CLP-induced Septic Shock Research Articles

Ponciri Fructus Immaturus ethanol extract attenuates septic shock through inhibition of the STAT1 signaling pathway.

Sepsis is a systemic inflammatory disease to infections and results in tissue damage and multiple organ failure. Ponciri Fructus Immaturus (PFI) is widely used in traditional medicine for allergic inflammation and gastrointestinal disorders. However, the effect of PFI on sepsis is still unknown. This study investigated the anti-inflammatory and antiseptic effects of PFI ethanol extract (PFIE) in LPS-stimulated J774 macrophages and mice with CLP- or LPS-induced sepsis, respectively. PFIE attenuates the LPS-induced production of the proinflammatory mediator NO by inhibiting the expression of iNOS in J774 cells. Real-time RT-PCR data and ELISA showed that the mRNA and protein levels of TNF-α, IL-1β, and IL-6 increased in LPS-stimulated J774 cells. However, this induction was significantly suppressed in PFIE pre-treated J774 cells. We also found that PFIE administration increased the survival rate of mice with LPS- and CLP-induced sepsis. Decreased serum levels of AST, ALT, and CK were observed after administration of PFIE, which was associated with reduced production of proinflammatory factors, such as NO, TNF-α, IL-1β, and IL-6. Moreover, PFIE suppressed the phosphorylation and nuclear translocation of STAT1 in LPS-stimulated J774 cells, suggesting that PFIE can inhibit LPS- and CLP-induced septic shock by suppressing the STAT1 activation. These findings provide the potential therapeutic relevance of PFIE in treating acute inflammatory disease.

The role of CXCL10 in the pathogenesis of experimental septic shock.

IntroductionThe chemokine CXCL10 is produced during infection and inflammation to activate the chemokine receptor CXCR3, an important regulator of lymphocyte trafficking and activation. The goal of this study was to assess the contributions of CXCL10 to the pathogenesis of experimental septic shock in mice.MethodsSeptic shock was induced by cecal ligation and puncture (CLP) in mice resuscitated with lactated Ringer’s solution and, in some cases, the broad spectrum antibiotic Primaxin. Studies were performed in CXCL10 knockout mice and mice treated with anti-CXCL10 immunoglobulin G (IgG). Endpoints included leukocyte trafficking and activation, core body temperature, plasma cytokine concentrations, bacterial clearance and survival.ResultsCXCL10 was present at high concentrations in plasma and peritoneal cavity during CLP-induced septic shock. Survival was significantly improved in CXCL10 knockout (CXCL10KO) mice and mice treated with anti-CXCL10 IgG compared to controls. CXCL10KO mice and mice treated with anti-CXCL10 IgG showed attenuated hypothermia, lower concentrations of interleukin-6 (IL-6) and macrophage inhibitory protein-2 (MIP-2) in plasma and lessened natural killer (NK) cell activation compared to control mice. Compared to control mice, bacterial burden in blood and lungs was lower in CXCL10-deficient mice but not in mice treated with anti-CXCL10 IgG. Treatment of mice with anti-CXCL10 IgG plus fluids and Primaxin at 2 or 6 hours after CLP significantly improved survival compared to mice treated with non-specific IgG under the same conditions.ConclusionsCXCL10 plays a role in the pathogenesis of CLP-induced septic shock and could serve as a therapeutic target during the acute phase of septic shock.

STAT1-Deficient Mice Are Resistant to Cecal Ligation and Puncture–Induced Septic Shock

STAT1 (signal transducer and activator of transcription 1) is a member of the JAK-STAT signaling family and plays a key role in facilitating gene transcription in response to activation of the types I and II interferon (IFN) receptors. TYK2 is essential for type I, but not type II, IFN-induced STAT1 activation. Previous studies show that STAT1-deficient mice are resistant to endotoxin-induced shock. The goal of the present study was to assess the response of STAT1- and TYK2-deficient mice to septic shock caused by cecal ligation and puncture (CLP). End points included survival, core temperature, organ injury, systemic cytokine production, and bacterial clearance. Results showed that survival rates were significantly higher in STAT1 knockout (STAT1KO) mice compared with wild-type controls (80% vs. 10%). The improved survival of STAT1KO mice was associated with less hypothermia, metabolic acidosis, hypoglycemia, and hepatocellular injury. Plasma interleukin 6, MIP-2, CXCL10, and IFN-α concentrations were significantly lower in STAT1KO mice than in wild-type mice. In the absence of antibiotic treatment, blood and lung bacterial counts were significantly lower in STAT1KO mice than in controls. However, treatment with antibiotics ablated that difference. A survival advantage was not observed in TYK2-deficient mice compared with control. However, CLP-induced hypothermia and systemic interleukin 6 and CXCL10 production were significantly attenuated in TYK2-deficient mice. These results indicate that STAT1 activation is an important factor in the pathogenesis of CLP-induced septic shock and is associated with the development of systemic inflammation and organ injury. TYK2 activation also appears to contribute to CLP-induced inflammation, but to a lesser extent than STAT1.

Therapeutic efficacy of CXCR3 blockade in an experimental model of severe sepsis

IntroductionIn our previous studies we demonstrated that CXC chemokine receptor 3 (CXCR3) participates in the regulation of lymphocyte trafficking during cecal ligation and puncture (CLP)-induced sepsis. In this study, we evaluated the effects of treatment with anti-CXCR3 immunoglobulin (IgG) and antibiotics on outcome during septic shock caused by CLP.MethodsC57BL/6J mice were treated with neutralizing IgG against CXCR3 plus Primaxin either 24 hours prior to, 2 hours after or 6 hours after CLP. Control mice received nonspecific IgG plus Primaxin in the same regimen. Survival, core body temperature, bacterial clearance and systemic cytokine production were evaluated.ResultsOur results show that treatment with anti-CXCR3 IgG plus Primaxin significantly improved survival when administered 24 hours prior to CLP (50% vs. 10%), 2 hours after CLP (55% vs. 10%) or 6 hours after CLP (55% vs. 25%) compared with mice receiving nonspecific IgG plus Primaxin. Treatment with anti-CXCR3 plus Primaxin 24 hours prior to CLP attenuated hypothermia and IL-6 and macrophage inflammatory protein 2 (MIP-2) production but did not alter bacterial clearance. Treatment with anti-CXCR3 IgG and Primaxin 2 hours after CLP did not improve bacterial clearance and systemic cytokine production compared with mice treated with IgG and Primaxin, whereas 6 hours after CLP the bacterial clearance and IL-6 and MIP-2 concentrations, both in plasma and peritoneal lavage fluid, were significantly improved in mice receiving anti-CXCR3 IgG and Primaxin compared with mice that only received nonspecific IgG and Primaxin.ConclusionThe results from this study indicate that neutralization of CXCR3 prior to, 2 hours after or 6 hours after the initiation of CLP-induced septic shock improves survival and attenuates CLP-induced inflammation and physiologic dysfunction.

The New Vitamin E Derivative, ETS-GS, Protects Against Cecal Ligation and Puncture-Induced Systemic Inflammation in Rats

Sepsis-related systemic inflammation frequently occurs in the critical care setting. Systemic inflammation is implicated in the progression of organ injury, which is associated with a high mortality rate. Recently, vitamin E and glutamic acid have been reported to attenuate inflammation. We therefore investigated whether the vitamin E derivative, ETS-GS, could inhibit the secretion of cytokines and high-mobility group box1 (HMGB1), and thereby reduce organ damage in a rat model of cecal ligation and puncture (CLP)-induced sepsis. Male Wistar rats weighing 250-300g were used. Rats received water or ETS-GS (10mg/kg) by oral administration for 3weeks, and then sepsis was induced by CLP under sevoflurane anesthesia. Serum levels of interleukin-6, tumor necrosis factor-α, and HMGB1 were determined at 3, 6, and 12h after CLP; lung histology was assessed at 12h. Histology results showed markedly reduced interstitial edema and leukocytic infiltration in lung tissue harvested at 12h in ETS-GS-treated mice compared with untreated controls. ETS-GS treatment also attenuated the CLP-induced increase in serum levels of cytokines and HMGB1. To investigate the mechanisms by which ETS-GS exerts its anti-inflammatory effects, the phosphorylation of Akt, IκBα, and mitogen-activated protein kinase (MAPK) was assessed in mouse macrophage RAW264.7 cells stimulated with LPS, with and without ETS-GS. In these in vitro studies, ETS-GS-induced phosphoinositide 3-kinase (PI3K)-Akt phosphorylation and inhibited IκBα and MAPK phosphorylation. ETS-GS blocked the CLP-induced septic shock response and protected against acute lung injury. This mechanism appeared to be mediated by the induction of PI3K-Akt and the inhibition of IκBα and MAPK phosphorylation. Given these results, ETS-GS shows promise as a potential therapeutic agent for sepsis.

The role of interferon-γ in the pathogenesis of acute intra-abdominal sepsis

Several studies indicate that IFN-γ facilitates systemic inflammation during endotoxin-induced shock. However, the pathobiology of IFN-γ in clinically relevant models of septic shock, such as CLP, is not well understood. In this study, the role of IFN-γ in the pathogenesis of CLP-induced septic shock was evaluated by examining IFN-γ production at the tissue and cellular levels. The impact of IFN-γ neutralization on systemic inflammation, bacterial clearance, and survival was also determined. Following CLP, concentrations of IFN-γ in plasma and peritoneal lavage fluid were low in comparison with concentrations of IL-6 and MIP-2, as was IFN-γ mRNA expression in liver and spleen. The overall percentage of IFN-γ+ splenocytes was <5% after CLP and not statistically different from control mice. Intracellular IFN-γ was present in a large proportion of peritoneal exudate cells after CLP, primarily in infiltrating myeloid cells and NK cells. i.p. myeloid cell activation was decreased in IFN-γKO mice, and plasma concentrations of IL-6 and MIP-2 were significantly lower in IFN-γKO mice and in mice treated with anti-IFN-γ compared with controls, but bacterial clearance was not affected. IFN-γKO mice were resistant to CLP-induced mortality when treated with systemic antibiotics. However, neutralization of IFN-γ with blocking antibodies did not improve survival significantly. These studies show that IFN-γ facilitates the proinflammatory response during CLP-induced septic shock. However, neutralization of IFN-γ did not improve survival uniformly.

Effect of Tempol, a Membrane-Permeable Radical Scavenger, on Mesenteric Blood Flow and Organ Injury in a Murine Cecal Ligation and Puncture Model of Septic Shock

Background: Interventions that reduce the generation or the effects of reactive oxygen species exert beneficial effects in a variety of models of septic shock. We investigatedthe effect of tempol, a low-molecular-weight membrane-permeable radical scavenger, on mesenteric blood flow and organ injury in a murine cecal ligation and puncture (CLP) model of septic shock. Materials and Methods: Forty-four Swiss albino mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and subjected to CLP (except for the sham-operated animals). The animals were divided randomly into 4 groups: the 1st group was sham operated (sham-operated group, n = 10); the 2nd group underwent CLP and was injected with saline (CLP + saline group, n = 12); the 3rd group was sham operated and treated with tempol (10 mg/kg, i.p., sham-treated + tempol group, n = 10); the 4th group underwent CLP and was treated with tempol (10 mg/kg, i.p., CLP + tempol group, n = 12). Mesenteric arterial blood flow (MABF) was measured by Doppler ultrasound. Poly(adenosine 5′-diphosphate-ribose) polymerase (PARP) activity was examined in the liver, lung, and kidneys. Results: In the CLP + saline group, the MABF was significantly lower than in the sham-operated group (p < 0.001). After tempol administration, MABF values significantly increased (p < 0.05). We observed significantly stronger PARP-positive staining in the lungs and kidney glomeruli in the CLP + saline group than in those of the sham-operated group (p_lung = 0.0148, p_glomeruli = 0.0025). A marked reduction in PARP activity was found in the lung and kidney glomeruli of the CLP + tempol group (p_lung = 0.0026, p_glomeruli = 0.0085). There was no significant effect of CLP on PARP activity in the liver and kidney tubuli (p_liver > 0.05, p_tubuli > 0.05). Conclusion: Tempol improved MABF in a CLP-induced septic shock model. Although tempol could not prevent the activation of PARP in the liver and kidney tubuli, it did attenuate PARP activation in the lung and kidney glomeruli.

NK but Not CD1-Restricted NKT Cells Facilitate Systemic Inflammation during Polymicrobial Intra-Abdominal Sepsis

Evidence suggests that NK and NKT cells contribute to inflammation and mortality during septic shock caused by cecal ligation and puncture (CLP). However, the specific contributions of these cell types to the pathogenesis of CLP-induced septic shock have not been fully defined. The goal of the present study was to determine the mechanisms by which NK and NKT cells mediate the host response to CLP. Control, NK cell-deficient, and NKT cell-deficient mice underwent CLP. Survival, cytokine production, and bacterial clearance were measured. NK cell trafficking and interaction with myeloid cells was also studied. Results show that mice treated with anti-asialoGM1 (NK cell deficient) or anti-NK1.1 (NK/NKT cell deficient) show less systemic inflammation and have improved survival compared with IgG-treated controls. CD1 knockout mice (NKT cell deficient) did not demonstrate decreased cytokine production or improved survival compared with wild type mice. Trafficking studies show migration of NK cells from blood and spleen into the inflamed peritoneal cavity where they appear to facilitate the activation of peritoneal macrophages (F4-80(+)GR-1(-)) and F4-80(+)Gr-1(+) myeloid cells. These findings indicate that NK but not CD1-restricted NKT cells contribute to acute CLP-induced inflammation. NK cells appear to mediate their proinflammatory functions during septic shock, in part, by migration into the peritoneal cavity and amplification of the proinflammatory activities of specific myeloid cell populations. These findings provide new insights into the mechanisms used by NK cells to facilitate acute inflammation during septic shock.

The Survival Time Post-Cecal Ligation and Puncture in Sinoaortic Denervated Rats

Arterial baroreflex (ABR) function is an important determinant factor in prognosis of many cardiovascular diseases. The present work was designed to study the relationship between ABR function and the survival time of septic shock in a cecal ligation and puncture (CLP) rat model. The dysfunction of ABR was introduced by sinoaortic denervation (SAD). It was found that the survival time after CLP was significantly reduced in SAD rats compared with sham-operated rats (12.7 +/- 2.92 hours versus 15.0 +/- 4.01 hours; P < 0.05). Furthermore, significant differences were also seen when the results were expressed by Kaplan-Meier survival curves. Compared with the baseline values, both noradrenaline and adrenaline significantly increased in both SAD and Sham groups after CLP, but we found the baseline of noradrenaline was significantly elevated in SAD rats. In addition, the TNF-alpha, noradrenaline, and adrenaline levels of the SAD group were significantly higher than those of the Sham group at 5 hours post-CLP. In conclusion, the present work demonstrates that ABR function was related to the survival time in CLP-induced lethal shock model. The loss of inhibition in the sympathetic activity and in the release of some inflammatory cytokines during CLP-induced septic shock related to baroreflex and/or chemoreflex dysfunction may be the mechanisms involved in the poorer prognosis in septic shock.