescence and heart nitrotyrosine staining. Interestingly, MEG sodium succinate and FeTPPS improved endotoxin-induced myocardial contractile dysfunction, which was associated with reduced degradation of nuclear factor kappa B inhibitory protein I-kappa-B, plasma TNF-alpha levels, and microvascular endothelial cell-leukocyte activation. CONCLUSIONS: These observations suggest that the beneficial effects of MEG and FeTPPS on endotoxin-induced myocardial contractile dysfunction could be related to the unique effects of these compounds on cardiovascular inflammation processes.     

Induction of early-phase tolerance to endotoxin-induced mucosal injury, xanthine oxidase activation, and bacterial translocation by pretreatment with endotoxin.

The goal of this study was to determine whether tolerance would develop to endotoxin-induced mucosal injury, xanthine oxidase activation, and bacterial translocation. To accomplish this goal, four groups of mice were studied: 1) mice receiving ip injections of saline 96 and 24 hr prior to sacrifice, 2) mice receiving ip injections of saline 96 and endotoxin (0.1 mg) 24 hr prior to sacrifice, 3) mice receiving ip injections of endotoxin 96 and 24 hr prior to sacrifice, and 4) mice receiving ip injections of endotoxin 96 hr and saline 24 hr prior to sacrifice. In contrast to the saline control animals or mice sacrificed 96 hr after a single dose of endotoxin, mice sacrificed 24 hr after receiving a single dose of endotoxin had evidence of mucosal injury, elevated levels of ileal xanthine oxidase activity, and an 81% incidence of bacterial translocation. Mice sacrificed 24 hr after a second dose of endotoxin were largely protected against the toxic effects of endotoxin. Thus tolerance to endotoxin-induced bacterial translocation does develop and is associated with tolerance to endotoxin-induced ileal mucosal injury and xanthine oxidase activation.     

Changes in activity of µ- and m-calpains and signs of neuroinflammation in the hippocampus and striatum of rats after single intraperitoneal injection of subseptic dose of endotoxin

Some mechanisms of neuronal degeneration in endotoxinemia are already well described, but need to be detailed. In this study, we tested the effect of a single intraperitoneal injection of a LPS sub-septic dose (1 mg/kg of animal weight) on calpain activity in the striatum and hippocampus. We showed, that in the hippocampus the day after LPS administration an increase in production of IL-1β and TNF-α mRNA, followed by elevated mRNA expression and activity of µ- and m-calpains without signs of microglia activation is observed. In striatal cells, the day after LPS injection an increase in expression of IL-1β, TNF-α, IBA-1, m-calpain and calpastatin mRNA is revealed, which only intensifies over time. The elicited changes are accompanied by a decrease in motor behavior, which can be considered as a sign of sickness behavior. In the hippocampus, 180 days after LPS administration expression of TNF-α, content and activity of µ-calpain are increased. In the striatum, elevation in expression of TNF-α, IBA-1, µ- and m-calpain mRNA, with hyperactivation of only m-calpain, is observed. Significantly reduced motor activity can be a consequence of LPS-induced neuronal death. A long-lasting endotoxin activates microglia that damage neurons via proinflammation cytokines and calpain hyperactivation. The endotoxin hypothesis of neurodegeneration is unproven, but if correct, then neurodegeneration may be reduced by decreasing endotoxin-induced neuroinflammation and m-calpain hyperactivation. Therefore, the drugs, that decrease endotoxin-induced neuroinflammation and differently inhibit µ- or m-calpain, can be used to prevent or reduce the severity of neurodegeneration.

     

Induction of T-kininogen and tumor necrosis factor-alpha by macrophage migration inhibitory factor in vivo

Administration of lipopolysaccharide (LPS) induces inflammation and tissue injuries that occasionally results in disseminated intravascular coagulation (DIC). This process is believed to be mediated by vasoactive molecules such as kinins and leads to endothelial damage and obstruction of the microcirculation. In this study, we evaluated the involvement of T-kininogen and macrophage migration inhibitory factor (MIF) in endotoxin-induced systemic inflammation. T-Kininogen is a protein unique to the rat and known as an acute-phase protein in response to endotoxins. Similarly, MIF functions as a proinflammatory cytokine and glucocorticoid-induced immunoregulator. First, we examined the effects of anti-MIF antibody on Wistar King male rats (ca 400 g) treated with intraperitoneal injection of LPS. At 6 hours after LPS injection (5 mg/kg), the platelet counts had decreased from 85 +/- 12.8 (x 10(4)/microL) to 8.8 +/- 2.6 (x 10(4)/microL). We treated these rats with the anti-rat MIF antibody (5 mg gamma G immunoglobulin [IgG] fraction/kg) 2 hours prior to LPS injection. This treatment prevented the decrease in platelet counts (45.6 +/- 5.6 [x 10(4)/microL]). Next, we examined the potential of MIF for production of T-kininogen. Intraperitoneal injection of rat MIF significantly upregulated the serum content of T-kininogen at the dose of 500 microg MIF/head. These results imply that MIF and T-kininogen might function in concert in the event of endotoxin-induced inflammation.     

Polyethylene glycol-superoxide dismutase prevents endotoxin-induced cardiac dysfunction

RATIONALE: Sepsis produces significant mitochondrial and contractile dysfunction in the heart, but the role of superoxide-derived free radicals in the genesis of these abnormalities is not completely understood. OBJECTIVES: The study was designed to test the hypothesis that superoxide scavenger administration prevents endotoxin-induced cardiac mitochondrial and contractile dysfunction. METHODS: Four groups of rats were studied, and animals were injected with either saline, endotoxin, endotoxin plus polyethylene glycol-adsorbed-superoxide dismutase (PEG-SOD; a free-radical scavenger), or PEG-SOD alone. Animals were killed 48 h after injections. We then measured cardiac mitochondrial generation of reactive oxygen species (ROS), formation of free-radical reaction products (protein carbonyls, lipid aldehydes, nitrotyrosine), mitochondrial function, and cardiac contractile function. MEASUREMENTS AND MAIN RESULTS: Endotoxin elicited increases in cardiac mitochondrial ROS formation (p < 0.001), increases in cardiac levels of free-radical reaction products, reductions in mitochondrial ATP generation (p < 0.001), and decrements in cardiac pressure-generating capacity (p < 0.01). Administration of PEG-SOD blocked formation of free-radical reaction products, prevented mitochondrial dysfunction, and preserved cardiac contractility. For example, mitochondrial ATP generation was 923 +/- 50, 392 +/- 32, 753 +/- 25, and 763 +/- 36 nmol/min/mg, respectively, for control, endotoxin, endotoxin + PEG-SOD, and PEG-SOD groups (p < 0.001). In addition, cardiac systolic pressure generation at a diastolic pressure of 15 mm Hg averaged 110 +/- 11, 66 +/- 7, 129 +/- 10 and 124 +/- 5 mm Hg, respectively, for control, endotoxin, endotoxin + PEG-SOD, and PEG-SOD groups (p < 0.01). CONCLUSION: These data indicate that superoxide-derived oxidants play a critical role in the development of cardiac mitochondrial and contractile dysfunction in endotoxin-induced sepsis.     

Caspase inhibition prevents cardiac dysfunction and heart apoptosis in a rat model of sepsis

Despite intensive therapy, severe septic shock is commonly associated with myocardial dysfunction and death in humans. No new therapies have proven efficiency against cardiovascular alterations in sepsis. Here, we addressed the question of a beneficial effect of pharmacological inhibition of caspases on myocardial dysfunction following endotoxin treatment. Hearts from rats treated with endotoxin (10 mg/kg, intravenously) were isolated 4 h posttreatment for analysis. Assessment of myocardial contractility ex vivo and detection of apoptosis were performed. Hearts from endotoxin-treated rats displayed multiple caspase activities and also typical apoptosis pattern as detected by TUNEL, DNA fragmentation assays, and cytochrome c release as compared with control rats. z-VAD.fmk (3 mg/kg, intravenously), a broad spectrum caspase inhibitor (but not the irrelevant peptide z-FA.fmk), in coinjection with endotoxin, not only reduced caspase activities and nuclear apoptosis but also completely prevented endotoxin-induced myocardial dysfunction evaluated 4 h and even 14 h after endotoxin challenge. These data indicate that caspase activation plays an important role in myocardial cell dysfunction. Moreover, these results suggest that inhibitors of caspases may have important therapeutic applications in sepsis.     

重组人生长激素对心肌梗死大鼠心功能及心肌炎性因子表达的影响

目的 观察重组人生长激素(rhGH)对大鼠急性心肌梗死后心功能及心肌炎性因子表达的影响. 方法 结扎大鼠左冠状动脉前降支建立心肌梗死模型,将术后24 h存活的大鼠随机分为对照组和rhGH组,另设假手术组.rhGH组给予rhGH 2 mg.kg-1.d-1皮下注射,对照组和假手术组给予等体积的生理盐水皮下注射,4周后观察rhGH对大鼠心功能及心肌细胞肿瘤坏死因子-α(TNF-α)、白介素-1p(IL-1p)、白介素-6(IL-6)、白介素-10(IL-10)mRNA表达的影响. 结果 与假手术组比较,对照组心肌组织中促炎性细胞因子TNF-α、IL-1β、IL-β和IL-10 mRNA表达(假手术组分别为0.10±0.02、0.08±0.01、0.18±0.01和0.14±0.05;梗死区分别为0.77±0.15、0.93±0.17、1.10±0.14和0.73±0.11;非梗死区分别为0.88±0.14、0.95±0.17、1.18±0.11和0.83±0.16)明显升高.与对照组比较,rhGH组心肌组织中TNF-α、IL-1β、IL-6 mRNA表达(梗死区分别为0.35±0.10、0.36±0.10、0.43±0.11;非梗死区分别为0.51±0.10、0.42±0.11、0.51±0.12)明显下降,IL-10 mRNA表达(梗死区为1.18±0.18;非梗死区为1.21±0.22)升高,P＜0.05.心脏超声显示rhGH组左室功能明显改善. 结论 早期rhGH治疗改善了心肌梗死大鼠心肌炎性因子表达和心脏功能.rhGH有效改善心功能与其降低心肌细胞促炎细胞因子和升高抗炎因子的作用有关.     

Endotoxin potentiates ozone-induced pulmonary chemokine and inflammatory responses

Urban air consists of a combination of environmental pollutants. Recent studies have suggested that normally innocuous doses of a particular pollutant may be rendered more toxic to the lung if primed by earlier events. Pulmonary inflammation has been observed in humans and in many animal species after endotoxin and ozone exposures. The present study was designed to test the hypothesis that inhalation of low levels of endotoxin following ozone exposure will potentiate ozone-induced lung injury. We exposed 8-week-old C57BL/6J mice to 1 ppm ozone for 24 hours; inhalation of low-dose endotoxin (37.5 EU) for 10 minutes; or 1 ppm ozone immediately followed by endotoxin inhalation (37.5 EU). The mice were examined 4 or 24 hours post exposure. After 24 hours of recovery, significant increases were measured in bronchoalveolar lavage (BAL) fluid levels of protein and lavageable polymorphonuclear neutrophils (PMNs) after coexposure to ozone followed immediately by endotoxin inhalation as compared to exposures individually. Messages encoding macrophage inflammatory protein (MIP)-1beta, MIP-1alpha, MIP-2, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-1alpha, IL-1beta, IL-1Ra, IL-6, and Macrophage Migration Inhibitory Factor (MIF) were significantly elevated 24 hours post ozone followed by endotoxin as compared to exposure to ozone or endotoxin individually. These results demonstrate that preexposure to ozone, which primarily attacks the epithelium, can cause sensitization to a secondary stimulus through a mechanism that culminates in a greater and prolonged onset of inflammatory cell recruitment, pulmonary edema, and increased expression of chemokine and cytokine messages.     

Recruited inflammatory cells mediate endotoxin-induced lung maturation in preterm fetal lambs

RATIONALE: Chorioamnionitis is paradoxically associated with a decreased incidence of respiratory distress syndrome in preterm infants. In preterm lambs, intraamniotic endotoxin and interleukin 1 (IL-1) induce lung inflammation followed by lung maturation. OBJECTIVE: To test if inflammatory cells are required to mediate induced lung maturation. METHODS: Lung inflammation was induced by intraamniotic injection of endotoxin or IL-1. Inflammatory cell recruitment to the lung was inhibited by an anti-CD18 blocking antibody given intramuscularly to the fetus. Preterm lambs were delivered at 124-d gestation (term = 150 d) 2 or 7 d after exposure to endotoxin/IL-1 or endotoxin/IL-1 + anti-CD18 antibody. MEASUREMENTS: Lung inflammation was measured by bronchoalveolar lavage fluid cell count, inflammatory scoring of lung parenchyma, and expression of proinflammatory cytokines and inducible nitric oxide synthase. Lung maturation was quantitated by surfactant protein mRNA expression, saturated phosphatidylcholine pool size, and pressure-volume curves. MAIN RESULTS: Inhibition of CD18 significantly reduced endotoxin-induced but not IL-1-induced fetal lung inflammatory cell recruitment and activation as well as expression of proinflammatory cytokines. Compared with control lungs, both endotoxin and IL-1 induced lung maturation. Anti-CD18 antibody administration inhibited only endotoxin-induced but not IL-1-induced increases in surfactant protein mRNA and surfactant saturated phosphatidylcholine. Exposure to anti-CD18 antibody moderated endotoxin-induced increases in lung volumes but had no effect on IL-1-induced increases in lung volumes. CONCLUSIONS: (1) Endotoxin- but not IL-1-induced inflammatory cell recruitment in the preterm fetal lamb lung is CD18 dependent; (2) recruited inflammatory cells mediate some aspects of fetal lung maturation.     

Pulmonary endothelium as a site of synthesis and storage of interleukin-6 in experimental congestive heart failure

BACKGROUND: Pulmonary endothelium is an early upstream hemodynamic target of left ventricular dysfunction. Interleukin 6 (IL-6) is a pro-inflammatory cytokine reported to increase in congestive heart failure (CHF) patients. AIMS: We sought to determine the origin of IL-6, IL-6 receptor (IL-6R) and gp130 in experimental CHF. METHODS: We used rats with coronary artery ligation as an experimental model of either compensated or decompensated heart failure. Lung and aorta samples were analysed by RT-PCR, ELISA and immunohistochemistry for IL-6 and its receptors. RESULTS: IL-6 mRNA expression increased in the lung of rats with decompensated heart failure and was positively correlated with infarct severity whereas IL-6R mRNA decreased in the lung of myocardial infarction rats and gp130 mRNA remained unchanged. In contrast, there were no changes in IL-6 mRNA expression in the aorta and left ventricular myocardium. IL-6 peptide content as determined by ELISA and Western Blot in lung tissue was 2-fold higher in decompensated heart failure as compared to control rats. These data were confirmed by immunohistochemistry showing a preferential endothelial localization of IL-6 in the CHF lung. IL-6 peptide was also present in the pleural effusion of decompensated heart failure and was positively correlated with IL-6 mRNA expression in the lungs of decompensated HF rats. Pulmonary IL-6 overexpression was associated with nuclear translocation of NF-kappaB and cytosolic degradation of IkappaB. CONCLUSION: Dysfunctional pulmonary endothelium is a source of synthesis and storage of IL-6 in an experimental model of CHF.     

Endotoxin-induced activation of the coagulation cascade in humans: effect of acetylsalicylic acid and acetaminophen.

During Gram-negative septic shock, lipopolysaccharide (LPS, endotoxin) induces tissue factor (TF) expression. TF expression is mediated by nuclear factor kappaB and amplified by activated platelets. TF forms a highly procoagulant complex with activated coagulation factor VII (FVIIa). Hence, we hypothesized that aspirin, which inhibits LPS-induced, nuclear factor kappaB-dependent TF expression in vitro and platelet activation in vivo, may suppress LPS-induced coagulation in humans. Therefore, we studied the effects of aspirin on systemic coagulation activation in the established and controlled setting of the human LPS model. Thirty healthy volunteers were challenged with LPS (4 ng/kg IV) after intake of either placebo or aspirin (1000 mg). Acetaminophen (1000 mg) was given to a third group to control for potential effects of antipyresis. Neither aspirin nor acetaminophen inhibited LPS-induced coagulation. However, LPS increased the percentage of circulating TF(+) monocytes by 2-fold. This increase was associated with a decrease in FVIIa levels, which reached a minimum of 50% 24 hours after LPS infusion. Furthermore, LPS-induced thrombin generation increased plasma levels of circulating polymerized, but not cross-linked, fibrin (ie, thrombus precursor protein), whereas levels of soluble fibrin were unaffected. In summary, a single 1000-mg dose of aspirin did not decrease LPS-induced coagulation. However, our study showed, for the first time, that LPS increases TF(+) monocytes, substantially decreases FVIIa levels, and enhances plasma levels of thrombus precursor protein, which may be a useful marker of fibrin formation in humans.     

Gene knockout or inhibition of macrophage migration inhibitory factor alleviates lipopolysaccharide-induced liver injury via inhibiting inflammatory response

BackgroundLiver injury is one of the most common complications during sepsis. Macrophage migration inhibitory factor (MIF) is an important proinflammatory cytokine. This study explored the role of MIF in the lipopolysaccharide (LPS)-induced liver injury through genetically manipulated mouse strains.MethodsThe model of LPS-induced liver injury was established in wild-type and Mif-knockout C57/BL6 mice. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBil) were detected, and the expressions of MIF, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were measured. Liver histopathology was conducted to assess liver injury. Moreover, the inhibitions of MIF with (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) and 4-iodo-6-phenylpyrimidine (4-IPP) were used to evaluate their therapeutic potential of liver injury.ResultsCompared with wild-type mice, the liver function indices and inflammation factors presented no significant difference in the Mif^−/− mice. After 72 h of the LPS-induced liver injury, serum levels of ALT, AST, and TBil as well as TNF-α and IL-1β were significantly increased, but the knockout of Mif attenuated liver injury and inflammatory response. In liver tissue, mRNA levels of TNF-α, IL-1β and NF-κB p65 were remarkably elevated in LPS-induced liver injury, while the knockout of Mif reduced these levels. Moreover, in LPS-induced liver injury, the inhibitions of MIF with ISO-1 and 4-IPP alleviated liver injury and slightly attenuated inflammatory response. Importantly, compared to mice with LPS-induced liver injury, Mif knockout or MIF inhibitions significantly prolonged the survival of the mice.ConclusionsIn LPS-induced liver injury, the knockout of Mif or MIF inhibitions alleviated liver injury and slightly attenuated inflammatory response, thereby prolonged the survival of the mice. Targeting MIF may be an important strategy to protect the liver from injury during sepsis.     

楤木皂苷抑制内毒素导致的心肌损伤和焦亡

目的 内毒素（LPS）导致的心功能障碍是脓毒血症患者死亡的重要原因。本研究旨在探讨采用楤木皂苷（saponins extracted from Aralia taibaiensis,sAT）能否抑制LPS导致的心肌损伤和心肌细胞焦亡。 方法 将雄性C57/BL6小鼠随机分为:正常对照组,sAT对照组,LPS组和LPS+sAT组,每组8只。采用sAT [240 mg/(kg·d),7天]灌胃后腹腔注射LPS (15 mg/kg),于24小时后检测心脏功能,同时测定各组炎症因子水平;Western blot检测caspase-11的表达。细胞学实验采用H9C2心肌细胞给予LPS结合常规焦亡刺激物—霍乱毒素B型亚单位（CTB）诱导心肌细胞焦亡,检测sAT对H9C2细胞存活率及细胞培养上清中IL-1β,LDH水平和细胞caspase-11水平的影响。 结果 与对照组相比,LPS导致心功能指标显著降低,血浆CK-MB显著升高,死亡率增高,心肌组织中炎症反应显著增强（P < 0.05）。给予sAT处理可显著改善LPS所导致的心功能障碍并抑制炎症反应,与LPS组相比,降低LPS组小鼠的死亡率（P < 0.05）。LPS可导致心肌组织出现细胞焦亡特征,表现为心肌caspase-11活化,IL-1β水平升高和血浆LDH水平显著升高;sAT处理可有效抑制LPS导致的心肌细胞焦亡。细胞学实验证实,LPS结合CTB可诱导H9C2心肌细胞焦亡,sAT处理可有效抑制细胞caspase-11活化,有效降低细胞培养上清中的IL-1β和LDH水平,显著提高细胞存活率。 结论 sAT能够抑制LPS导致的心肌损伤和心功能障碍,并有效抑制心肌细胞焦亡。     

Mechanisms of endotoxin-induced NO, IL-6, and TNF-alpha production in activated rat hepatic stellate cells: role of p38 MAPK

Compelling experimental evidence indicates that the interactions between endotoxin and hepatic stellate cells (HSCs) can play a significant role in the pathogenesis of liver disease. Endotoxin-induced release of a multifunctional mediator NO (via inducible NO synthase) and the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-6 by HSCs could be an important mechanism of pathological changes in the liver. However, the signaling mechanisms of these effects are poorly understood. In this study, we found that endotoxin causes activation of mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated protein kinase [ERK] 1 and 2, p38, and c-Jun NH2-terminal kinase [JNK]) and nuclear factor kappaB (NF-kappaB) and production of H(2)O(2) in culture-activated HSCs. However, only p38 and NF-kappaB were found to be responsible for the synthesis of NO, IL-6, and TNF-alpha. Exogenous H(2)O(2) caused modest stimulation of TNF-alpha synthesis, did not affect the synthesis of NO or IL-6, and did not activate NF-kappaB or MAPKs. Inhibition of p38 and NF-kappaB activation by SB203580 and pyrrolidine dithiocarbamate, respectively, blocked endotoxin-induced H(2)O(2), NO, TNF-alpha, and IL-6 synthesis. Inhibition of ERK1/2 and JNK phosphorylation did not alter these effects of endotoxin. Whereas SB203580 inhibited endotoxin-induced NF-kappaB activation, pyrrolidine dithiocarbamate did not affect p38 phosphorylation in endotoxin-stimulated cells. In conclusion, endotoxin-induced synthesis of NO, TNF-alpha, and IL-6 in HSCs is mediated by p38 and NF-kappaB, with involvement of H(2)O(2) in TNF-alpha production.