Endotoxin Shock Research Articles

USP38 Couples Histone Ubiquitination and Methylation via KDM5B to Resolve Inflammation.

Chromatin modifications, such as histone acetylation, ubiquitination, and methylation, play fundamental roles in maintaining chromatin architecture and regulating gene transcription. Although their crosstalk in chromatin remodeling has been gradually uncovered, the functional relationship between histone ubiquitination and methylation in regulating immunity and inflammation remains unclear. Here, it is reported that USP38 is a novel histone deubiquitinase that works together with the histone H3K4 modifier KDM5B to orchestrate inflammatory responses. USP38 specifically removes the monoubiquitin on H2B at lysine 120, which functions as a prerequisite for the subsequent recruitment of demethylase KDM5B to the promoters of proinflammatory cytokines Il6 and Il23a during LPS stimulation. KDM5B in turn inhibits the binding of NF‐κB transcription factors to the Il6 and Il23a promoters by reducing H3K4 trimethylation. Furthermore, USP38 can bind to KDM5B and prevent it from proteasomal degradation, which further enhances the function of KDM5B in the regulation of inflammation‐related genes. Loss of Usp38 in mice markedly enhances susceptibility to endotoxin shock and acute colitis, and these mice display a more severe inflammatory phenotype compared to wild‐type mice. The studies identify USP38‐KDM5B as a distinct chromatin modification complex that restrains inflammatory responses through manipulating the crosstalk of histone ubiquitination and methylation.

Atraric Acid Exhibits Anti-Inflammatory Effect in Lipopolysaccharide-Stimulated RAW264.7 Cells and Mouse Models.

Lichens, composite organisms resulting from the symbiotic association between the fungi and algae, produce a variety of secondary metabolites that exhibit pharmacological activities. This study aimed to investigate the anti-inflammatory activities of the secondary metabolite atraric acid produced by Heterodermia hypoleuca. The results confirmed that atraric acid could regulate induced pro-inflammatory cytokine, nitric oxide, prostaglandin E2, induced nitric oxide synthase and cyclooxygenase-2 enzyme expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Meanwhile, atraric acid downregulated the expression of phosphorylated IκB, extracellular signal-regulated kinases (ERK) and nuclear factor kappa B (NFκB) signaling pathway to exhibit anti-inflammatory effects in LPS-stimulated RAW264.7 cells. Based on these results, the anti-inflammatory effect of atraric acid during LPS-induced endotoxin shock in a mouse model was confirmed. In the atraric acid treated-group, cytokine production was decreased in the peritoneum and serum, and each organ damaged by LPS-stimulation was recovered. These results indicate that atraric acid has an anti-inflammatory effect, which may be the underlying molecular mechanism involved in the inactivation of the ERK/NFκB signaling pathway, demonstrating its potential therapeutic value for treating inflammatory diseases.

Non-cytotoxic doses of shikonin inhibit lipopolysaccharide-induced TNF-α expression via activation of the AMP-activated protein kinase signaling pathway.

Shikonin has been reported to exhibit a wide variety of medical functions. However, the strong non-selective cytotoxicity of shikonin can restrict its clinical application. The aim of the present study was to investigate the effects of shikonin at non-cytotoxic doses on the pro-inflammation functions of monocytes and macrophages. The present results suggested that the non-cytotoxic doses of shikonin effectively inhibited lipopolysaccharide (LPS)-induced reactive oxygen species production, NF-κB activation and TNF-α expression in RAW 264.7 mouse macrophages via AMP-activated protein kinase (AMPK) signaling pathway. In addition, the non-cytotoxic doses of shikonin downregulated LPS-induced TNF-α expression via AMPK signaling activation in primary murine bone marrow-derived macrophages, and also in monocytes cultured ex vivo from patients with chronic obstructive pulmonary disease (COPD). The present in vivo results indicated that the low-toxic dose of shikonin suppressed LPS-induced endotoxin shock and TNF-α expression in mice. Collectively, the present results may provide clinical and translational relevance for treating COPD and other TNF-α-related inflammatory disorders.

Toll-Like Receptor 4 Signaling in the Ileum and Colon of Gnotobiotic Piglets Infected with Salmonella Typhimurium or Its Isogenic ∆rfa Mutants.

Salmonella Typhimurium is a Gram-negative bacterium that causes enterocolitis in humans and pigs. Lipopolysaccharide (LPS) is a component of the outer leaflet of Gram-negative bacteria that provokes endotoxin shock. LPS can be synthesized completely or incompletely and creates S (smooth) or R (rough) chemotypes. Toll-like receptors (TLR) 2, 4, and 9 initiate an inflammatory reaction to combat bacterial infections. We associated/challenged one-week-old gnotobiotic piglets with wild-type S. Typhimurium with S chemotype or its isogenic ∆rfa mutants with R chemotype LPS. The wild-type S. Typhimurium induced TLR2 and TLR4 mRNA expression but not TLR9 mRNA expression in the ileum and colon of one-week-old gnotobiotic piglets 24 h after challenge. The TLR2 and TLR4 stimulatory effects of the S. Typhimurium ∆rfa mutants were related to the completeness of their LPS chain. The transcription of IL-12/23 p40, IFN-γ, and IL-6 in the intestine and the intestinal and plasmatic levels of IL-12/23 p40 and IL-6 but not IFN-γ were related to the activation of TLR2 and TLR4 signaling pathways. The avirulent S. Typhimurium ∆rfa mutants are potentially useful for modulation of the TLR2 and TLR4 signaling pathways to protect the immunocompromised gnotobiotic piglets against subsequent infection with the virulent S. Typhimurium.

Bivalent Inhibitor with Selectivity for Trimeric MMP-9 Amplifies Neutrophil Chemotaxis and Enables Functional Studies on MMP-9 Proteoforms.

A fundamental part of the immune response to infection or injury is leukocyte migration. Matrix metalloproteinases (MMPs) are a class of secreted or cell-bound endopeptidases, implicated in every step of the process of inflammatory cell migration. Hence, specific inhibition of MMPs is an interesting approach to control inflammation. We evaluated the potential of a bivalent carboxylate inhibitor to selectively inhibit the trimeric proteoform of MMP-9 and compared this with a corresponding monovalent inhibitor. The bivalent inhibitor efficiently inhibited trimeric MMP-9 (IC50 = 0.1 nM), with at least 500-fold selectivity for MMP-9 trimers over monomers. Surprisingly, in a mouse model for chemotaxis, the bivalent inhibitor amplified leukocyte influxes towards lipopolysaccharide-induced inflammation. We verified by microscopic and flow cytometry analysis increased amounts of neutrophils. In a mouse model for endotoxin shock, mice treated with the bivalent inhibitor had significantly increased levels of MMP-9 in plasma and lungs, indicative for increased inflammation. In conclusion, we propose a new role for MMP-9 trimers in tempering excessive neutrophil migration. In addition, we have identified a small molecule inhibitor with a high selectivity for the trimeric proteoform of MMP-9, which will allow further research on the functions of MMP-9 proteoforms.

TAOK1 positively regulates TLR4-induced inflammatory responses by promoting ERK1/2 activation in macrophages

Thousand and one amino acid kinase 1 (TAOK1) is a member of Ste20-like kinases, but its function in regulating inflammatory responses remains largely unknown. In this study, we identify TAOK1 as a positive regulator of TLR4-triggered inflammatory responses in macrophages. TAOK1 increases LPS-induced production of pro-inflammatory cytokine such as IL-6, TNF-α and IL12p40 in macrophages. TAOK1 deficient mice showed decreased susceptibility to endotoxin shock, with less pro-inflammatory cytokine production than control mice. TAOK1 promotes LPS-induced activation of ERK1/2 by constitutively interacting with TRAF6 and TPL2. These finding unravel the important role of TAOK1 as a positive regulator of TLR4-induced inflammatory responses.

Two new terpenoids from Reduning Injection

ObjectiveTo study the antipyretic and anti-inflammatory constituents from the active fraction of Reduning (RDN) Injection. MethodsIn this study, the active fraction of RDN Injection was screened by the LPS-induced mouse endotoxin shock model. The chemical constituents were isolated by chromatography on HP-20 macroporous adsorptive resins, silica gel, ODS columns and reverse phase MPLC and HPLC repeatedly, and their structures were elucidated based on spectroscopic analysis (HR-ESI-MS, NMR, ECD) and chemical methods. Meanwhile, we evaluated the anti-inflammatory activities of the isolates by measuring their inhibitory effects on TNF-α production in LPS stimulated RAW 264.7 macrophages. ResultsThe 95% ethanol eluate of RDN Injection by the macroporous adsorption resin column was proved to be the antipyretic and anti-inflammatory active fraction of this injection. A novel iridoid, named jasminoide A (1), and a new guaiane sesquiterpenoid, named (1R,7R,8S,10R)-7,8,11-trihydroxy-4-guaien-3-one (2), were isolated from Reduning injection, and compound 2 can inhibit TNF-α production with IC50 values of 72.24 µmol/L. ConclusionIn this study, two new terpenoids were isolated from Reduning Injection, and compound 2 showed inhibitory activity against LPS-activated TNF-α production in RAW 264.7 cells in vitro.

The on-off action of Forkhead protein O3a in endotoxin tolerance of Kupffer cells depends on the PI3K/AKT pathway

BackgroundThe endotoxin tolerance (ET) of Kupffer cells (KCs) is an important protective mechanism for limiting endotoxin shock. As a key anti-inflammatory molecule, the roles and mechanism of Forkhead protein O3a (Foxo3a) in ET of KCs are not yet well understood. MethodsET and nonendotoxin tolerance (NET) KCs models were established in vitro and in vivo. The levels of cytokines were detected by enzyme-linked immunosorbent assay (ELISA). The protein expression and phosphorylation levels were detected by western blotting (WB). Changes in the localization of nuclear factor kappa B (NF-κB) and Foxo3a in KCs were detected by immunofluorescence assays. KCs apoptosis and survival rates were detected by flow cytometry and an automatic cell counter, respectively. ResultsThe activity of NF-κB and the levels of p-Foxo3a and tumor necrosis factor (TNF-α) in the ET group were significantly lower than those in the NET group, while the levels of Foxo3a and interleukin 10 (IL-10) in the ET group were significantly higher than those in the NET group. Overexpression of Foxo3a or the use of a phosphatidylinositol-3-hydroxykinase (PI3K) inhibitor suppressed the activation of NF-κB by decreasing the levels of p-Foxo3a by inhibiting the activity of PI3K/AKT, which improved the tolerance of KCs and mice to endotoxin. In contrast, silencing Foxo3a or the use of a PI3K agonist reduced the tolerance of KCs and mice to endotoxin. The PI3K agonist counteracted the inhibitory effects of Foxo3a overexpression on NF-κB, impairing the tolerance of KCs to endotoxin. ConclusionsThe on-off action of Foxo3a in the ET of KCs depends on the PI3K/AKT pathway.

Cathelicidin deficiency exacerbates cardiac dysfunction in lipopolysaccharide-induced endotoxaemic mice.

The therapeutic potential of the antimicrobial peptide cathelicidin (Camp) administration in sepsis has been widely investigated. However, little is known about the pathophysiological roles of cathelicidin in septic cardiomyopathy. In a lipopolysaccharide (LPS)-induced endotoxaemic model, we found that the mRNA and protein expression of cardiac cathelicidin were induced in C57BL/6J wild-type (WT) mice upon LPS challenge, accompanied by increased circulating cathelicidin levels. We showed that this peptide was mainly derived from neutrophils and monocytes/macrophages. Camp deficiency exacerbated LPS-induced myocardial depression, while the administration of CRAMP (the mature form of mouse cathelicidin) decreased the LPS-induced mortality in a D-galactosamine hydrochloride (D-GalN)-sensitized endotoxin shock model. In vivo, LPS-treated Camp knockout mice had a significant higher protein level of myocardial and circulating tumour necrosis factor-alpha (TNF-α), a major contributing factor to septic cardiomyopathy, compared to LPS-treated WT mice, while CRAMP administration inhibited LPS-induced TNF-α production in the heart and plasma in D-GalN-sensitized endotoxaemic mice. In vitro, CRAMP treatment suppressed LPS-induced Tnf-α mRNA expression in cultured neonatal mouse cardiomyocytes and reduced TNF-α secretion in the culture supernatant. The inhibitory effects of CRAMP on TNF-α production may be related to its neutralizing ability of LPS, since CRAMP application had no effects on another toll-like receptor 4 ligand paclitaxel-induced Tnf-α mRNA expression in cardiomyocytes. These findings suggest that LPS-induced cathelicidin protects the heart against myocardial depression partly through the inhibition of TNF-α production via neutralizing LPS.

CD163 deficiency facilitates lipopolysaccharide‐induced inflammatory responses and endotoxin shock in mice

ObjectivesSeptic (or endotoxin) shock is a severe systemic inflammatory disease caused by bacteraemia or endotoxaemia. Although it is known that increased serum levels of CD163 are observed in septic/endotoxin shock patients, the exact function and significance of CD163 in macrophage activation remain unclear. Therefore, in the current study, we tested whether CD163 contributes to the pathogenesis of endotoxin shock in mice.Methods and resultsIn samples obtained from autopsy, the number of CD163‐positive macrophages was increased in the kidney, liver, heart, bone marrow and spleen of patients who had died from septic/endotoxin shock when compared to patients who had died from other causes. The animal study revealed a significantly lower survival rate in CD163‐deficient mice after lipopolysaccharide (LPS) injection. Several cytokines and oxidative stress‐related molecules were significantly elevated in the sera of LPS‐induced endotoxin shock mice models. Higher concentrations of IL‐6, TNF‐α, IL‐1β, nitrite (NO2‐) and nitrate (NO3‐) and a lower concentration of IL‐10 were observed in CD163‐deficient mice treated with LPS. Similar results were observed in CD163‐deficient LPS‐stimulated macrophages. Furthermore, in an antitype II collagen antibody‐induced arthritis (CAIA), rheumatoid arthritis model, inflammation and bone erosion scores as well as the expression of IL‐6 and IL‐1β were significantly increased in CD163‐deficient mice.ConclusionsCD163 was suggested to be involved in the regulation of inflammatory cytokine expression in septic/endotoxin shock and CAIA.

Real-time in vivo Imaging of LPS-induced Local Inflammation and Drug Deposition in NF-κB Reporter Mice.

Wound, biomaterial, and surgical infections are all characterized by a localized and excessive inflammation, motivating the development of in vivo methods focused on the analysis of local immune events. However, current inflammation models, such as the commonly used in vivo models of endotoxin-induced inflammation are based on systemic, usually intraperitoneal, administration of lipopolysaccharide (LPS), causing endotoxin shock. Here, we describe a model of LPS-induced local inflammation in NF-κB-RE-Luc reporter mice. LPS, alone or with added therapeutic substances, is delivered locally via a hydrogel which is deposited subcutaneously, providing a spatially defined environment, enabling in vivo bioimaging analyses of local NF-κB activation. Evaluation of drug efficacy can be analyzed longitudinally in the same mouse, and using fluorescently labeled drugs, local drug deposition can be simultaneously analyzed, and correlated to the site of inflammation. Finally, the protocol can also be used to study retention and systemic release of the drug from locally deposited gels and other biomaterials.

Успешная терапия эндотоксинового шока и полиорганной дисфункции с помощью последовательной целенаправленной экстракорпоральной терапии у больного после комбинированной кардиохирургической операции

Успешная терапия эндотоксинового шока и полиорганной дисфункции с помощью последовательной целенаправленной экстракорпоральной терапии у больного после комбинированной кардиохирургической операции

Natural Naphthohydroquinone Dimer Rubioncolin C Exerts Anti-Tumor Activity by Inducing Apoptotic and Autophagic Cell Death and Inhibiting the NF-κB and Akt/mTOR/P70S6K Pathway in Human Cancer Cells

Naphthohydroquinone dimers isolated from Rubia plants have garnered more attention due to their distinctive chemical structures and intriguing bioactivities. In our previous studies, we obtained ten naphthohydroquinone dimers containing seven novel ones and found that most of them possessed anti-tumor activities, especially rubioncolin C. However, the underlying mechanism remains unknown. In this study, we focused on rubioncolin C and found that it could inhibit the growth of cancer cell lines with IC50 values between 1.14 and 9.93 μM. Further experiments demonstrated that rubioncolin C induced apoptotic and autophagic cell death and inhibited the Akt/mTOR/P70S6K signaling pathway in HCT116 and HepG2 cells. Moreover, we observed that rubioncolin C inhibited the TNF-α- and LPS-induced NF-κB activation upstream of the p65 protein, which contributed to rubioncolin C-induced cell death. Rubioncolin C could also prevent LPS-induced endotoxin shock in vivo. Moreover, rubioncolin C suppressed tumor growth through inducing apoptosis and autophagy and inactivating NF-κB in vivo. These findings clarify the anti-tumor mechanism of rubioncolin C using biochemical techniques and pharmacological models and might contribute to the future development of rubioncolin C as a new therapeutic agent for treating cancer.

The endotoxin influence on the deformability of red blood cells. (in vitro)

Experiments with endotoxin is an interesting direction for medical applications. It is known that certain abnormalities in the microcirculation in endotoxin shock are related to a reduction of the surface electrical charge and the deformability of red blood cells. The intravascular coagulation of blood cells or “sludge phenomenon” has been observed in endotoxin and other types of shock. To gain a more comprehensive insight into the effect of endotoxin on erythrocyte membranes an assessment was made of the light dispersion in electric field by erythrocyte suspension. Its adoption enables to investigate better the dynamics of endotoxin interaction with erythrocyte membranes, changes in deformability and the like.

Gentiopicroside (GENT) protects against sepsis induced by lipopolysaccharide (LPS) through the NF-κB signaling pathway.

Sepsis is a high-mortality disease without effective therapeutic options. The hyperactivation of the monocyte-macrophage system, especially M1 macrophages, triggers the onset of septic shock. Gentiopicroside (GENT), the main active component in the traditional Chinese medicinal herb Radix Gentianae, has been shown to have anti-inflammatory properties. Nevertheless, this anti-inflammatory effect has not been fully elucidated. In vitro, we stimulated primary bone marrow-derived macrophages (BMMs) or peritoneal elucidated macrophages (PEMs) by lipopolysaccharide (LPS) and interferon (IFN)-γ and pre-treated with GENT and we tested the cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF) α production by enzyme linked immunosorbent assay (ELISA) or real-time quantitative PCR (qPCR). Further, we determined the NF-κB-mediated inflammatory pathway such as IKKα/β and p65 phosphorylation by Western blot. Then we detected the p65 nuclear localization by immunofluorescent staining. Moreover, NF-κB inhibitor and p65-targeted siRNAs were further used to validate the anti-inflammatory mechanism of GENT. In vivo, GENT (50 mg/kg) was administered intragastrically before and after LPS (40 mg/kg) injection. The death time were recorded and the serum levels of IL-1β, IL-6 and TNFα were tested by ELISA, and the IL-1β, IL-6 and TNFα mRNA expression in the lung were test by qPCR and the M1 infiltration in the lung were determined by F4/80 and INOS immunofluorescent staining. In vitro, we observed that GENT reduced the inflammatory cytokine production of BMMs stimulated by (LPS)/IFN-γ and ameliorated the phosphorylation of IKKα/β and p65, the degradation of IκBα, and the translocation of p65 into the nucleus. We did not find GENT has any effect on MAPK signaling under LPS/IFN-γ stimulation. NF-κB inhibitor and p65 siRNAs eliminated the inhibition effect of GENT. In vivo, we observed GENT prevented mice from dying in the LPS-induced shock model and decreased the serum levels of IL-1β and IL-6, the mRNA expression of IL-1β, IL-6 and TNFα in lung tissue, and the amount of M1 macrophage infiltration in the lung. GENT prevented LPS/IFN-γ-induced inflammatory cytokine production by macrophages through the NF-κB signaling pathway in vitro and protected against the endotoxin shock induced by LPS in vivo.

Anti-Inflammatory Effects of Shenfu Injection against Acute Lung Injury through Inhibiting HMGB1-NF-κB Pathway in a Rat Model of Endotoxin Shock.

Shenfu injection (SFI), a Chinese herbal medicine with substances extracted from Ginseng Radix et Rhizoma Rubra and Aconiti Lateralis Radix Praeparata, is widely used as an anti-inflammatory reagent to treat endotoxin shock in China. However, the mechanism of SFI in endotoxin shock remains to be illuminated. High mobility group box 1 (HMGB1), a vital inflammatory factor in the late stage of endotoxin shock, may stimulate multiple signalling cascades, including κB (NF-κB), a nuclear transcription factor, as well as tumour necrosis factor (TNF)-α and interleukin (IL)-1β, among others in the overexpression of downstream proinflammatory cytokines. An investigation into the effects of SFI on the inhibition of the HMGB1-NF-κB pathway revealed the contribution of SFI to acute lung injury (ALI) in a rat model of endotoxin shock. To assess the anti-inflammatory activity of SFI, 5 ml/kg, 10 ml/kg, or 15 ml/kg of SFI was administered to different groups of rats following an injection of LPS, and the mean arterial pressure (MAP) at 5 h and the survival rate at 72 h were measured. 24 h after LPS injection, we observed pathological changes in the lung tissue and measured the mRNA expression, production, translocation, and secretion of HMGB1, as well as the expression of the NF-κB signal pathway-related proteins inhibitor of NF-κB (IκB)-α, P50, and P65. We also evaluated the regulation of SFI on the secretion of inflammatory factors including interleukin-1 beta (IL-1β) and TNF-α. SFI effectively prevented the drop in MAP, relieved lung tissue damage, and increased the survival rate in the endotoxin shock model in dose-dependent manner. SFI inhibited the transcription, expression, translocation, and secretion of HMGB1, increased the expression of toll-like receptor (TLR4), increased the production of IκB-α, and decreased the levels of P65, P50, and TNF-α in the lung tissue of endotoxin shock rats in a dose-dependent manner. Furthermore, SFI decreased the secretion of proinflammatory cytokines TNF-α and IL-1β. In summary, SFI improves the survival rate of endotoxin shock, perhaps through inhibiting the HMGB1-NF-κB pathway and thus preventing cytokine storm.

Non-invasive in vivo imaging of caspase-1 activation enables rapid and spatiotemporal detection of acute and chronic inflammatory disorders

Inflammasome plays a critical role in diverse inflammatory disorders, including cancers and Alzheimer's disease. It is induced by various pathogenic insults and activates caspase-1, a hallmark executor of inflammasome. Here, we developed an activatable fluorescence probe for visualization of active caspase-1. This caspase-1 probe is biocompatible, efficiently delivered into cells and tissues, and specifically emits fluorescence upon caspase-1 activation as assessed in in vitro and in vivo models of inflammatory conditions. We demonstrated efficient in vivo imaging of caspase-1 activation in early stages of various inflammatory conditions of mice models, including endotoxin shock, inflammatory bowel disorder, transplanted cancer, and Alzheimer's disease. Notably, the caspase-1 probe enables detection of neuroinflammation in vivo two months earlier than cognitive impairments occur in Alzheimer's disease model. We detected significant fluorescence emitted from inflamed sites, as well as their draining lymph nodes, by macroscopic imaging analysis within 30 min after systemic injection of the probe. This novel synthetic probe could be applied for efficient and rapid detection of caspase-1 activity in a spatiotemporal way by non-invasive imaging.

P4605Landiolol, a beta-blocker, optimizes cardiac energetics and peripheral microhemodynamics better than ivabradine while reducing heart rate at hemodynamic resuscitation of experimental septic shock

Abstract Background Septic shock is associated with sympathetic over-activation characterized by tachycardia, which is associated with poor survival. Though several kinds of drugs are available to reduce heart rate (HR), it is not clear which drug should be used to reduce HR effectively in initial hemodynamic resuscitation in septic shock. Purpose The aim of this study was to experimentally investigate comparative effects of landiolol (a β-blocker) and ivabradine (an If-channel inhibitor) on initial hemodynamic resuscitation in a canine model of endotoxin shock. Methods We used 19 anesthetized dogs. Seven dogs were allocated to control (CT) group, six dogs to landiolol (BB) group, and six dogs to ivabradine (IVA) group. In all the groups, endotoxin shock was induced by intravenous infusion of Escherichia coli lipopolysaccharide (4 mg/kg) over 1 h. After induction of endotoxin shock, we started hemodynamic resuscitation to restore mean arterial pressure (AP) and cardiac output (CO) by infusing noradrenaline (NA) and Ringer acetate solution (RiA). Dose of NA and RiA were automatically titrated with use of a computer-controlled drug infusion system that we developed previously. Hemodynamic resuscitation was continued for 4 h. During resuscitation, the BB group was given landiolol infusion at a low-dose range (1–10 μg/kg/min, manually titrated), while the IVA group was given ivabradine at 0.25 mg/kg bolus-injected every 2 h. Results The drug infusion system automatically restored AP to 70 mmHg and CO to greater than 90% of baseline level in all the groups. The restored AP and CO were not significantly different among the three groups (Figure A, B). There were no significant differences in the computer-controlled infusion rate of NA and cumulated volume of RiA infused among the three groups (Figure C). During the first 2 h of resuscitation, HR was significantly reduced in the BB and IVA groups in comparison to the CT group (Figure D). However, only in the BB group, cardiac oxygen consumption was significantly lower than that in the CT group (Figure E). Blood lactate level decreased significantly after hemodynamic resuscitation only within the BB group (Figure F). Figure 1 Conclusions In the initial hemodynamic resuscitation of septic shock, both low-dose landiolol infusion and ivabradine injection may be used to reduce HR. However, to optimize cardiac energetics and peripheral microhemodynamics, low-dose landiolol infusion may be preferable to ivabradine injection. Acknowledgement/Funding This work was supported by JSPS KAKENHI Grant Number 15K01307, 18K12126.

P4141Lack of IkBNS promotes cholate-containing high-fat diet-induced inflammation and atherogenesis in low-density lipoprotein (LDL) receptor-deficient mice

Abstract Background IκBNS is one of the nuclear IκB proteins and regulates a subset of Toll-like receptor (TLR) dependent genes. LPS acts as extremely strong stimulator of innate immunity. We tried to investigate whether stimulation of innate immunity could promote atherosclerosis in the IκBNS-deficient atherogenic mice. However all IκBNS-deficient mice died of LPS challenge at a dose of which almost all wild-type mice survived, because IκBNS-deficient mice are highly sensitive to LPS-induced endotoxin shock. Then, we decided to use a cholate-containing high fat diet (HFD(CA(+))), which has been widely used as an atherogenic diet in mice. Furthermore, HFD(CA(+)) has been shown to induce TLR4 mediated early inflammatory response. The present study aims to clarify the lack of IκBNS promotes atherogenesis in LDL receptor-deficient (LDLr−/−) mice fed HFD(CA(+)) compared with those fed a cholate-free HFD (HFD(CA(−)). Methods and results Mice that lacked IκBNS (IκBNS−/−) were crossed with LDLr−/− mice and formation of atherosclerotic lesions was analyzed after 6 weeks consumption of HFD(CA(+)) or HFD(CA(−)). The extent of atherosclerosis in the aorta (en face) was significantly increased in IκBNS−/−/LDLr−/−(CA(+)) mice compared with others after 6-week consumption of HFD (p<0.01) (Figure). Interestingly, HFD(CA(−)) did not induce significant atherosclerotic lesions in IκBNS−/−/LDLr−/− compared with LDLr−/− mice after 6-week consumption (Figure). Immunostaining of aortic root lesion revealed that HFD(CA(+)) significantly increased positive area of Mac-3 (macrophage) by 1.5-fold (p=0.01) and TLR4, interleukin-6 (IL-6) expression by 1.7-fold (P<0.05) and 1.5-fold (p<0.05) respectively in IκBNS−/−/LDLr−/− (CA(+)) compared to LDLr−/− (CA(+)) mice. Furthermore, active STAT3 (pSTAT3)-positive cells were significantly increased by 1.7-fold in the atherosclerotic lesions of IκBNS−/−/LDLr−/− (CA(+)) compared with LDLr−/− (CA(+)) mice (p<0.01). TLR4 positive areas, IL-6 positive areas, and pSTAT3 positive cells were overlapped with Mac-3, indicating that TLR4-IL-6-STAT3 axis was activated in macrophages in IκBNS−/−/LDLr−/− (CA(+)) mice. On the other hand, HFD(CA(−)) could not induce any difference in these immunoreactivities of arteriosclerotic lesions between IκBNS−/−/LDLr−/− (CA(−)) compared with LDLr−/− (CA(−)) mice. These findings suggest that IκBNS deficiency and HFD(CA(+)) promote atherogenesis in LDLr−/− mice via TLR4/IL-6/STAT3 pathway. Finally, we show the monocytes from peripheral blood of IκBNS−/−/LDLr−/− (CA(+)) mice were found to contain the most mounts of Ly6Chi among four groups, suggesting that lack of IκBNS enhances inflammation in the response HFD(CA(+)) feeding and thereby influence atherogenesis in IκBNS−/−/LDLr−/− mice. Aortic root atherosclerotic lesions Conclusions The present study is the first to demonstrate that the activation of innate immune system using HFD(CA(+)) induced significant inflammation and atherogenesis in IκBNS−/−/LDLr−/− compared with LDLr−/− mice.

Protection from Endotoxin Shock by Selective Targeting of Proinflammatory Signaling to the Nucleus Mediated by Importin Alpha 5.

Endotoxin shock is induced by LPS, one of the most potent virulence factors of the Gram-negative bacteria that cause sepsis. It remains unknown if either proinflammatory stress-responsive transcription factors (SRTFs), ferried to nucleus by importin α5, or lipid-regulating sterol regulatory element binding proteins (SREBPs), transported to the nucleus by importin β1, mediate endotoxin shock. A novel cell-penetrating peptide targeting importin α5 while sparing importin β1 protected 80% of animals from death in response to a high dose of LPS. This peptide suppresses inflammatory mediators, liver glycogen depletion, endothelial injury, neutrophil trafficking, and apoptosis caused by LPS. In d-galactosamine-pretreated mice challenged by 700-times lower dose of LPS, rapid death through massive apoptosis and hemorrhagic necrosis of the liver was also averted by the importin α5–selective peptide. Thus, using a new tool for selective suppression of nuclear transport, we demonstrate that SRTFs, rather than SREBPs, mediate endotoxin shock.