Lung Injury In Septic Mice Research Articles

Curcumin alleviates septic lung injury in mice by inhibiting TXNIP/TRX-1/GPX4-mediated ferroptosis

To investigate whether curcumin alleviates septic lung injury by inhibiting ferroptosis through modulating the TXNIP/TRX-1/GPX4 pathway. Male C57BL/6 mice were randomly divided into Sham group, cecal ligation puncture (CLP)-induced sepsis group, CLP with curcumin treatment (50, 100, and 200 mg/kg) groups, and CLP with both curcumin (200 mg/kg) and TRX-1 inhibitor PX-12 (25 mg/kg) treatment group. Inflammatory factors, MDA, MPO, and GSH levels in the lung tissue of the mice were detected. Beas-2B cells stimulated with lipopolysaccharide (LPS; 1 μg/mL) were treated with 2.5, 5, or 10 μmol/L curcumin or with 10 μmol/L curcumin combined with 5 μmol/L PX-12, and the changes in MDA, Fe2+ and ROS levels were assessed. Western blotting was performed to detect the protein expressions of TXNIP, TRX-1, GPX4 and X-CT in both the mouse lung tissues and Beas-2B cells. The mice with CLP-induced sepsis showed severe lung injury with elevated expressions of IL-6, IL-1β, TNF-α, MDA and MPO and decreased GSH expression. In Beas-2B cells, LPS stimulation significantly increased MDA and Fe2+ levels and ROS release, increased TXNIP protein expression, and lowered the protein expression levels of TRX-1, GPX4 and X-CT, and these changes were also observed in the septic mice. Curcumin treatments at different concentrations obviously alleviated lung injury in the septic mice and reduced LPS-induced injury in Beas-2B cells. Curcumin significantly decreased the release of inflammatory factors, MDA and MPO, increased GSH level, lowered Fe2+, MDA and ROS levels, increased TXNIP protein expression, and lowered the protein expressions of TRX-1, GPX4 and X-CT in both septic mouse lung tissues and LPS-stimulated Beas-2B cells. The protective effect of curcumin was effectively blocked by PX-12 treatment. Curcumin inhibits ferroptosis and alleviates septic lung injury in mice by elevating TRX-1 and GPX4 and decreasing TXNIP in the lung tissue.

Suppression of Skp2 contributes to sepsis-induced acute lung injury by enhancing ferroptosis through the ubiquitination of SLC3A2

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The inflammatory cytokine storm causes systemic organ damage, especially acute lung injury in sepsis. In this study, we found that the expression of S-phase kinase-associated protein 2 (Skp2) was significantly decreased in sepsis-induced acute lung injury (ALI). Sepsis activated the MEK/ERK pathway and inhibited Skp2 expression in the pulmonary epithelium, resulting in a reduction of K48 ubiquitination of solute carrier family 3 member 2 (SLC3A2), thereby impairing its membrane localization and cystine/glutamate exchange function. Consequently, the dysregulated intracellular redox reactions induced ferroptosis in pulmonary epithelial cells, leading to lung injury. Finally, we demonstrated that intravenous administration of Skp2 mRNA-encapsulating lipid nanoparticles (LNPs) inhibited ferroptosis in the pulmonary epithelium and alleviated lung injury in septic mice. Taken together, these data provide an innovative understanding of the underlying mechanisms of sepsis-induced ALI and a promising therapeutic strategy for sepsis.

Histone lactylation-regulated METTL3 promotes ferroptosis via m6A-modification on ACSL4 in sepsis-associated lung injury

Elevated lactate levels are a significant biomarker of sepsis and are positively associated with sepsis-related mortality. Sepsis-associated lung injury (ALI) is a leading cause of poor prognosis in clinical patients. However, the underlying mechanisms of lactate's involvement in sepsis-associated ALI remain unclear. In this study, we demonstrate that lactate regulates N6-methyladenosine (m6A) modification levels by facilitating p300-mediated H3K18la binding to the METTL3 promoter site. The METTL3-mediated m6A modification is enriched in ACSL4, and its mRNA stability is regulated through a YTHDC1-dependent pathway. Furthermore, short-term lactate stimulation upregulates ACSL4, which promotes mitochondria-associated ferroptosis. Inhibition of METTL3 through knockdown or targeted inhibition effectively suppresses septic hyper-lactate-induced ferroptosis in alveolar epithelial cells and mitigates lung injury in septic mice. Our findings suggest that lactate induces ferroptosis via the GPR81/H3K18la/METTL3/ACSL4 axis in alveolar epithelial cells during sepsis-associated ALI. These results reveal a histone lactylation-driven mechanism inducing ferroptosis through METTL3-mediated m6A modification. Targeting METTL3 represents a promising therapeutic strategy for patients with sepsis-associated ALI.

Preparation of Dexamethasone-Loaded Nanoparticles and Therapeutic Adoption for Acute Lung Injury in Septic Mice Through the TLR9 Pathway

This research aimed to better exert the efficacy of dexamethasone (DEX) and fabricate an intercellular adhesion molecule A (ILDMA) monoclonal antibody (mAb)-modified nanostructured lipid carrier (NSLC). The anionic DEX NSLC was fabricated by the aqueous solvent diffusion methodology using DEX as the model drug and in combination with various types of lipids. Using N,N’-succinimidyl carbonate as the link, anti-ILDMA mAb-modified anion DEX NSLC (ILDM/DEX/NSLC) and anti-IgG mAb-modified cation DEX NSLC (IgG/DEX/NSLC) were prepared. The total lipid content was controlled unchanged, 3% mass ratio of glyceryl monostearate (MS) in the original prescription was replaced with 3% mass ratio of octadecylamine (ODA), and ILDM/DEX/ODA-NSLC and IgG/DEX/ODA-NSLC were fabricated in the same way. The four NSLCs prepared in the experiment were round in shape and uniform in size. The nanoparticles with a size of approximately 230 nm were similar, and the zeta potentials were (−29.8±21.5) mV, (−27.9±1.6) mV, (36.8±0.8) mV, and (33.7±2.9) mV, respectively. In vitro drug release demonstrated a cumulative release rate of more than 55% of DEX NSLC. The inhibitory rate of DEX NSLC on the activated human vascular endothelial cell line (EAhy926 cell) was dose dependent, and ILDM/DEX/NSLC could transport DEX to activated endothelial cells more efficiently, thus enhancing the intervention ability on diseased endothelium. For the establishment of a sepsis-induced acute lung injury (ALI) mouse model, ILDM/DEX/NSLC was highly distributed in the lung of the model, and its infiltration effect on inflammatory cells was superior to that of other drugs (P < 0.05). Meanwhile, ILDM/DEX/NSLC could more markedly repair the pathological features in the mouse model than other drugs did (P < 0.05). The nanodrug inhibited the protein level of TLR9 in mouse lung tissue to the maximum extent (P < 0.05), thereby enhancing the survival rate of the mice.

Tomatidine activates autophagy to improve lung injury and inflammation in sepsis by inhibiting NF-κB and MAPK pathways.

Sepsis-induced acute lung injury (ALI) is a life-threatening medical condition with high mortality and morbidity. Autophagy is involved in the pathophysiological process of sepsis-induced ALI, including inflammation, which indicates that regulating autophagy may be beneficial for this disease. Tomatidine, a natural compound abundant in unripe tomatoes, has been reported to have anti-inflammatory, anti-tumorigenic, and lipid-lowering effects. However, the biological functions and mechanisms of tomatidine in sepsis-induced ALI remain unknown. The principal objective of this study was to investigate the effect of tomatidine on sepsis-induced ALI. Cecal ligation and puncture (CLP) was used to induce septic lung injury in mice, and 10mg/kg tomatidine was intraperitoneally injected into mice 2h after the operation. The results of hematoxylin and eosin staining and assessment of lung edema and total protein levels in bronchoalveolar lavage fluid (BALF) demonstrated that tomatidine alleviated CLP-induced severe lung injuries such as hemorrhage, infiltration of inflammatory cells, and interstitial and alveolar edema in mice. Additionally, the levels of proinflammatory cytokines in BALF and lung tissues were measured by enzyme-linked immunosorbent assay (ELISA), and the results showed that tomatidine inhibited CLP-induced inflammatory damage to lungs. Moreover, the results of western blotting showed that tomatidine promoted autophagy during CLP-induced ALI. Mechanistically, immunofluorescence staining and western blotting were used to measure the protein levels of TLR4, phosphorylated NF-κB, phosphorylated IκBα, and phosphorylated MAPKs, showing that tomatidine inactivated NF-κB and MAPK signaling in lung tissues of CLP-induced ALI mice. In conclusion, tomatidine exerts protective effects against sepsis-induced severe damage to the lungs by inhibiting inflammation and activating autophagy in CLP-treated mice through inactivating the NF-κB and MAPK pathways, which may be an effective candidate for treating septic ALI.

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

To investigate the mechanism of tea polyphenols (TP) for regulating NLRP3 inflammasomes and alleviating acute lung injury in septic mice. 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-1β, 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. 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. 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.

Theacrine alleviates inflammation and lung injury in septic mice by mediating SIRT3

Purpose: To evaluate the effect of theacrine on sepsis progression and sepsis-induced lung injury and its mechanism of action.&#x0D; Methods: Lung injury model of lipopolysaccharide (LPS)-induced sepsis was made in the mice by injected with 3 mg/kg LPS dissolved in 50 μL PBS or only PBS (control group = 10). The remaining three groups (divided into ten mice per group) were administered theacrine (10, 20, and 40 mg/kg), by oral gavage 1 hour after LPS treatment. Hematoxylin and eosin (H&amp;E) staining was performed to confirm the effect of theacrine on lung injury. Quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) were conducted to determine inflammatory and oxidative stress factors. TUNEL as well as immunoblot assays were carried to confirm its effect on apoptosis and mechanism of action.&#x0D; Results: Theacrine significantly improved lung injury as well as lung relief score in LPS-induced sepsis mice (p &lt; 0.01) and it significantly reversed the increased levels of inflammatory cytokines in a dose-dependent manner in LPS-induced sepsis mice (p &lt; 0.01), In addition, theacrine administration significantly reduced the increased intensity of ROS and MDA levels, and significantly improved the levels of SOD in lung tissues (p &lt; 0.01). Furthermore, it improved LPS-induced apoptosis of lung tissue cells and alleviated lung injury by significantly activating SIRT3 pathway (p &lt; 0.01).&#x0D; Conclusion: Theacrine alleviates inflammation and lung injury in septic mice by mediating SIRT3, thereby making it a potential lead in the development of drugs against sepsis-related inflammation and lung injury.

Tangeretin attenuates acute lung injury in septic mice by inhibiting ROS-mediated NLRP3 inflammasome activation via regulating PLK1/AMPK/DRP1 signaling axis.

NLRP3 inflammasome-mediated pyroptosis of macrophage acts essential roles in the progression of sepsis-induced acute lung injury (ALI). Tangeretin (TAN), enriched in citrus fruit peel, presents anti-oxidative and anti-inflammatory effects. Here, we aimed to explore the potentially protective effect of TAN on sepsis-induced ALI, and the underlying mechanism of TAN in regulating NLRP3 inflammasome. The effect of TAN on sepsis-induced ALI and NLRP3 inflammasome-mediated pyroptosis of macrophage were examined in vivo and in vitro using a LPS-treated mice model and LPS-induced murine macrophages, respectively. The mechanism of TAN regulating the activation of NLRP3 inflammasome in sepsis-induced ALI was investigated with HE staining, Masson staining, immunofluorescent staining, ELISA, molecular docking, transmission electron microscope detection, qRT-PCR, and western blot. TAN could evidently attenuate sepsis-induced ALI in mice, evidenced by reducing pulmonary edema, pulmonary congestion and lung interstitial fibrosis, and inhibiting macrophage infiltration in the lung tissue. Besides, TAN significantly suppressed inflammatory cytokine IL-1β and IL-18 expression in the serum or bronchoalveolar lavage fluid (BALF) samples of mice with LPS-induced ALI, and inhibited NLRP3 inflammasome-mediated pyroptosis of macrophages. Furthermore, we found TAN inhibited ROS production, preserved mitochondrial morphology, and alleviated excessive mitochondrial fission in LPS-induced ALI in mice. Through bioinformatic analysis and molecular docking, Polo-like kinase 1 (PLK1) was identified as a potential target of TAN for treating sepsis-induced ALI. Moreover, TAN significantly inhibited the reduction of PLK1 expression, AMP-activated protein kinase (AMPK) phosphorylation, and Dynamin related protein 1 (Drp1) phosphorylation (S637) in LPS-induced ALI in mice. In addition, Volasertib, a specific inhibitor of PLK1, abolished the protective effects of TAN against NLRP3 inflammasome-mediated pyroptosis of macrophage and lung injury in the cell and mice septic models. TAN attenuates sepsis-induced ALI by inhibiting ROS-mediated NLRP3 inflammasome activation via regulating PLK1/AMPK/DRP1 signaling axis, and TAN is a potentially therapeutic candidate against ALI through inhibiting pyroptosis.

Shenhuangdan decoction alleviates sepsis-induced lung injury through inhibition of GSDMD-mediated pyroptosis

Ethnopharmacological relevanceSepsis-induced lung injury is closely associated with it remarkable morbidity and mortality. Shenhuangdan (SHD) decoction, a traditional Chinese medicine prescription, has been clinically proven to be an effective treatment for sepsis. However, the mechanism of SHD decoction in treating sepsis remains unclear. Aim of the studyThis study aimed to evaluate the therapeutic effect of SHD decoction on sepsis-induced lung injury and its underlying mechanism. Materials and methodsIn this study, we established a mouse model of sepsis by cecum ligation and puncture (CLP) surgery. Firstly, seven-day survival analysis and histological staining of lung tissue were used to evaluate the therapeutic effect of SHD decoction on lung injury in septic mice. Multifactor microarray was used to detect cytokine expression changes in serum and bronchoalveolar lavage fluid (BALF). Subsequently, the main components in medicated serum of SHD decoction were inspected by Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The material basis of SHD decoction and potential interaction mechanisms were analysed by systemic pharmacology. To confirm the reliability of network pharmacology for predicting outcomes, we used molecular docking techniques to identify interactions between the core components and targets of SHD. Finally, TUNEL staining, immunofluorescence and western blotting were used to explore the mechanism of SHD decoction through the inhibition of GSDMD-mediated pyroptosis in septic mice. ResultsSHD was found to be effective in reducing the mortality and alleviating lung pathological damage in septic mice. Multifactor microarray results showed that SHD can reduce the expression of inflammation factors (IL-18, IL-1β, IL-5, IL-6 and TNF-α) in serum and BALF of septic mice. There were 22 major blood-entry components detected by UPLC-MS/MS. Then, combined with the network pharmacological analysis, it is evident that the main components of SHD for sepsis are Renshen-ginsenoside Rh2, Danshen-tanshinone IIA and Dahuang-rhein. The main targets were IL-1β and caspase-1, which were related to GSDMD-mediated pyroptosis signalling pathway. Molecular docking exhibited that Renshen-ginsenoside Rh2, Danshen-tanshinone IIA and Dahuang-rhein can closely bind to GSDMD, IL-1β and caspase-1. In addition, TUNEL staining and immunohistochemistry demonstrated that SHD alleviated the expression of GSDMD protein. The western blotting showed that SHD significantly inhibited the protein expression levels of NLRP3, GSDMD, GSDMD-N, cleved caspase-1, caspase-1 and ASC in lung tissue. ConclusionsOur study revealed that SHD improves CLP-induced lung injury by blocking the GSDMD-mediated pyroptosis signalling pathway in septic mice. This study provides evidence to support that SHD had a potential therapeutic effect on sepsis.

Geldanamycin ameliorates multiple organ dysfunction and microthrombosis in septic mice by inhibiting the formation of the neutrophil extracellular network by activating heat shock factor 1 HSF1.

Sepsis and septic shock are common critical illnesses in the intensive care unit with a high mortality rate. Geldanamycin (GA) has a broad spectrum of antibacterial and antiviral activity and has inhibitory effects on various viruses. However, whether GA affects sepsis due to infections remains unknown. In this study, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen and creatinine in serum; neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in the urine, cytokines (tumour necrosis factor alpha, interleukin-1β and interleukin-6) in the bronchoalveolar lavage fluid and myeloperoxidase in the lung tissues were measured using enzyme-linked immunosorbent assay kits. Pathological injury was measured by hematoxylin and eosin staining and neutrophils were measured by flow cytometry analysis; related expressions were analysed by qPCR, western blot and immunofluorescence assay. The results showed that GA significantly ameliorated cecum ligation and puncture (CLP)-triggered liver, kidney and lung injury in septic mice. In addition, we found that GA dose-dependently inhibited microthrombosis and alleviated coagulopathy in septic mice. Further molecular mechanism analysis suggests that GA may act through upregulation of heat shock factor 1 and tissue-type plasminogen activator. In conclusion, our study elucidated the protective effects of GA in a mouse model established using CLP, and the results reveal that GA may be a promising agent for sepsis.

Berberine Alleviates Acute Lung Injury in Septic Mice by Modulating Treg/Th17 Homeostasis and Downregulating NF-κB Signaling.

A common complication of sepsis is acute lung injury (ALI), which is associated with an acute onset, rapid disease changes, and high mortality. Regulatory T (Treg) and T helper 17 (Th17) cells comprise CD4+ T cell subsets, which strongly influence inflammation during ALI. In this study, we investigated the effect of berberine (BBR), an antioxidant, anti-inflammatory, and immunomodulatory drug, on the inflammatory response and immune state in mice with sepsis. A mouse model of cecal ligation and puncture (CLP) was established. The mice were intragastrically administered 50 mg/kg BBR. We used histological techniques to evaluate inflammatory tissue injury and flow cytometry for analyzing Treg/Th17 levels. We also assessed NF-κB signaling pathways by Western blotting assays and immunofluorescence staining. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the content of cytokines. Treatment with BBR considerably mitigated lung injury while improving survival, post-cecal ligation, and puncture (CLP). Treatment with BBR ameliorated pulmonary edema and hypoxemia in septic mice and inhibited the NF-κB signaling pathway. BBR also increased Treg cells and decreased Th17 proportions in the spleen and lung tissue of CLP-treated mice. Blocking Treg cells weakened the protective effect of BBR on sepsis-associated lung injury. Overall, these results suggested that BBR is a potential therapeutic agent for sepsis.

Up-regulated CD38 by daphnetin alleviates lipopolysaccharide-induced lung injury via inhibiting MAPK/NF-κB/NLRP3 pathway

BackgroundSepsis is a life-threatening organ dysfunction syndrome resulted from severe infection with high morbidity and mortality. Cluster of differentiation 38 (CD38) is a multifunctional type II transmembrane glycoprotein widely expressed on the surface of various immunocytes membranes that mediates host immune response to infection and plays an important role in many inflammatory diseases. Daphnetin (Daph), isolated from the daphne genus plant, is a natural coumarin derivative that possesses anti-inflammatory and anti-apoptotic effects. The current study aimed to investigate the role and mechanism of Daph in alleviating lipopolysaccharide (LPS)-induced septic lung injury, and to explore whether the protective effect of Daph in mice and cell models was related to CD38.MethodsFirstly, network pharmacology analysis of Daph was performed. Secondly, LPS-induced septic lung injury in mice were treated with Daph or vehicle control respectively and then assessed for survival, pulmonary inflammation and pathological changes. Lastly, Mouse lung epithelial cells (MLE-12 cells) were transfected with CD38 shRNA plasmid or CD38 overexpressed plasmid, followed by LPS and Daph treatment. Cells were assessed for viability and transfection efficiency, inflammatory and signaling.ResultsOur results indicated that Daph treatment improved survival rate and alleviated pulmonary pathological damage of the sepsis mice, as well as reduced the excessive release of pro-inflammatory cytokines IL-1β, IL-18, IL-6, iNOS and chemokines MCP-1 regulated by MAPK/NF-κB pathway in pulmonary injury. Daph treatment decreased Caspase-3 and Bax, increased Bcl-2, inhibited nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome‐mediated pyroptosis in lung tissues of septic lung injury. Also, Daph treatment reduced the level of excessive inflammatory mediators, inhibited apoptosis and pyroptosis in MLE-12 cells. It is noteworthy that the protective effect of Daph on MLE-12 cells damage and death was assisted by the enhanced expression of CD38.ConclusionsOur results demonstrated that Daph offered a beneficial therapeutic effect for septic lung injury via the up-regulation of CD38 and inhibition of MAPK/NF-κB/NLRP3 pathway.6xvKSfv8oa4hBEdywDEkeuVideo

RBM3 is associated with acute lung injury in septic mice and patients via the NF-κB/NLRP3 pathway.

Sepsis refers to host response disorders caused by infection, leading to life-threatening organ dysfunction. RNA-binding motif protein 3 (RBM3) is an important cold-shock protein that is upregulated in response to mild hypothermia or hypoxia. In this study, we aimed to investigate whether RBM3 is involved in sepsis-associated acute lung injury (ALI). Intraperitoneal injection of LPS (10mg/kg) was performed in wild type (WT) and RBM3 knockout (KO, RBM3-/-) mice to establish an in vivo sepsis model. An NLRP3 inflammasome inhibitor, MCC950 (50mg/kg), was injected intraperitoneally 30min before LPS treatment. Serum, lung tissues, and BALF were collected 24h later for further analysis. In addition, we also collected serum from sepsis patients and healthy volunteers to detect their RBM3 expression. The results showed that the expression of RBM3 in the lung tissues of LPS-induced sepsis mice and the serum of patients with sepsis was significantly increased and positively correlated with disease severity. In addition, RBM3 knockout (KO) mice had a low survival rate, and RBM3 KO mice had more severe lung damage, inflammation, lung cell apoptosis, and oxidative stress than WT mice. LPS treatment significantly increased the levels of nucleotide binding and oligomerization domain-like receptor family 3 (NLRP3) inflammasomes and mononuclear cell nuclear factor-κB (NF-κB) in the lung tissues of RBM3 KO mice. However, these levels were only slightly elevated in WT mice. Interestingly, MCC950 improved LPS-induced acute lung injury in WT and RBM3 KO mice but inhibited the expression of NLRP3, caspase-1, and IL-1β. In conclusion, RBM3 was overexpressed in sepsis patients and LPS-induced mice. RBM3 gene deficiency aggravated sepsis-associated ALI through the NF-κB/NLRP3 pathway.

Neutrophil Extracellular Traps Induce Alveolar Macrophage Pyroptosis by Regulating NLRP3 Deubiquitination, Aggravating the Development of Septic Lung Injury.

Uncontrolled inflammation is a typical feature of sepsis-related lung injury. The key event in the progression of lung injury is Caspase-1-dependent alveolar macrophage (AM) pyroptosis. Similarly, neutrophils are stimulated to release neutrophil extracellular traps (NETs) to participate in the innate immune response. This study aims to illustrate the specific mechanisms by which NETs activate AM at the post-translational level and maintain lung inflammation. We established a septic lung injury model by caecal ligation and puncture. We found elevated NETs and interleukin-1b (IL-1β) levels in the lung tissues of septic mice. Western blot and immunofluorescence analyses was utilized to determine whether NETs promote AM pyroptosis and whether degrading NETs or targeting the NLRP3 inflammasome had protective effects on AM pyroptosis and lung injury. Flow cytometric and co-immunoprecipitation analysesverified intracellular reactive oxygen species (ROS) levels and the binding of NLRP3 and ubiquitin (UB) molecules, respectively. Increased NETs production and IL-1β release in septic mice were correlated with the degree of lung injury. NETs upregulated the level of NLRP3, followed by NLRP3 inflammasome assembly and caspase-1 activation, leading to AM pyroptosis executed by the activated fragment of full-length gasdermin D (FH-GSDMD). However, the opposite effect was observed in the context of NETs degradation. Furthermore, NETs markedly elicited an increase in ROS, which facilitated the activation of NLRP3 deubiquitination and the subsequent pyroptosis pathway in AM. Removal of ROS could promote the binding of NLRP3 and ubiquitin, inhibit NLRP3 binding to apoptosis-associated spotted proteins (ASC) and further alleviate the inflammatory changes in the lungs. In summary, these findings indicate that NETs prime ROS generation, which promotes NLRP3 inflammasome activation at the post-translational level to mediate AM pyroptosis and sustain lung injury in septic mice.

High Concentration Hydrogen Mitigates Sepsis-Induced Acute Lung Injury in Mice by Alleviating Mitochondrial Fission and Dysfunction.

Background: Multiple organ failure (MOF) is the main cause of early death in septic shock. Lungs are among the organs that are affected in MOF, resulting in acute lung injury. A large number of inflammatory factors and stress injury in sepsis can lead to alterations in mitochondrial dynamics. Numerous studies have confirmed that hydrogen can alleviate sepsis in the animal model. The purpose of this experiment was to explore the therapeutic effect of high concentration (67%) hydrogen on acute lung injury in septic mice and its mechanism. Methods: The moderate and severe septic models were prepared by cecal ligation and puncture. Hydrogen with different concentrations was inhaled for one hour at 1 h and 6 h after the corresponding surgery. The arterial blood gas of mice during hydrogen inhalation was monitored in real time, and the 7-day survival rate of mice with sepsis was recorded. The pathological changes of lung tissues and functions of livers and kidneys were measured. The changes of oxidation products, antioxidant enzymes and pro-inflammatory cytokines in lungs and serums were detected. Mitochondrial function was measured. Results: The inhalation of 2% or 67% hydrogen improves the 7-day survival rate and reduces acute lung injury as well as liver and kidney injury in sepsis. The therapeutic effect of 67% hydrogen inhalation on sepsis was related to increasing antioxidant enzyme activity, reducing oxidation products and pro-inflammatory cytokines in lungs and serums. Compared with the Sham group, mitochondrial dysfunction was alleviated in hydrogen groups. Conclusions: Hydrogen inhalation by high or low concentration can both significantly improve sepsis; however, a high concentration demonstrates a better protective effect. High concentration hydrogen inhalation can significantly improve the mitochondrial dynamic balance and reduce the lung injury in septic mice.

Circular RNA protein tyrosine kinase 2 aggravates pyroptosis and inflammation in septic lung tissue by promoting microRNA-766/eukaryotic initiation factor 5A axis-mediated ATP efflux

Sepsis is characterized by an acute inflammatory response to infection, often with multiple organ failures, especially severe lung injury. This study was implemented to probe circular RNA (circRNA) protein tyrosine kinase 2 (circPTK2)-associated regulatory mechanisms in septic acute lung injury (ALI). A cecal ligation and puncture-based mouse model and an lipopolysaccharides (LPS)-based alveolar type II cell (RLE-6TN) model were generated to mimic sepsis. In the two models, inflammation- and pyroptosis-related genes were measured. The degree of lung injury in mice was analyzed by hematoxylin and eosin (H&E) staining and the apoptosis was by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining. In addition, pyroptosis and toxicity were detected in cells. Finally, the binding relationship between circPTK2, miR-766, and eukaryotic initiation factor 5A (eIF5A) was detected. Data indicated that circPTK2 and eIF5A were up-regulated and miR-766 was down-regulated in LPS-treated RLE-6TN cells and lung tissue of septic mice. Lung injury in septic mice was ameliorated after inhibition of circPTK2. It was confirmed in the cell model that knockdown of circPTK2 effectively ameliorated LPS-induced ATP efflux, pyroptosis, and inflammation. Mechanistically, circPTK2 mediated eIF5A expression by competitively adsorbing miR-766. Taken together, circPTK2/miR-766/eIF5A axis ameliorates septic ALI, developing a novel therapeutic target for the disease.

Memantine nitrate MN-08 suppresses NLRP3 inflammasome activation to protect against sepsis-induced acute lung injury in mice

Sepsis is a life-threatening organ dysfunction with devastating consequences, prominent among which is lung damage. Memantine, an N-methyl-D-aspartic acid receptor (NMDAR) antagonist, is able to alleviate acute lung injury (ALI). Nitric oxide (NO) suppresses NLRP3 inflammasome activation against lipopolysaccharide (LPS)-induced septic shock. MN-08, a novel nitrate derivative of memantine, possesses both the ability to antagonize NMDAR and release NO. In the present study, we aimed to investigate the protective effects of MN-08 against LPS-induced systemic inflammation and septic lung injury in mice, and to explore the underlying mechanisms of MN-08 in LPS-induced mice and THP-1 macrophages. MN-08 significantly increased the survival rate of septic mice, alleviated LPS-induced sepsis in mice via improving systemic inflammatory response syndrome and immune dysfunction, and attenuated pulmonary injury and inflammatory infiltration. More importantly, the therapeutic benefit of MN-08 for sepsis was greater than that of memantine and dexamethasone. Mechanistically, MN-08 exerted anti-inflammatory activity through inhibiting nuclear translocation of NF-κB, activation of the MAPK signaling pathway and the signaling transduction of STAT3/NF-κB. In addition, MN-08 suppressed NLRP3 inflammasome activation. Taken together, our studies demonstrate that MN-08 may be a promising therapeutic agent for sepsis-induced acute lung injury.

NAT10 regulates neutrophil pyroptosis in sepsis via acetylating ULK1 RNA and activating STING pathway

Emerging evidence suggests that pyroptosis is involved in sepsis. However, the role of neutrophil pyroptosis in sepsis and the mechanisms remains elusive. We find that N-acetyltransferase 10 (NAT10), an acetyltransferase responsible for the N4-acetylation of Cytidine (ac4C) in mRNA, is significantly downregulated in neutrophils from septic mice. Neutrophil-specific over-expression of NAT10 improves the survival and ameliorates lung injury in septic mice by inhibiting neutrophil pyroptosis. Notably, UNC-52-like kinase 1 (ULK1) is identified as the target of NAT10 in neutrophils. The decreased expression of NAT10 resultes in the decay of ULK1 transcripts and therefore the reduced expression of ULK1. As a regulator of STING phosphorylation, the loss of ULK1 enhances the activation of STING-IRF3 signaling and subsequently the elevated pyroptosis-inducing NLRP3 inflammasome in neutrophils. While over-expression of NAT10 restrains pyroptosis in neutrophils as well as septic lethality in mice by reversing the ULK1-STING-NLRP3 axis. The decreased expression of NAT10 are also observed in sepsis patients and its correlation with clinical severity is found. Collectively, our findings disclose that NAT10 is a negative regulator of neutrophil pyroptosis and its downregulation contributes to the progress of sepsis by exacerbating pyroptosis via the ULK1-STING-NLRP3 axis, therefore revealing a potential therapeutic target for sepsis.

Activation of CD4+ T Cell-Derived Cannabinoid Receptor 2 Signaling Exacerbates Sepsis via Inhibiting IL-10.

The cannabinoid receptor 2 (CB2) is a receptor mainly expressed in immune cells and believed to be immunosuppressive in infective or inflammatory models. However, its role in sepsis has not been fully elucidated. In this study, we delineate the function and mechanism of CB2 in the cecal ligation and puncture-induced septic model in mice. The activation of CB2 signaling with HU308 led to decreased survival rates and more severe lung injury in septic mice, and lower IL-10 levels in peritoneal lavage fluid were observed in the CB2 agonist group. The mice with conditional knockout of CB2-encoding gene CNR2 in CD4+ T cells (CD4 Cre CNR2fl/fl) improved survival, enhanced IL-10 production, and ameliorated pulmonary damage in the sepsis model after CB2 activation. In addition, double-knockout of the CNR2 gene (Lyz2 Cre CD4 Cre CNR2fl/fl) decreased the susceptibility to sepsis compared with Lyz2 Cre CNR2fl/fl mice. Mechanistically, the blockade of IL-10 with the anti-IL-10 Ab abolished its protection in CD4 Cre CNR2fl/fl mice. In accordance with the animal study, in vitro results revealed that the lack of CNR2 in CD4+ cells elevated IL-10 production, and CB2 activation inhibited CD4+ T cell-derived IL-10 production. Furthermore, in the clinical environment, septic patients expressed enhanced CB2 mRNA levels compared with healthy donors in PBMCs, and their CB2 expression was inversely correlated with IL-10. These results suggested that the activation of CD4+ T cell-derived CB2 increased susceptibility to sepsis through inhibiting IL-10 production.

Fudosteine attenuates acute lung injury in septic mice by inhibiting pyroptosis via the TXNIP/NLRP3/GSDMD pathway

There is a dearth of effective pharmacotherapies for sepsis-induced acute lung injury/acute respiratory distress syndrome (ALI/ARDS) to which oxidative stress and excessive inflammation are major contributors. We hypothesized that fudosteine, a cysteine derivative, may protect against sepsis-induced ALI/ARDS given its anti-oxidant capacity. This study aimed to investigate the effects and mechanisms of fudosteine in a mouse model of sepsis-induced ALI. Sepsis was induced by cecal ligation and puncture (CLP). The intragastrical administration of fudosteine (25 mg/kg, 50 mg/kg, and 100 mg/kg) dose-dependently decreased proinflammatory cytokine levels in bronchoalveolar lavage fluid (BALF) and serum and reduced BALF/serum albumin and lung wet/dry weight ratios in septic mice. The lung injury score was significantly lowered by fudosteine [e.g., 0.18 ± 0.03 (100 mg/kg) vs. 0.42 ± 0.03 (CLP), P < 0.0001]. Fudosteine also reduced the biomarkers of lung epithelial injury in BALF and markedly improved oxidative stress indicators in lung tissues [e.g., malondialdehyde: 337.70 ± 23.78 (100 mg/kg) vs. 686.40 ± 28.36 (CLP) nmol/mg protein, P < 0.0001]. Lung tissue transcriptomics analyses revealed suppressed inflammatory responses and oxidative stress with fudosteine and the involvement of the inflammasome and pyroptosis pathways. Western blot analyses indicated that fudosteine inhibited the sepsis-induced activation of gasdermin D (GSDMD) and caspase-1 and the upregulation of thioredoxin-interacting protein (TXNIP), nucleotide-binding domain, leucine-rich repeat-containing receptor, pyrin domain-containing-3 (NLRP3), and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Fudosteine therefore protects against sepsis-induced ALI in mice, and the inhibition of pyroptosis via the TXNIP/NLRP3/GSDMD pathway may be an underlying mechanism.