Post Cecal Ligation And Puncture Research Articles

Shengjiang San alleviated sepsis-induced lung injury through its bidirectional regulatory effect

BackgroundSepsis is a life-threatening organ dysfunction caused by dysregulated host responses to infection, for which effective therapeutic strategies are still absent. Shengjiang San (SJS), a well-known Traditional Chinese Medicine formula, has been widely used clinically. However, its role in sepsis-induced lung injury remains unclear.MethodsTo explore its specific mechanism, we firstly established a sepsis animal model using cecal ligation and puncture (CLP) and treated MH-S cells with LPS plus ATP. Then, UPLC/Q-TOF–MS/MS was utilized to identify its active ingredients. Network pharmacology analysis was performed to uncover the potential mechanism. HE staining and biochemical analysis were conducted to validate its therapeutic effect. ELISA was applied to detect the release of pro-inflammatory and anti-inflammatory cytokines. Western blot was utilized to detect the protein levels of GSDMD, NLRP3, P65, ASC and caspase-1.ResultsSJS could dramatically increase the survival rate of sepsis. In addition, it is able to inhibit the pro-inflammatory cytokines release at day 1 post CLP while promote their production at day 7, indicating SJS could attenuate uncontrolled inflammatory response in the early stage and improve immunosuppression in the late phase. Network pharmacology analysis showed that pyroptosis is the crucial action SJS exerted in the protection of sepsis-induced lung injury. Western blot data implicated SJS could attenuate pyroptosis in early sepsis while enhance in the late phase.ConclusionsSJS acted to alleviate sepsis-induced lung injury through its bidirectional regulatory effect.

Alpha-Chymotrypsin Protects Against Acute Lung, Kidney, and Liver Injuries and Increases Survival in CLP-Induced Sepsis in Rats Through Inhibition of TLR4/NF-κB Pathway.

Inflammation and oxidative stress play a major role in the development of sepsis and its associated complications, leading to multiple organ failure and death. The lungs, liver, and kidneys are among the early affected organs correlated with mortality in sepsis. Alpha-chymotrypsin (α-ch) is a serine protease that exerts anti-inflammatory, anti-edematous, and anti-oxidant properties.PurposeThis study was undertaken to elucidate if the anti-inflammatory and anti-oxidant effects of α-ch observed in previous studies can alleviate lung, liver, and kidney injuries in a cecal ligation and puncture (CLP)-induced sepsis model, and thus decrease mortality.Materials and MethodsSeptic animals were given α-ch 2 h post CLP procedure. Sepsis outcomes were assessed in the lungs, liver, and kidneys. Separate animal groups were investigated for a survival study.ResultsCLP resulted in 0% survival, while α-chymotrypsin post-treatment led to 50% survival at the end of the study. Administration of α-chymotrypsin resulted in a significant attenuation of sepsis-induced elevated malonaldehyde (MDA) and total nitrite/nitrate (NOx) levels. In addition, there was a significant increase in reduced glutathione (GSH) content and superoxide dismutase (SOD) activity in the lungs, liver, and kidneys. Administration of α-ch reduced elevated tissue expression of toll-like receptor-4 (TLR4), nuclear factor kappa-B (NF-κB), myeloperoxidase (MPO), and inducible nitric oxide synthase (iNOS). Alpha-chymotrypsin resulted in a significant reduction in serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). Alpha-chymotrypsin attenuated the rise in serum creatinine, cystatin C, blood urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels that was observed in the septic group. In addition, α-ch significantly reduced the lung wet/dry weight ratio, total protein content, and leukocytic counts in bronchoalveolar lavage fluid (BALF). Histopathological examination of the lungs, liver, and kidneys confirmed the protective effects of α-ch on those organs.Conclusionα-ch has protective potential against sepsis through lowering tissue expression of TLR4, NF-κB, MPO, and iNOS leading to decreased oxidative stress and inflammatory signals induced by sepsis. This effect appeared to alleviate the damage to the lungs, liver, and kidneys and increase survival in rats subjected to sepsis.

Circular RNA mmu_circ_0001295 from hypoxia pretreated adipose-derived mesenchymal stem cells (ADSCs) exosomes improves outcomes and inhibits sepsis-induced renal injury in a mouse model of sepsis

ABSTRACT Microvascular dysfunction causes mortality in the presence of sepsis and multi-organ failure. Previous studies have demonstrated that exogenous administration of exosomes from adipose-derived mesenchymal stem cells (ADSCs) protects against sepsis, improves organ function, decreases vascular leakage and increases survival. However, the underlying regulatory mechanism was largely unknown. Therefore, in this study, a mouse sepsis model based on cecal ligation and puncture (CLP) was constructed. Exosomes from various ADSCs were intravenously administered at 4 h post CLP. Treatment with ADSC exosomes (Exo), particularly those with hypoxic pretreatment (HExo), enhanced survival, suppressed renal vascular leakage and decreased kidney dysfunction in septic mice. HExo ameliorated sepsis-induced increases in chemokine and cytokine plasma levels. Furthermore, the HExo circRNA content, determined through next-generation sequencing, revealed abundant mmu_circ_0001295. Further studies demonstrated that downregulation of exosomal mmu_circ_0001295 suppressed the exosomes’ protective effects against sepsis. HExo prevented microvascular dysfunction, thus potentially improving sepsis outcomes via mmu_circ_0001295 delivery. In summary, the data indicated that HExo elongate sepsis-induced renal injury through delivering mmu_circ_0001295.

Intravenous calcitriol administration modulates mesenteric lymph node CD4+ T-cell polarization and attenuates intestinal inflammation in obese mice complicated with polymicrobial sepsis.

Sepsis is a lethal syndrome with T-cell dysregulation, imbalanced inflammatory reactions, and gastrointestinal dysfunction. Obesity coexistent with sepsis can cause more-deleterious disease outcomes. Vitamin D is a nutrient with immunomodulatory ability and helps maintain intestinal homeostasis. This study investigated treatment with calcitriol on mesenteric lymph node (MLN) CD4+ T-cell polarization and intestinal injury in obese mice with sepsis. Mice received a high-fat diet for 10 weeks; then, mice were separated into an obese control group without sepsis and sepsis groups that underwent cecal ligation and puncture (CLP). Septic mice were subdivided into a group that was injected with saline (SS group) or a group that was injected with calcitriol (SD group) via a tail vein 1 h after CLP. Obese mice with sepsis were euthanized at 12 or 24 h post CLP. Sepsis resulted in increased percentages of type 2 T helper (Th2), Th17, and regulatory T (Treg) cells in MLNs. Also, inflammation-associated genes were upregulated and tight junction genes downregulated in the intestines after CLP. Compared with the SS group, the SD group exhibited reduced Th2, Th17, and Treg percentages in MLNs. Also, intestinal inflammatory chemokine expressions were reduced, whereas MUC2, ZO-1, and occludin had increased after CLP. Lower inflammatory cytokine levels in peritoneal lavage fluid in the ileum were also noted in the SD group. Intravenous calcitriol treatment after sepsis can elicit more-balanced CD4 T-cell subsets in lymph nodes near the intestines and alleviate intestinal inflammation and injury in obese mice complicated with sepsis.

Immunophenotypic characterization and therapeutics effects of human bone marrow- and umbilical cord-derived mesenchymal stromal cells in an experimental model of sepsis

Sepsis is a complicated multi-system disorder characterized by a dysregulated host response to infection. Despite substantial progress in the understanding of mechanisms of sepsis, translation of these advances into clinically effective therapies remains challenging. Mesenchymal Stromal Cells (MSCs) possess immunomodulatory properties that have shown therapeutic promise in preclinical models of sepsis. The therapeutic effects of MSCs may vary depending on the source and type of these cells. In this comparative study, the gene expression pattern and surface markers of bone marrow-derived MSCs (BM-MSCs) and umbilical cord-derived MSCs (UC-MSCs) as well as their therapeutic effects in a clinically relevant mouse model of polymicrobial sepsis, cecal ligation and puncture (CLP), were investigated. The results showed remarkable differences in gene expression profile, surface markers and therapeutic potency in terms of enhancing survival and pro/anti-inflammatory responses between the two MSC types. BM-MSCs improved survival concomitant with an enhanced systemic bacterial clearance and improved inflammatory profile post CLP surgery. Despite some improvement in the inflammatory profile of the septic animals, treatment with UC-MSCs did not enhance survival or bacterial clearance. Overall, the beneficial therapeutic effects of BM-MSCs over UC-MSCs may likely be attributed to their pro-inflammatory function, and to some extent anti-inflammatory features, reflected in their gene expression pattern enhancing macrophage polarization to M1/M2 phenotypes resulting in a balanced pro- and anti-inflammatory response against polymicrobial sepsis.

SHP2 inhibitor PHPS1 ameliorates acute kidney injury by Erk1/2-STAT3 signaling in a combined murine hemorrhage followed by septic challenge model

BackgroundHypovolemic shock and septic challenge are two major causes of acute kidney injury (AKI) in the clinic setting. Src homology 2 domain-containing phosphatase 2 (SHP2) is one of the major protein phosphatase tyrosine phosphatase (PTPs), which play a significant role in maintaining immunological homeostasis by regulating many facets of immune cell signaling. In this study, we explored whether SHP2 signaling contributed to development of AKI sequential hemorrhage (Hem) and cecal ligation and puncture (CLP) and whether inactivation of SHP2 through administration of its selective inhibitor, phenylhydrazonopyrazolone sulfonate 1 (PHPS1), attenuated this injury.MethodsMale C57BL/6 mice were subjected to Hem (a “priming” insult) followed by CLP or sham-Hem plus sham-CLP (S/S) as controls. Samples of blood and kidney were harvested at 24 h post CLP. The expression of neutrophil gelatinase-associated lipocalin (NGAL), high mobility group box 1 (HMGB1), caspase3 as well as SHP2:phospho-SHP2, extracellular-regulated kinase (Erk1/2): phospho-Erk1/2, and signal transducer and activator of transcription 3 (STAT3):phospho-STAT3 protein in kidney tissues were detected by Western blotting. The levels of creatinine (Cre) and blood urea nitrogen (BUN) in serum were measured according to the manufacturer’s instructions. Blood inflammatory cytokine/chemokine levels were detected by ELISA.ResultsWe found that indices of kidney injury, including levels of BUN, Cre and NGAL as well as histopathologic changes, were significantly increased after Hem/CLP in comparison with that in the S/S group. Furthermore, Hem/CLP resulted in elevated serum levels of inflammatory cytokines/chemokines, and induced increased levels of HMGB1, SHP2:phospho-SHP2, Erk1/2:phospho-Erk1/2, and STAT3:phospho-STAT3 protein expression in the kidney. Treatment with PHPS1 markedly attenuated these Hem/CLP-induced changes.ConclusionsIn conclusion, our data indicate that SHP2 inhibition attenuates AKI induced by our double-hit/sequential insult model of Hem/CLP and that this protective action may be attributable to its ability to mitigate activation of the Erk1/2 and STAT3 signaling pathway. We believe this is a potentially important finding with clinical implications warranting further investigation.

Correction of immunosuppression in aged septic rats by human ghrelin and growth hormone through the vagus nerve-dependent inhibition of TGF-\u03b2 production

BackgroundCo-administration of human ghrelin and growth hormone (GH) reverse immunosuppression in septic aged animals, but the mechanism remains elusive. Here, we hypothesize that ghrelin and GH co-treatment restores the immune response in aged septic rats by inhibiting the production of transforming growth factor-β (TGF-β), an immunoregulatory cytokine, through the vagus nerve.MethodsMale aged Fischer rats (22–23-month-old) were made septic by cecal ligation and puncture (CLP) with or without dissecting the vagus nerve (vagotomy). Human ghrelin and GH or vehicle (PBS) were administrated subcutaneously at 5 h post CLP. After 20 h of CLP, serum and spleens were harvested.ResultsSerum TGF-β levels were increased in septic aged rats, while ghrelin and GH treatment significantly reduced its levels. Expression of TGF-β in the spleen was upregulated after sepsis, while ghrelin and GH treatment significantly inhibited its expression. TNF-α and IL-6 levels were significantly reduced after ex vivo LPS stimulation of splenocytes from rats that underwent CLP compared to sham rats; while these levels were significantly higher in splenocytes from ghrelin and GH-treated CLP rats compared to vehicle-treated CLP rats. Ghrelin and GH treatment reduced program death receptor-1 (PD-1) expression, increased human leukocyte antigen-DR (HLA-DR) expression, attenuated lymphopenia, and cleaved caspase-3 levels in the spleen of septic aged rats. Vagotomy diminished the beneficial effects of ghrelin and GH treatment in septic rats. In vitro, the addition of ghrelin, GH, or ghrelin and GH together had no effect on restoring immune response in splenocytes from CLP rats following LPS stimulation, indicating the requirement of the vagus nerve for ghrelin and GH’s effect.ConclusionsGhrelin and GH attenuate immunosuppression in aged septic rats through the vagus nerve-dependent inhibition of TGF-β production.

Novel Endothelial Cell Targeted Peptide Nanoformulation for Inhibiting von Willebrand Factor Secretion to Reduce Thrombotic Complications in Sepsis

RationaleMicrovascular thrombosis and disseminated intravascular coagulation (DIC) are primary determinants of ischemia, organ failure and mortality/morbidity in sepsis. Interestingly, elevated plasma level of von Willebrand factor (vWF), the glue to which platelets adhere to form a clot, is associated with reduced survival and poor outcome in septic patients. Previously, we demonstrated that endothelial cell (EC) Gα12 is required for soluble N‐ethylmaleimide‐sensitive‐fusion factor attachment protein α (αSNAP)‐dependent vWF secretion from Weibel Palade bodies. Here, we report that sterically stabilized simple micelles (SSM) containing cell‐permeable myristoylated αSNAP binding domain (Myr‐SBD) peptide based on the unique N‐terminal Gα12 sequence that binds to αSNAP, inhibits vWF secretion.MethodsStructural complex of αSNAP/SBD was determined by blind‐ and data‐driven docking using AutoDock and HADDOCK, respectively. After incorporating Myr‐SBD into SSM (Myr‐SBD‐SSM) by film hydration method, micelle size and shape were confirmed by dynamic light scattering and transmission electron microscopy (TEM), respectively. Intracellular delivery of Myr‐SBD‐SSM was evaluated by fluorescence microscopy and fluorometry in cultured ECs. Cecal ligation and puncture (CLP) in C57BL/6 mice (14–16 wks, 25 g) was used to induce severe inflammation, microvascular thrombosis and DIC, and blood was collected 6 h and 24 h post CLP. The effect of Myr‐SBD‐SSM on plasma vWF, platelet count and inflammatory cytokines was assessed following addition of 0, 2 or 4 μmol Myr‐SBD/kg given i.v. at the time of surgery. Bleeding was assessed by tail‐snip assay. Glomerular fibrin deposition was scored by IHC analysis at 24 h post CLP.ResultsThe αSNAP/SBD structure showed the potential interaction between Arg of SBD peptide and Glu139 of αSNAP by H‐bond. Spherical shape of Myr‐SBD‐SSM (14.1 ± 2.7 nm in diameter) was confirmed by negative staining TEM. Addition of Myr‐SBD‐SSM to ECs resulted in the intracellular accumulation of Myr‐SBD which blocked thrombin‐induced vWF secretion. Intravenous delivery of Myr‐SBD‐SSM to mice at the time of CLP blunted the increase in plasma vWF and associated microvascular thrombosis in the kidney glomerulus. Importantly, Myr‐SBD‐SSM treatment resulted in increased platelet and WBC counts 6 h after surgery and improved survival compared to untreated or scrambled peptide treated mice with severe vascular inflammation suggesting Myr‐SBD‐SSM reduced clotting, platelet consumption and systemic inflammation in intraperitoneal sepsis. Moreover, Myr‐SBD‐SSM treatment did not increase tail‐snip bleeding time suggesting it did not prevent hemostasis.ConclusionOur studies demonstrate that Myr‐SBD‐SSM nanoformulation reduces vWF secretion and attenuates the thrombotic complications of severe inflammation.Support or Funding InformationThis work was supported by MPI R01 HL125356 (RDM, XD), Michael Reese Research and Education Foundation (RDM) and American Heart Association (MB, LR).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 16947: Mitochondrial Calcium Uniporter Channel Blockade Ameliorates Sepsis Induced Cardiomyopathy

Introduction: Mitochondria serve as critical Ca 2+ sinks, regulating the duration and magnitude of c Ca2+ transients and c Ca2+ entry into the mitochondrion. Mitochondrial calcium uniporter (MCU) channel is activated in conditions of inflammation and oxidative stress.MCU and its activity are potential therapeutic targets for cardiac hypertrophy and heart failure. Hypothesis: Blunting of mCa2+ uptake can contribute to increased cCa2+ transients associated with excitation-contraction coupling and offer cardio protection following sepsis induced stress. Methods: MCU C96A KI generated using CRISPR-Cas9 d10 nickase mRNA co-injected with gRNA into wild-type(C57BL6) zygotes and then implanted into pseudo-pregnant female mice.To elucidate mechanistic role of mCa2+ in modulating cardiac contractility and myocardial bioenergetics, adult cardiomyocytes were isolated from mouse model and subjected to hypoxia/re-oxygenation injury.Polymicrobial Sepsis in 6-week-old C57BL/6J mice were created using Cecal ligation and puncture (CLP) and MCU oxidation at 1, 6, 12 and 24 hours post injury was compared with Sham and control mice (n=15). MCU protein quantification was done with western blot, functional assessment of Reactive oxygen species (ROS) by confocal laser microscopy, echocardiography with Vevo2100 imaging system. Mice were pretreated with MCU blocking agent Ru360 1 hr prior to CLP and compared with the control group at 6 hours. Results: Cardiomyocytes exposed to hypoxia/re-oxygenation(h/R ) stress exhibited increased MitoSOX red fluorescence.MCU oxidation was ubiquitously induced in all tissues post CLP surgery and was most significant at 6 hrs (Fig 1a). Oxidation of MCU was marked by increase in ROS in tissue sections of small Intestine of CLP versus the Sham group(p<0.001). (Fig 1 b &c). Pretreatment with Ru360 ameliorated MCU oxidation at 6 hours post CLP injury. MCUC96AKI had significantly decreased ejection fraction and fractional shortening compared to the wild type mice. Further the survival in MCU C96A KI was significantly better than the WT or MCU KO mice ( p<0.05). Conclusions: Inhibition of mitochondrial calcium uniporter is a potential therapeutic target and protects from sepsis induced cardiomyopathy by mitigating mitophagy.

Reduced silent information regulator 1 signaling exacerbates sepsis-induced myocardial injury and mitigates the protective effect of a liver X receptor agonist

Myocardial injury and dysfunction are critical manifestations of sepsis. Previous studies have reported that liver X receptor (LXR) activation is protective during sepsis. However, whether LXR activation protects against septic heart injury and its underlying mechanisms remain elusive. This study was designed to determine the role of LXR activation in the septic heart with a focus on SIRT1 (silent information regulator 1) signaling. Male cardiac-specific SIRT1 knockout mice (SIRT1-/-) and their wild-type littermates were subjected to sepsis by cecal ligation and puncture (CLP) in the presence or absence of LXR agonist T0901317. The survival rate of mice was recorded during the 7-day period post CLP. Our results demonstrated that SIRT1-/- mice suffered from exacerbated mortality and myocardial injury in comparison with their wild-type littermates. Meanwhile, T0901317 treatment improved mice survival, accompanied by significant ameliorations of myocardial injury and dysfunction in wild-type mice but not in SIRT1-/- mice. Furthermore, the levels of myocardial inflammatory cytokines (TNF-α, IL-6, IL-1β, MCP-1, MPO and HMGB1), oxidative stress (ROS generation, MDA), endoplasmic-reticulum (ER) stress (protein levels of CHOP, GRP78, GRP94, IRE1α, and ATF6), and cardiac apoptosis following CLP were inhibited by T0901317 treatment in wild-type mice but not in SIRT1-/- mice. Mechanistically, T0901317 enhanced SIRT1 signaling and the subsequent deacetylation and activation of antioxidative FoxO1 and anti-ER stress HSF1, as well as the deacetylation and inhibition of pro-inflammatory NF-ΚB and pro-apoptotic P53, thereby alleviating sepsis-induced myocardial injury and dysfunction. Our data support the promise of LXR activation as an effective strategy for relieving heart septic injury.

Abstract 159: Synthetic High Density Lipoprotein - a Potential Therapy for Sepsis

Introduction: Sepsis is the leading cause of death in critically ill patients with mortality rates exceeding 30%. The ability of endogenous HDL to neutralize and facilitate elimination of lipopolysaccharides (LPS) is known and synthetic HDLs (sHDL) have been administered in sepsis models with varying success. In the current study we optimized composition of sHDL, apolipoprotein A-I (apoA-I) mimetic peptide-phospholipid particles, to maximize their protective effect against endothelial dysfunction in sepsis, and examined changes in HDL and apoA-I levels in patients with sepsis to select sHDL doses for evaluation in a sepsis animal model. Methods/Results: We first measured HDL cholesterol (HDL-C) and apoA-I in patients at time of intake to the intensive care unit, observing HDL to be 45% lower in septic patients compared to non-septic patients (p<0.01), with HDL levels in non-surviving septic patients being significantly lower compared to those in surviving septic patients (p<0.01). Next, we tested a new generation of sHDL, ETC642, for its efficacy on cecal ligation and puncture (CLP)-challenged mice. We administered sHDL to mice 2h post CLP and found that sHDL treatment significantly increased plasma HDL-C and decreased BUN, plasma IL-6 and IL-10 levels. More importantly, sHDL therapy improved 7d survival rate greater than 2-fold over PBS treated mice (p=0.01). To understand the mechanisms underlying sHDL protection, we investigated the effect of sHDL on endotoxin-induced inflammatory response in vitro . sHDL not only neutralized LPS/LTA but also suppressed TLR4-/TLR2-NF-kB signaling in macrophages, and inhibited LPS/LTA/TNF-α induced endothelial cell activation. Conclusions: In this study, we show that HDL-C levels are markedly decreased in septic patients, which is associated with poor prognosis. We demonstrate that sHDL treatment increases HDL levels and effectively protects against CLP-induced septic death. We further established that sHDL therapy provides multiple protective mechanisms through LPS/LTA detoxification, suppressing inflammatory signaling in macrophages and inhibiting endothelial activation. Together, our findings suggest that targeting the vasculature via sHDL can be an effective platform for sepsis therapy.

Advanced age enhances the sepsis-induced up-regulation of the γ- and α7-nicotinic acetylcholine receptors in different parts of the skeletal muscles

BackgroundThe muscle mass decreases with age, leading to frailty and weakness; however, the role of acetylcholine receptors in this process has not been properly studied. In this article, we hypothesize that diaphragmatic as well as peripheral muscle weakness may be caused by the up-regulation of gamma and alpha 7 nicotinic acetylcholine receptors (nAChRs) on muscle cell membranes. MethodAdult male rats were randomly divided into sham and sepsis groups. Sepsis was induced by cecal ligation and puncture (CLP). Blood specimens and biopsies of tibialis anterior muscle and diaphragm were obtained at 24h post CLP. ResultsUp-regulation of gamma and alpha 7 nAChRs was detected in both sham and septic groups; however, this response was more robust in septic animals. Compared to tibialis anterior muscle, the diaphragm expressed a higher number of both receptor types. ConclusionsMuscle weakness in old age and sepsis may have common molecular underpinnings. Loss of diaphragmatic strength may explain hypoxia and respiratory failure often encountered in frail elderly.

Abstract 308: Corticosteroid Therapy Benefits Septic Mice With Adrenal Insufficiency but Harms Septic Mice Without Adrenal Insufficiency

Rationale: Corticosteroid (CS) therapy is frequently used in septic patients. However, the efficacy of CS therapy is highly controversial. The heterogeneity of septic patients, the technical difficulties in identifying adrenal insufficiency and a lack of adrenal insufficiency animal model present significant barriers to address this longstanding clinical issue. Objectives: In this study, we utilized scavenger receptor BI (SR-BI) null mice as the first relative adrenal insufficiency animal model to evaluate the efficacy of CS therapy and assess the pathogenesis caused by adrenal insufficiency. Methods: Cecal ligation and puncture (CLP) was conducted on SR-BI-/- and wild type control mice, and then CS was administered 2 or 18h post CLP. Measurements and Main Results: We demonstrated that CS supplementation protects against CLP-induced sepsis in SR-BI-/- mice but causes more septic death in wild type controls. Importantly, we identified a cocktail of CS that provides more efficient protection than an individual CS, and the septic mice could be rescued 18h post CLP by the CS cocktail; we further demonstrated that adrenal insufficiency impairs both innate and adaptive immunity. Conclusions: This preclinical study provides a proof-of-concept that corticosteroid benefits septic patients with adrenal insufficiency but harms septic patients without adrenal insufficiency, and suggests that it is necessary to determine the status of adrenal insufficiency and only prescribe corticosteroid for septic patients with adrenal insufficiency.

Preclinical evaluation of the mitochondria‐targeted antioxidant MitoTEMPO on the renal microcirculation in a pediatric model of sepsis‐induced renal injury

Microcirculatory failure, mitochondrial dysfunction and oxidant generation are thought to be key events during sepsis that lead to multi-organ failure. Acute kidney injury is a leading cause of mortality in septic children. We developed a clinically relevant cecal ligation and puncture (CLP) sepsis model in rat pups 17-19 days old to identify and evaluate new therapeutic approaches. Analysis of the renal microcirculation using intravital video microscopy at 18 h post CLP or sham surgery showed a significant decrease in the percentage of continuously flowing capillaries (from 75% to 40%) and an increase in capillaries with no flow (from 5% to 30%). Microcirculatory failure was associated with a 2-fold increase in mitochondrial superoxide generation in renal tubules. We then performed dose-response studies to evaluate the effectiveness of the mitochondria-targeted antioxidant MitoTEMPO to reduce mitochondria superoxide and to improve renal microcirculation. MitoTEMPO was administered at doses of 1, 3, or 10 mg/kg (ip) at the time of CLP or sham surgery. Superoxide and perfusion were measured at 18 h. MitoTEMPO produced dose-dependent decreases in mitochondrial superoxide and increases in microcirculatory perfusion with 3 mg/kg being the lowest most efficacious dose to preserve the renal microcirculation. These findings suggest a previously unrecognized link between mitochondrial oxidants in the renal tubule epithelium and the regulation of microvascular perfusion. The protective effect of MitoTEMPO in the kidney suggests that agents like it should be evaluated further for their effectiveness at reducing sepsis-induced multi-organ failure. Supported by T32 GM106999 and AHA PRE20450050.

Effect of compound hypertonic saline solution on septic rats

To study the effect of compound hypertonic saline solution ( HSD ) on sepsis. 133 male Wistar rats were divided into four groups, sham operation group ( n = 15 ), cecal ligation and puncture ( CLP ) group ( n = 45 ), CLP plus normal saline ( NS ) group ( n = 45 ), and CLP plus HSD group ( n = 28 ). A rat model of sepsis was reproduced by CLP, and the rats in sham operation group received celiotomy without ligation and puncture. All rats in four groups received subcutaneous injection of 30 mL/kg 0.9% sodium chloride after laparotomy. The rats in CLP plus NS group and CLP plus HSD group received infusion of 5 mL/kg 0.9% sodium chloride or 7.5% sodium chloride/6% dextran post CLP via jugular vein for 3 hours, with the infusion rate of 0.4 mL×kg(-1)×min(-1). The survival rate of each group was observed 9 hours and 18 hours after laparotomy. Mean arterial pressure ( MAP ) at 0, 9, 18 hours were monitored. Blood specimens were collected from all rats 0, 9 and 18 hours after laparotomy, respectively, for measurement of the plasma levels of tumor necrosis factor-α ( TNF-α), interleukin-1β ( IL-1β ), and procalcitonin ( PCT ). The rats were all sacrificed, and their lung tissues were harvested for the neutrophil count in bronchoalveolar lavage fluid ( BALF ), myeloperoxidase ( MPO ) activity in lung tissue, wet/dry weight ratio ( W/D ) of lung, and pathological changes in lung tissue. There was no death in the sham operation group. The survival rates at 9 hours and 18 hours were 62.2% and 31.1% in the CLP group, 57.8% and 35.6% in the CLP plus NS group, 85.7% and 64.3% in the CLP plus HSD group, and they were all significantly higher compared with those of the CLP group and the CLP plus NS group ( P<0.05 or P<0.01 ). MAP levels in the CLP group and the CLP plus NS group were significantly lower than those in sham operation group, and the plasma levels of TNF-α, IL-1β and PCT were significantly higher compared with those of sham operation group, while there was no difference between CLP group and the CLP plus NS group. MAP and the plasma levels of TNF-α, IL-1β and PCT in the CLP plus HSD group were significantly improved compared with those of the CLP plus NS group at 9 hours and 18 hours [ MAP ( mmHg, 1 mmHg = 0.133 kPa ) at 9 hours: 102±5 vs. 94±6, 18 hours: 90±2 vs. 72±3; TNF-α ( ng/L ) at 9 hours: 284.19±57.18 vs. 329.67±45.79, 18 hours: 263.46±42.58 vs. 349.68±52.40; IL-1β ( ng/L ) at 9 hours: 219.28±39.21 vs. 263.47±32.36, 18 hours: 195.98±39.06 vs. 250.10±41.57; PCT ( μg/L ) at 9 hours: 2.32±0.37 vs. 4.52±0.75, 18 hours: 2.89±0.62 vs. 5.02±0.84; P<0.05 or P<0.01 ]. The ratio of neutrophils in BALF, MPO activity and lung W/D at 18 hours in the CLP group and the CLP plus NS group were significantly higher than those of the sham operation group, while they were all significantly lower in the CLP plus HSD group than those of the CLP group and the CLP plus NS group [ ratio of neutrophils in BALF: 0.094±0.019 vs. 0.148±0.062, 0.151±0.055; MPO ( U/g ): 1.19±0.45 vs. 2.31±0.79, 2.64±0.69; lung W/D ratio: 4.02±0.63 vs. 5.14±0.59, 5.12±0.83, all P<0.05 ]. Under light microscope, no pathobiological changes were found in sham operation group. The lung tissues in the CLP group and the CLP plus NS group showed congestion, edema, infiltrating inflammatory changes, while the inflammatory changes in the lung tissue in the CLP plus HSD group were significantly alleviated. HSD can obviously ameliorate the circulatory failure in septic rats, alleviate immune disturbance and acute lung injury, and improve the survival rate of rats with sepsis.

Betulinic acid attenuates lung injury by modulation of inflammatory cytokine response in experimentally-induced polymicrobial sepsis in mice

Sepsis commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity and mortality. Septic ALI is characterized by excessive production of proinflammatory mediators. It remained refractory to present therapies and new therapies need to be developed to improve further clinical outcomes. Betulinic acid (BA), a pentacyclic lupane group triterpenoid has been shown to have anti-inflammatory activities in many studies. However, its therapeutic efficacy in polymicrobial septic ALI is yet unknown. Therefore, we investigated the effects of BA on septic ALI using cecal ligation and puncture (CLP) model in mice. Vehicle or BA (3, 10, and 30mg/kg) was administered intraperitoneally, 3 times (0, 24 and 48h) before CLP and CLP was done on 49thh of the study. Survival rate was observed till 120h post CLP. Lung tissues were collected for analysis by sacrificing mice 18h post CLP. BA at 10 and 30mg/kg dose significantly reduced sepsis-induced mortality and lung injury as implied by attenuated lung histopathological changes, decreased protein and neutrophils infiltration. BA also decreased lung NF-κB expression, cytokine, intercellular adhesion molecule-1, monocyte chemoattractant protein-1 and matrix metalloproteinase-9 levels. These evidences suggest that, the protective effects of BA on lungs are associated with defending action against inflammatory response and BA could be a potential modulatory agent of inflammation in sepsis-induced ALI.

Adenosine A1 receptor antagonist, L-97-1, improves survival and protects the kidney in a rat model of cecal ligation and puncture induced sepsis

Previously it was reported that combining antibiotics with L-97-1, an adenosine A1 receptor antagonist, significantly improves survival and blocks acute lung injury induced by Yersinia pestis CO 99 in a rat model of pneumonic plague. In the current studies using a conscious rat model of cecal ligation and puncture (CLP) sepsis, L-97-1 was administered in daily intravenous infusions in combination with antibiotics to simulate the use of L-97-1 as an anti-sepsis therapeutic in the clinical setting. In these studies, when administered at 12 h following CLP, in combination with broad spectrum antibiotics, ceftriaxone and clindamycin, L-97-1 improves 7 day (d) survival [25%, 35%, and 75% for L-97-1 (10mg/kg/h, 12.5mg/kg/h, and 15mg/kg/h, respectively) versus (vs.) 25% for antibiotics alone] in a dose-dependent manner. The addition of L-97–1, 15mg/kg/h to antibiotics significantly increased 7 d survival following CLP compared to therapy with either antibiotics alone (P=0.002) or L-97-1 at 15mg/kg/h alone (P<0.001) and was not significantly different than survival in sham CLP animals (Log-rank (Mantel-Cox) test with Bonferroni׳s correction for multiple comparisons). Moreover, in these studies, in combination with antibiotics L-97-1 dose-dependently protects the kidney, significantly improving renal function at 24h post CLP at 10mg/kg/h (P<0.001), 12.5mg/kg/h (P<0.0001), and 15mg/kg/h (P<0.0001) vs. antibiotics alone (ANOVA followed by Tukey׳s post-hoc test for pair-wise comparisons). The results of these studies support efficacy for L-97-1 as an anti-sepsis therapeutic.

Effects of glycemic control on glucose utilization and mitochondrial respiration during resuscitated murine septic shock.

BackgroundThis study aims to test the hypothesis whether lowering glycemia improves mitochondrial function and thereby attenuates apoptotic cell death during resuscitated murine septic shock.MethodsImmediately and 6 h after cecal ligation and puncture (CLP), mice randomly received either vehicle or the anti-diabetic drug EMD008 (100 μg · g-1). At 15 h post CLP, mice were anesthetized, mechanically ventilated, instrumented and rendered normo- or hyperglycemic (target glycemia 100 ± 20 and 180 ± 50 mg · dL-1, respectively) by infusing stable, non-radioactive isotope-labeled 13C6-glucose. Target hemodynamics was achieved by colloid fluid resuscitation and continuous i.v. noradrenaline, and mechanical ventilation was titrated according to blood gases and pulmonary compliance measurements. Gluconeogenesis and glucose oxidation were derived from blood and expiratory glucose and 13CO2 isotope enrichments, respectively; mathematical modeling allowed analyzing isotope data for glucose uptake as a function of glycemia. Postmortem liver tissue was analyzed for HO-1, AMPK, caspase-3, and Bax (western blotting) expression as well as for mitochondrial respiratory activity (high-resolution respirometry).ResultsHyperglycemia lowered mitochondrial respiratory capacity; EMD008 treatment was associated with increased mitochondrial respiration. Hyperglycemia decreased AMPK phosphorylation, and EMD008 attenuated both this effect as well as the expression of activated caspase-3 and Bax. During hyperglycemia EMD008 increased HO-1 expression. During hyperglycemia, maximal mitochondrial oxidative phosphorylation rate was directly related to HO-1 expression, while it was unrelated to AMPK activation. According to the mathematical modeling, EMD008 increased the slope of glucose uptake plotted as a function of glycemia.ConclusionsDuring resuscitated, polymicrobial, murine septic shock, glycemic control either by reducing glucose infusion rates or EMD008 improved glucose uptake and thereby liver tissue mitochondrial respiratory activity. EMD008 effects were more pronounced during hyperglycemia and coincided with attenuated markers of apoptosis. The effects of glucose control were at least in part due to the up-regulation of HO-1 and activation of AMPK.Electronic supplementary materialThe online version of this article (doi:10.1186/2197-425X-2-19) contains supplementary material, which is available to authorized users.

Effects of Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA) pretreatment on septic rats

To evaluate the effects of Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA) injection on the survival rate of rats post cecal ligation and puncture (CLP), Sprague-Dawley (SD) rats were subcutaneously injected with 0.125 ml, 0.25 ml or 0.5 ml PA-MSHA for 8 days or 16 days before CLP. The survival rate and physiological appearance of rats in each group were monitored daily post CLP. The expression of Toll-like receptor 4 (TLR4) and cytokines related to inflammation was evaluated. We found that the 0.5 ml-8d (0.5 ml PA-MSHA injected for 8 days) group had the highest 7-day survival rate (91.7%), which was significantly improved compared with the CLP-only group (33.3%). Furthermore, our results showed that PA-MSHA effectively increased serum pro-inflammatory mediators (TNF-α, IL-1β and IL-6) at the early stage (8 days) but increased anti-inflammatory mediators (IL-4 and IL-10) at the late stage (16 days). PA-MSHA significantly up-regulated the mRNA expression of TLR4 at 8 and 16 days. After PA-MSHA pretreatment, CLP had no marked effect on the levels of most inflammatory factors. To explore potential protective mechanisms of PA-MSHA against CLP, we examined the effect of PA-MSHA on murine macrophage-like RAW264.7 cells and found that PA-MSHA induced endotoxin tolerance. In conclusion, this study suggested that precisely controlling the dosage and time of PA-MSHA administration can effectively increase the rat survival rate post CLP, which may be mediated through regulating inflammatory mediators and inducing endotoxin tolerance.

Preclinical evaluation of the mitochondria‐targeted antioxidant mitoquinone to treat sepsis‐induced acute kidney injury

Sepsis is caused by a severe microbial infection and can lead to acute kidney injury (AKI). This results in a high mortality rate of 70%. No specific therapies exist for septic AKI. To test if mitochondrial injury contributes to septic AKI, we used a clinically relevant cecal ligation and puncture (CLP) murine model. At 4h and 18h after CLP, the activity of manganese superoxide dismutase was decreased 50%, suggesting decreased mitochondrial antioxidant defense. Intravital microscopy showed a 50% fall in perfusion of renal capillaries at 18h after CLP (P &lt; 0.05 versus control mice). Also, renal mitochondrial superoxide levels were more than doubled at 18h. We hypothesize that protecting the renal mitochondria using mitochondrial specific mitoquinone (MitoQ) will attenuate sepsis induced AKI. MitoQ (10mg/kg, i.p) given at the time of sepsis restored renal perfusion, decreased mitochondrial superoxide and preserved renal morphology at 18h post CLP. MitoQ also improved survival rate. In addition, MitoQ administered in a clinically relevant delayed dosing paradigm at 6h post CLP, also protected the renal microcirculation and inhibited mitochondrial oxidant generation. These data show that mitochondrial superoxide, a previously unrecognized mediator of septic AKI, can be targeted by mitochondrial antioxidants to attenuate septic AKI, even with delayed treatment. Supported by NIH DK075991 and AHA 12PRE12040174.