Intravenous Injection Of Lipopolysaccharide Research Articles

Oxycodone attenuates vascular leak and lung inflammation in a clinically relevant two-hit rat model of acute lung injury

BackgroundOxycodone is a synthetic opioid receptor agonist that exerts antinociceptive activity via κ-, μ- and δ-opioid receptors (KOR, MOR and DOR, respectively). Activation of MOR has been reported to provide protection against acute lung injury (ALI). We hypothesized that pretreatment with oxycodone would attenuate lung injury at the level of alveolar tight junctions (TJs) and aquaporins (AQPs) and investigated this possibility in a two-hit model of ALI induced by lipopolysaccharide (LPS) and mechanical ventilation (MV). MethodMale Sprague Dawley rats and A59 cells were divided into 6 groups: the control group, ALI group, oxycodone-pretreated group, and oxycodone/κ-, μ-, or δ-opioid receptor antagonist-pretreated groups. The rats were pretreated with oxycodone 30 min before intravenous injection of LPS and then allowed to recover for 24 h prior to MV, establishing a two-hit model of ALI. The cells were similarly treated with oxycodone (with or without antagonists) 30 min after exposure to lipopolysaccharide. The cells were cyclically stretched 24 h later to mirror the in vivo MV protocol. ResultsOxycodone alleviated the histological lung changes in the rats with ALI and decreased pulmonary microvascular permeability both in vivo and in vitro. Oxycodone upregulated the expression of claudin-5, ZO-1, AQP1, and AQP5 but downregulated the expression of TNF-α, IL-1β, TLR4, NF-κB, MMP9, and caspase-3 and suppressed endothelial apoptosis in vivo and in vitro. These protective effects of oxycodone were partly eliminated by KOR and MOR antagonists but not by DOR antagonists. ConclusionOxycodone pretreatment appears to act via κ- and μ-opioid receptors to ameliorate LPS- and MV-induced lung injury by suppressing inflammation and apoptosis, and this protective effect might be mediated through the inhibition of the TLR4/NF-κB pathways.

In utero heat stress alters the postnatal innate immune response of pigs.

The effects of in utero heat stress (IUHS) range from decreased growth performance to altered behavior, but the long-term impact of IUHS on postnatal innate immune function in pigs is unknown. Therefore, the study objective was to determine the effects of early gestation IUHS on the immune, metabolic, and stress response of pigs subjected to an 8 hr lipopolysaccharide (LPS) challenge during postnatal life. Twenty-four pregnant gilts were exposed to thermoneutral (TN; n = 12; 17.5 ± 2.1 °C) or heat stress (HS; n = 12; cyclic 26 to 36 °C) conditions from days 6 to 59 of gestation, and then TN conditions (20.9 ± 2.3 °C) from day 60 of gestation to farrowing. At 12 wk of age, 16 IUHS and 16 in utero thermoneutral (IUTN) pigs were selected, balanced by sex and given an intravenous injection of LPS (2 µg/kg BW mixed with sterile saline [SAL] and injected at 2 µL/kg BW) or SAL (2 µL/kg BW). Body temperature was monitored every 30 min, and blood was obtained at 0, 1, 2, 3, 4, 6, and 8 hr following the LPS challenge. Blood samples were analyzed for glucose, insulin, non-esterified fatty acids (NEFA), cortisol, and cytokine concentrations. In addition, white blood cell counts were determined at 0 and 4 hr. Hour 0 data were used as covariates. Body temperature was increased (P < 0.01) in LPS (40.88 ± 0.08 °C) vs. SAL (39.83 ± 0.08 °C) pigs. Eosinophils tended to be decreased overall (P = 0.09; 43.9%) in IUHS vs. IUTN pigs. Glucose concentrations were reduced overall (P = 0.05; 5.9%) in IUHS vs. IUTN pigs. The NEFA concentrations tended to be greater (P = 0.07; 143.4%) in IUHS-LPS pigs compared with all other treatments, and IUTN-LPS pigs tended to have greater (127.4%) circulating NEFA concentrations compared with IUTN-SAL and IUHS-SAL pigs. Cortisol was increased (P = 0.04) in IUHS-LPS compared with IUTN-LPS pigs at 3 hr (21.5%) and 4 hr (64.3%). At 1 hr, tumor necrosis factor α was increased (P = 0.01; 115.1%) in IUHS-LPS compared with IUTN-LPS pigs. Overall, interleukin-1β (IL-1β) and interleukin-6 (IL-6) were greater (P < 0.04; 281.3% and 297.8%, respectively) in IUHS-LPS pigs compared with all other treatments, and IUTN-LPS pigs had increased IL-1β and IL-6 concentrations compared with IUTN-SAL and IUHS-SAL pigs. In summary, IUHS altered the postnatal cytokine, metabolic, and physiological stress response of pigs during postnatal life, which may have negative implications toward the innate immune response of IUHS pigs to pathogens.

Acute inflammation and psychomotor slowing: Experimental assessment using lipopolysaccharide administration in healthy humans

Data from clinical and cross-sectional studies suggest that inflammation contributes to psychomotor slowing and attentional deficits found in depressive disorder. However, experimental evidence is still lacking. The aim of this study was to clarify the effect of inflammation on psychomotor slowing using an experimental and acute model of inflammation, in which twenty-two healthy volunteers received an intravenous injection of lipopolysaccharide (LPS, dose: 0.8 ​ng/kg body weight) and of placebo, in a randomized order following a double-blind within-subject crossover design. A reaction time test and a go/no-go test were conducted 3 ​h after the LPS/placebo injection and interleukin (IL)-6 and tumor necrosis factor (TNF)-α concentrations were assessed. No effect of experimental inflammation on reaction times or errors for either test was found. However, inflammation was related to worse self-rated performance and lower effort put in the tasks. Exploratory analyses indicated that reaction time fluctuated more over time during acute inflammation. These data indicate that acute inflammation has only modest effects on psychomotor speed and attention in healthy subjects objectively, but alters the subjective evaluation of test performance. Increased variability in reaction time might be the first objective sign of altered psychomotor ability and would merit further investigation.

The Effect of Atrial Septal Defect in the Treatment of ARDS with Left Ventricular Dysfunction Simulating Severe COVID-19.

To explore the effect of atrial septal defect (ASD) and venoarterial extracorporeal membrane oxygenation (VA-ECMO) in the treatment of ARDS combined with left ventricular dysfunction (LVD) to find a new effective method for treating severe COVID-19 patients. Five large animal ARDS models of sheep were established by intravenous injection of Lipopolysaccharide. ASD was made under general anesthesia and VA-ECMO was simulated by extracorporeal circulation machine. The oxygenation of peripheral blood, systemic circulation, and cardiac function were observed under conditions of closed and opened ASD, and the significance of ASD shunt in improving cardiopulmonary function was evaluated. With ASD closed, the atrial shunts disappeared, the peripheral artery pressure of oxygen(PaO2): 141.2±21.4mmHg, the oxygenation index (PaO2/FiO2): 353.0±53.5, the mean blood pressure (MAP): 49.3±13.5 mmHg, the heart was full; with ASD opened, the left-to-right shunt was observed, PaO2: 169.3±18.9mmHg, PaO2/FiO2: 423.3±47.3, MAP: 68.2±16.1 mmHg, the range of cardiac motion significantly increased, heart beat was powerful, and systemic circulation significantly improved. Statistical analysis showed that there were significant differences between opened and closed ASD (P < .01). ASD plus VA-ECMO is an effective method for the treatment of ARDS combined with LVD, which is the main cause of death in severe COVID-19 patients. However, further clinical validation is needed.

Dexmedetomidine alleviates LPS‑induced acute lung injury via regulation of the p38/HO‑1 pathway.

Acute lung injury (ALI) is a common critical illness in clinical anesthesia and the intensive care unit that can cause acute hypoxic respiratory insufficiency. Despite various therapeutic regimes having been investigated, there is currently no effective pharmacotherapy available to treat ALI. Previous studies have reported that the NOD-like receptor protein 3 (NLRP3) signaling pathway plays an important role in the inflammatory response and is involved in the pathogenesis of ALI. Moreover, dexmedetomidine (Dex), an α2-adrenergic receptor activating agent, has been routinely used as an adjuvant therapy in treating inflammatory diseases, including ALI. However, the precise pathological mechanisms of Dex in ALI remain to be elucidated. Thus, the present study aimed to investigate the effects of the p38/heme oxygenase 1 (HO-1) signaling pathways in the pathological mechanisms of Dex in ALI. Newborn male Sprague-Dawley rats (n=48) were randomly divided into four groups (n=12 each), and an intravenous injection of lipopolysaccharide (LPS) was used to successfully induce the ALI model, with increased pulmonary damage, cell apoptosis, interleukin-1β (IL-1β) secretion and edema fluid in lungs. Moreover, the mRNA and protein expression levels of NLRP3 were significantly upregulated, while that of HO-1 were downregulated by LPS treatment. Furthermore, the levels of phosphorylated p38 were also upregulated in ALI rats. It was demonstrated that Dex administration significantly alleviated LPS-induced ALI, downregulated the secretion of IL-1β, decreased the expression of NLRP3, inhibited the phospho-activation of p38 and increased HO-1 expression. In addition, pharmacological inhibition of p38 using the inhibitor SB20380 further enhanced the effect of Dex. Collectively, these preliminarily results identified the effects of Dex intervention on the pathogenesis of ALI via the regulation of p38/HO-1 signaling pathways, which impacted the inflammatory effects, thus providing a theoretical basis and novel evidence for the development of new targets for clinical treatment of ALI.

Immunological and behavioral responses to in vivo lipopolysaccharide administration in young and healthy obese and normal-weight humans

Obesity is associated with an increase prevalence of neuropsychiatric symptoms and diseases, such as depression. Based on the facts that pro-inflammatory cytokines are able to modulate behavior, and that obesity is characterized by a chronic low-grade inflammatory state, inflammation has been hypothesized to contribute to the neuropsychiatric comorbidity in obese individuals. However, a causal link between inflammation and the development of neuropsychiatric symptoms is hard to establish in humans. Here, we used an inflammatory stimulus, i.e. the intravenous injection of lipopolysaccharide (LPS), in a double-blind placebo-controlled design, to determine the vulnerability of obese individuals to inflammation-induced behavioral changes. The hypothesis was that obese individuals would show heightened behavioral response compared to normal-weight subjects for the same inflammatory stimulus, reflecting an increased sensitivity to the behavioral effects of pro-inflammatory cytokines. LPS (dose 0.8 ng/kg body weight, adjusted for estimated blood volume in obese subjects) and placebo (saline) were intravenously injected in 14 obese healthy subjects and 23 normal-weight healthy subjects in a within-subject, randomized, crossover design. LPS administration induced, in both groups, an acute increase in blood concentrations of cytokines (interleukin-6, tumor necrosis factor-α, and IL-10), as well as in body temperature, cortisol, norepinephrine, sickness symptoms, fatigue, negative mood, and state anxiety. There were little differences in the immune and behavioral responses to LPS between obese and normal-weight subjects, but the cortisol response to LPS was strongly attenuated in obese individuals. Higher percentage of body fat was related to a lower cortisol response to LPS. Taken together, the population of young and healthy obese individuals in this study did not exhibit an increased behavioral sensitivity to cytokines, but an attenuated cortisol response to the immune challenge. Future studies will need to determine whether additional physiological and psychological factors interact with the state of obesity to increase the risk for inflammation-induced neuropsychiatric symptoms.

In vivo metabolomic and lipidomic response of healthy humans to intravenous lipopolysaccharide challenge

IntroductionAcute inflammation is a complex protective response to injury or infection. However, systemic hyperinflammatory states can lead to septic shock. Lipopolysaccharide (LPS) is a component of gram‐negative bacteria that is widely used for inducing inflammatory responses in animal models. However, many of the critical molecular networks involved in the LPS response in humans remain largely unknown.ObjectiveTo better characterize the molecular underpinnings of acute inflammation in humans through real‐time biochemical and network appraisals of in vivo challenge with LPS.MethodsHealthy men were intravenously injected with 0.6 ng/kg LPS at Pennsylvania State University. Plasma markers of inflammation (IL‐6, TNFα, and CRP) were measured prior to and 1, 2, 4, 8, 24, and 72 h post injection. Untargeted metabolomic and lipidomic profiling using liquid chromatography tandem mass spectrometry was conducted on the plasma from 10 men. Deuterated internal standards (SPLASH, Avanti Polar Lipids) were added to plasma to facilitate semi‐quantitative analysis of individual lipid molecular species. Statistics were performed in R (version 1.1.463) and Microsoft Excel (version 16.30).ResultsOf the 1847 metabolites reliably detected, 265 remained significant (FDR‐adjusted p &lt; 0.05) after analysis. Of these metabolites, 197 increased, 51 decreased, and 17 both increased and decreased (depending on the point in the time course). Both metabolomics and lipidomics revealed that molecular species of lipid classes including phospholipids (PL), lyso‐PLs, sphingolipids (SL), diglycerides (DG), and triglycerides (TG) had largest number of metabolites altered after LPS challenge. Notably, levels of 50 lyso‐PLs including lyso‐phosphatidylcholine (PC) and lyso‐phosphatidylethanolamine (PE) molecular species were increased in plasma. Additionally, there were significant elevations in certain PL molecular species representing PC, PE, phosphatidylserine, and phosphatidylinositol. Levels of 29 molecular species of SL increased with the majority representing sphingomyelin and ceramide classes. Neutral lipids including DG and TG were also markedly elevated. Pathway analyses highlighted glycerophospholipid metabolism (p &lt; 0.0001) and sphingolipid metabolism (p &lt; 0.001) as highly significant. These data also suggest the activation of multiple lipases including phospholipase A2, phospholipase C and phospholipase D play a critical role in the inflammatory response induced by LPS.DiscussionIntravenous LPS injection profoundly alters lipid metabolism in healthy adult men. These data may facilitate the identification of biochemical lipid networks and the development of biomarkers associated with inflammatory health/disease status.Support or Funding InformationFunding source: NIH R01 AT008621

Protective effect of low-dose hydrocortisone on myocardium in early septic shock

To investigate the effects and mechanisms of low-dose hydrocortisone on myocardial injury in early septic shock rats. Seventy-two healthy male Sprague-Dawley (SD) rats were divided into Sham group, lipopolysaccharide (LPS) model group (LPS group) and low dose hydrocortisone intervention group (LD group) according to the random number table method, with 24 rats in each group. The rat model of septic shock was produced by intravenous injection of LPS at 20 mg/kg. Sham group was injected with an equal amount of physiological saline. The LD group was injected 5 mg/kg of hydrocortisone via right femoral vein after model establishment. Sham group and LPS group were injected with an equal amount of physiological saline. Blood pressure and heart rate (HR) of rats in each group were continuously monitored. In each group, 8 rats were sacrificed for arterial blood gas analysis at 0, 3 and 6 hours after model establishment, and the level of plasma N-terminal B-type brain natriuretic peptide precursor (NT-proBNP) was detected by enzyme linked immunosorbent assay (ELISA). The expressions of cleaved-caspase-3 and nuclear factor-κB p65 (NF-κB p65) were detected by Western Blot. Myocardial tissue was harvested 6 hours after model establishment, the histopathological changes were observed by hematoxylin eosin (HE) staining, and the apoptosis rate of myocardial cells was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL). After LPS injection, mean arterial pressure (MAP) decreased significantly at 1 hour, then gradually increased, and was significantly higher than Sham group at 6 hours. There was no significant change in HR, and the difference was not statistically significant compared with Sham group. Blood lactic acid (Lac), base excess (BE), plasma NT-proBNP level, myocardial tissue caspase-3 and NF-κB p65 expression increased with the extension of time, all reach the peak in 6 hours, and significantly higher than Sham group. After early treatment with low-dose hydrocortisone in septic shock, MAP showed an increasing trend and Lac, BE decreased slowly. At 6 hours, MAP, Lac and BE were significantly lower than those in the LPS group [MAP (mmHg, 1 mmHg = 0.133 kPa): 98.6±7.5 vs. 106.1±8.5, Lac (mmol/L): 1.29±0.08 vs. 2.42±0.37, BE (mmol/L): 4.45±0.57 vs. 8.18±1.03, all P < 0.05]. The level of plasma NT-proBNP, and the expressions of caspase-3 and NF-κB p65 in myocardial tissue were significantly lower than those in LPS group at 3 hours and 6 hours after low-dose hydrocortisol treatment [NT-proBNP (ng/L): 2 740.56±97.31 vs. 4 425.60±743.32 at 3 hours, 2 638.81±205.12 vs. 4 993.01±373.78 at 6 hours; caspase-3/GAPDH: 0.567±0.045 vs. 0.841±0.162 at 3 hours, 0.496±0.071 vs. 1.116±0.172 at 6 hours; NF-κB p65/GAPDH: 0.852±0.734 vs. 1.232±0.115 at 3 hours, 0.783±0.047 vs. 1.383±0.215 at 6 hours, all P < 0.05]. HE staining results showed that myocardial cells in the LPS group were broken and inflammatory cells infiltrated. The myocardial histopathological changes in LD group were significantly less than those in LPS group. TUNEL staining showed that the apoptosis of myocardial cells in LPS group increased, and the apoptosis rate was significantly higher than that in Sham group [(82.41±1.57)% vs. (5.77±0.69)%, P < 0.05]. The apoptosis rate in LD group was significantly lower than that in LPS group [(27.82±1.77)% vs. (82.41±1.57)%, P < 0.05]. Low-dose hydrocortisone plays a protective role in the myocardial injury of early septic shock, and its mechanism may be related to the inhibition of caspase-3 and NF-κB p65 expression, the reduction of apoptosis rate and myocardial inhibition.

Biological motion during inflammation in humans

Biological motion is a powerful perceptual cue that can reveal important information about the inner state of an individual. Activation of inflammatory processes likely leads to changes in gait, posture, and mobility patterns, but the specific characteristics of inflammation-related biological motion have not been characterized. The aim of this study was to determine the effect of inflammation on gait and motion in humans. Systemic inflammation was induced in 19 healthy volunteers with an intravenous injection of lipopolysaccharide (2 ng/kg body weight). Biological motion parameters (walking speed, stride length and time, arm, leg, head, and shoulder angles) were assessed during a walking paradigm and the timed-up-and-go test. Cytokine concentrations, body temperature, and sickness symptoms were measured. During inflammation, compared to placebo, participants exhibited shorter, slower, and wider strides, less arm extension, less knee flexion, and a more downward-tilting head while walking. They were also slower and took a shorter first step in the timed-up-and-go test. Higher interleukin-6 concentrations, stronger sickness symptoms, and lower body temperature predicted the inflammation-related alterations in biological motion. These findings show that biological motion contains clear information about the inflammatory status of an individual, and may be used by peers or artificial intelligence to recognize that someone is sick or contagious.

Effects of intrauterine infusion of bacterial lipopolysaccharides on the mammary gland inflammatory response in goats

This study aimed to determine if intrauterine-infused lipopolysaccharides (LPS) can be translocated to the mammary glands and induce an inflammatory response. Thirty-seven goats were divided into two experiments. Nineteen goats (control group, n = 9; LPS group, n = 10) were subjected to intravenous injection of LPS, and eighteen goats (control group, n = 8; LPS group, n = 10) were subjected to intrauterine infusion of LPS. Milk and blood samples were collected before and after the LPS challenge, to measure the blood leukocyte count (BLC), plasma LPS-binding protein (LBP), milk yield, milk somatic cell count (SCC), lactoferrin (LF), milk lactoperoxidase (LPO) activity, and pro- and anti-inflammatory cytokines in plasma and milk. Mammary gland tissues were collected from the parenchyma before and after the LPS challenge, for immunohistochemistry of LPS. In the intravenous injection experiment, the BLC (P < 0.001) and milk yield (P = 0.009) were lower, whereas the LF concentration (P < 0.001) and milk LPO activity (P < 0.001) were higher in the LPS group compared to that in the control group. LPS was detected in the mammary gland 3 and 24 h after intravenous injection of LPS. In the intrauterine infusion experiment, the mean concentrations of IL-1β and IL-6 in milk were higher in the LPS group compared to that in the control group (P = 0.004 and P = 0.017, respectively), whereas there were no changes in milk yield or SCC. LPS was detected in the connective tissues and interepithelial spaces of the alveoli of the mammary glands 24 h after intrauterine infusion of LPS. We conclude that intrauterine-infused LPS can be translocated to the mammary glands from the uterus, however, the amount of translocated LPS might not be enough to induce symptoms of clinical or subclinical mastitis.

Dexmedetomidine-mediated protection against septic liver injury depends on TLR4/MyD88/NF-κB signaling downregulation partly via cholinergic anti-inflammatory mechanisms

BackgroundUncontrolled inflammatory responses exacerbate the pathogenesis of septic acute liver injury (ALI), posing a lethal threat to the host. Dexmedetomidine (DEX) has been reported to possess protective properties in inflammatory conditions. This study aimed to investigate whether DEX pretreatment exhibits hepatoprotection against ALI induced by lipopolysaccharide (LPS) in rats and determine its possible molecular mechanism. MethodsSeptic ALI was induced by intravenous injection of LPS. The rats received DEX intraperitoneally 30 min before LPS administration. α-Bungarotoxin (α-BGT), a specific α7 nicotinic acetylcholine receptor (α7nAChR) antagonist, was administered intraperitoneally 1 h before LPS exposure. The role of the vagus nerve was verified by performing unilateral cervical vagotomy or sham surgery before sepsis. ResultsThe expression of α7nAChR, toll-like receptor 4 (TLR4), high mobility group box 1 (HMGB1), and cleaved caspase-3 increased, peaking 24 h during sepsis. DEX enhanced α7nAChR activation and reduced TLR4 expression upon challenge with LPS. DEX significantly prevented LPS-induced ALI, which was associated with increased survival, the mitigation of pathological changes, the attenuation of inflammatory cytokine expression and apoptosis, and the downregulation of TLR4/MyD88/NF-κB pathway. Moreover, the hepatoprotective effect of DEX was abolished by α-BGT. Further investigation established that vagotomy, compared to sham surgery, triggered more severe pathogenic manifestations and higher proinflammatory cytokine levels. The inhibitory effects of DEX were shown in sham-operated rats but not in vagotomized rats. ConclusionsOur data highlight the pivotal function of α7nAChR and intact vagus nerves in protecting against LPS-induced ALI through inhibiting the TLR4/MyD88/NF-κB signaling pathway upon pretreatment with DEX.

Irisin alleviates pulmonary epithelial barrier dysfunction in sepsis-induced acute lung injury via activation of AMPK/SIRT1 pathways

ObjectiveAlveolar epithelial barrier dysfunction in response to inflammatory reaction contributes to pulmonary edema in acute lung injury(ALI).Irisin,a newly-found myokine,exerts the anti-inflammatory effects. This study aims to investigate the protective effects of irisin on lipopolysaccharide (LPS)-induced ALIin vivo and in vitro, and to explore its underlying mechanism. MethodsMale SD rats and A549 cells were divided into 4 groups: control group, LPS group, Irisin pretreated group, and Irisin/Compound C(a special inhibitor of AMPK)-treated group. The ALI model was established by intravenous injection of LPS in rats, and LPS challenge in A549 cells. Pulmonary specimens were harvested for microscopic examination of the pathological changes, and the expression of AMPK,SIRT1,NF-κB, p66Shc and caspase-3 in lung tissues. The pulmonary permeability were examined by wet/dry lung weight ratio(W/D) and lung permeability index(LPI). The apoptotic index, and the expression of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), monocyte chemoattractant activating protein-1 (MCP-1), tight junctions (occludin,ZO-1) were determined both in lung tissue and A549 cells. ResultsIrisin alleviated lung histological changes and decreased pulmonary microvascular permeability in LPS-induced rats. Irisin up-regulated the expression of occludin, ZO-1,AMPK,SIRT1, down-regulated the expression of TNF-α,IL-1β,MCP-1,NF-κB, p66Shc caspase-3, and decreased the apoptotic index in LPS-induced rats and A549 cells. All these protective effects of irisin could be reversed by Compound C. ConclusionIrisin improved LPS-induced alveolar epithelial barrier dysfunction via suppressing inflammation and apoptosis, and this protective effect might be mediated by activating AMPK/SIRT1 pathways.

Antithrombin plus alpha-1 protease inhibitor does not affect coagulation and inflammation in two murine models of acute lung injury

BackgroundIn acute respiratory distress syndrome (ARDS), uncontrolled production of activators of coagulation and proinflammatory mediators results in a shift from an adequate local innate immune response to hypercoagulability and inflammation. This study aimed to investigate whether the protease inhibitors antithrombin (AT) and alpha-1 protease inhibitor (A1PI) may attenuate an exaggerated pulmonary immune response.MethodsLung injury was induced either by single intranasal administration of lipopolysaccharide (LPS) (5 mg/kg) in BALB/c mice or by combination of an intravenous injection of LPS (10 mg/kg) with subsequent injurious ventilation using high tidal volumes (12–15 ml/kg) for 4 h in RccHan Wistar rats. Animals received either a single bolus of AT (250 IU/kg) or A1PI (60 mg/kg) alone or in combination, with or without intravenous low-dose heparin (100 U/kg). Control animals received saline. Additional controls received neither LPS, nor ventilation, nor treatment. Endpoints were local and systemic markers of coagulation, e.g., thrombin–antithrombin complexes (TATc), and inflammation, e.g., interleukin-6.ResultsBoth lung injury models resulted in a pronounced immune response within the pulmonary compartment shown by elevated levels of markers of coagulation and inflammation. The two-hit lung injury model also induced profound systemic coagulopathy and inflammation. Monotherapy with AT or A1PI did not reduce pulmonary coagulopathy or inflammation in any lung injury model. Nor did combination therapy with AT and A1PI result in a decrease of coagulation or inflammatory parameters. AT markedly reduced systemic levels of TATc in the two-hit lung injury model. Systemic inflammation was not affected by the different interventions. Additional administration of heparin did not lead to macroscopic bleeding incidences.ConclusionsIn two different murine models of acute lung injury, neither single therapy with AT or A1PI nor combination of both agents attenuates the pronounced pulmonary coagulation or inflammatory response.

Comparison of the TLR4/NFκB and NLRP3 signalling pathways in major organs of the mouse after intravenous injection of lipopolysaccharide

Context: Lipopolysaccharide (LPS) is often used to induce immunoinflammatory reactions. TLR4/NFκB and NLRP3 signalling are major factors for inflammation. Dexamethasone (DXM) has an anti-immunoinflammatory effect.Objective: To investigate the inflammatory reaction in pathological changes of organs and the expression of inflammatory signalling during LPS infection.Materials and methods: ICR mice were divided into control group (n = 9), LPS group (n = 15) and LPS + DXM group (n = 14). LPS (10 mg/kg) was injected intravenously in LPS group and LPS + DXM group, normal saline was injected to the control group; DXM (0.5 mg/kg) was given by intragastric administration. 12 h after LPS, the blood was collected and the organs were isolated for biochemical analysis, protein expression, and morphological examination.Results: The results showed that BUN, Cre, ALT, AST in the LPS group increased distinctly by 81.42, 67.84, 40.53 and 36.05%, respectively, and CK, ALP, TP and ALB decreased by 71.37, 60.6, 12.57 and 19.73%, respectively, compared with the control group. In the morphologic observation, local necrosis in the liver, arterial vasodilation in the heart and kidney, alveolar secretions and pulmonary interstitial in the lungs, and mucosal shedding in the small and large intestines, the expression of TLR4-NFκB signalling were up-regulated distinctly whereas NLRP3 signalling was less broadly affected. DXM can decrease BUN and Cre, downregulate the expression of TLR4-NFκB signalling, but has no effect on the organ damage based on morphology.Conclusion: Acute injuries induced by LPS are extensive. The inflammatory damage in small and large intestines, liver and kidney was more severe than other organs. TLR4-NFκB signalling was the major response to LPS stress.

Platelet α-granules modulate the inflammatory response under systemic lipopolysaccharide injection in mice.

Beyond their role in hemostasis and thrombosis, platelets are also important mediators of inflammation by the release of hundreds of factors stored in their α-granules. Mutations in Nbeal2 cause gray platelet syndrome (GPS), characterized by the lack of platelet α-granules. This study aims to evaluate the immunological (proinflammatory) effects of platelet α-granules. We performed an experiment using Nbeal2-/- mice, the mouse model of GPS. Systemic inflammation was induced by intravenous injection of lipopolysaccharide (LPS). Inflammatory response was assessed by quantification of inflammatory soluble factors and platelet biological response modifiers. The lack of Nbeal2 (in Nbeal2 -/- mice, compared with controls) significantly reduced the recruitment of circulating neutrophils and monocytes. Moreover, after LPS injection, there was a significant increase in neutrophil and monocyte counts in control animals, compared with Nbeal2 -/- mice. The control of inflammation, evaluated by the production of anti-inflammatory cytokines, appeared to be greater in Nbeal2-/- mice compared with controls. Conversely, the production of certain inflammatory-soluble mediators known to characterize normal platelet secretion, such as soluble CD40 ligand (sCD40L), was decreased under experimental inflammation in Nbeal2 -/- mice. These results show that α-granules play a direct role in platelet-mediated inflammation balance, confirming the need to further investigate platelet-associated inflammatory pathophysiology and inflammatory adverse events related to blood transfusion.

265 - Electron paramagnetic resonance detection of nitric oxide produced in different tissues during fever and antipyresis

Fever is a regulated increase in body temperature and a component of the acute phase response, triggered mainly after the invasion of pathogens in the body. The nitric oxide (NO•) is a molecule abundant in the body that acts as an important signaling molecule in several biological processes, such as neurotransmission, smooth muscle relaxation, blood pressure regulation, defense mechanisms, and may also be related to the signaling process during fever. Wistar rats received oral pre-treatment with dipyrone, ibuprofen, celecoxib or acetaminophen 30 min prior to intravenous injection of lipopolysaccharide (LPS) or vehicle, which led to a reduction in fever in all treated animals. The animals were euthanized 5 h after the administration of LPS. For the estimation of the production of NO•, the concentration of nitrosyl hemoglobin (HbNO) in the blood was measured by Electron Paramagnetic Resonance. For the quantification of NO• in hypothalamus, liver and brown adipose tissue, rats received injection of iron-citrate and DETC 1 h prior the euthanasia. The concentration of HbNO was 15-fold higher in the animals of LPS group compared to controls. Among the treated groups, only dipyrone and acetaminophen reduced the concentration of HbNO (-66% and -73%, respectively). The administration of iron-citrate and DETC inhibited the fever in rats, while it was observed an increase in the Fe-DETC-NO signal in liver and brown adipose tissue of these animals, which was prevented with the treatment with dipyrone and acetaminophen. These data suggest that there is a higher amount of NO• produced in peripheral tissues during fever, which may contributes for the maintenance of fever, and that reduction of the bioavailability of NO• by dipyrone or acetaminophen may contribute to the mechanism of action of these drugs.

Effects of dexmedetomidine on alveolar fluid clearance in rats with acute lung injury induced by lipopolysaccharide

Objective To investigate effects of dexmedetomidine(Dex) on alveolar fluid clearance (AFC) in rats with acute lung injury induced by lipopolysaccharide (LPS). Methods Forty-eight male Wistar rats were randomly divided into normal saline control (NS) group, LPS group, and Dex treatment (LPS+Dex) group. The rats in NS group were intravenously injected with 0.9% normal saline (5 ml/kg) followed by influsion of 0.9% normal saline (5 μg·kg-1·h-1). The rats in LPS group were intravenously injected with LPS (10 mg/kg) followed by influsion of 0.9% normal saline (5 μg·kg-1·h-1). The rats in LPS+Dex group were intravenously injected with LPS (10 mg/kg) followed by influsion of Dex (5 μg·kg-1·h-1). The rats were sacrificed 6 h after intravenous injection of LPS or NS, PaO2 was measured, AFC was determined by Evans blue-tagged albumin concentration changes. Histology of the lungs were assayed with H-E staining. The wet/dry weight ratio (W/D) of the lung was determined. The levels of TNF-α and IL-1β in the lung tissues were assessed by ELISA. The protein levels of potassium sodium adenosine triphosphate enzyme (Na-K-ATPase), alveolar epithelial sodium channel (ENaC) were detected by Western blot and immunohistochemistry. Results Compared with NS group, PaO2 was lower in LPS group, and this drop was reversed by Dex (in LPS+Dex group)(P<0.01). The W/D and the concentrations of TNF-α and IL-1β in the pulmonary tissues were increased in the LPS group, relative to the NS group, but the increases were compromised in the LPS+Dex group(P<0.01). AFC was reduced in LPS group, relative to NS group, but the reduction was compensat in LPS+Dex group(P<0.01). The expression of Na-K-ATPase and ENaC was lower in the LPS group than the NS group, but they were normalized in the LPS+Dex group(P<0.01). Conclusions Our findings suggest that dexmedetomidine may alleviate pulmonary edema by increasing AFC and the expression of Na-K-ATPase and ENaC in rats with acute lung injury induced by lipopolysaccharide. Key words: Dexmedetomidine; Acute lung injury; Alveolar fluid clearance; Potassium sodium adenosine triphosphate enzyme; Alveolar epithelial sodium channel

The macrophages in the pancreatic islets are activated and sense pathogen associated products in blood

Abstract Using RNAseq, we examined the transcriptome of endocrine macrophage in the islets of Langerhans in NOD (non-obese diabetic) and in two non-diabetic controls: NOD.Rag1−/− and B6.g7. The NOD mice spontaneously develop diabetes at 20–24 weeks of age. By gene expression analysis we found that at 3 weeks of age the macrophages from all 3 strains of mice were already activated. They expressed high levels of MHC-II, costimulatory molecules, and the pro-inflammatory cytokines IL-1b, TNF, both at protein and transcript levels. Leveraging the ImmGen database, we found that this activation signature is common for resident macrophages from barrier surfaces of the body, in particular lung and gastrointestinal tract. In addition to the basal level activation found in all islet macrophages at 3 weeks of age, we found an elevated activation signature in NOD when comparing to non-diabetic control. This finding indicates that the diabetic inflammatory process was already underway at as early as 3 weeks of age. These results were confirmed by single cell qRT-PCR that showed a distinct activated population of Cxcl9, Ccl5, Ly6a high (~28%) macrophages in NOD, but not in NOD.Rag1−/−. Additionally, supporting the idea of a barrier phenotype in islet macrophages, we found that they responded to intravenous injection of lipopolysaccharide by rapid upregulation of inflammatory gene expression. In imaging experiments we showed that macrophages were constantly extending filopodias into the blood lumen and capturing microparticles. In conclusion, the islet macrophages are in a highly activated steady state, sense pathogen associated products in blood, and may have a protective barrier function in the endocrine pancreas.

The Influence of Intravenous Lipopolysaccharide Injection on TLR4 Transcription Levels in Duck (Anas domestica) Liver, Spleen and Bursa of Fabricius

The Influence of Intravenous Lipopolysaccharide Injection on TLR4 Transcription Levels in Duck (Anas domestica) Liver, Spleen and Bursa of Fabricius

Cordycepin alleviates lipopolysaccharide-induced acute lung injury via Nrf2/HO-1 pathway

AimsThe present study is to investigate the protective effect of cordycepin on inflammatory reactions in rats with acute lung injury (ALI) induced by lipopolysaccharide (LPS), as well as the underlying mechanism. MethodsWistar rat model of ALI was induced by intravenous injection of LPS (30 mg/kg body weight). One hour later, intravenous injection of cordycepin (1, 10 or 30 mg/kg body weight) was administered. The wet-to-dry weight ratio of lung tissues and myeloperoxidase activity in the lung tissues were measured. The contents of nitrite and nitrate were measured by reduction method, while chemiluminescence was used to determine the content of superoxide. Quantitative real-time polymerase chain reaction and Western blotting were used to determine the expression of mRNA and protein, respectively. Colorimetry was performed to determine the enzymatic activity of heme oxygenase-1 (HO-1). Nuclear translocation of Nrf2 was identified by Western blotting. The plasma contents of cytokines were measured by enzyme-linked immunosorbent assay. ResultsCordycepin enhanced the expression and enzymatic activity of HO-1 in ALI rats, and activated Nrf2 by inducing the translocation of Nrf2 from cytoplasm to nucleus. In addition, cordycepin regulated the secretion of TNF-α, IL-6 and IL-10 via HO-1, and suppressed inflammation in lung tissues of ALI rats by inducing the expression of HO-1. HO-1 played important roles in the down-regulation of superoxide levels in lung tissues by cordycepin, and HO-1 expression induced by cordycepin affected nitrite and nitrate concentrations in plasma and iNOS protein expression in lung tissues. Cordycepin showed protective effect on injuries in lung tissues. ConclusionThe present study demonstrates that cordycepin alleviates inflammation induced by LPS via the activation of Nrf2 and up-regulation of HO-1 expression.