Cecal Ligation And Puncture Challenge Research Articles

Deletion of MAPL ameliorates septic cardiomyopathy by mitigating mitochondrial dysfunction

AimMitochondrial dysfunction is a critical factor in the pathogenesis of septic cardiomyopathy (SCM). Mitochondrial anchored protein ligase (MAPL), a small ubiquitin-like modifier (SUMO) E3 ligase, plays a significant role in mitochondrial function. However, the role of MAPL in SCM remains unclear.MethodsTo investigate the role of MAPL in SCM, cardiomyocyte-specific MAPL knockout mice were generated. A cecal ligation and puncture (CLP) procedure was employed to induce a sepsis-like condition.ResultsThe expression of MAPL in heart tissues and H9C2 cardiomyocytes was elevated following CLP challenge or lipopolysaccharide (LPS) stimulation. MAPL deficiency ameliorated CLP-induced cardiac injury, dysfunction, and inflammation, and also improved the survival rate of mice following CLP operation. Additionally, MAPL deficiency or knockdown inhibited LPS-induced cardiomyocyte apoptosis, improved mitochondrial structural abnormalities, and increased ATP production. Furthermore, MAPL knockdown mitigated LPS-induced reductions in mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) production. Mechanistically, the expression of dynamin-related protein 1 (drp1) in the mitochondria of heart tissues or H9C2 cardiomyocytes was elevated under septic conditions. Accordingly, the SUMOylation of drp1 in heart tissues or H9C2 cardiomyocytes was increased under sepsis conditions, which was reduced by MAPL knockout or knockdown.ConclusionOur results reveal that MAPL promotes cardiac injury/dysfunction and inflammation in SCM. Deficiency or knockdown of MAPL alleviates SCM by reducing drp1 SUMOylation as well as drp1-mediated mitochondrial dysfunction. These findings suggest that targeting MAPL may represent a therapeutic strategy for patients with SCM.

Tim-3 Blockade Decreases the Apoptosis of CD8+ T Cells and Reduces the Severity of Sepsis in Mice

IntroductionThe T cell immunoglobulin and mucin domain 3 (Tim-3) mediated immunosuppressive pathway has been shown to play an essential role in the development of sepsis. However, the influence of Tim-3 blockade during sepsis and the possible effects on T cells’ function remains largely unknown. Our study investigates the role of Tim-3 in cecal ligation and puncture (CLP)-induced sepsis in mice. MethodsSepsis was induced in C57BL/6 male mice via CLP. The expression of Tim-3 in CD8+ T cells after CLP challenge was measured. A dose of 50 μg anti-Tim-3 antibodies was injected intraperitoneally 30 min after surgery. Postoperative survival, bacterial clearance in the blood and peritoneal lavage fluid, cytokine secretion in the blood, and lung and liver histology were evaluated. In addition, the apoptosis of immune cells in the spleen and thymus was examined, respectively. ResultsTim-3 expression was elevated in the splenic CD8+ T cells of septic mice. At the early stage of CLP-induced sepsis, blocking Tim-3 with anti-Tim-3 antibodies reduced the severity of sepsis. The anti-Tim-3 antibodies alleviated the morphology of lung and liver injuries in septic mice. The anti-Tim-3 antibodies also reduced the severity of the inflammatory responses and lymphocyte apoptosis in septic mice. ConclusionsAnti-Tim-3 antibodies might be a potential therapeutic strategy for sepsis.

Palmitoylation Regulates Extracellular Vesicle Production and Cargo Contents

DHHC21 belongs to a family of palmitoyl acyltransferases (PATs) and catalyzes the addition of palmitic acid to cysteine residue(s) of a protein. This reversible posttranslational modification dynamically controls protein localization and function, thus regulating various cellular processes. Extracellular vesicles (EVs) are membrane-encapsulated particles that serve as a critical component in intercellular communication. Recent studies have shown that palmitoylated proteins are highly enriched in EVs, however, there is limited information regarding the role of PATs in EV production and function. In this study, we investigated whether and how DHHC21 regulates EV formation and cargo content during septic inflammation caused by cecal ligation and puncture (CLP). EVs were isolated from the plasma of wild-type (WT) and DHHC21 functional deficient (Zdhhc21dep/dep) mice via ultracentrifugation followed by size-exclusion chromatography 24 hours after sham or CLP challenge. Nanoparticle tracking analysis showed no significant difference in the plasma level of EVs between WT and Zdhhc21 dep/dep mice under basal conditions. However, DHHC21 loss-of-function greatly reduced sepsis-induced increase in EV production. Next, palmitoylated proteins in EVs were separated using resin-assisted capture and quantitatively analyzed using liquid chromatography with tandem mass spectrometry. A total of 548 palmitoylated proteins were identified. A clustered heatmap revealed that the EV palmitoyl-protein profiles were remarkably different between CLP-challenged and sham-treated WT mice. Gene ontology (GO) PANTHER pathway analysis on sepsis-altered EV palmitoylated proteins showed that those proteins were highly associated with integrin signaling, inflammation, blood coagulation, and plasminogen activation. More importantly, DHHC21 was involved in regulating EV palmitoyl-protein profile during sepsis, as evidenced by a distinctive protein expression pattern of EVs in Zdhhc21dep/dep mice after CLP. These findings suggest that DHHC21 contributes to the regulation of EV production and cargo protein content during sepsis.

Immunomodulatory dose of clindamycin in combination with ceftriaxone improves survival and prevents organ damage in murine polymicrobial sepsis.

Sepsis is a life-threatening organ dysfunction resulting from inflammatory responses instigated by toxins secreted by bacteria. Immunomodulatory effect of clindamycin is earlier reported in a murine lipopolysaccharide (LPS)-induced sepsis model. There are no studies demonstrating the immunomodulatory effect of clindamycin in combination with ceftriaxone in a clinically relevant murine polymicrobial sepsis model induced by cecal ligation and puncture (CLP). Ceftriaxone is combined to control the bacterial growth. Following 3h of CLP challenge, Swiss albino mice were administered vehicle, ceftriaxone alone (100mg/kg, subcutaneously), and in combination with clindamycin at immunomodulatory dose (200mg/kg, intraperitoneally). Survival was assessed for 5days, and bacterial count and biochemical and physiological parameters were measured after 18h of CLP challenge. Ceftriaxone alone caused significant reduction in bacterial count in blood, peritoneal fluid, lung, liver, and kidney homogenate which was not further substantially reduced by ceftriaxone and clindamycin combination. Day 5 survival was greatly improved by combination compared with ceftriaxone alone which was also evident through marked drop in blood glucose, total white blood cell (WBC) count, and body temperature. The combination group significantly mitigated the cytokine (tumor necrosis factor (TNF)-α and interleukin (IL)-6) and myeloperoxidase (MPO) levels in plasma, lung, liver, and kidney of CLP-challenged mice, which further helped in significantly suppressing the elevated levels of liver and kidney function parameters. Clindamycin at immunomodulatory dose in combination with ceftriaxone attenuated organ damage and improved survival of septic mice by suppressing infection, inflammatory responses, and oxidative stress.

Aqueous extract of <i>Aconitum carmichaelii</i> Debeaux attenuates sepsis-induced acute lung injury via regulation of TLR4/NF-ΚB pathway

Purpose: To investigate the therapeutic effect of aqueous extract of Aconitum carmichaelii Debeaux (AEACD) on sepsis-induced acute lung injury (ALI), as well as explore the underlying mechanism of action.
 Methods: C57BL/6 mice were treated with AEACD by gavage (4.0 g/kg/day) for 5 days before cecal ligation and puncture (CLP) challenge. After 24 h, the pathological morphology of lung tissue and the biochemical parameters in bronchoalveolar lavage fluid (BALF) were determined by H&E staining and enzyme-linked immunosorbent assay (ELISA). Furthermore, the total protein content and lactate dehydrogenase (LDH) level of BALF, as well as the oxidative biomarkers (malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD)) were evaluated in the lung homogenates by ELISA assay. The levels of pro-inflammatory cytokines, TNFα, IL-1β, and IL-6, in lung tissue were measured by qRT-PCR or ELISA. Finally, key proteins in Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway in lung tissue were evaluated by western blot.
 Results: CLP challenge induced abnormal changes in the histological structures of lung tissue, lung wet-to-dry weight (W/D) ratio, protein content and LDH levels of BALF, which were remarkably reversed by AEACD. In addition, AEACD decreased MDA levels, and increased GSH levels and SOD activity in the lung tissue of CLP–treated mice (p < 0.05). Furthermore, AEACD attenuated the CLP challengeinduced upregulation of TNFα, IL-1β, and IL-6. Finally, AEACD inactivated TLR4/NF-κB pathway by upregulating IκBα and downregulating TLR4 and phosphorylated-p65 levels.
 Conclusion: AEACD administration protects mice against sepsis-induced ALI through the regulation of oxidative stress and inflammatory responses in lung tissues. The underlying mechanism occurs by inhibiting TLR4/NF-κB signaling pathway.
 Keywords: Aconitum carmichaelii Debeaux, Acute lung injury, Sepsis, TLR4, NF-κB

Azithromycin in Combination with Ceftriaxone Reduces Systemic Inflammation and Provides Survival Benefit in a Murine Model of Polymicrobial Sepsis.

Sepsis is a life-threatening systemic inflammatory condition triggered as a result of an excessive host immune response to infection. In the past, immunomodulators have demonstrated a protective effect in sepsis. Azithromycin (a macrolide antibiotic) has immunomodulatory activity and was therefore evaluated in combination with ceftriaxone in a clinically relevant murine model of sepsis induced by cecal ligation and puncture (CLP). First, mice underwent CLP and 3 h later were administered the vehicle or a subprotective dose of ceftriaxone (100 mg/kg of body weight subcutaneously) alone or in combination with an immunomodulatory dose of azithromycin (100 mg/kg intraperitoneally). Survival was monitored for 5 days. In order to assess the immunomodulatory activity, parameters such as plasma and lung cytokine (interleukin-6 [IL-6], IL-1β, tumor necrosis factor alpha) concentrations, the plasma glutathione (GSH) concentration, plasma and lung myeloperoxidase (MPO) concentrations, body temperature, blood glucose concentration, and total white blood cell count, along with the bacterial load in blood, peritoneal lavage fluid, and lung homogenate, were measured 18 h after CLP challenge. Azithromycin in the presence of ceftriaxone significantly improved the survival of CLP-challenged mice. Further, the combination attenuated the elevated levels of inflammatory cytokines and MPO in plasma and lung tissue and increased the body temperature and blood glucose and GSH concentrations, which were otherwise markedly decreased in CLP-challenged mice. Ceftriaxone produced a significant reduction in the bacterial load, while coadministration of azithromycin did not produce a further reduction. Therefore, the survival benefit offered by azithromycin was due to immunomodulation and not its antibacterial action. The findings of this study indicate that azithromycin, in conjunction with appropriate antibacterial agents, could provide clinical benefits in sepsis.

Inhibition of the mTOR Pathway Exerts Cardioprotective Effects Partly through Autophagy in CLP Rats.

Background Sepsis-induced myocardial dysfunction is a severe clinical problem. Recent studies have indicated that autophagy and myocardial energy depletion play a major role in myocardial dysfunction during sepsis, a mechanistic target of rapamycin (mTOR) as a master sensor of energy status and autophagy mediator; however, there are little data describing its role during sepsis in the heart. Methods Cecal ligation and puncture (CLP) or sham operation (SHAM) was performed in rats. After treatment, pathological changes were determined by H&E staining, cardiac functions by echocardiography, the distribution of microtubule-associated protein light chain 3 (LC-3) type II and hypoxia-inducible factor 1α (HIF-1a) by immunohistochemical staining, and autophagic vacuoles by transmission electron microscopy. Moreover, the mTOR signaling pathway and LC3II, p62, and HIF-1a expression were measured by western blotting. Results Rapamycin alleviated the pathological damage of myocardial tissue, attenuated cardiac dysfunction (left ventricular ejection fraction (LVEF), p < 0.05; fractional shortening (FS), p < 0.05), and reduced HIF-1a expression (p < 0.05). Expectedly, rapamycin decreased the activity of the mTOR pathway in both sham-operated rats (p < 0.0001) and CLP rats (p < 0.01). Interestingly, we also found inhibition of the mTOR pathway in CLP rats compared with sham-operated rats; phosphorylation of both mTOR (p < 0.001) and pS6K1 (p < 0.01) was significantly suppressed following CLP challenge. Furthermore, autophagic processes were elevated by CLP; the ratio of LC3II/LC3I (p < 0.05) was increased while p62 expression (p < 0.001) was decreased significantly; there were also more autophagic vacuoles in CLP rats; and rapamycin could further elevate the autophagic processes compared with CLP rats (LC3II/LC3I, p < 0.05; P62, p < 0.05). Conclusion Inhibition of the mTOR pathway has cardioprotective effects on myocardial dysfunction during sepsis induced by CLP, which is partly mediated through autophagy.

Accelerated Autophagy of Cecal Ligation and Puncture-Induced Myocardial Dysfunction and Its Correlation with Mammalian Target of Rapamycin Pathway in Rats.

Background:Recent studies have indicated that autophagy is involved in sepsis-induced myocardial dysfunction. This study aimed to investigate the change of autophagy in cecal ligation and puncture (CLP)-induced myocardium dysfunction and its relationship with mammalian target of rapamycin (mTOR) pathway.Methods:Totally, 12 rats were randomly divided into CLP group or sham-operated (SHAM) group. Cardiac tissues were harvested 18 h after CLP or sham operation. Pathology was detected by hematoxylin and eosin staining, cardiac functions by echocardiography, distribution of microtubule-associated protein light chain 3 type II (LC3II) by immunohistochemical staining, and autophagic vacuoles by transmission electron microscopy. Moreover, phosphorylation of mTOR (p-mTOR), phosphorylation of S6 kinase-1 (PS6K1), and LC3II and p62 expression were measured by western blotting. Pearson's correlation coefficient was used to analyze the correlation of two parameters.Results:The results by pathology and echocardiography revealed that there was obvious myocardial injury in CLP rats (left ventricle ejection fraction: SHAM 0.76 ± 0.06 vs. CLP 0.59 ± 0.11, P < 0.01; fractional shortening: SHAM 0.51 ± 0.09 vs. CLP 0.37 ± 0.06, P < 0.05). We also found that the autophagy process was elevated by CLP, the ratio of LC3II/LC3I was increased (P < 0.05) while the expression of p62 was decreased (P < 0.05) in the CLP rats, and there were also more autophagosomes and autolysosomes in the CLP rats. Furthermore, the mTOR pathway in CLP myocardium was inhibited when compared with the sham-operated rats; p-mTOR (P < 0.01) and PS6K1 (P < 0.05) were both significantly suppressed following CLP challenge. Interestingly, we found that the mTOR pathway was closely correlated with the autophagy processes. In our study, while p-mTOR in the myocardium was significantly correlated with p62 (r = 0.66, P = 0.02), PS6K1 was significantly positively correlated with p62 (r = 0.70, P = 0.01) and negatively correlated with LC3II (r = −0.71, P = 0.01).Conclusions:The autophagy process in the myocardium was accelerated in CLP rats, which was closely correlated with the inhibition of the mTOR pathway.

PICK1 deficiency exacerbates sepsis-associated acute lung injury and impairs glutathione synthesis via reduction of xCT.

The role of oxidative stress has been well documented in the development of sepsis-induced acute lung injury (ALI). Protein interaction with C-kinase 1 (PICK1) participates in oxidative stress-related neuronal diseases. However, its function in lung infections and inflammatory diseases is not known. We therefore sought to investigate whether PICK1 is involved in sepsis-induced ALI. Cecal ligation and puncture (CLP) was performed in anesthetized wild type (WT) and PICK1 knock out (KO, PICK1-/-) mice with C57BL/6 background. At the time of CLP, mice were given fluid resuscitation. Mouse lungs were harvested at 24 and 72 h for Western blot analysis, qRT-PCR, BALF analysis, Hematoxylin and Eosin staining, TUNEL staining, maleimide staining, flow cytometry analysis, GCL, GSH, GSSG and cysteine levels measurement. A marked elevation of PICK1 mRNA and protein level were demonstrated in lung tissue, which was accompanied by increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and consumption of glutathione (GSH). N-acetylcysteine (NAC), buthionine sulfoximine (BSO) and GSH-monoethyl ester (GSH-MEE) were injected into mice via caudal vein to regulate glutathione (GSH) level in lung. Alterations of lung GSH content induced PICK1 level change after CLP challenge. In PICK1-/- underwent with CLP, lung injury and survival were significantly aggravated compared with wild-type mice underwent with CLP. Concomitantly, CLP-induced lung cell apoptosis was exacerbated in PICK1-/- mice. The level of xCT, other than PKCα, in lung tissue was significantly lowered in PICK1-/- but not in wild type that underwent CLP surgery. Meanwhile, Nrf2 activation, which dominating xCT expression, was inhibited in PICK1-/- but not in wild type mice that underwent CLP surgery, as well. Moreover, higher level of PICK1 was detected in PBMCs of septic patients than healthy controls. Taken together, PICK1 plays a pivotal role in sepsis-induced ALI by regulating GSH synthesis via affecting the substrate-specific subunit of lung cystine/glutamate transporter, xCT.

Modulation of mitogen‑activated protein kinase attenuates sepsis‑induced acute lung injury in acute respiratory distress syndrome rats.

Sepsis is the most important predisposing cause inducing acute respiratory distress syndrome (ARDS); however, the mechanism of sepsis leading to the development of ARDS remains to be elucidated. Suppression of the mitogen‑activated protein kinase (MAPK) signal by blocking the phosphorylation of Jun N‑terminal kinase (JNK) and p38 in lung tissues could alleviate acute lung injury induced by sepsis. MAPK signaling may have a crucial role in development of the sepsis‑induced acute lung injury. The specific inhibitors of JNK and p38 MAPK, SP600125 and SB203580, were administrated by intragastric injection 4h before induction of ARDS after cecal ligation and puncture (CLP). Rats were sacrificed at 1, 6 or 24h after CLP challenge. The histological evaluation, lung water content, and biochemical analysis were performed. The results revealed that the JNK and p38 MAPK inhibitor improved lung permeability, attenuated system inflammation, further alleviated the lung injury induced by sepsis. In conclusion, JNK and p38 MAPK signaling are essential for the development of ARDS following sepsis. Further studies are needed to illuminate the detailed mechanisms of JNK and p38 MAPK signaling in sepsis‑induced ARDS.

The lack of NK and mCD8+ T cells in IL-15KO mice confers resistance to septic shock

Abstract Septic shock remains one of the leading causes of death in the USA. However, the immunological mechanisms of septic shock are still not well understood. Interleukin (IL)-15 is an essential mediator for natural killer (NK) and memory (m) CD8+T cell survival, as indicated by the deficits of these cells in IL-15 knockout (KO) mice. As NK and CD8+T cells are implicated in the pathogenesis of septic shock, we hypothesized that IL-15KO mice will be resistant to septic shock due to their lack of NK and mCD8+T cells. IL-15KO mice showed a significant survival advantage over wild type (WT) control mice during septic shock caused by cecal ligation and puncture (CLP) or LPS challenge. IL-15KO mice displayed less sepsis-induced hypothermia and attenuated plasma IL-6 production in our sepsis models. Treatment with low-dose IL-15 superagonist (SA) regenerated NK and mCD8+T cells in IL-15KO mice and reestablished mortality in these mice during CLP- and LPS-induced septic shock. However, if NK and CD8+T cell regeneration was prevented by anti-asialoGM1 and anti-CD8α prior to IL-15 SA treatment, IL-15 SA failed to reestablish sepsis-induced mortality in IL-15 KO mice. When given to WT mice 4 days prior to CLP or LPS challenge, M96, an IL-15 neutralizing IgG, caused 80.8% depletion of splenic NK cells and conferred protection against septic shock. However, when given shortly prior to CLP or LPS challenge, M96 did not deplete splenic NK cells and failed to confer protection against septic shock. In conclusion, IL-15 contributes to the lethality of septic mice by maintaining NK and mCD8+ T cell populations, but does not play a direct role in septic shock. Thus, NK and mCD8+ T cells may represent effective therapeutic targets for septic shock.

PHARMACOLOGICAL SIRT1 ACTIVATION IMPROVES MORTALITY AND MARKEDLY ALTERS TRANSCRIPTIONAL PROFILES THAT ACCOMPANY EXPERIMENTAL SEPSIS.

The sirtuin family consists of seven NAD+-dependent enzymes affecting a broad array of regulatory protein networks by primarily catalyzing the deacetylation of key lysine residues in regulatory proteins. The enzymatic activity of SIRT1 can be enhanced by small molecule activators known as SIRT1 activator compounds (STACs). We tested the therapeutic potential of the STAC SRT3025 in two preclinical models of severe infection, the murine cecal ligation and puncture (CLP) model to induce peritonitis and intratracheal installation of Streptococcus pneumoniae to induce severe bacterial pneumonia. SRT3025 provided significant survival benefits over vehicle control in both the peritonitis and pneumococcal pneumonia models when administered with appropriate antimicrobial agents. The survival benefit of SRT3025 in the CLP model was absent in SIRT1 knockout showing the SIRT1 dependency of SRT3025's effects. SRT3025 administration promoted bacterial clearance and significantly reduced inflammatory cytokines from the lungs of animals challenged with S. pneumoniae. SRT3025 treatment was also accompanied by striking changes in the transcription profiles in multiple inflammatory and metabolic pathways in liver, spleen, small bowel, and lung tissue. Remarkably, these organ-specific changes in the transcriptome analyses were similar following CLP or pneumococcal challenge despite different sets of pathogens at disparate sites of infection. Pharmacologic activation of SIRT1 modulates the innate host response and could represent a novel treatment strategy for severe infection.

Vagus nerve electrical stimulation inhibits serum levels of S100A8 protein in septic shock rats.

The vagus nerve and the released acetylcholine exert anti-inflammatory effects and inhibit septic shock. However, their detailed mechanisms remain to be elucidated. The present study aimed to investigate the effects of vagus nerve electrical stimulation on serum S100A8levels in septic shock rats. A total of36male Sprague-Dawley rats were randomly divided into six equal groups: i)Sham group, receiving sham operation; ii)CLP group, subjected to cecal ligation and puncture (CLP) to establish a model of polymicrobial sepsis; iii)VGX group, subjected to CLP and bilateral cervical vagotomy; iv)STM group, subjected to CLP, bilateral cervical vagotomy and electrical stimulation on the left vagus nerve trunk; v)α‑bungarotoxin (BGT) group was administered α‑BGT prior to electrical stimulation; vi)Anti‑receptor for advanced glycation end products (RAGE) group, administered intraperitoneal injection of anti‑RAGE antibody prior to electrical stimulation. The right carotid artery was cannulated to monitor mean artery pressure (MAP). The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured to assess the liver function. Serum S100A8and advanced glycation end product (AGE) levels were measured using enzyme‑linked immunosorbent assays. The expression of hepatic RAGE was determined by western blotting. The present study revealed that Sprague‑Dawley rats exhibited progressive hypotension and significantly increased serum AST and ALT levels following CLP challenge compared with the sham group. The levels of S100A8and AGEs, and the protein expression of hepatic RAGE were significantly increased following CLP compared with the sham group. Vagus nerve electrical stimulation significantly prevented the development of CLP‑induced hypotension, alleviated the hepatic damage, reduced serum S100A8 and AGEs production, and reduced the expression of hepatic RAGE. The inhibitory effect of vagus nerve electrical stimulation was reversed by pre-treatment with α-BGT and enhanced by pre-treatment with anti-RAGE antibody. In conclusion, vagus nerve electrical stimulation may produce a protective effect on septic shock in rats by decreasing the production of S100A8.

Salidroside rescued mice from experimental sepsis through anti-inflammatory and anti-apoptosis effects

BackgroundSalidroside (SDS) is the main effective component of Rhodiola rosea L with a variety of pharmacologic properties. The objective of this study was to investigate the efficacy of SDS in the treatment of experimental sepsis in mice and explore the possible underlying action mechanisms. MethodsSepsis was induced in C57BL/6 male mice via cecal ligation and puncture (CLP). The animals were divided into three groups as follows: sham, CLP, and CLP plus SDS. SDS (50 mg/kg) was injected intraperitoneally 1 h after operation. Postoperative survival of the mice, bacterial clearance in blood and peritoneal lavage fluid, cytokine secretion in blood, and histology of lung were evaluated. In addition, apoptosis of immune cells in the spleen and thymus were examined, respectively. ResultsSDS administration prolonged the survival of the septic mice, inhibited the proinflammatory responses, and enhanced bacterial clearance. It also alleviated the pathologic changes in the lung and inhibited the apoptosis of immune cells in the spleen and thymus after CLP challenge. ConclusionsSDS exerts a protective effect in CLP-induced sepsis by attenuating the proinflammatory responses, enhancing bacterial clearance, and preserving adaptive immunity. SDS may be a promising therapeutic strategy for the treatment of sepsis.

Endotoxin Tolerance Drives Neutrophil To Infectious Site

The objective of this randomized animal study and laboratory investigation was to investigate whether lipopolysaccharide tolerance redirects neutrophil migration between organs. Male BALB/c mice received subcutaneous injections of lipopolysaccharide (1 mg/kg) for 5 days, followed by cecal ligation and puncture (CLP). Cytokines and adhesion molecules were measured after tolerance and CLP challenge. Increased numbers of neutrophils were observed in the peritoneal cavity of tolerant mice, which was associated with increased levels of adhesion molecules and chemokines. In contrast, nontolerant mice accumulated higher numbers of neutrophils in the lungs compared with those in the peritoneal cavity. Neutrophil function accessed by hydrogen peroxide production from neutrophils recovered from peritoneal cavity showed that tolerance increased the capacity to produce hydrogen peroxide. Mortality was reduced in tolerant animals. This study demonstrated that tolerance reduces leukocyte accumulation in the lung after CLP by redirecting neutrophils to the site of infection.

Effect of salidroside on lung injury by upregulating peroxisome proliferator-activated receptor γ expression in septic rats.

Successful drug treatment for sepsis-related acute lung injury (ALI) remains a major clinical problem. Thus, the aim of the present study was to investigate the beneficial effects of salidroside on ameliorating cecal ligation and puncture (CLP)-induced lung inflammation. Rats underwent CLP surgery to induce ALI and 800 mg/kg salidroside (i.v.) was administered 24 h after the CLP challenge. Subsequently, biochemical changes in the blood and lung tissues, as well as morphological and histological alterations in the lungs, that were associated with inflammation and injury were analysed. CLP was shown to significantly increase the serum levels of plasma tumour necrosis factor-α and interleukin-6, -1β and-10. In addition, CLP increased pulmonary oedema, thickened the alveolar septa and caused inflammation in the lung cells. These changes were ameliorated by the administration of 800 mg/kg salidroside (i.v.) 24 h after the CLP challenge. This post-treatment drug administration also significantly attenuated the lipopolysaccharide-induced activation of nuclear factor-κβ and increased the release of peroxisome proliferator-activated receptor γ in the lung tissue. Therefore, salidroside administered following the induction of ALI by CLP significantly prevented and reversed lung tissue injuries. The positive post-treatment effects of salidroside administration indicated that salidroside may be a potential candidate for the management of lung inflammation in CLP-induced endotoxemia and septic shock.

Simultaneous Targeting of IL-1 and IL-18 Is Required for Protection against Inflammatory and Septic Shock

Sepsis is one of the leading causes of death around the world. The failure of clinical trials to treat sepsis demonstrates that the molecular mechanisms are multiple and are still insufficiently understood. To clarify the long disputed hierarchical contribution of several central inflammatory mediators (IL-1β, IL-18, caspase [CASP] 7, CASP1, and CASP11) in septic shock and to explore their therapeutic potential. LPS- and tumor necrosis factor (TNF)-induced lethal shock, and cecal ligation and puncture (CLP) were performed in genetically or pharmacologically targeted mice. Body temperature and survival were monitored closely, and plasma was analyzed for several markers of cellular disintegration and inflammation. Interestingly, deficiency of both IL-1β and IL-18 additively prevented LPS-induced mortality. The detrimental role of IL-1β and IL-18 was confirmed in mice subjected to a lethal dose of TNF, or to a lethal CLP procedure. Although their upstream activator, CASP1, and its amplifier, CASP11, are considered potential therapeutic targets because of their crucial involvement in endotoxin-induced toxicity, CASP11- or CASP1/11-deficient mice were not, or hardly, protected against a lethal TNF or CLP challenge. In line with our results obtained in genetically deficient mice, only the combined neutralization of IL-1 and IL-18, using the IL-1 receptor antagonist anakinra and anti-IL-18 antibodies, conferred complete protection against endotoxin-induced lethality. Our data point toward the therapeutic potential of neutralizing IL-1 and IL-18 simultaneously in sepsis, rather than inhibiting the upstream inflammatory caspases.

PD-L1 Blockade Attenuated Sepsis-Induced Liver Injury in a Mouse Cecal Ligation and Puncture Model

Liver plays a major role in hypermetabolism and produces acute phase proteins during systemic inflammatory response syndrome and it is of vital importance in host defense and bacteria clearance. Our previous studies indicated that programmed death-1 (PD-1) and its ligand programmed death ligand-1 (PD-L1) are crucial modulators of host immune responses during sepsis. Our current study was designed to investigate the role of PD-L1 in sepsis-induced liver injury by a mouse cecal ligation and puncture (CLP) model. Our results indicated that there was a significant increase of PD-L1 expression in liver after CLP challenge compared to sham-operated controls, in terms of levels of mRNA transcription and immunohistochemistry. Anti-PD-L1 antibody significantly alleviated the morphology of liver injury in CLP mice. Anti-PD-L1 antibody administration decreased ALT and AST release in CLP mice, decreased the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 mRNA in liver after sepsis challenge. Thus, anti-PD-L1 antibody might have a therapeutic potential in attenuating liver injury in sepsis.

Transgenic Expression of FoxM1 Promotes Endothelial Repair following Lung Injury Induced by Polymicrobial Sepsis in Mice

Enhancing endothelial barrier integrity for the treatment of acute lung injury (ALI) is an emerging novel therapeutic strategy. Our previous studies have demonstrated the essential role of FoxM1 in mediating endothelial regeneration and barrier repair following lipopolysaccharide-induced lung injury. However, it remains unclear whether FoxM1 expression is sufficient to promote endothelial repair in experimental models of sepsis. Here, employing the FoxM1 transgenic (FoxM1 Tg) mice, we showed that transgenic expression of FoxM1 promoted rapid recovery of endothelial barrier function and survival in a clinically relevant model of sepsis induced by cecal ligation and puncture (CLP). We observed lung vascular permeability was rapidly recovered and returned to levels similar to baseline at 48 h post-CLP challenge in FoxM1 Tg mice whereas it remained markedly elevated in WT mice. Lung edema and inflammation were resolved only in FoxM1 Tg mice at 24 h post-CLP. 5-bromo-2-deoxyuridine incorporation assay revealed a drastic induction of endothelial proliferation in FoxM1 Tg lungs at 24h post-CLP, correlating with early induction of expression of FoxM1 target genes essential for cell cycle progression. Additionally, deletion of FoxM1 in endothelial cells, employing the mouse model with endothelial cell-restricted disruption of FoxM1 (FoxM1 CKO) resulted in impaired endothelial repair following CLP challenge. Together, these data suggest FoxM1 expression in endothelial cells is necessary and sufficient to mediate endothelial repair and thereby promote survival following sepsis challenge.

Inhibition of Janus Kinase 2 and Signal Transduction and Activator of Transcription 3 Protect Against Cecal Ligation and Puncture-Induced Multiple Organ Damage and Mortality

Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway plays an important role in sepsis, transducing a multitude of inflammatory signals. To date, knowledge of JAK/STAT pathway in sepsis is limited. This study was to investigate the potential role of JAK/STAT pathway in mediating multiple organ damage and mortality in septic rats. Our data showed that inhibition of JAK2/STAT3 attenuated cecal ligation and puncture-induced multiple organ damage and mortality in 48 hours in rats. A total of 98 male Wistar rats were randomly divided into 4 groups as follows: (1) normal control group (n = 10); (2) cecal ligation and puncture (CLP) group (n = 40), which was further divided into 2, 6, 24, 48-hour post-CLP groups; (3) AG490 (8.0 mg/kg, Calbiochem, La Jolla, CA) treatment group (n = 24), which was further divided into 2, 6, 24, 48-hour post-CLP groups; (4) rapamycin (0.4 mg/kg, Calbiochem, Calbiochem, La Jolla, CA) treatment group (n = 24), which was further divided into 2, 6, 24, 48-hour post-CLP groups; CLP was performed to induce experimental sepsis. AG490 (8 mg/kg) or rapamycin (0.4 mg/kg) was injected subcutaneously 0.5 hour before CLP in respective group. Animals were killed at destined time after CLP (not including the death rate observation group), and specimens of serum, liver, and lungs were harvested and stored in liquid nitrogen for subsequent analyses. In an additional experiment, 88 animals were randomly divided into three groups to compare the survival rate, including CLP group (n = 40), AG490 treatment group (8 mg/kg, n = 24), and rapamycin treatment group (0.4 mg/kg, n = 24). Mortality of rats in each group was recorded up to 48 hours after the procedure. After CLP challenge, myeloperoxidase (MPO), aspartate transaminase, and alanine aminotransferase levels, as well as activation of JAK2 and STAT3, were markedly increased. Administration of AG490 or rapamycin significantly decreased activation of JAK2 and STAT3, as well as high mobility group box-1 protein, MPO, alanine aminotransferase levels (p < 0.05 or p < 0.01). In addition, treatment with AG490 or rapamycin significantly improved the 48-hour survival rate from 37.5% (15 of 40) to 66.7% (16 of 24) and 70.8% (17 of 24), respectively (both p < 0.05). JAK2/STAT3 pathway might play a role in the development of multiple organ damage in septic rats, which suggested a potential strategy to control sepsis.