Pellino1 orchestrates gut-kidney axis to perpetuate septic acute kidney injury through activation of STING pathway and NLRP3 inflammasome.

AIMS: Intestinal barrier dysfunction is the initial and propagable factor of sepsis in which acute kidney injury (AKI) has been considered as a common life-threatening complication. Our recent study identifies the regulatory role of Pellino1 in tubular death under inflammatory conditions in vitro. The objective of our current study is to explore the impact of Pellino1 on gut-kidney axis during septic AKI and uncover the molecular mechanism (s) underlying this process. MATERIALS AND METHODS: Immunohistochemistry (IHC) was conducted to evaluate Pellino1 and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) levels in renal biopsies from critically ill patients with a clinical diagnosis of sepsis. Functional and mechanistic studies were characterized in septic models of the Peli-knockout (Peli1-/-) mice by histopathological staining, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunofluorescence, biochemical detection, CRISPR/Cas9-mediated gene editing and intestinal organoid. KEY FINDINGS: Pellino1, together with NLRP3, are highly expressed in renal biopsies from critically ill patients diagnosed with sepsis and kidney tissues of septic mice. The Peli1-/- mice with sepsis become less prone to develop AKI and have markedly compromised NLRP3 activation in kidney. Loss of Peli1 endows septic mice refractory to intestinal inflammation, barrier permeability and enterocyte apoptosis that requires stimulator of interferons genes (STING) pathway. Administration of STING agonist DMXAA deteriorates AKI and mortality of septic Peli1-/- mice in the presence of kidney-specific NLRP3 reconstitution. SIGNIFICANCE: Our studies suggest that Pellino1 has a principal role in orchestrating gut homeostasis towards renal pathophysiology, thus providing a potential therapeutic target for septic AKI.

Analysis of risk factors affecting the prognosis of patients with sepsis and construction of nomogram prediction model.

OBJECTIVE: This study analyzed the clinical data of 200 sepsis patients, exploring the risk factors that affect patient prognosis and providing the basis for clinically targeted intervention to improve patient prognosis. PATIENTS AND METHODS: 200 septic patients were admitted to Yulin Second Hospital, and they were divided into a survival group of 151 patients and a death group of 49 patients, according to their clinical outcomes on admission. The relevant clinical parameters within 24 h of admission were collected, and the independent risk factors affecting the prognosis of septic patients were analyzed by multivariate Logistic regression. R language 4.21 software was used to construct a nomogram prediction model. The receiver operating characteristic curve was used to evaluate the discrimination of the nomogram model, and decline curve analysis was drawn to evaluate the effectiveness of the model. RESULTS: In the nomogram prediction model, age, the Acute Physiology and Chronic Health Scoring System Domain (APACHE II) score, the Sequential Organ Failure Assessment (SOFA) score, C-reactive protein (CRP), total bilirubin, albumin (Alb), urea nitrogen, creatinine, and lactate (Lac) were independent risk factors for death in septic patients. The area under the receiver operating characteristic (ROC) curve for predicting the prognosis of septic patients was 0.597-1.000, and the calibration curve tends to be the ideal curve. The model had good discrimination and calibration and had high accuracy in evaluating septic patients. The modeling curves in the decline curve analysis (DCA) were all above the two extreme curves, which had good clinical value. CONCLUSIONS: Nine clinical variables have been found to be independent risk factors for death in septic patients. The prediction model established based on this has good accuracy, discrimination, and consistency in predicting the prognosis of sepsis patients.

4-phenylbutyric acid improves sepsis-induced cardiac dysfunction by modulating amino acid metabolism and lipid metabolism via Comt/Ptgs2/Ppara.

INTRODUCTION: Cardiac dysfunction after sepsis the most common and severe sepsis-related organ failure. The severity of cardiac damage in sepsis patients was positively associated to mortality. It is important to look for drugs targeting sepsis-induced cardiac damage. Our previous studies found that 4-phenylbutyric acid (PBA) was beneficial to septic shock by improving cardiovascular function and survival, while the specific mechanism is unclear. OBJECTIVES: We aimed to explore the specific mechanism and PBA for protecting cardiac function in sepsis. METHODS: The cecal ligation and puncture-induced septic shock models were used to observe the therapeutic effects of PBA on myocardial contractility and the serum levels of cardiac troponin-T. The mechanisms of PBA against sepsis were explored by metabolomics and network pharmacology. RESULTS: The results showed that PBA alleviated the sepsis-induced cardiac damage. The metabolomics results showed that there were 28 metabolites involving in the therapeutic effects of PBA against sepsis. According to network pharmacology, 11 hub genes were found that were involved in lipid metabolism and amino acid transport following PBA treatment. The further integrated analysis focused on 7 key targets, including Comt, Slc6a4, Maoa, Ppara, Pparg, Ptgs2 and Trpv1, as well as their core metabolites and pathways. In an in vitro assay, PBA effectively inhibited sepsis-induced reductions in Comt, Ptgs2 and Ppara after sepsis. CONCLUSIONS: PBA protects sepsis-induced cardiac injury by targeting Comt/Ptgs2/Ppara, which regulates amino acid metabolism and lipid metabolism. The study reveals the complicated mechanisms of PBA against sepsis.

Proteome characterization of liver-kidney comorbidity after microbial sepsis.

Sepsis is a life-threatening condition that occurs when the body responds to an infection but subsequently triggers widespread inflammation and impaired blood flow. These pathologic responses can rapidly cause multiple organ dysfunction or failure either one by one or simultaneously. The fundamental common mechanisms involved in sepsis-induced multiple organ dysfunction remain unclear. Here, employing quantitative global and phosphoproteomics, we examine the liver's temporal proteome and phosphoproteome changes after moderate sepsis induced by cecum ligation and puncture. In total, 4593 global proteins and 1186 phosphoproteins according to 3275 phosphosites were identified. To characterize the liver-kidney comorbidity after sepsis, we developed a mathematical model and performed cross-analyses of liver and kidney proteome data obtained from the same set of mice. Beyond immune response, we showed the commonly disturbed pathways and key regulators of the liver-kidney comorbidity are linked to energy metabolism and consumption. Our data provide open resources to understand the communication between the liver and kidney as they work to fight infection and maintain homeostasis.

Aldosterone levels do not predict 28-day mortality in patients treated for COVID-19 in the intensive care unit.

The immunotropic effects of aldosterone might play a role in COVID-19, as SARS-CoV-2 reportedly uses angiotensin-converting enzyme 2 receptors as an entry point into cells. Aldosterone function is closely linked to its action on mineralocorticoid receptors in kidneys; it increases the renal retention of sodium and the excretion of potassium, which increases blood pressure. Despite the large number of studies examining the effect of Ang-II and its blockers on the course of COVID-19 infection, there is still uncertainty about the role of aldosterone. The aim of the study was to assess the correlation of aldosterone, urea, creatinine, C-reactive protein (CRP), and procalcitonin (PCT) levels with 28 days of mortality in patients treated for COVID19 in an intensive care unit (ICU). This cross-selection study involved 115 adult patients who were divided into two groups: those who died within a 28-day period (n = 82) and those who survived (n = 33). The correlation of aldosterone, urea, creatinine, C-reactive protein (CRP), and procalcitonin (PCT) levels with 28 days of mortality in patients treated for COVID-19 were performed. The patients' age, sex, scores from the APACHE II, SAPS II, and SOFA scales and comorbidities like HA, IHD and DM were also analyzed. Remarkably, the individuals who survived for 28 days were of significantly lower mean age and achieved notably lower scores on the APACHE II, SAPS II, and SOFA assessment scales. Statistically significantly higher CRP levels were observed on days 3, 5, and 7 in individuals who survived for 28 days. Creatinine levels in the same group were also statistically significantly lower on days 1, 3, and 5 than those of individuals who died within 28 days. The investigation employed both univariate and multivariate Cox proportional hazard regression models to explore factors related to mortality. In the univariate analysis, variables with a p value of less than 0.50 were included in the multivariate model. Age, APACHE II, SAPS II, and SOFA demonstrated significance in univariate analysis and were considered to be associated with mortality. The outcomes of the multivariate analysis indicated that age (HR = 1.03, p = 0.033) served as a robust predictor of mortality in the entire study population. In conclusion the plasma aldosterone level is not associated with ICU mortality in patients with COVID-19. Other factors, including the patient's age, creatinine or CRP contribute to the severity and prognosis of the disease. This study was retrospectively registered in the Australian New Zealand Clinical Trials Registry (ANZCTR) with registration no. ACTRN12621001300864 (27/09/2021: https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=382563&isReview=true ).

TAK-242 improves sepsis-associated acute kidney injury in rats by inhibiting the TLR4/NF-κB signaling pathway.

OBJECTIVE: This study was designed to observe the effect of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway activity on sepsis-associated acute kidney injury (SA-AKI), thereby providing new considerations for the prevention and treatment of SA-AKI. METHODS: The rats were divided into Sham, cecal ligation and puncture (CLP), CLP + vehicle, and CLP + TAK-242 groups. Except the Sham group, a model of CLP-induced sepsis was established in other groups. After 24 h, the indicators related to kidney injury in blood samples were detected. The pathological changes in the kidneys were observed by hematoxylin-eosin staining, and tubular damage was scored. Oxidative stress-related factors, mitochondrial dysfunction-related indicators in each group were measured; the levels of inflammatory factors in serum and kidney tissue of rats were examined. Finally, the expression of proteins related to the TLR4/NF-κB signaling pathway was observed by western blot. RESULTS: Compared with the CLP + vehicle and CLP + TAK-242 groups, the CLP + TAK-242 group reduced blood urea nitrogen (BUN), creatinine (Cr), cystatin-C (Cys-C), reactive oxygen species (ROS), malondialdehyde (MDA), and inflammatory factors levels (p < 0.01), as well as increased superoxide dismutase (SOD) activity of CLP rats (p < 0.01). Additionally, TAK-242 treatment improved the condition of CLP rats that had glomerular and tubular injuries and mitochondrial disorders (p < 0.01). Further mechanism research revealed that TAK-242 can inhibit the TLR4/NF-κB signaling pathway activated by CLP (p < 0.01). Above indicators after TAK-242 treatment were close to those of the Sham group. CONCLUSION: TAK-242 can improve oxidative stress, mitochondrial dysfunction, and inflammatory response by inhibiting the activity of TLR4/NF-κB signaling pathway, thereby preventing rats from SA-AKI.

Peroxiredoxin 3 has a crucial role in the macrophage polarization by regulating mitochondrial homeostasis.

Acute lung injury (ALI) is one of the life-threatening complications of sepsis, and macrophage polarization plays a crucial role in the sepsis-associated ALI. However, the regulatory mechanisms of macrophage polarization in ALI and in the development of inflammation are largely unknown. In this study, we demonstrated that macrophage polarization occurs in sepsis-associated ALI and is accompanied by mitochondrial dysfunction and inflammation, and a decrease of PRDX3 promotes the initiation of macrophage polarization and mitochondrial dysfunction. Mechanistically, PRDX3 overexpression promotes M1 macrophages to differentiate into M2 macrophages, and enhances mitochondrial functional recovery after injury by reducing the level of glycolysis and increasing TCA cycle activity. In conclusion, we identified PRDX3 as a critical hub integrating oxidative stress, inflammation, and metabolic reprogramming in macrophage polarization. The findings illustrate an adaptive mechanism underlying the link between macrophage polarization and sepsis-associated ALI.

Carrimycin ameliorates lipopolysaccharide and cecal ligation and puncture-induced sepsis in mice.

Carrimycin (CA), sanctioned by China's National Medical Products Administration (NMPA) in 2019 for treating acute bronchitis and sinusitis, has recently been observed to exhibit multifaceted biological activities, encompassing anti-inflammatory, antiviral, and anti-tumor properties. Despite these applications, its efficacy in sepsis treatment remains unexplored. This study introduces a novel function of CA, demonstrating its capacity to mitigate sepsis induced by lipopolysaccharide (LPS) and cecal ligation and puncture (CLP) in mice models. Our research employed in vitro assays, real-time quantitative polymerase chain reaction (RT-qPCR), and RNA-seq analysis to establish that CA significantly reduces the levels of pro-inflammatory cytokines, namely tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1ß), and interleukin 6 (IL-6), in response to LPS stimulation. Additionally, Western blotting and immunofluorescence assays revealed that CA impedes Nuclear Factor Kappa B (NF-κB) activation in LPS-stimulated RAW264.7 cells. Complementing these findings, in vivo experiments demonstrated that CA effectively alleviates LPS- and CLP-triggered organ inflammation in C57BL/6 mice. Further insights were gained through 16S sequencing, highlighting CA's pivotal role in enhancing gut microbiota diversity and modulating metabolic pathways, particularly by augmenting the production of short-chain fatty acids in mice subjected to CLP. Notably, a comparative analysis revealed that CA's anti-inflammatory efficacy surpasses that of equivalent doses of aspirin (ASP) and TIENAM. Collectively, these findings suggest that CA exhibits significant therapeutic potential in sepsis treatment. This discovery provides a foundational theoretical basis for the clinical application of CA in sepsis management.

Age-associated accumulation of B cells promotes macrophage inflammation and inhibits lipolysis in adipose tissue during sepsis.

Non-canonical lipolysis induced by inflammatory cytokines or Toll-like receptor ligands is required for the regulation of inflammation during endotoxemia and sepsis. Canonical lipolysis induced by catecholamines declines during aging due to factors including an expansion of lymphocytes, pro-inflammatory macrophage polarization, and an increase in chronic low-grade inflammation; however, the extent to which the non-canonical pathway of lipolysis is active and impacted by immune cells during aging remains unclear. Therefore, we aimed to define the extent to which immune cells from old mice influence non-canonical lipolysis during sepsis. We identified age-associated impairments of non-canonical lipolysis and an accumulation of dysfunctional B1 B cells in the visceral white adipose tissue (vWAT) of old mice. Lifelong deficiency of B cells results in restored non-canonical lipolysis and reductions in pro-inflammatory macrophage populations. Our study suggests that targeting the B cell-macrophage signaling axis may resolve metabolic dysfunction in aged vWAT and attenuate septic severity in older individuals.

Thrombocytopenia Independently Leads to Changes in Monocyte Immune Function.

BACKGROUND: While platelets have well-studied hemostatic functions, platelets are immune cells that circulate at the interface between the vascular wall and white blood cells. The physiological implications of these constant transient interactions are poorly understood. Activated platelets induce and amplify immune responses, but platelets may also maintain immune homeostasis in healthy conditions, including maintaining vascular integrity and T helper cell differentiation, meaning that platelets are central to both immune responses and immune quiescence. Clinical data have shown an association between low platelet counts (thrombocytopenia) and immune dysfunction in patients with sepsis and extracorporeal membrane oxygenation, further implicating platelets as more holistic immune regulators, but studies of platelet immune functions in nondisease contexts have had limited study. METHODS: We used in vivo models of thrombocytopenia and in vitro models of platelet and monocyte interactions, as well as RNA-seq and ATAC-seq (assay for transposase-accessible chromatin with sequencing), to mechanistically determine how resting platelet and monocyte interactions immune program monocytes. RESULTS: Circulating platelets and monocytes interact in a CD47-dependent manner to regulate monocyte metabolism, histone methylation, and gene expression. Resting platelet-monocyte interactions limit TLR (toll-like receptor) signaling responses in healthy conditions in an innate immune training-like manner. In both human patients with sepsis and mouse sepsis models, thrombocytopenia exacerbated monocyte immune dysfunction, including increased cytokine production. CONCLUSIONS: Thrombocytopenia immune programs monocytes in a manner that may lead to immune dysfunction in the context of sepsis. This is the first demonstration that sterile, endogenous cell interactions between resting platelets and monocytes regulate monocyte metabolism and pathogen responses, demonstrating platelets to be immune rheostats in both health and disease.

Oxidative Stress in Sepsis: A Focus on Cardiac Pathology.

This study aims to analyze post-mortem human cardiac specimens, to verify and evaluate the existence or extent of oxidative stress in subjects whose cause of death has been traced to sepsis, through immunohistological oxidative/nitrosative stress markers. Indeed, in the present study, i-NOS, NOX2, and nitrotyrosine markers were higher expressed in the septic death group when compared to the control group, associated with also a significant increase in 8-OHdG, highlighting the pivotal role of oxidative stress in septic etiopathogenesis. In particular, 70% of cardiomyocyte nuclei from septic death specimens showed positivity for 8-OHdG. Furthermore, intense and massive NOX2-positive myocyte immunoreaction was noticed in the septic group, as nitrotyrosine immunostaining intense reaction was found in the cardiac cells. These results demonstrated a correlation between oxidative and nitrosative stress imbalance and the pathophysiology of cardiac dysfunction documented in cases of sepsis. Therefore, subsequent studies will focus on the expression of oxidative stress markers in other organs and tissues, as well as on the involvement of the intracellular pattern of apoptosis, to better clarify the complex pathogenesis of multi-organ failure, leading to support the rationale for including therapies targeting redox abnormalities in the management of septic patients.

Differential mRNA profiles reveal the potential roles of genes involved in lactate stimulation in mouse macrophages.

Lactate is a glycolysis end product, and its levels are markedly associated with disease severity, morbidity, and mortality in sepsis. It modulates key functions of immune cells, including macrophages. In this investigation, transcriptomic analysis was performed using lactic acid, sodium lactate, and hydrochloric acid-stimulated mouse bone marrow-derived macrophages (iBMDM), respectively, to identify lactate-associated signaling pathways. After 24 h of stimulation, 896 differentially expressed genes (DEG) indicated were up-regulation, whereas 792 were down-regulated in the lactic acid group, in the sodium lactate group, 128 DEG were up-regulated, and 41 were down-regulated, and in the hydrochloric acid group, 499 DEG were up-regulated, and 285 were down-regulated. Subsequently, clinical samples were used to further verify the eight genes with significant differences, among which Tssk6, Ypel4, Elovl3, Trp53inp1, and Cfp were differentially expressed in patients with high lactic acid, indicating their possible involvement in lactic acid-induced inflammation and various physiological diseases caused by sepsis. However, elongation of very long chain fatty acids protein 3 (Elovl3) was negatively correlated with lactic acid content in patients. The results of this study provide a necessary reference for better understanding the transcriptomic changes caused by lactic acid and explain the potential role of high lactic acid in the regulation of macrophages in sepsis.

Exploring the role of antioxidants in sepsis-associated oxidative stress: a comprehensive review.

Sepsis is a potentially fatal condition characterized by organ dysfunction caused by an imbalanced immune response to infection. Although an increased inflammatory response significantly contributes to the pathogenesis of sepsis, several molecular mechanisms underlying the progression of sepsis are associated with increased cellular reactive oxygen species (ROS) generation and exhausted antioxidant pathways. This review article provides a comprehensive overview of the involvement of ROS in the pathophysiology of sepsis and the potential application of antioxidants with antimicrobial properties as an adjunct to primary therapies (fluid and antibiotic therapies) against sepsis. This article delves into the advantages and disadvantages associated with the utilization of antioxidants in the therapeutic approach to sepsis, which has been explored in a variety of animal models and clinical trials. While the application of antioxidants has been suggested as a potential therapy to suppress the immune response in cases where an intensified inflammatory reaction occurs, the use of multiple antioxidant agents can be beneficial as they can act additively or synergistically on different pathways, thereby enhancing the antioxidant defense. Furthermore, the utilization of immunoadjuvant therapy, specifically in septic patients displaying immunosuppressive tendencies, represents a promising advancement in sepsis therapy.

Endothelial cell-derived extracellular vesicles expressing surface VCAM1 promote sepsis-related acute lung injury by targeting and reprogramming monocytes.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a common life-threatening syndrome with no effective pharmacotherapy. Sepsis-related ARDS is the main type of ARDS and is more fatal than other types. Extracellular vesicles (EVs) are considered novel mediators in the development of inflammatory diseases. Our previous research suggested that endothelial cell-derived EVs (EC-EVs) play a crucial role in ALI/ARDS development, but the mechanism remains largely unknown. Here, we demonstrated that the number of circulating EC-EVs was increased in sepsis, exacerbating lung injury by targeting monocytes and reprogramming them towards proinflammatory macrophages. Bioinformatics analysis and further mechanistic studies revealed that vascular cell adhesion molecule 1 (VCAM1), overexpressed on EC-EVs during sepsis, activated the NF-κB pathway by interacting with integrin subunit alpha 4 (ITGA4) on the monocyte surface, rather than the tissue resident macrophage surface, thereby regulating monocyte differentiation. This effect could be attenuated by decreasing VCAM1 levels in EC-EVs or blocking ITGA4 on monocytes. Furthermore, the number of VCAM1+ EC-EVs was significantly increased in patients with sepsis-related ARDS. These findings not only shed light on a previously unidentified mechanism underling sepsis-related ALI/ARDS, but also provide potential novel targets and strategies for its precise treatment.

Antiseptic Functions of CGK012 against HMGB1-Mediated Septic Responses.

High mobility group box 1 (HMGB1), a protein with important functions, has been recognized as a potential therapeutic target for the treatment of sepsis. One possible mechanism for this is that inhibiting HMGB1 secretion can exert antiseptic effects, which can restore the integrity of the vascular barrier. (7S)-(+)-cyclopentyl carbamic acid 8,8-dimethyl-2-oxo-6,7-dihydro-2H,8H-pyrano[3,2-g]chromen-7-yl-ester (CGK012) is a newly synthesized pyranocoumarin compound that could function as a novel small-molecule inhibitor of the Wnt/ß-catenin signaling pathway. However, no studies have yet determined the effects of CGK012 on sepsis. We investigated the potential of CGK012 to attenuate the excessive permeability induced by HMGB1 and enhance survival rates in a mouse model of sepsis with reduced HMGB1 levels following lipopolysaccharide (LPS) treatment. In both LPS-stimulated human endothelial cells and a mouse model exhibiting septic symptoms due to cecal ligation and puncture (CLP), we assessed proinflammatory protein levels and tissue damage biomarkers as indicators of reduced vascular permeability. CGK012 was applied after induction in human endothelial cells exposed to LPS and the CLP-induced mouse model of sepsis. CGK012 effectively mitigated excessive permeability and suppressed HMGB1 release, resulting in improved vascular stability, decreased mortality, and enhanced histological conditions in the mouse model of CLP-induced sepsis. In conclusion, our findings indicate that CGK012 treatment in mice with CLP-induced sepsis diminished HMGB1 release and increased the survival rate, suggesting its potential as a pharmaceutical intervention for sepsis.

[Chlorogenic acid alleviates acute kidney injury in septic mice by inhibiting NLRP3 inflammasomes and the caspase-1 canonical pyroptosis pathway].

OBJECTIVE: To investigate the role of caspase-1-medicated canonical pyroptosis pathway in chlorogenic acid (CGA) treatment of acute kidney injury (AKI) in mice. METHOD: Twenty-four C57Bl/6J mice were randomized into sham-operated group, cecal ligation and puncture (CLP) group, CLP+dexamethasone group (CLP+DXM group), and CLP+CGA group (n=6) and subjected to either sham operation (laparotomy only) or CLP. After modeling the mice received intravenous infusion of 10 mg/kg normal saline (in sham and CLP groups), 1 µg/kg dexamethasone or 15 mg/kg of chlorogenic acid for 6 h delivered using an intravenous pump. Eight hours after the infusion, renal morphology and histology, renal cell apoptosis, and the renal function parameters such as urea nitrogen (BUN), creatinine (Scr), and kidney injury molecule 1 (KIM-1) were compared among the 4 groups; the 7-day survival rates of the mice were recorded, and the expressions of NLRP3 inflammasomes and key proteins of the caspase-1 pathway in the renal tissue were detected. RESULTS: CGA treatment significantly improved the 7-day survival rate, reduced renal pathologies of the septic mice (P < 0.05), and lowered the levels of BUN, Scr, KIM-1, NLRP3 inflammasome and expressions of key proteins of the caspase-1 pathway. CONCLUSION: CGA alleviates AKI in mice with CLP-induced sepsis by inhibiting NLRP3 inflammasomes and the caspase-1 signaling pathway.

[Tea polyphenols ameliorates acute lung injury in septic mice by inhibiting NLRP3 inflammasomes].

OBJECTIVE: To investigate the mechanism of tea polyphenols (TP) for regulating NLRP3 inflammasomes and alleviating acute lung injury in septic mice. METHODS: Sixty C57BL/6 mice were randomly assigned into sham-operated, cecal ligation and puncture (CLP) and CLP +TP treatment groups, and survival of the mice was recorded after modeling in each group. The lung wet/dry weight ratio and myeloperoxidase (MPO) activity were determined, and lung injury of the mice was evaluated using HE staining and acute lung injury score. The expressions of IL-1ß, TNF-α, IL-6, NLRP3, caspase-1 p10, ASC, MPO, and caspase-8 in the lung tissue were detected using ELISA, Western blotting, or immunohistochemical staining. MDA and H2O2 levels in the lungs were detected to evaluate the level of oxidative stress. Immunofluorescence assay was used to investigate the co-localization of NLRP3 and NOX4. RESULTS: The postoperative mortality rate at 72 h, lung wet/dry weight ratio, MPO level and acute lung injury scores were significantly lower in CLP+TP group than in CLP group (P < 0.05). Treatment with TP significantly reduced the expressions of NLRP3-related inflammatory factors (P < 0.05) and lowered MDA and H2O2 levels in the lung tissue of the septic mice (P < 0.05). Immunofluorescence co-staining showed a lower level of NOX4 and NLRP3 co-localization in CLP+TP group than in CLP group. CONCLUSION: TP inhibits NLRP3 inflammasome-associated inflammation to alleviate CLP-induced acute lung injury in mice through a regulatory mechanism that inhibits NOX4 expression and reduces oxidative stress in the lung tissue.

YBX-1 alleviates sepsis-stimulated lung epithelial cell injury.

OBJECTIVE: To explore the role of Y-box binding protein 1 (YBX-1) in the lipopolysaccharide (LPS)-stimulated inflammation and oxidative stress of BEAS-2B cell line and clarify the underlying mechanism. METHODS: LPS-stimulated BEAS-2B cells were used as a cell model of sepsis-stimulated acute lung injury (ALI). Immunoblot and quantitative polymerase chain reaction assays were used to detect the expression of YBX-1 in LPS-stimulated BEAS-2B cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, TdT-mediated dUTP nick end labeling, and immunoblot assays were conducted to determine the effects of YBX-1 on cell survival. JC-1 staining and adenosine triphosphate production were used to detect the effects of YBX-1 on mitochondrial function. Immunostaining and enzyme-linked immunosorbent serologic assay were performed to examine the effects of YBX-1 on the inflammation and oxidative stress of cells. Immunoblot assay was conducted to confirm the mechanism. RESULTS: YBX-1 was lowly expressed in LPS-stimulated BEAS-2B cells and enhanced the survival of LPS-stimulated lung epithelial cells. In addition, YBX-1 improved mitochondrial function of LPS-stimulated BEAS-2B cells. YBX-1 inhibited the inflammation and oxidative stress of LPS-stimulated BEAS-2B cells. Mechanically, YBX-1 inhibited mitogen-activated protein kinase (MAPK) axis, thereby alleviating sepsis-stimulated ALI. CONCLUSION: YBX-1 alleviated inflammation and oxidative stress of LPS-stimulated BEAS-2B cells via MAPK axis.

Identification of HK3 as a promising immunomodulatory and prognostic target in sepsis-induced acute lung injury.

BACKGROUND: Sepsis is a life-threatening global disease with a significant impact on human health. Acute lung injury (ALI) has been identified as one of the primary causes of mortality in septic patients. This study aimed to identify candidate genes involved in sepsis-induced ALI through a comprehensive approach combining bioinformatics analysis and experimental validation. METHODS: The datasets GSE65682 and GSE32707 obtained from the Gene Expression Omnibus database were merged to screen for sepsis-induced ALI related differentially expressed genes (DEGs). Functional enrichment and immune infiltration analyses were conducted on DGEs, with the construction of protein-protein interaction (PPI) networks to identify hub genes. In vitro and in vivo models of sepsis-induced ALI were used to study the expression and function of hexokinase 3 (HK3) using various techniques including Western blot, real-time PCR, immunohistochemistry, immunofluorescence, Cell Counting Kit-8, Enzyme-linked immunosorbent assay, and flow cytometry. RESULTS: The results of bioinformatics analysis have identified HK3, MMP9, and S100A8 as hub genes with diagnostic and prognostic significance for sepsis-induced ALI. The HK3 has profound effects on sepsis-induced ALI and exhibits a correlation with immune regulation. Experimental results showed increased HK3 expression in lung tissue of septic mice, particularly in bronchial and alveolar epithelial cells. In vitro studies demonstrated upregulation of HK3 in lipopolysaccharide (LPS)-stimulated lung epithelial cells, with cytoplasmic localization around the nucleus. Interestingly, following the knockdown of HK3 expression, lung epithelial cells exhibited a significant decrease in proliferation activity and glycolytic flux, accompanied by an increase in cellular inflammatory response, oxidative stress, and cell apoptosis. CONCLUSIONS: It was observed for the first time that HK3 plays a crucial role in the progression of sepsis-induced ALI and may be a valuable target for immunomodulation and therapy.Bioinformatics analysis identified HK3, MMP9, and S100A8 as hub genes with diagnostic and prognostic relevance in sepsis-induced ALI. Experimental findings showed increased HK3 expression in the lung tissue of septic mice, particularly in bronchial and alveolar epithelial cells. In vitro experiments demonstrated increased HK3 levels in lung epithelial cells stimulated with LPS, with cytoplasmic localization near the nucleus. Knockdown of HK3 expression resulted in decreased proliferation activity and glycolytic flux, increased inflammatory response, oxidative stress, and cell apoptosis in lung epithelial cells.

Acetyl-CoA synthetase 2 induces pyroptosis and inflammation of renal epithelial tubular cells in sepsis-induced acute kidney injury by upregulating the KLF5/NF-κB pathway.

BACKGROUND: Pyroptosis of the renal tubular epithelial cells (RTECs) and interstitial inflammation are central pathological characteristics of acute kidney injury (AKI). Pyroptosis acts as a pro-inflammatory form of programmed cell death and is mainly dependent on activation of the NLRP3 inflammasome. Previous studies revealed that acetyl-CoA synthetase 2 (ACSS2) promotes inflammation during metabolic stress suggesting that ACSS2 might regulate pyroptosis and inflammatory responses of RTECs in AKI. METHODS AND RESULTS: The expression of ACSS2 was found to be significantly increased in the renal epithelial cells of mice with lipopolysaccharide (LPS)-induced AKI. Pharmacological and genetic strategies demonstrated that ACSS2 regulated NLRP3-mediated caspase-1 activation and pyroptosis through the stimulation of the KLF5/NF-κB pathway in RTECs. The deletion of ACSS2 attenuated renal tubular pathological injury and inflammatory cell infiltration in an LPS-induced mouse model, and ACSS2-deficient mice displayed impaired NLRP3 activation-mediated pyroptosis and decreased IL-1ß production in response to the LPS challenge. In HK-2 cells, ACSS2 deficiency suppressed NLRP3-mediated caspase-1 activation and pyroptosis through the downregulation of the KLF5/NF-κB pathway. The KLF5 inhibitor ML264 suppressed NF-κB activity and NLRP3-mediated caspase-1 activation, thus protecting HK-2 cells from LPS-induced pyroptosis. CONCLUSION: Our results suggested that ACSS2 regulates activation of the NLRP3 inflammasome and pyroptosis by inducing the KLF5/NF-κB pathway in RTECs. These results identified ACSS2 as a potential therapeutic target in AKI.