Preconditioning with ß-hydroxybutyrate attenuates lung ischemia-reperfusion injury by suppressing alveolar macrophage pyroptosis through the SIRT1-FOXO3 signaling pathway.

The complex pathogenesis of lung ischemia-reperfusion injury (LIRI) was examined in a murine model, focusing on the role of pyroptosis and its exacerbation of lung injury. We specifically examined the levels and cellular localization of pyroptosis within the lung, which revealed alveolar macrophages as the primary site. The inhibition of pyroptosis by VX-765 reduced the severity of lung injury, underscoring its significant role in LIRI. Furthermore, the therapeutic potential of ß-hydroxybutyrate (ß-OHB) in ameliorating LIRI was examined. Modulation of ß-OHB levels was evaluated by ketone ester supplementation and 3-hydroxybutyrate dehydrogenase 1 (BDH-1) gene knockout, along with the manipulation of the SIRT1-FOXO3 signaling pathway using EX-527 and pCMV-SIRT1 plasmid transfection. This revealed that ß-OHB exerts lung-protective and anti-pyroptotic effects, which were mediated through the upregulation of SIRT1 and the enhancement of FOXO3 deacetylation, leading to decreased pyroptosis markers and lung injury. In addition, ß-OHB treatment of MH-S cells in vitro showed a concentration-dependent improvement in pyroptosis, linking its therapeutic benefits to specific cell mechanisms. Overall, this study highlights the significance of alveolar macrophage pyroptosis in the exacerbation of LIRI and indicates the potential of ß-OHB in mitigating injury by modulating the SIRT1-FOXO3 signaling pathway.

MFSD7C protects hemolysis-induced lung impairments by inhibiting ferroptosis.

Hemolysis drives susceptibility to lung injury and predicts poor outcomes in diseases, such as malaria and sickle cell disease (SCD). However, the underlying pathological mechanism remains elusive. Here, we report that major facilitator superfamily domain containing 7 C (MFSD7C) protects the lung from hemolytic-induced damage by preventing ferroptosis. Mechanistically, MFSD7C deficiency in HuLEC-5A cells leads to mitochondrial dysfunction, lipid remodeling and dysregulation of ACSL4 and GPX4, thereby enhancing lipid peroxidation and promoting ferroptosis. Furthermore, systemic administration of MFSD7C mRNA-loaded nanoparticles effectively prevents lung injury in hemolytic mice, such as HbSS-Townes mice and PHZ-challenged 7 C-/- mice. These findings present the detailed link between hemolytic complications and ferroptosis, providing potential therapeutic targets for patients with hemolytic disorders.

CircMAPK1 induces cell pyroptosis in sepsis-induced lung injury by mediating KDM2B mRNA decay to epigenetically regulate WNK1.

BACKGROUND: Macrophage pyroptosis is a pivotal inflammatory mechanism in sepsis-induced lung injury, however, the underlying mechanisms remain inadequately elucidated. METHODS: Lipopolysaccharides (LPS)/adenosine triphosphate (ATP)-stimulated macrophages and cecal ligation and puncture (CLP)-induced mouse model for sepsis were established. The levels of key molecules were examined by qRT-PCR, Western blotting, immunohistochemistry (IHC) and ELISA assay. The subcellular localization of circMAPK1 was detected by RNA fluorescence in situ hybridization (FISH). Cell viability, LDH release and caspase-1 activity were monitored by CCK-8, LDH assays, and flow cytometry. The bindings between KDM2B/H3K36me2 and WNK1 promoter was detected by chromatin immunoprecipitation (ChIP) assay and luciferase assay, and associations among circMAPK1, UPF1 and KDM2B mRNA were assessed by RNA pull-down or RNA immunoprecipitation (RIP) assays. The pathological injury of lung tissues was evaluated by lung wet/dry weight ratio and hematoxylin and eosin (H&E) staining. RESULTS: CircMAPK1 was elevated in patients with septic lung injury. Knockdown of circMAPK1 protected against LPS/ATP-impaired cell viability and macrophage pyroptosis via WNK1/NLRP3 axis. Mechanistically, loss of circMAPK1 enhanced the association between KDM2B and WNK1 promoter to promote the demethylation of WNK1 and increase its expression. CircMAPK1 facilitated KDM2B mRNA decay by recruiting UPF1. Functional experiments showed that silencing of KDM2B or WNK1 counteracted circMAPK1 knockdown-suppressed macrophage pyroptosis. In addition, silencing of circMAPK1 alleviated CLP-induced lung injury in mice via KDM2B/WNK1/NLRP3 axis. CONCLUSION: CircMAPK1 exacerbates sepsis-induced lung injury by destabilizing KDM2B mRNA to suppress WNK1 expression, thus facilitating NLRP3-driven macrophage pyroptosis.

Sulfotransferase homolog 2 receptors blockade on monocyte subsets along with their inflammatory cytokines for septic lung injury.

PURPOSE OF THE STUDY: To observe the dynamic changes in monocyte subsets during septic lung injury and to assess the anti-inflammatory role of the sulfotransferase homolog 2 (ST2) receptor. MATERIALS AND METHODS: Dynamic changes of monocyte subsets from patients with septic lung injury and mice post-cecal ligation and puncture (CLP) were monitored. ST2 receptors on mice monocytes and concentrations of IL-33, IL-1ß, IL-12, and IL-27 from peripheral blood or culture supernatant were detected. RESULTS: CD14lowCD16- (Mo0) and CD14++CD16+ (Mo2) monocyte subsets were significantly expanded in patients with sepsis-related acute respiratory distress syndrome. In sepsis model mice, monocyte counts, particularly of Ly6Cint and CDLy6Cint+hi monocytes, were significantly increased. The mean optical density value of TNF-α after CLP mainly increased after 24 h, whereas that of IL-6 was significantly increased at all time points assessed after CLP. The levels of IL-1ß, IL-12, IL-27, and IL-33 increased to variable degrees at 6, 12, 24, and 48h after CLP, and ST2+ monocytes were significantly expanded in sepsis model mice compared to sham-operated mice. ST2 receptor blockade suppressed IL-1ß and IL-12 production in cell culture. CONCLUSIONS: Changes in monocyte subsets expressing the ST2 receptor play an important role in septic lung injury by modulating inflammatory cytokine secretion.

NAT10-mediated ac4C acetylation of TFRC promotes sepsis-induced pulmonary injury through regulating ferroptosis.

BACKGROUND: Sepsis-induced pulmonary injury (SPI) is a common complication of sepsis with a high rate of mortality. N4-acetylcytidine (ac4C) is mediated by the ac4C "writer", N-acetyltransferase (NAT)10, to regulate the stabilization of mRNA. This study aimed to investigate the role of NAT10 in SPI and the underlying mechanism. METHODS: Twenty-three acute respiratory distress syndrome (ARDS) patients and 27 non-ARDS volunteers were recruited. A sepsis rat model was established. Reverse transcription-quantitative polymerase chain reaction was used to detect the expression of NAT10 and transferrin receptor (TFRC). Cell viability was detected by cell counting kit-8. The levels of Fe2+, glutathione, and malondialdehyde were assessed by commercial kits. Lipid reactive oxygen species production was measured by flow cytometric analysis. Western blot was used to detect ferroptosis-related protein levels. Haematoxylin & eosin staining was performed to observe the pulmonary pathological symptoms. RESULTS: The results showed that NAT10 was increased in ARDS patients and lipopolysaccharide-treated human lung microvascular endothelial cell line-5a (HULEC-5a) cells. NAT10 inhibition increased cell viability and decreased ferroptosis in HULEC-5a cells. TFRC was a downstream regulatory target of NAT10-mediated ac4C acetylation. Overexpression of TFRC decreased cell viability and promoted ferroptosis. In in vivo study, NAT10 inhibition alleviated SPI. CONCLUSION: NAT10-mediated ac4C acetylation of TFRC aggravated SPI through promoting ferroptosis.

Macrophage extracellular vesicle-packaged miR-23a-3p impairs maintenance and angiogenic capacity of human endothelial progenitor cells in neonatal hyperoxia-induced lung injury.

BACKGROUND: Premature infants requiring mechanical ventilation and supplemental oxygen for respiratory support are at increased risk for bronchopulmonary dysplasia (BPD), wherein inflammation have been proposed as a driver of hyperoxia-induced injuries, including persistent loss of endothelial progenitor cells (EPCs), impaired vascularization and eventual alveolar simplification in BPD lungs. However, the underlying mechanisms linking these phenomena remain poorly defined. METHODS: We used clodronate liposomes to deplete macrophages in a mouse model of neonatal hyperoxia-induced lung injury to evaluate if EPC loss in BPD lungs could be an effect of macrophage infiltration. We further generated in vitro culture systems initiated with cord blood (CB)-derived CD34+ EPCs and neonatal macrophages either polarized from CB-derived monocytes or isolated from tracheal aspirates of human preterm infants requiring mechanical ventilation and oxygen supplementation, to identify EV-transmitted molecular mechanism that is critical for inhibitory actions of hyperoxic macrophages on EPCs. RESULTS: Initial experiments using mouse model identified the crucial role of macrophage infiltration in eliciting significant reduction of c-Kit+ EPCs in BPD lungs. Further examination of this concept in human system, we found that hyperoxia-exposed neonatal macrophages hamper human CD34+ EPC maintenance and impair endothelial function in the differentiated progeny via the EV transmission of miR-23a-3p. Notably, treatment with antagomiR-23a-3p to silence miR-23a-3p in vivo enhances c-Kit+ EPC maintenance, and increases capillary density, and consequently mitigates simplified alveolarization in BPD lungs. CONCLUSION: Our findings highlight the importance of pulmonary intercellular communication in the pathophysiology of BPD, by identifying a linkage through vesicle transfer of miR-23a-3p from hyperoxic macrophages to EPCs, and thus demonstrating potential for novel therapeutic target in BPD.

Short-peptide-based enteral nutrition affects rats MDP translocation and protects against gut-lung injury via the PepT1-NOD2-beclin-1 pathway in vivo.

BACKGROUND: Peptide transporter 1 (PepT1) transports bacterial oligopeptide products and induces inflammation of the bowel. Nutritional peptides compete for the binding of intestinal bacterial products to PepT1. We investigated the mechanism of short-peptide-based enteral nutrition (SPEN) on the damage to the gut caused by the bacterial oligopeptide product muramyl dipeptide (MDP), which is transported by PepT1. The gut-lung axis is a shared mucosal immune system, and immune responses and disorders can affect the gut-respiratory relationship. METHODS AND RESULTS: Sprague-Dawley rats were gavaged with solutions containing MDP, MDP + SPEN, MDP + intact-protein-based enteral nutrition (IPEN), glucose as a control, or glucose with GSK669 (a NOD2 antagonist). Inflammation, mitochondrial damage, autophagy, and apoptosis were explored to determine the role of the PepT1-nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-beclin-1 signaling pathway in the small intestinal mucosa. MDP and proinflammatory factors of lung tissue were explored to determine that MDP can migrate to lung tissue and cause inflammation. Induction of proinflammatory cell accumulation and intestinal damage in MDP gavage rats was associated with increased NOD2 and Beclin-1 mRNA expression. IL-6 and TNF-α expression and apoptosis were increased, and mitochondrial damage was severe, as indicated by increased mtDNA in the MDP group compared with controls. MDP levels and expression of proinflammatory factors in lung tissue increased in the MDP group compared with the control group. SPEN, but not IPEN, eliminated these impacts. CONCLUSIONS: Gavage of MDP to rats resulted in damage to the gut-lung axis. SPEN reverses the adverse effects of MDP. The PepT1-NOD2-beclin-1 pathway plays a role in small intestinal inflammation, mitochondrial damage, autophagy, and apoptosis.

Epithelial-mesenchymal transition in chemoradiation-induced lung damage: Mechanisms and potential treatment approaches.

Pulmonary injury is one of the key restricting factors for the therapy of malignancies with chemotherapy or following radiotherapy for chest cancers. The lung is a sensitive organ to some severely toxic antitumor drugs, consisting of bleomycin and alkylating agents. Furthermore, treatment with radiotherapy may drive acute and late adverse impacts on the lung. The major consequences of radiotherapy and chemotherapy in the lung are pneumonitis and fibrosis. Pneumonitis may arise some months to a few years behind cancer therapy. However, fibrosis is a long-term effect that appears years after chemo/or radiotherapy. Several mechanisms such as oxidative stress and severe immune reactions are implicated in the progression of pulmonary fibrosis. Epithelial-mesenchymal transition (EMT) is offered as a pivotal mechanism for lung fibrosis behind chemotherapy and radiotherapy. It seems that pulmonary fibrosis is the main consequence of EMT after chemo/radiotherapy. Several biological processes, consisting of the liberation of pro-inflammatory and pro-fibrosis molecules, oxidative stress, upregulation of nuclear factor of κB and Akt, epigenetic changes, and some others, may participate in EMT and pulmonary fibrosis behind cancer therapy. In this review, we aim to discuss how chemotherapy or radiotherapy may promote EMT and lung fibrosis. Furthermore, we review potential targets and effective agents to suppress EMT and lung fibrosis after cancer therapy.

Flavone attenuates nicotine-induced lung injury in rats exposed to gamma radiation via modulating PI3K/Nrf2 and FoxO1/NLRP3 inflammasome.

Prolonged exposure to different occupational or environmental toxicants triggered oxidative stress and inflammatory reactions mediated lung damage. This study was designed to explore the influence and protective impact of flavone on lung injury in rats intoxicated with nicotine (NIC) and exposed to radiation (IR). Forty rats were divided into four groups; group I control, group II flavone; rats were administered with flavone (25 mg/kg/day), group III NIC + IR; rats were injected intraperitoneally with NIC (1 mg/kg/day) and exposed to γ-IR (3.5 Gy once/week for 2 weeks) while group IV NIC + IR + flavone; rats were injected with NIC, exposed to IR and administered with flavone. Redox status parameters and histopathological changes in lung tissue were evaluated. Nuclear factor-kappa B (NF-κB), forkhead box O-class1 (FoxO1) and nucleotide-binding domain- (NOD-) like receptor pyrin domain-containing-3 (NLRP3) gene expression were measured in lung tissues. Moreover, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and phosphatidylinositol three kinase (PI3K) were measured using ELISA kits. Our data demonstrates, for the first time, that flavone protects the lung from NIC/IR-associated cytotoxicity, by attenuating the disrupted redox status and aggravating the antioxidant defence mechanism via activation of the PI3K/Nrf2. Moreover, flavone alleviates pulmonary inflammation by inhibiting the inflammatory signaling pathway FOXO1/NF-κB/NLRP3- Inflammasome. Collectively, the obtained results exhibited a notable efficiency of flavone in alleviating lung injury induced by NIC and IR via modulating PI3K/Nrf2 and FoxO1/NLRP3 Inflammasome.

Evaluation of the protective effect of Curcuma longa and PPARγ agonist, pioglitazone on paraquat-induced lung injury in rats.

BACKGROUND: The inhalation of paraquat (PQ), one of the most widely used herbicides in the world, can result in lung injury. Curcuma longa (Cl) has long history in traditional and folk medicine for the treatment of a wide range of disorders including respiratory diseases. AIM: The aim of the present work was to evaluate the preventive effect of Cl on inhaled PQ-induced lung injury in rats. METHODS: Male Wistar rats were divided into 8 groups (n = 7), one group exposed to saline (control) and other groups exposed to PQ aerosol. Saline (PQ), Cl extract, (two doses), curcumin (Cu), pioglitazone (Pio), and the combination of Cl-L + Pio and dexamethasone (Dex) were administered during the exposure period to PQ. Total and differential white blood cell (WBC) counts, oxidant and antioxidant indicators in the bronchoalveolar lavage (BALF), interleukin (IL)-10, and tumor necrosis alpha (TNF-α) levels in the lung tissues, lung histologic lesions score, and air way responsiveness to methacholine were evaluated. RESULTS: WBC counts (Total and differential), malondialdehyde level, tracheal responsiveness (TR), IL-10, TNF-α and histopathological changes of the lung were markedly elevated but total thiol content and the activities of catalase and superoxide dismutase were decreased in the BALF in the PQ group. Both doses of Cl, Cu, Pio, Cl-L + Pio, and Dex markedly improved all measured variables in comparison with the PQ group. CONCLUSION: CI, Pio, and Cl-L + Pio improved PQ-induced lung inflammation and oxidative damage comparable with the effects of Dex.

Silibinin Mitigates Vanadium-induced Lung Injury via the TLR4/MAPK/NF-κB Pathway in Mice.

BACKGROUND/AIM: Silibinin, has been investigated for its potential benefits and mechanisms in addressing vanadium pentoxide (V2O5)-induced pulmonary inflammation. This study explored the anti-inflammatory activity of silibinin and elucidate the mechanisms by which it operates in a mouse model of vanadium-induced lung injury. MATERIALS AND METHODS: Eight-week-old male BALB/c mice were exposed to V2O5 to induce lung injury. Mice were pretreated with silibinin at doses of 50 mg/kg and 100 mg/kg. Histological analyses were performed to assess cell viability and infiltration of inflammatory cells. The expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß) and activation of the MAPK and NF-[Formula: see text]B signaling pathways, as well as the NLRP3 inflammasome, were evaluated using real-time PCR, western blot analysis, and immunohistochemistry. Whole blood analysis was conducted to measure white blood cell counts. RESULTS: Silibinin treatment significantly improved cell viability, reduced inflammatory cell infiltration, and decreased the expression of pro-inflammatory cytokines in V2O5-induced lung injury. It also notably suppressed the activation of the MAPK and NF-[Formula: see text]B signaling pathways, along with a marked reduction in NLRP3 inflammasome expression levels in lung tissues. Additionally, silibinin-treated groups exhibited a significant decrease in white blood cell counts, including neutrophils, lymphocytes, and eosinophils. CONCLUSION: These findings underscore the potent anti-inflammatory effects of silibinin in mice with V2O5-induced lung inflammation, highlighting its therapeutic potential. The study not only confirms the efficacy of silibinin in mitigating inflammatory responses but also provides a foundational understanding of its role in modulating key inflammatory pathways, paving the way for future therapeutic strategies against pulmonary inflammation induced by environmental pollutants.

Melatonin alleviates hyperoxia-induced lung injury through elevating MSC exosomal miR-18a-5p expression to repress PUM2 signaling.

Mesenchymal stem cells (MSC)-derived exosomes (Exo) are a possible option for hyperoxia-induced lung injury (HLI). We wanted to see if melatonin (MT)-pretreated MSC-derived exosomes (MT-Exo) were more effective against HLI, and we also tried to figure out the underlying mechanism. HLI models were established by hyperoxia exposure. HE staining was adopted to analyze lung pathological changes. MTT and flow cytometry were used to determine cell viability and apoptosis, respectively. The mitochondrial membrane potential (MMP) was analyzed using the JC-1 probe. LDH, ROS, SOD, and GSH-Px levels were examined by the corresponding kits. The interactions between miR-18a-5p, PUM2, and DUB3 were analyzed by molecular interaction experiments. MT-Exo could effectively inhibit hyperoxia-induced oxidative stress, inflammatory injury, and apoptosis in lung epithelial cells, while these effects of MT-Exo were weakened by miR-18a-5p knockdown in MSCs. miR-18a-5p reduced PUM2 expression in MLE-12 cells by directly targeting PUM2. In addition, PUM2 inactivated the Nrf2/HO-1 signaling pathway by promoting DUB3 mRNA decay post-transcriptionally. As expected, PUM2 overexpression or DUB3 knockdown abolished the protective effect of MT-Exo on hyperoxia-induced lung epithelial cell injury. MT-Exo carrying miR-18a-5p reduced hyperoxia-mediated lung injury in mice through activating Nrf2/HO-1 pathway. MT reduced PUM2 expression and subsequently activated the DUB3/Nrf2/HO-1 signal axis by increasing miR-18a-5p expression in MSC-derived exosomes to alleviate HLI.

[2-DG improves lung ischemia/reperfusion injury by inhibiting NLRP3-mediated pyroptosis in rats].

The aim of this study was to investigate whether the protective effect of 2-deoxyglucose (2-DG) on lung ischemia/reperfusion (I/R) injury is mediated by inhibiting nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)-mediated pyroptosis in rats. Male Sprague-Dawley rats were randomly divided into control group, 2-DG group, lung I/R injury group (I/R group) and 2-DG+I/R group. 2-DG (0.7 g/kg) was intraperitoneally injected 1 h prior to lung ischemia. The tissue structure was measured under light microscope. Lung injury parameters were detected. The contents of malondialdehyde (MDA), myeloperoxidase (MPO) and lactate were determined by commercially available kits. ELISA was used to detect the levels of IL-1ß and IL-18. Western blot, qRT-PCR and immunofluorescence staining were used to measure the expression changes of glycolysis and pyroptosis related indicators. The results showed that there was no significant difference in the parameters between the control group and the 2-DG group. However, the lung injury parameters, oxidative stress response, lactic acid content, IL-1ß, and IL-18 levels were significantly increased in the I/R group. The protein expression levels of glycolysis and pyroptosis related indicators including hexokinase 2 (HK2), pyruvate kinase 2 (PKM2), NLRP3, Gasdermin superfamily member GSDMD-N, cleaved-Caspase1, cleaved-IL-1ß and cleaved-IL-18, and the gene expression levels of HK2, PKM2 and NLRP3 were markedly up-regulated in the I/R group compared with those in the control group. The expression of HK2 and NLRP3 was also increased detected by immunofluorescence staining. Compared with the I/R group, the 2-DG+I/R group exhibited significantly improved alveolar structure and inflammatory infiltration, reduced lung injury parameters, and decreased expression of glycolysis and pyroptosis related indicators. These results suggest that 2-DG protects against lung I/R injury possibly by inhibiting NLRP3-mediated pyroptosis in rats.

TNKS1BP1 mediates AECII senescence and radiation induced lung injury through suppressing EEF2 degradation.

BACKGROUND: Although recent studies provide mechanistic understanding to the pathogenesis of radiation induced lung injury (RILI), rare therapeutics show definitive promise for treating this disease. Type II alveolar epithelial cells (AECII) injury in various manner results in an inflammation response to initiate RILI. RESULTS: Here, we reported that radiation (IR) up-regulated the TNKS1BP1, causing progressive accumulation of the cellular senescence by up-regulating EEF2 in AECII and lung tissue of RILI mice. Senescent AECII induced Senescence-Associated Secretory Phenotype (SASP), consequently activating fibroblasts and macrophages to promote RILI development. In response to IR, elevated TNKS1BP1 interacted with and decreased CNOT4 to suppress EEF2 degradation. Ectopic expression of EEF2 accelerated AECII senescence. Using a model system of TNKS1BP1 knockout (KO) mice, we demonstrated that TNKS1BP1 KO prevents IR-induced lung tissue senescence and RILI. CONCLUSIONS: Notably, this study suggested that a regulatory mechanism of the TNKS1BP1/CNOT4/EEF2 axis in AECII senescence may be a potential strategy for RILI.

Necroptosis in alveolar epithelial cells drives lung inflammation and injury caused by SARS-CoV-2 infection.

COVID-19, caused by SARS-CoV-2 infection, results in irreversible or fatal lung injury. We assumed that necroptosis of virus-infected alveolar epithelial cells (AEC) could promote local inflammation and further lung injury in COVID-19. Since CD8+ lymphocytes induced AEC cell death via cytotoxic molecules such as FAS ligands, we examined the involvement of FAS-mediated cell death in COVID-19 patients and murine COVID-19 model. We identified the occurrence of necroptosis and subsequent release of HMGB1 in the admitted patients with COVID-19. In the mouse model of COVID-19, lung inflammation and injury were attenuated in Fas-deficient mice compared to Fas-intact mice. The infection enhanced Type I interferon-inducible genes in both groups, while inflammasome-associated genes were specifically upregulated in Fas-intact mice. The treatment with necroptosis inhibitor, Nec1s, improved survival rate, lung injury, and systemic inflammation. SARS-CoV-2 induced necroptosis causes cytokine induction and lung damage, and its inhibition could be a novel therapeutic strategy for COVID-19.

Role of sex as a biological variable in neonatal alveolar macrophages.

The lung macrophages play a crucial role in health and disease. Sexual dimorphism significantly impacts the phenotype and function of tissue-resident macrophages. The primary mechanisms responsible for sexually dimorphic outcomes in bronchopulmonary dysplasia (BPD) remain unidentified. We tested the hypothesis that biological sex plays a crucial role in the transcriptional state of alveolar macrophages, using neonatal murine hyperoxia-induced lung injury as a relevant model for human BPD. The effects of neonatal hyperoxia exposure (95 % FiO2, PND1-5: saccular stage) on the lung myeloid cells acutely after injury and during normoxic recovery were measured. Alveolar macrophages (AM) from room air- and hyperoxia exposed from male and female neonatal murine lungs were subjected to bulk-RNA Sequencing. AMs are significantly depleted in the hyperoxia-exposed lung acutely after injury, with subsequent recovery in both sexes. The transcriptome of the alveolar macrophages is impacted by neonatal hyperoxia exposure and by sex as a biological variable. Pathways related to DNA damage and interferon-signaling were positively enriched in female AMs. Metabolic pathways related to glucose and carbohydrate metabolism were positively enriched in the male AMs, while oxidative phosphorylation was negatively enriched. These pathways were shared with monocytes and airway macrophages from intubated male and female human premature neonates.

Dexmedetomidine's protective mechanism against hyperoxic injury in neonatal rats.

Bronchopulmonary dysplasia (BPD) is a common serious complication of premature babies. No effective means control it. Hyperoxia damage is one of the important mechanisms of BPD. The reaserach confirmed pyroptosis existed in BPD. Dexmedetomidine is a new, high-specific α2 receptor agonist. Previous research foundation found that dexmedetomidine has a protective effect on BPD. To investigate how dexmedetomidine improves hyperoxic lung injury in neonatal mice by regulating pyroptosis. Neonatal rats were randomly divided into four groups: normal control group, hyperoxic injury group, air plus dexmedetomidine group, and hyperoxia plus dexmedetomidine group. After seven days the lungs of rats in each group were extracted, and the wet-to-dry weight ratio of the lung was measured. The lung injury in rats was observed using hematoxylin-eosin staining. Additionally, the expression and localization of nucleotide-binding oligomerization domain-like receptor thermal protein domain associated protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), and gasdermin D (GSDMD) proteins were examined in the lungs of rats using immunofluorescence staining. The mRNA levels of NLRP3, ASC, caspase-1, and interleukin 18 (IL-18) in the lungs of rats were determined using real-time PCR. Moreover, the protein levels of NLRP3, ASC, caspase-1/cleaved caspase-1, interleukin 1beta (IL-1ß), IL-18, and tunor necrosis factor alpha (TNF-α) were detected in lungs of rats using Western blot. The extent of mitochondrial damage in lung tissues of each group was observed by transmission electron microscopy. The lung tissue injury of the neonatal rats was significantly improved in the hyperoxia plus dexmedetomidine group compared to the hyperoxic injury group. Furthermore, the expressions of pyroptosis-related proteins such as NLRP3, ASC, cleaved-caspase-1, and GSDMD were significantly decreased, along with the expressions of inflammatory factors in lung tissues. By inhibiting the NLRP3/caspase-1/GSDMD pyroptosis pathway, dexmedetomidine reduces the activation and release of inflammatory factors and provides a protective effect against hyperoxic lung injury in neonatal mice.

Integration of microbiomics, metabolomics, and transcriptomics reveals the therapeutic mechanism underlying Fuzheng-Qushi decoction for the treatment of lipopolysaccharide-induced lung injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Fuzheng-Qushi decoction (FZQS) is a practical Chinese herbal formula for relieving cough and fever. Therefore, the action and specific molecular mechanism of FZQS in the treatment of lung injury with cough and fever as the main symptoms need to be further investigated. AIMS OF THE STUDY: To elucidate the protective effects of FZQS against lung injury in mice and reveal its potential targets and key biological pathways for the treatment of lung injury based on transcriptomics, microbiomics, and untargeted metabolomics analyses. MATERIALS AND METHODS: Lipopolysaccharide (LPS) was used to induce a mouse model of lung injury, followed by the administration of FZQS. ELISA was used to detect IL-1ß, IL-6, IL-17A, IL-4, IL-10, and TNF-α, in mouse lung tissues. Macrophage polarization and neutrophil activation were measured by flow cytometry. RNA sequencing (RNA-seq) was applied to screen for differentially expressed genes (DEGs) in lung tissues. RT-qPCR and Western blot assays were utilized to validate key DEGs and target proteins in lung tissues. 16S rRNA sequencing was employed to characterize the gut microbiota of mice. Metabolites in the gut were analyzed using untargeted metabolomics. RESULTS: FZQS treatment significantly ameliorated lung histopathological damage, decreased pro-inflammatory cytokine levels, and increased anti-inflammatory cytokine levels. M1 macrophage levels in the peripheral blood decreased, M2 macrophage levels increased, and activated neutrophils were inhibited in mice with LPS-induced lung injury. Importantly, transcriptomic analysis showed that FZQS downregulated macrophage and neutrophil activation and migration and adhesion pathways by reversing 51 DEGs, which was further confirmed by RT-qPCR and Western blot analysis. In addition, FZQS modulated the dysbiosis of the gut microbiota by reversing the abundance of Corynebacterium, Facklamia, Staphylococcus, Paenalcaligenes, Lachnoclostridium, norank_f_Muribaculaceae, and unclassified_f_Lachnospiraceae. Meanwhile, metabolomics analysis revealed that FZQS significantly regulated tryptophan metabolism by reducing the levels of 3-Indoleacetonitrile and 5-Hydroxykynurenine. CONCLUSION: FZQS effectively ameliorated LPS-induced lung injury by inhibiting the activation, migration, and adhesion of macrophages and neutrophils and modulating gut microbiota and its metabolites.

The deubiquitinase OTUD1 deubiquitinates TIPE2 and plays a protective role in sepsis-induced lung injury by targeting TAK1-mediated MAPK and NF-κB signaling.

Ovarian tumor domain-containing protease 1 (OTUD1) is a critical negative regulator that promotes innate immune homeostasis and is extensively involved in the pathogenesis of sepsis. In this study, we performed a powerful integration of multiomics analysis and an experimental mechanistic investigation to elucidate the immunoregulatory role of OTUD1 in sepsis at the clinical, animal and cellular levels. Our study revealed the upregulation of OTUD1 expression and the related distinctive alterations observed via multiomics profiling in clinical and experimental sepsis. Importantly, in vivo and in vitro, OTUD1 was shown to negatively regulate inflammatory responses and play a protective role in sepsis-induced pathological lung injury by mechanistically inhibiting the activation of the transforming growth factor-beta-activated kinase 1 (TAK1)-mediated mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathways in the present study. Subsequently, we probed the molecular mechanisms underlying OTUD1's regulation of NF-κB and MAPK pathways by pinpointing the target proteins that OTUD1 can deubiquitinate. Drawing upon prior research conducted in our laboratory, it has been demonstrated that tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) performs a protective function in septic lung injury and septic encephalopathy by suppressing the NF-κB and MAPK pathways. Hence, we hypothesized that TIPE2 might be a target protein of OTUD1. Additional experiments, including Co-IP, immunofluorescence co-localization, and Western blotting, revealed that OTUD1 indeed has the ability to deubiquitinate TIPE2. In summary, OTUD1 holds potential as an immunoregulatory and inflammatory checkpoint agent, and could serve as a promising therapeutic target for sepsis-induced lung injury.