Stattic ameliorates the cecal ligation and puncture-induced cardiac injury in septic mice via IL-6-gp130-STAT3 signaling pathway.

AIM: Sepsis-induced cardiac dysfunction is the leading cause of higher morbidity and mortality with poor prognosis in septic patients. Our recent previous investigation provides evidence of the hallmarks of signal transducer and activator of transcription3 (STAT3) activation in sepsis and targeting of STAT3 with Stattic, a small-molecule inhibitor of STAT3, has beneficial effects in various septic tissues. We investigated the possible cardioprotective effects of Stattic on cardiac inflammation and dysfunction in mice with cecal ligation and puncture (CLP)-induced sepsis. MAIN METHODS: A polymicrobial sepsis model was induced by CLP in mice and Stattic (25 mg/kg) was intraperitoneally given at one and twelve hours after CLP operation. The cecum was exposed in sham-control mice without CLP. After 18 h of surgery, electrocardiogram (ECG) for anaesthized mice was registered followed by collecting of samples of blood and tissues for bimolecular and histopathological assessments. Myeloperoxidase, a marker of neutrophil infiltration, was assessed immunohistochemically. KEY FINDINGS: CLP profoundly impaired cardiac functions as evidenced by ECG changes in septic mice as well as elevation of cardiac enzymes, and inflammatory markers with myocardial histopathological and immunohistochemical alterations. While, Stattic markedly reversed the CLP-induced cardiac abnormalities and restored the cardiac function by its anti-inflammatory activities. SIGNIFICANCE: Stattic treatment had potential beneficial effects against sepsis-induced cardiac inflammation, dysfunction and damage. Its cardioprotective effects were possibly attributed to its anti-inflammatory activities by targeting STAT3 and downregulation of IL-6 and gp130. Our investigations suggest that Stattic could be a promising target for management of cardiac sepsis and inflammation-related cardiac damage.

Targeting sting to reduce sepsis-induced acute intestinal injury.

BACKGROUND: Sepsis is a dysregulated host response to infection syndrome leading to life-threatening organ dysfunction. Sepsis-induced intestinal dysfunction is a key element in the progression to multisystem organ failure. The stimulator of interferon genes is an intracellular protein implicated in intestinal injury in sepsis. H151, a small molecule inhibitor of stimulator of interferon genes, has not yet been studied as a potential therapeutic in sepsis. We hypothesize that H151 therapeutically reduces sepsis-induced acute intestinal injury. METHODS: Male mice underwent cecal ligation and puncture and were treated with intraperitoneal H151 (10 mg/kg body weight) or vehicle. Intestines and serum were collected for analysis 20 hours after cecal ligation and puncture. Oral gavage of mice with FITC-dextran was performed 15 hours after cecal ligation and puncture. Five hours after gavage, serum was collected, and intestinal permeability was assessed. Mice were monitored for 10 days after cecal ligation and puncture to assess survival. RESULTS: Zonula occludens 1 tight junctional protein expression was reduced after cecal ligation and puncture and recovered with H151 treatment. This was associated with a 62.3% reduction in intestinal permeability as assessed by fluorimetry. After cecal ligation and puncture, treatment with H151 was associated with a 58.7% reduction in intestinal histopathologic injury (P < .05) and a 56.6% reduction in intestinal apoptosis (P < .05). Intestinal myeloperoxidase activity was decreased by 70.8% after H151 treatment (P < .05). Finally, H151 improved 10-day survival from 33% to 80% after cecal ligation and puncture (P = .011). CONCLUSION: H151, a novel stimulator of interferon genes inhibitor, reduces intestinal injury, inflammation, and permeability when administered as a treatment for cecal ligation and puncture-induced sepsis. Thus, targeting stimulator of interferon genes shows promise as a therapeutic strategy to ameliorate sepsis-induced acute intestinal injury.

Critical roles of sepsis-reshaped fecal virota in attenuating sepsis severity.

Because studies on all fecal organisms (bacteria, fungi, and viruses) in sepsis are rare and bacteriophages during sepsis might have adapted against gut bacteria with possible pathogenicity, cecal ligation and puncture (CLP; a sepsis mouse model) was evaluated. In fecal bacteriome, sepsis increased Bacteroides and Proteobacteria but decreased Firmicutes, while fecal virome demonstrated increased Podoviridae when compared with sham feces. There was no difference in the fungal microbiome (predominant Ascomycota in both sham and CLP mice) and the abundance of all organisms between sepsis and control groups. Interestingly, the transfers of feces from CLP mice worsened sepsis severity when compared with sham fecal transplantation, as evaluated by mortality, renal injury (serum creatinine and histology), liver damage (liver enzyme and histology), spleen apoptosis, serum cytokines, endotoxemia, and bacteremia. In contrast, the transfers of fecal viral particles from sepsis mice, but not from sham mice, attenuated inflammation in CLP sepsis possibly through the decrease in several fecal pathogenic bacteria (such as Proteobacteria, Gammaproteobacteria, and Prevotellaceae) as evaluated by fecal microbiome analysis. Perhaps the isolation of favorable bacteriophages in sepsis feces and increased abundance ex vivo before oral treatment in a high concentration are beneficial.

Vaspin Alleviates Sepsis-Induced Cardiac Injury and Cardiac Inflammation by Inhibiting Kallikrein 7 in Mice.

Background: Vaspin is an important adipokine that is involved in cardiovascular diseases. This study is aimed at investigating whether vaspin participates in sepsis-induced cardiac injury and explored the possible mechanism. Methods: First, cecal ligation and puncture (CLP) and lipopolysaccharide (LPS) were used to establish a mouse model of sepsis, and cardiac vaspin expression was examined. In addition, after pretreatment with vaspin or phosphate-buffered saline (PBS), wild-type (WT) mice underwent CLP to establish a septic model and received sham as a control. Finally, WT mice and kallikrein 7 (KLK7-/-) mice were underwent CLP with or without vaspin pretreatment. Results: Mice that underwent CLP and were administered LPS exhibited increased vaspin expression in both the heart and serum compared with sham- or saline-treated mice. In CLP mice, pretreatment with vaspin reduced mortality and alleviated the expression of cardiac injury markers and cardiac dysfunction. In addition, vaspin reduced the cardiac levels of CD45+ cells and CD68+ cells, alleviated the cardiac inflammatory response, and reduced cardiomyocyte apoptosis. The protective effects of vaspin on CLP mice were masked by the deletion of KLK7, which was demonstrated to be a downstream signal of vaspin. Conclusions: Vaspin alleviates cardiac inflammation and plays a protective role in sepsis-induced cardiac injury by reducing KLK7 expression.

Machine learning and murine models explain failures of clinical sepsis trials.

BACKGROUND: Multiple clinical trials failed to demonstrate the efficacy of hydrocortisone, ascorbic acid, and thiamine (HAT) in sepsis. These trials were dominated by patients with pulmonary sepsis and have not accounted for differences in the inflammatory responses across varying etiologies of injury/illness. Hydrocortisone, ascorbic acid, and thiamine have previously revealed tremendous benefits in animal peritonitis sepsis models (cecal ligation and puncture [CLP]) in contradiction to the various clinical trials. The impact of HAT remains unclear in pulmonary sepsis. Our objective was to investigate the impact of HAT in pneumonia, consistent with the predominate etiology in the discordant clinical trials. We hypothesized that, in a pulmonary sepsis model, HAT would act synergistically to reduce end-organ dysfunction by the altering the inflammatory response, in a unique manner compared with CLP. METHODS: Using Pseudomonas aeruginosa pneumonia, a pulmonary sepsis model (pneumonia [PNA]) was compared directly to previously investigated intra-abdominal sepsis models. Machine learning applied to early vital signs stratified animals into those predicted to die (pDie) versus predicted to live (pLive). Animals were then randomized to receive antibiotics and fluids (vehicle [VEH]) vs. HAT). Vitals, cytokines, vitamin C, and markers of liver and kidney function were assessed in the blood, bronchoalveolar lavage, and organ homogenates. RESULTS: PNA was induced in 119 outbred wild-type Institute of Cancer Research mice (predicted mortality approximately 50%) similar to CLP. In PNA, interleukin 1 receptor antagonist in 72-hour bronchoalveolar lavage was lower with HAT (2.36 ng/mL) compared with VEH (4.88 ng/mL; p = 0.04). The remaining inflammatory cytokines and markers of liver/renal function showed no significant difference with HAT in PNA. PNA vitamin C levels were 0.62 mg/dL (pDie HAT), lower than vitamin C levels after CLP (1.195 mg/dL). Unlike CLP, PNA mice did not develop acute kidney injury (blood urea nitrogen: pDie, 33.5 mg/dL vs. pLive, 27.6 mg/dL; p = 0.17). Furthermore, following PNA, HAT did not significantly reduce microscopic renal oxidative stress (mean gray area: pDie, 16.64 vs. pLive, 6.88; p = 0.93). Unlike CLP where HAT demonstrated a survival benefit, HAT had no impact on survival in PNA. CONCLUSION: Hydrocortisone, ascorbic acid, and thiamine therapy has minimal benefits in pneumonia. The inflammatory response induced by pulmonary sepsis is unique compared with the response during intra-abdominal sepsis. Consequently, different etiologies of sepsis respond differently to HAT therapy.

c-Abl kinase regulates neutrophil extracellular trap formation and lung injury in abdominal sepsis.

Sepsis is associated with exaggerated neutrophil responses although mechanisms remain elusive. The aim of this study was to investigate the role of c-Abelson (c-Abl) kinase in neutrophil extracellular trap (NET) formation and inflammation in septic lung injury. Abdominal sepsis was induced by cecal ligation and puncture (CLP). NETs were detected by electron microscopy in the lung and by confocal microscopy in vitro. Plasma levels of DNA-histone complexes, interleukin-6 (IL-6) and CXC chemokines were quantified. CLP-induced enhanced phosphorylation of c-Abl kinase in circulating neutrophils. Administration of the c-Abl kinase inhibitor GZD824 not only abolished activation of c-Abl kinase in neutrophils but also reduced NET formation in the lung and plasma levels of DNA-histone complexes in CLP mice. Moreover, inhibition of c-Abl kinase decreased CLP-induced lung edema and injury. Administration of GDZ824 reduced CLP-induced increases in the number of alveolar neutrophils. Inhibition of c-Abl kinase also markedly attenuated levels of CXC chemokines in the lung and plasma as well as IL-6 levels in the plasma of septic animals. Taken together, this study demonstrates that c-Abl kinase is a potent regulator of NET formation and we conclude that c-Abl kinase might be a useful target to ameliorate lung damage in abdominal sepsis.

Metformin attenuates sepsis-induced neuronal injury and cognitive impairment.

BACKGROUND: Sepsis is considered to be a high-risk factor for cognitive impairment in the brain. The purpose of our study is to explore whether sepsis causes cognitive impairment and try to evaluate the underlying mechanisms and intervention measures. METHODS: Here, we used cecum ligation and puncture (CLP) to simulate sepsis. Open field, Novel Objective Recognition, and Morris Water Maze Test were used to detect cognitive function, long-term potentiation was used to assess of synaptic plasticity, and molecular biological technics were used to assess synaptic proteins, ELISA kits were used to detect inflammatory factors. Metformin was injected into the lateral ventricle of SD rats, and we evaluated whether metformin alleviated CLP-mediated cognitive impairment using behavioral, electrophysiological and molecular biological technology experiments. RESULTS: Here we report hippocampal-dependent cognitive deficits and synaptic dysfunction induced by the CLP, accompanied by a significant increase in inflammatory factors. At the same time, metformin was able to improve cognitive impairment induced by CLP in adult male rats. CONCLUSION: These findings highlight a novel pathogenic mechanism of sepsis-related cognitive impairment through activation of inflammatory factors, and these are blocked by metformin to attenuate sepsis-induced neuronal injury and cognitive impairment.

Transcriptomic Analysis Reveals Differential Expression of Genes between Lung Capillary and Post Capillary Venules in Abdominal Sepsis.

Lung endothelial cell dysfunction plays a central role in septic-induced lung injury. We hypothesized that endothelial cell subsets, capillary endothelial cells (capEC) and post capillary venules (PCV), might play different roles in regulating important pathophysiology in sepsis. In order to reveal global transcriptomic changes in endothelial cell subsets during sepsis, we induced sepsis in C57BL/6 mice by cecal ligation and puncture (CLP). We confirmed that CLP induced systemic and lung inflammation in our model. Endothelial cells (ECs) from lung capillary and PCV were isolated by cell sorting and transcriptomic changes were analyzed by bioinformatic tools. Our analysis revealed that lung capEC are transcriptionally different than PCV. Comparison of top differentially expressed genes (DEGs) of capEC and PCV revealed that capEC responses are different than PCV during sepsis. It was found that capEC are more enriched with genes related to regulation of coagulation, vascular permeability, wound healing and lipid metabolic processes after sepsis. In contrast, PCV are more enriched with genes related to chemotaxis, cell-cell adhesion by integrins, chemokine biosynthesis, regulation of actin filament process and neutrophil homeostasis after sepsis. In addition, we predicted some transcription factor targets that regulate a significant number of DEGs in sepsis. We proposed that targeting certain DEGs or transcriptional factors would be useful in protecting against sepsis-induced lung damage.

Activation of CB2 receptor inhibits pyroptosis and subsequently ameliorates cecal ligation and puncture-induced sepsis.

BACKGROUND: Cannabinoid receptor 2 (CB2), whose activities are upregulated during sepsis, may be related to the regulation of inflammatory programmed cell death called pyroptosis. The aim of this study is to investigate the role of CB2 activation in attenuation of inflammation through inhibiting pyroptosis in cecal ligation puncture (CLP)-induced sepsis andlipopolysaccharide (LPS) + ATP-stimulated macrophages. METHODS: C57BL/6 mice were subjected to CLP procedure and treated with CB2 agonist HU308 and CB2 antagonist AM630. Lung tissues were collected for analyses of lung W/D ratio, inflammatory factors levels, and pyroptosis-related protein expression. Murine bone-marrow-derived macrophages (BMDM) were treated with LPS and ATP to construct a septic model in vitro in the presence of HU308 and AM630 for assessment of cell injury, cytokine levels and pyroptosis-related protein expression accordingly. To verify the relationship between CB2 receptors and pyroptosis in the process of inflammatory response, BMDM were transduced with CB2 receptors knockdown lentiviral vectors in the presence of HU308 and AM630 for assessment of pyroptosis-related protein expression. RESULTS: CB2 activation ameliorated the release of inflammatory mediators. The results showed that CLP-induced pyroptosis was elevated, and CB2 agonist HU308 treatment inhibited the pyroptosis activity through a decrease of the protein levels of NLRP3 as well as caspase-1 and GSDMD activation. Similar results were obtained in BMDM after LPS and ATP treatment. Treatment with CB2 knockdown lentiviral particles prevented the HU308-induced decreases in cell pyroptosis, demonstrating that endogenous CB2 receptors are required for the cannabinoid-induced cell protection. CONCLUSIONS: CB2 receptors activation plays a protective role in sepsis through inhibition of pyroptosis. The effect of CB2 receptors against pyroptosis depends on the existence of endogenous CB2 receptors.

Senkyunolide I protect against lung injury via inhibiting formation of neutrophil extracellular trap in a murine model of cecal ligation and puncture.

BACKGROUND: Senkyunolide I (SEI), a component of a Chinese herb named Ligusticum Chuanxiong hort, which is included in the formulation of Xuebijing Injection, a medication used to treat sepsis in China. Our previous study showed that SEI was protective against sepsis-associated encephalopathy and the present study was performed to investigate the role of SEI in sepsis-induced lung injury in a murine model of cecal ligation and puncture (CLP). METHODS: SEI (36 mg/kg in 200 µl) or vehicle was administered immediately after CLP surgery. The lung injury was assessed 24 h later by histopathological tests, protein concentration in the bronchoalveolar lavage fluid (BALF), neutrophil recruitment in the lung tissue (myeloperoxidase fluorescence, MPO), pro-inflammatory cytokines and oxidative responses. Platelet activation was detected by CD42d/GP5 immunofluorescence and neutrophil extracellular trap (NET) were determined by immunofluorescence assays and enzyme linked immunosorbent assay (ELISA) of MPO-DNA. In vitro experiments were performed to detect the level of MPO-DNA complex released by SEI-treated neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) or co-cultured with platelets from CLP mice. RESULTS: SEI administration relieved the injury degree in CLP mice according to the histopathological tests (P < 0.05 compared with DMSO + CLP group). Protein level in the BALF and neutrophil infiltration were remarkably reduced by SEI after CLP surgery (P < 0.05 compared with DMSO + CLP group). TNF-α, IL-1ß and IL-6 were decreased in the plasma and lung tissues from CLP mice treated with SEI (P < 0.05 compared with DMSO + CLP group). The phosphorylation of JNK, ERK, p38 and p65 were all inhibited by SEI (P < 0.05 compared with DMSO + CLP group). Immunofluorescence of MPO showed that neutrophil number was significantly lower in SEI treated CLP mice than in vehicle treated CLP mice (P < 0.05). The CD42d/GP5 staining suggested that platelet activation was significantly reduced and the NET level in the lung tissue and plasma was greatly attenuated by SEI treatment (P < 0.05 compared with DMSO + CLP group). In vitro experiments showed that the MPO-DNA level stimulated by PMA was significantly reduced by SEI treatment (P < 0.05 compared with DMSO treatment). Co-culture neutrophils with platelets from CLP mice resulted in higher level of MPO-DNA complex, while SEI partly reversed such effects of platelet on NET formation. CONCLUSIONS: SEI was protective against lung injury induced by CLP in mice. The NET formation was significantly reduced by SEI treatment, which might be involved in the mechanism of the protective effect.

miR­214 ameliorates sepsis­induced acute kidney injury via PTEN/AKT/mTOR­regulated autophagy.

Previous studies have suggested that oxidative stress and autophagy results in acute kidney injury (AKI) during sepsis and microRNA (miR)­214 serves a vital role in the protection of kidneys subjected to oxidative stress. The present study aimed to test whether the renoprotection of miR­214 is related to autophagy in sepsis. The role of autophagy was investigated in a mouse model of cecal ligation and puncture (CLP). Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was used to analyze the expression of miR­214. The structure and function of kidneys harvested from the mice were evaluated. Kidney autophagy levels were detected with immunohistochemical, immunofluorescent and western blotting. It was found that miR­214 could alleviate AKI in septic mice by inhibiting the level of kidney autophagy. Furthermore, miR­214 inhibited autophagy by silencing PTEN expression in the kidney tissues of septic mice. These findings indicated that miR­214 ameliorated CLP­induced AKI by reducing oxidative stress and inhibiting autophagy through the regulation of the PTEN/AKT/mTOR pathway.

Atezolizumab alleviates the immunosuppression induced by PD­L1­positive neutrophils and improves the survival of mice during sepsis.

Atezolizumab can reduce immunosuppression caused by T lymphocyte apoptosis in various cancer types. The current study aimed to investigate whether this drug can also alleviate immunosuppression during sepsis. For that purpose, a C57BL/6 mouse sepsis model was generated. Mice were randomly assigned to three groups: Sham, cecal ligation and puncture (CLP) and atezolizumab groups. Atezolizumab was administered in vivo by intraperitoneal injection. The expression of programmed death ligand­1 (PD­L1) on neutrophils and programmed death­1 (PD­1) on T lymphocytes was evaluated, and endotoxin concentration, intestinal permeability, ileum histopathological score and tight junction protein expression were assessed to determine the extent of disease in each group. The rate of T lymphocyte apoptosis was determined to assess the effects of atezolizumab on T lymphocyte apoptosis in vivo and in vitro. Survival times were also recorded to compare mouse prognosis during sepsis. In the CLP group, the proportion of PD­L1+ neutrophils was significantly higher at 48, 72 and 96 h in blood, and at 24, 48, 72 and 96 h in bone marrow, compared with those of the sham group (P<0.05). The proportion of PD­1+ T lymphocytes was also upregulated at 72 h in blood. In the atezolizumab group, endotoxin concentration, intestinal permeability and ileum histopathological score were lower compared with those in the CLP group (P<0.05), whereas the expression of claudin­1 and occludin proteins on ileum was higher compared with that in the CLP group (P<0.05). Both in vivo and in vitro experiments indicated that the rate of T lymphocyte apoptosis following atezolizumab treatment was lower compared with that in the CLP group (P<0.05). Survival analysis demonstrated that mice in the atezolizumab group survived longer compared with those in the CLP group (P<0.05). The current study demonstrated that treatment with atezolizumab may be an effective method for treating immunosuppression induced by sepsis.

Astaxanthin Alleviates Ochratoxin A-Induced Cecum Injury and Inflammation in Mice by Regulating the Diversity of Cecal Microbiota and TLR4/MyD88/NF-κB Signaling Pathway.

Ochratoxin A (OTA) is a common environmental pollutant found in a variety of foods and grains, and excessive OTA consumption causes serious global health effects on animals and humans. Astaxanthin (AST) is a natural carotenoid that has anti-inflammatory, antiapoptotic, immunomodulatory, antitumor, antidiabetes, and other biological activities. The present study is aimed at investigating the effects of AST on OTA-induced cecum injury and its mechanism of action. Eighty C57 mice were randomly divided into four groups, including the control group, OTA group (5 mg/kg body weight), AST group (100 mg/kg body weight), and AST intervention group (100 mg/kg body weight AST+5 mg/kg body weight OTA). It was found that AST decreased the endotoxin content, effectively prevented the shortening of mouse cecum villi, and increased the expression levels of tight junction (TJ) proteins, consisting of occludin, claudin-1, and zonula occludens-1 (ZO-1). AST increased the number of goblet cells, the contents of mucin-2 (MUC2), and defensins (Defa5 and ß-pD2) significantly, while the expression of mucin-1 (MUC1) decreased significantly. The 16S rRNA sequencing showed that AST affected the richness and diversity of cecum flora, decreased the proportion of lactobacillus, and also decreased the contents of short-chain fatty acids (SCFAs) (acetate and butyrate). In addition, AST significantly decreased the expression of TLR4, MyD88, and p-p65, while increasing the expression of p65. Meanwhile, the expression of inflammatory factors including TNF-α and INF-γ decreased, while the expression of IL-10 increased. In conclusion, AST reduced OTA-induced cecum injury by regulating the cecum barrier function and TLR4/MyD88/NF-κB signaling pathway.

CD4 and CD8 T Cell Memory Interactions Alter Innate Immunity and Organ Injury in the CLP Sepsis Model.

The role of T cell memory in sepsis is poorly understood. Recent work has demonstrated that mice exposed to frequent antigenic stimulation, in contrast to laboratory mice, better recapitulate the human T cell repertoire. This difference may profoundly alter responses to inflammatory insults. We induced isolated T cell memory by inoculating C57Bl/6 mice with an anti-CD3ϵ activating antibody, a process we term "immune education." These mice were subjected to the cecal ligation and puncture (CLP) model of sepsis and responses were compared to those of isotype-treated controls. CLP-induced increases in 1) CD4 T cell production and serum levels of IFNγ, 2) CD8 T cell granzyme B levels, and 3) innate cell function were all more pronounced in educated mice than in control mice. Immune education increased CLP-induced liver injury and decreased survival. The differences in responses to CLP were not recapitulated in mice with either isolated CD4 or isolated CD8 T cell memory. Relative to controls, CLP in educated CD8-/- mice (isolated CD4 memory) increased monocyte-derived dendritic cells. Combined CD4 and CD8 memory did not increase monocyte-derived dendritic cells; this combination recapitulated increases in neutrophil and inflammatory monocyte numbers in educated wild-type mice. Induction of T cell memory prior to CLP alters immune responses, organ function, and survival. Both CD4 and CD8 memory T cells play important and independent roles in this response. These findings have profound implications for the development of murine models of human inflammatory disorders such as infection and sepsis.

Cardioprotective Effect of Tangeretin by Inhibiting PTEN/AKT/mTOR Axis in Experimental Sepsis-Induced Myocardial Dysfunction.

Sepsis aggregates undesirable immune response causing depression of ventricular myocardium and diastolic dysfunction. This present study examined the effect of a plant-derived flavone tangeretin (TG) on autophagy and reduction in myocardial dysfunction. The sepsis was induced by cecum ligation and puncture (CLP) in male Sprague-Dawley rats. Abnormal changes were seen in the heart after the sepsis induction. These abnormalities were analyzed based on the cardiac markers, namely Cardiac myosin light chain-1 (cMLC1) and Cardiac troponin I (cTnl), echocardiography, and plasma parameters, like Lactate dehydrogenase (LDH) and Creatinine kinase (CK). Microanatomy of the heart was studied using hematoxylin and eosin stained histopathological samples of cardiac tissue. Western blot technique was used to detect the nature and extent of protein with the amount of a specific RNA (gene expression) in the cardiac homogenate. Oxidative damage was analyzed using redox marker, reduced glutathione. This study successfully showed that TG attenuated sepsis-induced myocardial dysfunction by inhibiting myocardial autophagy via silencing the Phosphatase and tensin homolog (PTEN) expression and acting on the AKT/mTOR pathway. The present findings supported that TG is a novel cardioprotective therapeutic target for sepsis induced myocardial dysfunction.

Trained Innate Immunity by Repeated Low-Dose Lipopolysaccharide Injections Displays Long-Term Neuroprotective Effects.

Disrupted immune response is an important feature of many neurodegenerative conditions, including sepsis-associated cognitive impairment. Accumulating evidence has demonstrated that immune memory occurs in microglia, which has a significant impact on pathological hallmarks of neurological diseases. However, it remains unclear whether immune memory can cause subsequent alterations in the brain immune response and affect neurobehavioral outcomes in sepsis survivors. In the present study, mice received daily intraperitoneal injection of low-dose lipopolysaccharide (LPS, 0.1 mg/kg) for three consecutive days to induce immune memory (immune tolerance) and then were subjected to sham operation or cecal ligation and puncture (CLP) 9 months later, followed by a battery of neurobehavioral and biochemical studies. Here, we showed that repeated low-dose LPS injection-induced immune memory protected mice from sepsis-induced cognitive and affective impairments, which were accompanied by significantly decreased brain proinflammatory cytokines and immune response. In conclusion, our study suggests that modulation of brain immune responses by repeated LPS injections confers neuroprotective effects by preventing overactivated immune response in response to subsequent septic insult.

MicroRNA-34a Inhibition Alleviates Lung Injury in Cecal Ligation and Puncture Induced Septic Mice.

Sepsis is the leading cause of death in intensive care units. MicroRNA-34a (miR-34a) is involved in sepsis progression, while its underlying mechanisms on sepsis-induced lung injury remain obscure. Oxidative stress, pyroptosis, and inhibition of autophagy can result in organ injury. MiR-34a has been reported to regulate oxidative stress and autophagy via inhibiting silent information regulator T1 (SIRT1) and autophagy gene 4B (ATG4B) signaling. This study aimed at identifying the function of miR-34a in oxidative stress, inflammation, pyroptosis, and autophagy in sepsis-induced lung injury. Male 8-week-old C57BL/6 mice were subjected to cecal ligation and puncture and treated with miR-34a antagomir/agomir. Survival (n = 10), histopathological changes (n = 6), and lung wet-to-dry ratio (n = 6) were recorded and assayed. Other detection (n = 6) was performed to investigate the level of oxidative stress, inflammation, pyroptosis, and autophagy in lung tissues. Results showed that miR-34a down-regulation ameliorated lung injury in septic mice as reflected by decreased lung injury scores (decrease from 3.00 ± 0.32 to 2.00 ± 0.32) and wet-to-dry ratio (0.36-fold decrease). MiR-34a down-regulation also decreased reactive oxygen species accumulation (0.36-fold decrease), and promoted superoxide dismutase activity and the expression of SIRT1 (1.24-fold increase), heme oxygenase-1 and nuclear factor erythroid 2 like 2 to inhibit oxidative stress in septic mice. Moreover, miR-34a down-regulation suppressed inflammatory response and pyroptosis in septic mice, as evidenced by decreased level of pro-inflammatory factors including tumor necrosis factor α, interleukin-6 (IL-6), IL-1ß, and IL-18, activity of caspase-1 (0.51-fold decrease) and expression of nucleotide-binding domain and leucine-rich repeat protein-3 (0.48-fold decrease), apoptosis-associated speck-like protein containing a CARD, cleaved-caspase-1, and cleaved-gasdermin D (0.36-fold decrease), and increased level of anti-inflammatory factors IL-10. MiR-34a down-regulation also enhanced autophagy in septic mice as evidenced by more autolysosomes and elevated expressions of ATG4B (0.90-fold increase), beclin1, ATG9, and LC3 II/I. Among these experiments, miR-34a up-regulation showed opposite effects on oxidative stress, inflammatory response, pyroptosis, and autophagy in septic mice. Additionally, miR-34a could bind to the 3'-untranslated region of SIRT1 and ATG4B. In conclusion, our findings demonstrated that miR-34a was implicated in oxidative stress, inflammation, pyroptosis, and autophagy in the development of sepsis. MiR-34a inhibition had a potential to alleviate sepsis-induced lung injury.

A Non-canonical PDK1-RSK Signal Diminishes Pro-caspase-8-Mediated Necroptosis Blockade.

Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.

LncRNA-5657 silencing alleviates sepsis-induced lung injury by suppressing the expression of spinster homology protein 2.

BACKGROUND: LncRNAs are closely associated with many human major diseases, however, the roles of lncRNAs in sepsis-induced lung injury remain unclear. METHODS: High-throughput sequencing was used to detect the changes in lncRNA expression profile in alveolar macrophages after LPS stimulation. The BALF of patients with sepsis-induced lung injury was collected. Rats with CLP-induced septic lung injury were treated with sh-lncRNA-5657 via intravenous injection. NR8383 cells were transfected with lentiviral vectors expressing lncRNA-5657, lncRNA-5657 smart silencer, or si-spns2. The BALF cells expression levels of lncRNA-5657 and proinflammatory cytokines in BALF of patients and rats as well as in rat macrophages were measured. Changes in histopathologic score, lung wet/dry weight ratio, and spns2 expression in macrophages were examined. The relationship between lncRNA-5657 and its potential target gene spns2 was validated using a dual-luciferase reporter assay. RESULTS: The lncRNA expression profile of LPS-stimulated macrophages demonstrated that lncRNA-5657 showed the greatest fold-change. The BALF cells of patients with sepsis-induced ARDS and the lung tissue of rats with CLP-induced sepsis had significantly increased lncRNA-5657 levels. LncRNA-5657 silencing alleviated CLP-induced lung inflammation in rats. In NR8383 cells, lncRNA-5657 overexpression enhanced, whereas lncRNA-5657 silencing attenuated the expression of proinflammatory cytokines and spns2. A dual-luciferase reporter assay showed that lncRNA-5657 interacted with the promoter of the spns2 gene. Spns2 silencing alleviated LPS-induced inflammatory response and blocked the proinflammatory function of lncRNA-5657 in alveolar macrophages. CONCLUSION: LncRNA-5657 is closely associated with sepsis-induce lung injury. In vitro and in vivo data demonstrated that LncRNA-5657 silencing alleviates sepsis-induced lung injury by inhibiting lung inflammatory response via suppressing spns2 expression.