Losartan prevents sepsis-induced acute lung injury and decreases activation of nuclear factor kappaB and mitogen-activated protein kinases

Lack of specific and efficient therapy leads to the high mortality rate of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Losartan is a potent pharmaceutical drug for ALI/ARDS. However, the protective effects and mechanisms of losartan remain incompletely known. This study evaluates the effects of losartan on ALI/ARDS and further investigates the possible mechanisms of these protective effects. Mice received i.p. injections of the AT1 inhibitor losartan (15 mg/kg), or control vehicle, half hour after cecal ligation and puncture (CLP). Plasma TNF-alpha, IL-1beta, and IL-6 cytokines were assayed 6 h after CLP. Blood gas, wet/dry lung weight ratio, lung tissue histology for occurrence of ALI/ARDS, and survival were examined. Lastly, nuclear factor kappaB (NF-kappaB) activations, IkappaB-alpha degradations, phosphorylations of p38 MAPK, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase expressions were evaluated in lung tissue. Losartan treatment significantly attenuated TNF-alpha, IL-6, and IL-1beta 6 h after CLP. Furthermore, losartan prevented blood gas and histopathologic appearance of ALI/ARDS after sepsis and significantly improved survival. Finally, losartan given after sepsis led to inhibition of lung tissue NF-kappaB activation (P < 0.01 vs. CLP group), attenuated degradation of IkappaB-alpha, and inhibited phosphorylation of p38MAPK, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase, pathways critical for cytokine release. These data reveal that losartan exerts a protective effect on ALI/ARDS, and this protective effect may be dependent, at least in part, on NF-kappaB and MAPK mechanisms.     

Sophorolipids block lethal effects of septic shock in rats in a cecal ligation and puncture model of experimental sepsis

OBJECTIVE: Sophorolipids, a family of natural and easily chemoenzymatically modified microbial glycolipids, are promising modulators of the immune response. The potential of the therapeutic effect of sophorolipids was investigated in vivo in a rat model of sepsis and in vitro by analysis of nitric oxide and cytokine production. DESIGN: Prospective, randomized animal study. SETTING: Experimental laboratory. SUBJECTS: Male Sprague-Dawley rats, 200-240 g. INTERVENTIONS: Intra-abdominal sepsis was induced in vivo in 166 rats via cecal ligation and puncture (CLP); 60 rats were used to characterize the model. The remaining rats were treated with sophorolipids or vehicle (dimethylsulfoxide [DMSO]/physiologic saline) by intravenous (iv) tail vein or intraperitoneal (IP) injection immediately post-CLP (25/group). Survival rates were compared at 36 hrs after surgery. In vitro, macrophages were cultured in lipopolysaccharide (LPS) +/- sophorolipid and assayed for nitric oxide (NO) production and gene expression profiles of inflammatory cytokines. In addition, splenic lymphocytes isolated from CLP rats +/- sophorolipid treatment (three per group) were analyzed for cytokine production by RNase protection assay. MEASUREMENTS AND MAIN RESULTS: CLP with 16-gauge needles optimized sepsis induction and resultant mortality. Sophorolipid treatment improved rat survival by 34% (iv) and 14% (IP) in comparison with vehicle controls (p < .05 for iv treatment). Sophorolipids decreased LPS-induced macrophage NO production by 28% (p < .05). mRNA expression of interleukin (IL)-1beta was downregulated by 42.5 +/- 4.7% (p < .05) and transforming growth factor (TGF)-beta1 was upregulated by 11.7 +/- 1.5% (p < .05) in splenocytes obtained 6 hrs postsophorolipid treatment. LPS-treated macrophages cultured 36 hrs with sophorolipids showed increases in mRNA expression of IL-1alpha (51.7%), IL-1beta (31.3%), and IL-6 (66.8%) (p < .05). CONCLUSIONS: Administration of sophorolipids after induction of intra-abdominal sepsis significantly decreases mortality in this model. This may be mediated in part by decreased macrophage NO production and modulation of inflammatory responses.     

Antibiotics improve survival in sepsis independent of injury severity but do not change mortality in mice with markedly elevated interleukin 6 levels

Genetically identical mice have a heterogeneous response to antibiotic therapy in sepsis, with only a subset deriving therapeutic benefit. We sought to determine whether the severity of a septic insult correlates with the survival benefit conferred by antibiotics. We also sought to determine whether antibiotics given 12 h after injury alter survival in animals predicted to die based upon high interleukin (IL)-6 levels drawn 6 h earlier. Adult male ND4 mice (n = 363) were subjected to double-puncture cecal ligation and puncture (CLP) with a 19-, 21-, or 23-gauge needle. Animals were randomized to receive imipenem or 0.9% NaCl every 12 h after CLP for 5 days. Ten-day survival was 16%, 26%, and 52%, respectively, for untreated animals. Antibiotics decreased the absolute risk of death 17% to 23% regardless of injury severity. In a separate cohort, mice (n = 37) were subjected to single or double-puncture CLP with a 21-gauge needle. IL-6 levels were assayed 6 h postoperatively and mice were followed for survival. Levels greater than 14,000 pg/mL were identified as predicting a 100% mortality (7/7 animals dead). A third set of mice (n = 94) then underwent double-puncture CLP with either 21-, 23-, or 25-gauge needle and had IL-6 levels measured in a similar fashion. Animals were randomized to receive imipenem or 0.9% NaCl beginning 12 h postoperatively (6 h after IL-6 levels were drawn) and continued for 5 days or until death. Although antibiotics decreased mortality overall, all animals with IL-6 levels greater than 14,000 pg/mL (n = 13) died, regardless of whether they received antibiotics or the gauge of needle used. These results indicate that antibiotics improve outcome in murine sepsis, regardless of injury severity. Furthermore, there is a threshold IL-6 level that can be identified 6 h after sepsis above which animals are destined to die, and antibiotic treatment does not alter their outcome.     

Hyperbaric oxygen protects from sepsis mortality via an interleukin-10-dependent mechanism

OBJECTIVE: This study was performed to determine whether hyperbaric oxygen (HBO2) therapy is protective in cecal ligation and puncture (CLP)-induced sepsis and if protection is dependent on oxygen dosing. We also wished to determine whether HBO2 affected bacterial clearance or altered macrophage production of interleukin-10 (IL-10)s in the setting of CLP sepsis. Finally, we wished to determine whether the mechanism of HBO2 protection in sepsis was dependent on IL-10 production. DESIGN: Prospective, experimental study. SETTING: University experimental research laboratory. SUBJECTS: C57BL/6 and C57BL/6 IL-10 mice. INTERVENTIONS: Sepsis was induced by CLP. Mice were randomized to receive a 1.5-hr HBO2 treatment at either 1, 2.5, or 3 atmospheres absolute every 12 hrs or HBO2 at 2.5 atmospheres absolute every 24 hrs. Mice were also harvested at 24 hrs for determination of bacterial load and isolation and study of CD11b peritoneal macrophages. MEASUREMENTS AND MAIN RESULTS: Survival was monitored for 100 hrs after CLP +/- HBO2 treatment. HBO2 significantly improved survival when administered at 2.5 atmospheres absolute every 12 hrs. Other treatment schedules were not protective, and treatment at 3.0 atmospheres absolute significantly worsened survival outcome. Bacterial load was significantly reduced in splenic homogenates but not peritoneal fluid at 24 hrs. Macrophages isolated from HBO2-treated mice demonstrated enhanced IL-10 secretion in response to lipopolysaccharide as compared with CLP controls. Mice genetically deficient in IL-10 expression treated with HBO2 at 2.5 atmospheres absolute every 12 hrs were not protected from CLP-induced mortality. CONCLUSION: HBO2 may be protective in CLP sepsis within a window of oxygen dosing. The mechanism of HBO2 protection may be potentially linked in part to expression of IL-10, as peritoneal macrophages demonstrated enhanced IL-10 expression and IL-10 mice were not protected by HBO2 treatment.     

白细胞介素-1β在脓毒症鼠心肌中的表达及p38MAPK的调控作用

目的探讨白细胞介素-1β（IL-1β）在脓毒症鼠心肌损伤中的作用及p38MAPK的调控机制。方法采用盲肠结扎并穿刺（CLP）来制作脓毒症模型，并在不同时相点观察大鼠血清CPK-MB、IL-1β浓度及其mRNA在心肌的表达、心肌p38MAPK的活性。结果CLP术后血清IL-1β浓度进行性升高，CPK—MB显著提高。正常心肌组织微量表达IL-1β mRNA，脓毒症时可见大量表达，且p38MAPK明显激活。血清IL-1β的水平及其mRNA在心肌中的表达与CPK-MB呈显著正相关。应用p38MAPK抑制剂SB203580后，p38MAPK激活受抑，血清IL-1β浓度显著降低，IL-1β在心肌中的表达减少，心肌损害明显减轻。结论IL-1β的大量释放及其在心肌中显著表达是脓毒症鼠心肌损伤的原因之一，而通过调控p38MAPK信号通路可对心肌起保护作用。     

GLUTAMINE PREVENTS ACTIVATION OF NF-kappaB AND STRESS KINASE PATHWAYS, ATTENUATES INFLAMMATORY CYTOKINE RELEASE, AND PREVENTS ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) FOLLOWING SEPSIS

Glutamine (GLN) has been shown to attenuate cytokine release from LPS-stimulated human peripheral blood mononuclear cells; however, the in vivo antiinflammatory effect of GLN in polymicrobial sepsis and ARDS is unknown. This study evaluates the effect of GLN on inflammatory cytokine release and the pathways that may mediate antiinflammatory effects of GLN in the lung. Either 0.75 g/kg of GLN or saline placebo (SP) was administered to male rats 1 h after cecal ligation and puncture (CLP). NF-kappaB activation, IKBalpha degradation, phosphorylation of p38 MAPK, ERK, and MKP-1 expression were evaluated in lung tissue 6 h post-CLP. Lung tissue iNOS and eNOS, TNF-alpha, IL-6, and IL-18 cytokines were assayed. Last, lung histopathology for occurrence of ARDS and survival were examined. GLN given 1 h postsepsis led to inhibition of lung tissue NF-kappaB activation (P < 0.001 vs. SP), attenuated degradation of IKBalpha, and inhibited phosphorylation of p38 MAPK, and ERK, pathways critical for cytokine release. GLN treatment increased MKP-1 peptide expression and significantly attenuated TNF-alpha and IL-6 6 h after CLP. IL-18 was attenuated by GLN at multiple time points post-CLP. Further, GLN abrogated increases in lung iNOS expression and enhanced lung eNOS postsepsis. Finally, GLN prevented the histopathologic appearance of ARDS after sepsis and significantly improved survival. These data reveal that GLN exerts an antiinflammatory effect in sepsis that may be mediated via attenuation of multiple pathways of inflammation such as NF-kappaB, p38 MAPK, ERK, and MKP-1. GLN also showed an inhibition of increases in iNOS expression. The antiinflammatory effect of GLN was associated with attenuation of ARDS and mortality.     

Potential protective effect of NF-kappaB activity on the polymicrobial sepsis of rats preconditioning heat shock treatment

The present study was designed to investigate the role of NF-kappaB in influencing the outcome of sepsis modulated by previous heat shock treatment. Sepsis was induced in rats by cecum ligation and puncture (CLP) method, which manifests two distinct clinical phases: an initial hyperdynamic phase (9 h after CLP, early sepsis) followed by a hypodynamic phase (18 h after CLP, late sepsis). Rats of heated group were treated by whole body hyperthermia 24 h prior to the CLP operation. Lymphocytes were collected during the early and late sepsis phases. The expressions of Hsp72, p65 and I-kappa B were evaluated by Western blot and immunochemical analysis. NF-kappaB activity was detected by EMSA. The results showed that NF-kappaB activation was initiated during early sepsis and apparently suppressed during late stage of sepsis. Previously treated by heat shock, late-sepsis rats emerged with high preservation of p65 expression and NF-kappaB activity, while Hsp72 was over-expressed. In conclusion, down-regulation of NF-kappaB activity during late sepsis could be attenuated by pretreatment of heat shock through the preservation of p65 expression. The results may provide a mechanistic explanation for the improved outcome to polymicrobial sepsis of rats that are preconditioned with heat shock, as well as a novel highlight for therapeutic intervention of severe infection.     

Is prostacyclin responsible for producing the hyperdynamic response during early sepsis?

OBJECTIVE: Although polymicrobial sepsis is characterized by an early hyperdynamic phase (2-10 hrs after cecal ligation and puncture [CLP]), followed by a late hypodynamic phase (20 hrs after CLP), it remains unknown whether prostacyclin or prostaglandin I2 (PGI2) plays a significant role in modulating the hyperdynamic state during early sepsis. The aim of this study was to determine whether inhibition of PGI2 synthesis prevents the occurrence of the hyperdynamic response during early sepsis. DESIGN: Prospective, controlled animal study. SETTING: A university research laboratory. SUBJECTS: Adult male Sprague-Dawley rats were subjected to sepsis by CLP. INTERVENTIONS AND MEASUREMENTS: Blood samples were collected at 2, 5, 10, or 20 hrs after CLP, and plasma concentrations of PGI2, in the form of its stable product 6-keto-PGF1alpha, were measured by radioimmunoassay. In additional studies, a PGI2 synthase inhibitor, tranylcypromine, was administered subcutaneously at the time of CLP and again at 3 hrs after CLP. At 5 hrs after the onset of sepsis, the maximal rates of the left ventricular pressure rise (+dP/dtmax) and fall (-dP/dtmax) were determined by an in vivo heart performance analyzer. Microvascular blood flow in the liver, small intestine, and spleen was assessed by laser Doppler flowmetry. MAIN RESULTS: Plasma concentrations of 6-keto-PGF1alpha increased significantly at 2-20 hrs after CLP. At 5 hrs after the onset of sepsis, +/-dP/dt(max) and microvascular blood flow in the tested tissues increased significantly. Inhibition of PGI2 synthase activity did not prevent the occurrence of hypercardiovascular responses under such conditions. Moreover, the administration of tranylcypromine significantly reduced circulating concentrations of 6-keto-PGF1alpha at 5 hrs after CLP. CONCLUSIONS: Because inhibition of PGI2 production did not prevent the occurrence of the hyperdynamic and hypercardiovascular response during the early stage of sepsis, mediators other than PGI2 appear to play a major role in producing the hyperdynamic response under such conditions.     

The importance of systemic cytokines in the pathogenesis of polymicrobial sepsis and dehydroepiandrosterone treatment in a rodent model

The pathogenesis of sepsis is still undetermined to a large extent. It is an established fact that female gender is associated with a lower mortality and that sex steroid hormones influence the immunologic response. Dehydroepiandrosterone (DHEA) seems to have a protective immunologic effect in sepsis. It is still unknown in which way DHEA influences the pathogenesis of sepsis. Therefore, the effect of DHEA application on cytokine concentrations in tumor necrosis factor (TNF) receptor (TNF-RI(-/-)) and interleukin-6 (IL-6(-/-)) knockout mice was determined. In a model of polymicrobial sepsis induced by coecal ligation and puncture (CLP), the effect of DHEA on survival and cytokine concentrations was examined. For clarification of the role of TNF-RI, CLP was performed in TNF-RI knockout mice (TNF-RI(-/-)). In addition, IL-6 knockout mice (IL-6(-/-)) were used to clarify the role of IL-6. Furthermore, experiments were performed in mice that were not genetically modified (wild type, WT). The protective effect of DHEA could be confirmed in this CLP model. DHEA application was associated with a reduction in mortality in WT animals. Moreover, DHEA-treated animals demonstrated a reduction in systemic inflammatory effects, as determined by proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and the antiinflammatory cytokine IL-10. In this work, it was shown that the TNF-RI is essential for survival after CLP. DHEA application was associated with a reduction of mortality of 100% in TNF-RI(-/-) mice after CLP to 50%. This result engages, that the effect of DHEA is TNF-RI independent. However, the application of DHEA had no influence on the mortality in IL-6-/- mice. It can be concluded that the protective effect of DHEA in polymicrobial sepsis is mediated IL-6 dependently. DHEA reduces the systemic inflammation, measurable via the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and the antiinflammatory cytokine IL-10. IL-6 might be involved in the DHEA-mediated reduction of postseptic complications. In contrast, DHEA seems to be TNF-RI independent. Consequently, DHEA might be useful as an adjunct therapy for the immune modulation in sepsis.     

Harmful and protective roles of neutrophils in sepsis

The current studies demonstrate protective and harmful effects of neutrophils (PMN) during experimental sepsis after cecal ligation and puncture (CLP). It is known that CLP induces signaling defects in blood PMN. When PMN were depleted 12 h after CLP, there were dramatic reductions in levels of bacteremia, evidence for reduced liver and renal dysfunction, sharp reductions in serum levels of cytokines (IL-1beta, IL-6, IL-10, TNF-alpha, and IL-2), and improved survival. In contrast, PMN depletion before CLP resulted in substantial increases in bacteremia and no evidence for attenuation of liver and renal failure dysfunction. These data suggest that at the onset of sepsis, PMN are important in regulating the levels of bacteremia, whereas after the onset of sepsis, as they lose innate immune functions, their presence is associated with higher levels of bacteremia and intensified organ dysfunction.     

Caffeic acid phenethyl ester reduces mortality and sepsis-induced lung injury in rats

OBJECTIVE: Sepsis and ensuing multiorgan failure continue to be the major causes of mortality in intensive care units. Nuclear factor (NF)-kappaB activation is supposed to be one of the targets in the treatment of sepsis. We studied the effectiveness of caffeic phenethyl ester (CAPE), a known NF-kappaB inhibitor, in cecal ligation and puncture (CLP)-induced sepsis and lung injury. DESIGN: Randomized, controlled animal study. SETTING: Research laboratory of an academic institution. SUBJECTS: Female Sprague-Dawley rats. INTERVENTIONS: CLP was performed in all rats except the rats in control and sham+CAPE groups. CAPE was administered to rats at the time of operation in sham+CAPE and CAPE+sepsis 0 groups. CAPE was administered to rats in the CAPE+sepsis12 group 12 hrs after CLP. Eight rats from each group were killed 24 hrs after CLP. Blood was taken for assessment of interleukin-1, interleukin-6, interleukin-10, and tumor necrosis factor-alpha; the right lung was removed for histopathologic examination and the left lung for biochemical examination. Apoptosis, inducible nitric oxide synthase, heat shock protein 70, malondialdehyde, catalase, superoxide dismutase, and glutathione peroxidase were studied. The rest of the rats were observed for mortality. MEASUREMENTS AND MAIN RESULTS: Mortality was significantly decreased in groups that received CAPE compared with the sepsis group. All cytokine levels were similar to control levels only in the CAPE+sepsis12 group. Apoptosis, inducible nitric oxide synthase, and heat shock protein 70 evaluation were significantly changed between all groups in the following order: control < sham+CAPE< CAPE+sepsis12 < CAPE+sepsis 0 < sepsis. Malondialdehyde and catalase were increased in the sepsis group. CONCLUSIONS: CAPE reduced mortality in sepsis and improved histopathologic variables best when it was administered after the onset of sepsis.