Lung Ischemia-reperfusion Research Articles

Rebaudioside B Attenuates Lung Ischemia-reperfusion Injury Associated Apoptosis and Inflammation.

At present, no proven effective treatment is available for Lung Ischemiareperfusion Injury (LIRI). Natural compounds offer promising prospects for developing new drugs to address various diseases. This study sought to explore the potential of Rebaudioside B (Reb B) as a treatment compound for LIRI, both in vivo and in vitro. This study involved utilizing the human pulmonary alveolar cell line A549, consisting of epithelial type II cells, subjected to Oxygen-glucose Deprivation/recovery (OGD/R) for highthroughput in vitro cell viability screening. The aim was to identify the most promising candidate compounds. Additionally, an in vivo rat model of lung ischemia-reperfusion was employed to evaluate the potential protective effects of Reb B. Through high-throughput screening, Reb B emerged as the most promising natural compound among those tested. In the A549 OGD/R models, Reb B exhibited a capacity to enhance cell viability by mitigating apoptosis. In the in vivo LIRI model, pre-treatment with Reb B notably decreased apoptotic cells, perivascular edema, and neutrophil infiltration within lung tissues. Furthermore, Reb B demonstrated its ability to attenuate lung inflammation associated with LIRI primarily by elevating IL-10 levels while reducing levels of IL-6, IL-8, and TNF-α. The comprehensive outcomes strongly suggest Reb B's potential as a protective agent against LIRI. This effect is attributed to its inhibition of the mitochondrial apoptotic pathway and its ability to mitigate the inflammatory response.

Circulating mtNFPs Are Associated with ARDS after CPB and Regulate Endothelial Barrier through FPR2.

Cardiopulmonary bypass (CPB) increases the risk of acute respiratory distress syndrome (ARDS) due to endothelial cell (EC) barrier dysfunction. However, the specific role of mitochondrial N-formyl peptides (mtNFPs) in ARDS following CPB remains unexplored. Here, we investigated the differential expression of circulating mtNFPs in patients after CPB, focusing on the novel role of FPR2 in ECs. Levels of circulating mtNFPs were assessed using enzyme-linked immunosorbent assay (ELISA). Several mtNFPs (ND4, ND5, ND6, and Cox1) were significantly upregulated in patients with ARDS at day 1 post-CPB compared to patients without ARDS. Higher levels of ND6 were correlated with worst PaO2/FiO2 (r=-0.2219 and P<0.0001) and cardiac Troponin T (r=2.107 and P<0.0001). Utilizing patient-derived serum and a rat lung ischemia reperfusion injury (LIRI) model, we observed a positive correlation between serum ND6 concentration and ARDS, which is also associated with EC barrier dysfunction. In vitro experiments, using trans-endothelial electric resistance (TEER) measurements and fluorescence microscopy with FITC-labeled VE-cadherin, demonstrated that ND6 disrupts the EC barrier through FPR2. Furthermore, FPR2 controls the release of ND6 out of mitochondria and cytoplasm under hypoxia reoxygenation (HR). Activated FPR2 leads to upregulation of nuclear transcription factor-kappa B (NF-κB) by inducing IκBα phosphorylation, promoting ICAM1 and VCAM1 expression, thereby compromising EC barrier integrity. Circulating pro-inflammatory and barrier-disruptive mtNFPs, particularly ND6, are associated with ARDS in patients undergoing CPB. The novel ND6-FPR2 axis regulates inflammation and EC permeability through the NF-κB pathway.

Quercetin as a therapeutic agent activate the Nrf2/Keap1 pathway to alleviate lung ischemia-reperfusion injury

Lung ischemia-reperfusion injury (LIRI) causes oxidative stress, inflammation, and immune system activation. The Nrf2/Keap1/HO-1 pathway is important in cellular defense against these effects. Quercetin, a flavonoid with antioxidant, anti-inflammatory, and anti-cancer properties, has been investigated. Our aim in this study was to investigate the effect of quercetin on preventing lung ischemia-reperfusion injury and the role of the Nrf2/Keap1/HO-1 pathway. Sixty-four male Wistar rats were divided into four distinct groups(n = 16). Sham, lung ischemia-reperfusion (LIR), Saline + LIR, Quercetin + LIR (30 mg/kg i.p for a week before LIR). LIR groups were subjected to 60 min of ischemia (left pulmonary artery, vein, and bronchus) and 120 min of reperfusion. Our assessment encompassed a comprehensive analysis of various factors, including the evaluation of expression Nrf2, Keap1, and Heme Oxygenase-1 (HO-1) levels and NF-κB protein. Furthermore, we examined markers related to inflammation (interleukin-1β and tumor necrosis factor alpha), oxidative stress (malondialdehyde, total oxidant status, superoxide dismutase, glutathione peroxidase, total antioxidant capacity), lung edema (Wet/dry lung weight ratio and total protein concentration), apoptosis (Bax and Bcl2 protein), and histopathological alterations (intra-alveolar edema, alveolar hemorrhage, and neutrophil infiltration). Our results show that ischemia-reperfusion results in heightened inflammation, oxidative stress, apoptosis, lung edema, and histopathological damage. Quercetin showed preventive effects by reducing these markers, acting through modulation of the Nrf2/Keap1 pathway and inhibiting the NF-κB pathway. This anti-inflammatory effect, complementary to the antioxidant effects of quercetin, provides a multifaceted approach to cell protection that is important for developing therapeutic strategies against ischemia-reperfusion injury and could be helpful in preventive strategies against ischemia-reperfusion.

Heparanase-induced endothelial glycocalyx degradation exacerbates lung ischemia/reperfusion injury in male mice.

The endothelial glycocalyx (eGC) is a carbohydrate-rich layer on the vascular endothelium, and its damage can lead to endothelial and organ dysfunction. Heparanase (HPSE) degrades the eGC in response to cellular stress, but its role in organ dysfunction remains unclear. This study investigates HPSE's role in lung ischemia-reperfusion (I/R) injury. A left lung hilar occlusion model was used in B6 wildtype (WT) and HPSE genetic knockout (-/-) mice to induce I/R injury invivo. The left lungs were ischemic for 1 h followed by reperfusion for 4 h prior to investigations of lung function and eGC status. Data were compared between uninjured lungs and I/R-injured lungs in WT and HPSE-/- mice. WT lungs showed significant functional impairment after I/R injury, whereas HPSE-/- lungs did not. Inhibition or knockout of HPSE prevented eGC damage, inflammation, and cellular migration after I/R injury by reducing matrix metalloproteinase activities. HPSE-/- mice exhibited compensatory regulation of related gene expressions. HPSE facilitates eGC degradation leading to inflammation and impaired lung function after I/R injury. HPSE may be a therapeutic target to attenuate graft damage in lung transplantation.

Bone marrow mesenchymal stem cells-derived exosomal miR-381 alleviates lung ischemia-reperfusion injury by activating Treg differentiation through inhibiting YTHDF1 expression

AimOur study aimed to investigate whether BMSCs-derived exosomal miR-381 promotes Treg cell differentiation in lung ischemia-reperfusion injury (LIRI), and the underlying mechanism. MethodsThe in vitro and in vivo models of LIRI were established by hypoxia/reoxygenation (H/R) treatment and lung ischemia/reperfusion (I/R) surgery, respectively. BMSCs-derived exosomes were isolated and identified by western blot, nanoparticle tracking analysis, and transmission electron microscopy. Cell viability, proliferation, and apoptosis were assessed by CCK-8, EdU, and flow cytometry assay, respectively. IL-18 secretion level in lung microvascular endothelial cells (LMECs) and lung tissue homogenate was examined by ELISA. Treg cell differentiation was determined using flow cytometry. The relationships between miR-381, YTHDF1, and IL-18 were investigated using dual-luciferase reporter gene, RIP, and/or RNA pull-down assays. MeRIP assay was employed to determine m6A modification of IL-18 mRNA in LMECs. The ubiquitination level of Foxp3 protein in CD4+ T cells was analyzed by Co-IP assay. ResultsBMSCs-derived exosomes reduced LMECs injury and increased Treg cell differentiation in LIRI, whereas miR-381 inhibition in BMSCs weakened these impacts. Mechanistically, miR-381 inhibited IL-18 translation in LMECs by inhibiting YTHDF1 expression via binding to its 3’-UTR. As expected, YTHDF1 overexpression in LMECs abolished the effects of miR-381-overexpressed exosomes on LMECs injury and Treg cell differentiation. Moreover, LMECs-secreted IL-18 inhibited Treg cell differentiation by promoting the ubiquitination degradation of Foxp3 protein. ConclusionBMSCs-derived exosomal miR-381 suppressed IL-18 translation in LMECs through binding to YTHDF1 3’-UTR, thus suppressing the ubiquitination degradation of Foxp3 in CD4+ T cells, which promoted Treg cell differentiation and mitigated LIRI development.

Preconditioning with β-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 β-hydroxybutyrate (β-OHB) in ameliorating LIRI was examined. Modulation of β-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 β-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, β-OHB treatment of MH-S cells invitro 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 β-OHB in mitigating injury by modulating the SIRT1-FOXO3 signaling pathway.

Inhibition of ALOX12-12-HETE Alleviates Lung Ischemia-Reperfusion Injury by Reducing Endothelial Ferroptosis-Mediated Neutrophil Extracellular Trap Formation.

Lung ischemia-reperfusion injury (IRI) stands as the primary culprit behind primary graft dysfunction (PGD) after lung transplantation, yet viable therapeutic options are lacking. In the present study, we used a murine hilar clamp (1 h) and reperfusion (3 h) model to study IRI. The left lung tissues were harvested for metabolomics, transcriptomics, and single-cell RNA sequencing. Metabolomics of plasma from human lung transplantation recipients was also performed. Lung histology, pulmonary function, pulmonary edema, and survival analysis were measured in mice. Integrative analysis of metabolomics and transcriptomics revealed a marked up-regulation of arachidonate 12-lipoxygenase (ALOX12) and its metabolite 12-hydroxyeicosatetraenoic acid (12-HETE), which played a pivotal role in promoting ferroptosis and neutrophil extracellular trap (NET) formation during lung IRI. Additionally, single-cell RNA sequencing revealed that ferroptosis predominantly occurred in pulmonary endothelial cells. Importantly, Alox12-knockout (KO) mice exhibited a notable decrease in ferroptosis, NET formation, and tissue injury. To investigate the interplay between endothelial ferroptosis and NET formation, a hypoxia/reoxygenation (HR) cell model using 2 human endothelial cell lines was established. By incubating conditioned medium from HR cell model with neutrophils, we found that the liberation of high mobility group box1 (HMGB1) from endothelial cells undergoing ferroptosis facilitated the formation of NETs by activating the TLR4/MYD88 pathway. Last, the administration of ML355, a targeted inhibitor of Alox12, mitigated lung IRI in both murine hilar clamp/reperfusion and rat left lung transplant models. Collectively, our study indicates ALOX12 as a promising therapeutic strategy for lung IRI.

Exploring the research progression and evolutionary trends of lung ischemia-reperfusion injury: A bibliometric analysis from 1979 to 2023

BackgroundLung ischemia-reperfusion injury (LIRI) poses a significant challenge in various clinical scenarios. Despite extensive research on the pathogenesis and potential treatments of LIRI, there is a notable absence of bibliometric analysis. Materials and methodsWe summarized the results of LIRI research through two searches on the Web of Science, covering data from 1979 to 2023 with topic words “lung” and “reperfusion injury”. The collected data were analyzed and visualized based on country, author(s), and keywords by bibliometric software. The keyword “programmed cell death” was further added to explore the hotspot of the LIRI research field. ResultsThe initial analysis of 1648 research articles showed a total of 40 countries and 7031 researchers were involved in the publications, with America being the most productive country in the research field of LIRI. Keyword analysis revealed that the evolving focus of LIRI research has progressively transitioned from, lung transplantation, primary graft dysfunction, inflammation, oxidative stress, and ex vivo lung perfusion to cell death. Subsequently, 212 publications specifically addressing programmed cell death (PCD) in LIRI were identified, which clarified the recent hotspot of the LIRI field. ConclusionWith closer international cooperation and increasing research scale, the LIRI research focused mainly on the pathogenesis and potential therapeutic interventions for LIRI. PCD in LIRI is becoming a trending topic and will continue to be a hotspot in this field. Our study may offer valuable guidance for future research endeavors concerning LIRI.

Monomethyl fumarate attenuates lung Ischemia/Reperfusion injury by disrupting the GAPDH/Siah1 signaling cascade

Monomethyl fumarate (MMF), a potent anti-inflammatory agent used to treat multiple sclerosis, has demonstrated efficacy in various inflammatory and ischemia/reperfusion (IR) models; however, its impact on IR-induced acute lung injury (ALI) has not been explored. We investigated, for the first time, whether MMF attenuates lung IR injury through inhibition of the GAPDH/Siah1 signaling pathway. Rats were subjected to IR injury using an isolated perfused lung model, and proximity ligation assays were employed to evaluate the presence and distribution of the GAPDH/Siah1 complex. In vitro studies involved pretreating human primary alveolar epithelial cells (HPAECs) with MMF and/or inducing GAPDH overexpression or silencing, followed by exposure to hypoxia–reoxygenation. The findings revealed significantly reduced lung damage indicators, including edema, proinflammatory cytokines, oxidative stress and apoptosis, in MMF-treated rats. Notably, MMF treatment inhibited GAPDH/Siah1 complex formation and nuclear translocation, indicating that disruption of the GAPDH/Siah1 cascade was the primary cause of these improvements. Our in vitro studies on pretreated HPAECs corroborate these in vivo findings, further strengthening this interpretation. Our study results suggest that the protective effects of MMF against lung IR injury may be attributed, at least in part, to its ability to disrupt the GAPDH/Siah1 signaling cascade, thereby attenuating inflammatory and apoptotic responses. Given these encouraging results, MMF has emerged as a promising therapeutic candidate for the management of lung IR injury.

Cathepsin C from extracellular histone-induced M1 alveolar macrophages promotes NETosis during lung ischemia-reperfusion injury

Primary graft dysfunction (PGD) is a severe form of acute lung injury resulting from lung ischemia/reperfusion injury (I/R) in lung transplantation (LTx), associated with elevated post-transplant morbidity and mortality rates. Neutrophils infiltrating during reperfusion are identified as pivotal contributors to lung I/R injury by releasing excessive neutrophil extracellular traps (NETs) via NETosis. While alveolar macrophages (AMs) are involved in regulating neutrophil chemotaxis and infiltration, their role in NETosis during lung I/R remains inadequately elucidated. Extracellular histones constitute the main structure of NETs and can activate AMs. In this study, we confirmed the significant involvement of extracellular histone-induced M1 phenotype of AMs (M1-AMs) in driving NETosis during lung I/R. Using secretome analysis, public protein databases, and transwell co-culture models of AMs and neutrophils, we identified Cathepsin C (CTSC) derived from AMs as a major mediator in NETosis. Further elucidating the molecular mechanisms, we found that CTSC induced NETosis through a pathway dependent on NADPH oxidase-mediated production of reactive oxygen species (ROS). CTSC could significantly activate p38 MAPK, resulting in the phosphorylation of the NADPH oxidase subunit p47phox, thereby facilitating the trafficking of cytoplasmic subunits to the cell membrane and activating NADPH oxidase. Moreover, CTSC up-regulated and activated its substrate membrane proteinase 3 (mPR3), resulting in an increased release of NETosis-related inflammatory factors. Inhibiting CTSC revealed great potential in mitigating NETosis-related injury during lung I/R. These findings suggests that CTSC from AMs may be a crucial factor in mediating NETosis during lung I/R, and targeting CTSC inhition may represent a novel intervention for PGD in LTx.

Sevoflurane enhances autophagy via Rac1 to attenuate lung ischaemia‒reperfusion injury

Sevoflurane can attenuate lung ischaemia‒reperfusion injury (LIRI). However, the protective mechanism is unclear. In this study, we developed a LIRI model in vivo that animals (SD, n = 15) were subjected to the administration of 2.2 % sevoflurane 30 min before the onset of left pulmonary artery clamping for 45 min, which was then followed by 60 min of reperfusion treatment. Then, transcriptome sequencing was used to analyse lung tissues. Autophagy inhibition (3-MA) and Rac1-overexpression transfection plasmids were used in BEAS-2B cells, and BEAS-2B cells were subjected to hypoxia reoxygenation (H/R) and sevoflurane treatment. In both animal tissue and cells, inflammatory cytokines and apoptotic and autophagy molecules were measured by quantitative real-time PCR, western blotting and immunostaining. As a result, decreased arterial partial oxygen and damage to the histological structure of lung tissues were observed in LIRI model rats, and these effects were reversed by sevoflurane treatment. Activation of inflammation (elevated IL-1β, IL-6, and TNF-α) and apoptosis (elevated cleaved caspase3/caspase3 and Bax, degraded expression of Bcl2) and inhibition of autophagy (elevated P62, degraded expression of Beclin1 and LC3-II/LC3I) in the model group were ameliorated by sevoflurane. Transcriptome sequencing indicated that the PI3K/Akt pathway regulated by Rac1 plays an important role in LIRI. Furthermore, overexpression of Rac1 in a cell line inhibited the protective effect of sevoflurane in LIRI. Autophagy inhibition (3-MA) also prevented the protective effect of sevoflurane on inflammation and apoptosis.As shown in the present study, sevoflurane enhances autophagy via Rac1/PI3K/AKT signalling to attenuate lung ischaemia‒reperfusion injury.

Donor Inhalation of Nebulized Dexmedetomidine Alleviates Ischemia-Reperfusion Injury in Rat Lung Transplantation

Introduction: The occurrence of lung ischemia-reperfusion injury (LIRI) after lung transplantation results in primary graft dysfunction (PGD) in more than 50% of cases, which seriously affects the prognosis of recipients. Currently, donor lung protection is the focus of research on improving graft survival in lung transplant recipients. Dexmedetomidine (Dex) is a widely used general anesthesia adjuvant in clinical practice to alleviate ischemia-reperfusion injury in the lungs, liver, heart, kidneys, and brain. However, intravenous infusion of Dex can cause negative effects on the cardiovascular system. Inhaling nebulized Dex can directly act on the alveolar tissue and alleviate its cardiovascular inhibitory effect by reducing drug intake. This study aimed to investigate the effect of donor nebulized Dex inhalation on LIRI after lung transplantation in rats. Methods: We randomly divided the male Sprague-Dawley rats into donor rats and recipient rats, and allowed the donor rats to inhale nebulized Dex or physiological saline 15 min before surgery. The donor lung was refrigerated for 8 h before each single-lung transplant. After 2 h of reperfusion of the transplanted lung, serum and transplanted lung tissue were collected. The wet-to-dry weight ratio of the lung tissue was measured, arterial blood gas was detected, and histopathology changes, oxidative stress, inflammatory reactions, and apoptosis were evaluated. Results: Pretransplant inhalation of Dex through the donor’s lung reduced the injury of the transplanted lung, increased the levels of malondialdehyde and myeloperoxidase, and decreased the levels of superoxide dismutase and glutathione in the lung tissue. Moreover, nebulized Dex inhalation of the donor lung inhibited LIRI-induced tumor necrosis factor-α, interleukin-6, and inducible nitric oxide synthase expression and also suppressed nuclear factor kappa B phosphorylation. Nebulized Dex inhalation reduced the rate of cell apoptosis in the transplanted lung tissue by inhibiting the upregulation of Bax, downregulation of Bcl-2, and increase in caspase-3 lysis caused by LIRI. Conclusion: Inhalation of atomized Dex is a potential donor lung protection strategy, which can be used to reduce LIRI after lung transplantation and may be helpful to improve the occurrence of PGD and prognosis of lung transplant recipients.

Salidroside inhibits the ferroptosis to alleviate lung ischemia reperfusion injury via the JAK2/STAT3 signalling pathway

ObjectiveThe present study aims to investigate the protective potential of salidroside in both lung ischemia/reperfusion injury (LIRI) mice model and cell hypoxia/reoxygenation (H/R)model and the involvement of ferroptosis and JAK2/STAT3 pathway. Materials and methodsAfter we established the IR-induced lung injury model in mice, we administered salidroside and the ferroptosis inhibitor, ferrostatin-1, then assessed the lung tissue injury, ferroptosis (levels of reactive oxygen species level, malondialdehyde and glutathione), and inflammation in lung tissues. The levels of ferroptosis-related proteins (glutathione peroxidase 4, fibroblast-specific protein 1, solute carrier family 1 member 5 and glutaminase 2) in the lung tissue were measured with Western blotting. Next, BEAS-2B cells were used to establish an H/R cell model and treated with salidroside or ferrostatin-1 before the cell viability and the levels of lactate dehydrogenase (LDH), inflammatory factor, ferroptosis-related proteins were measured. The activation of the JAK2/STAT3 signaling pathway was measured with Western blotting, then its role was confirmed with STAT3 knockdown. ResultsRemarkably, salidroside was found to alleviate ferroptosis, inflammation, and lung injury in LIRI mice and the cell injury in H/R cell model. Severe ferroptosis were observed in LIRI mice models and H/R-induced BEAS-2B cells, which was alleviated by salidroside. Furthermore, salidroside could inhibit JAK2/STAT3 activation induced by LIRI. STAT3 knockdown could enhance the effect of salidroside treatment on H/R-induced cell damage and ferroptosis in vitro. ConclusionsSalidroside inhibits ferroptosis to alleviate lung ischemia reperfusion injury via the JAK2/STAT3 signaling pathway.

Metformin attenuates lung ischemia-reperfusion injury and necroptosis through AMPK pathway in type 2 diabetic recipient rats

BackgroundDiabetes mellitus (DM) can aggravate lung ischemia-reperfusion (I/R) injury and is a significant risk factor for recipient mortality after lung transplantation. Metformin protects against I/R injury in a variety of organs. However, the effect of metformin on diabetic lung I/R injury remains unclear. Therefore, this study aimed to observe the effect and mechanism of metformin on lung I/R injury following lung transplantation in type 2 diabetic rats.MethodsSprague–Dawley rats were randomly divided into the following six groups: the control + sham group (CS group), the control + I/R group (CIR group), the DM + sham group (DS group), the DM + I/R group (DIR group), the DM + I/R + metformin group (DIRM group) and the DM + I/R + metformin + Compound C group (DIRMC group). Control and diabetic rats underwent the sham operation or left lung transplantation operation. Lung function, alveolar capillary permeability, inflammatory response, oxidative stress, necroptosis and the p-AMPK/AMPK ratio were determined after 24 h of reperfusion.ResultsCompared with the CIR group, the DIR group exhibited decreased lung function, increased alveolar capillary permeability, inflammatory responses, oxidative stress and necroptosis, but decreased the p-AMPK/AMPK ratio. Metformin improved the function of lung grafts, decreased alveolar capillary permeability, inflammatory responses, oxidative stress and necroptosis, and increased the p-AMPK/AMPK ratio. In contrast, the protective effects of metformin were abrogated by Compound C.ConclusionsMetformin attenuates lung I/R injury and necroptosis through AMPK pathway in type 2 diabetic lung transplant recipient rats.

Transient heat stress protects from severe endothelial damage and dysfunction during prolonged experimental ex-vivo lung perfusion.

The pulmonary endothelium is the primary target of lung ischemia-reperfusion injury leading to primary graft dysfunction after lung transplantation. We hypothesized that treating damaged rat lungs by a transient heat stress during ex-vivo lung perfusion (EVLP) to elicit a pulmonary heat shock response could protect the endothelium from severe reperfusion injury. Rat lungs damaged by 1h warm ischemia were reperfused on an EVLP platform for up to 6h at a constant temperature (T°) of 37°C (EVLP37°C group), or following a transient heat stress (HS) at 41.5°C from 1 to 1.5h of EVLP (EVLPHS group). A group of lungs exposed to 1h EVLP only (pre-heating conditions) was added as control (Baseline group). In a first protocol, we measured lung heat sock protein expression (HSP70, HSP27 and Hsc70) at selected time-points (n=5/group at each time). In a second protocol, we determined (n=5/group) lung weight gain (edema), pulmonary compliance, oxygenation capacity, pulmonary artery pressure (PAP) and vascular resistance (PVR), the expression of PECAM-1 (CD31) and phosphorylation status of Src-kinase and VE-cadherin in lung tissue, as well as the release in perfusate of cytokines (TNFα, IL-1β) and endothelial biomarkers (sPECAM, von Willebrand Factor -vWF-, sE-selectin and sICAM-1). Histological and immunofluorescent studies assessed perivascular edema and formation of 3-nitrotyrosine (a marker of peroxinitrite) in CD31 lung endothelium. HS induced an early (3h) and persisting expression of HSP70 and HSP27, without influencing Hsc70. Lungs from the EVLP37°C group developed massive edema, low compliance and oxygenation, elevated PAP and PVR, substantial release of TNFα, IL-1β, s-PECAM, vWF, E-selectin and s-ICAM, as well as significant Src-kinase activation, VE-cadherin phosphorylation, endothelial 3-NT formation and reduced CD31 expression. In marked contrast, all these alterations were either abrogated or significantly attenuated by HS treatment. The therapeutic application of a transient heat stress during EVLP of damaged rat lungs reduces endothelial permeability, attenuates pulmonary vasoconstriction, prevents src-kinase activation and VE-cadherin phosphorylation, while reducing endothelial peroxinitrite generation and the release of cytokines and endothelial biomarkers. Collectively, these data demonstrate that therapeutic heat stress may represent a promising strategy to protect the lung endothelium from severe reperfusion injury.

Transient receptor potential vanilloid 4 channel inhibition attenuates lung ischemia-reperfusion injury in a porcine lung transplant model

ObjectiveTransient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel important in many physiological and pathophysiological processes, including pulmonary disease. Using a murine model, we previously demonstrated that TRPV4 mediates lung ischemia-reperfusion injury, the major cause of primary graft dysfunction after transplant. The current study tests the hypothesis that treatment with a TRPV4 inhibitor will attenuate lung ischemia-reperfusion injury in a clinically relevant porcine lung transplant model. MethodsA porcine left-lung transplant model was used. Animals were randomized to 2 treatment groups (n = 5/group): vehicle or GSK2193874 (selective TRPV4 inhibitor). Donor lungs underwent 30 minutes of warm ischemia and 24 hours of cold preservation before left lung allotransplantation and 4 hours of reperfusion. Vehicle or GSK2193874 (1 mg/kg) was administered to the recipient as a systemic infusion after recipient lung explant. Lung function, injury, and inflammatory biomarkers were compared. ResultsAfter transplant, left lung oxygenation was significantly improved in the TRPV4 inhibitor group after 3 and 4 hours of reperfusion. Lung histology scores and edema were significantly improved, and neutrophil infiltration was significantly reduced in the TRPV4 inhibitor group. TRPV4 inhibitor-treated recipients had significantly reduced expression of interleukin-8, high mobility group box 1, P-selectin, and tight junction proteins (occludin, claudin-5, and zonula occludens-1) in bronchoalveolar lavage fluid as well as reduced angiopoietin-2 in plasma, all indicative of preservation of endothelial barrier function. ConclusionsTreatment of lung transplant recipients with TRPV4 inhibitor significantly improves lung function and attenuates ischemia-reperfusion injury. Thus, selective TRPV4 inhibition may be a promising therapeutic strategy to prevent primary graft dysfunction after transplant.

Penehyclidine hydrochloride alleviates lung ischemia-reperfusion injury by inhibiting pyroptosis

ObjectiveThe aim of this research was to examine how penehyclidine hydrochloride (PHC) impacts the occurrence of pyroptosis in lung tissue cells within a rat model of lung ischemia-reperfusion injury.MethodsTwenty-four Sprague Dawley (SD) rats, weighing 250 g to 270 g, were randomly distributed into three distinct groups as outlined below: a sham operation group (S group), a control group (C group), and a test group (PHC group). Rats in the PHC group received a preliminary intravenous injection of PHC at a dose of 3 mg/kg. At the conclusion of the experiment, lung tissue and blood samples were collected and properly stored for subsequent analysis. The levels of malondialdehyde, superoxide dismutase, and myeloperoxidase in the lung tissue, as well as IL-18 and IL-1β in the blood serum, were assessed using an Elisa kit. Pyroptosis-related proteins, including Caspase1 p20, GSDMD-N, and NLRP3, were detected through the western blot method. Additionally, the dry-to-wet ratio (D/W) of the lung tissue and the findings from the blood gas analysis were also documented.ResultsIn contrast to the control group, the PHC group showed enhancements in oxygenation metrics, reductions in oxidative stress and inflammatory reactions, and a decrease in lung injury. Additionally, the PHC group exhibited lowered levels of pyroptosis-associated proteins, including the N-terminal segment of gasdermin D (GSDMD-N), caspase-1p20, and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3).ConclusionPre-administration of PHC has the potential to mitigate lung ischemia-reperfusion injuries by suppressing the pyroptosis of lung tissue cells, diminishing inflammatory reactions, and enhancing lung function. The primary mechanism behind anti-pyroptotic effect of PHC appears to involve the inhibition of oxidative stress.

Murine Left Pulmonary Hilar Clamp Model of Lung Ischemia Reperfusion Injury.

Ischemia reperfusion injury (IRI) during lung transplantation is a major risk factor for post-transplant complications, including primary graft dysfunction, acute and chronic rejection, and mortality. Efforts to study the underpinnings of IRI led to the development of a reliable and reproducible mouse model of left lung hilar clamping. This model involves a surgical procedure performed in an anesthetized and intubated mouse. A left thoracotomy is performed, followed by careful lung mobilization and dissection of the left pulmonary hilum. The hilar clamp involves reversible suture ligation of the pulmonary hilum with a slipknot, which stops the arterial inflow, venous outflow, and airflow through the left mainstem bronchus. Reperfusion is initiated by careful removal of the suture. Our laboratory uses 30 min of ischemia and 1 h of reperfusion for the experimental model in the current investigations. However, these time periods can be modified depending on the specific experimental question. Immediately prior to sacrifice, arterial blood gas can be obtained from the left ventricle after a 4 min period of right hilar clamping to ensure that the PaO2 values obtained are attributed to the injured left lung alone. We also describe a method to measure cell extravasation with flow cytometry, which involves intravenous injection of a fluorochrome-labeled antibody specific for the cell(s) to be studied prior to sacrifice. The left lung can then be harvested for flow cytometry, frozen or fixed, paraffin-embedded immunohistochemistry, and quantitative polymerase chain reaction. This hilar clamp technique allows fordetailed study of the cellular and molecular mechanisms underlying IRI. Representative results reveal decreased left lung oxygenation and histologic evidence of lung injury following hilar clamping. This technique can be readily learned and reproduced by personnel with and without microsurgical experience, leading to reliable and consistent results and serving as a widely adoptable model for studying lung IRI.

Lung Ischemia Reperfusion Injury as a Complication of Pulmonary Thrombectomy

Lung Ischemia Reperfusion Injury as a Complication of Pulmonary Thrombectomy

(269) - Inhibition of ALOX12-12-HETE Alleviates Lung Ischemia Reperfusion Injury by Reducing Endothelial Ferroptosis-Mediated Neutrophil Extracellular Traps Formation

(269) - Inhibition of ALOX12-12-HETE Alleviates Lung Ischemia Reperfusion Injury by Reducing Endothelial Ferroptosis-Mediated Neutrophil Extracellular Traps Formation