Attenuates Lung Ischemia-reperfusion Injury Research Articles

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.

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 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.

Post-ischemic treatment of nalmefene hydrochloride attenuated lung ischemia-reperfusion injury in rats via the Sirt1/Nrf2/HO-1 pathway with inhibition of ferroptosis

Objective: To study the effect and mechanism of post-ischemic treatment of nalmefene in alleviating the lung ischemia-reperfusion injury by inhibiting ferroptosis through activation of the Sirt 1/Nrf 2/HO-1 axis. Methods: A total of 60 rats were randomly divided into six groups equally (n=10): the sham group, the model group(I/R), the nalmefene group, the nalmefene+EX527 group, the nalmefene+ML385 group, the nalmefene+Fe-citrate group (nalmefene+Fe group). The sham group without drug treatment was not treated with ischemia-reperfusion. The pulmonary ischemia-reperfusion model was established by occlusion of the left pulmonary hilum in the model group without drug treatment. After ischemic treatment, the nalmefene group was injected with nalmefene (15 μg/kg) via the tail vein at 5 minutes before reperfusion. The nalmefene+EX527 group, the nalmefene+ML385 group, and the nalmefene+Fe group were injected intraperitoneally with EX527 (5 mg/kg), ML385 (30 mg/kg), Fe-citrate(15 mg/kg), respectively, 2 h before moulding and then injected with nalmefene (15 μg/kg) via the tail vein at 5 minutes before reperfusion. All rats were sacrificed three hours after reperfusion, and the specimens from the upper lobe of the left lung tissue were preserved. The degree of lung tissue injury and the wet/dry weight ratio were assessed in each group of rats. Fe 2+, MDA, TNF-α, and IL-6 content, GSH activity and the expression levels of Sirt1, Nrf2, HO-1, ACSL4 and GPX4 were determined. Results: Compared with the sham group, the wet/dry weight ratio, lung tissue injury score, ACSL 4 expression level, Fe 2+, TNF-α, IL-6 and MDA content, Sirt 1, Nrf 2, HO-1 messenger RNA and protein expression levels were significantly increased (P<0.01), while GPX 4 expression level and GSH activity were significantly decreased in the model group (P<0.01). Compared with the model group, wet/dry weight ratio, lung tissue injury score, ACSL 4 expression level, Fe 2+, TNF-α, IL-6, and MDA content decreased significantly (P<0.01), Nrf 2, HO-1 messenger RNA and protein, GPX 4 expression, and GSH activity were significantly increased in the nalmefene group and the nalmefene+EX527 group (P<0.01). Sirt 1 messenger RNA and protein expression increased significantly in the nalmefene (P<0.01) and the nalmefene+EX527 groups (P>0.05). In the nalmefene+ML385 group, the wet/dry weight ratio, lung tissue injury score, TNF-α and IL-6 content were decreased significantly (P<0.01), while Sirt 1 messenger RNA and protein expression levels were significantly increased (P<0.01), but there were no significant changes in Nrf 2, HO-1 messenger RNA and protein expression levels, ACSL 4 and GPX 4 expression levels, Fe 2+, MDA content, and GSH activity (P>0.05). In the nalmefene+Fe group, wet/dry weight ratio, lung-injury score, TNF-α, IL-6, MDA content were decreased significantly (P<0.01), messenger RNA and protein expression levels of Sirt 1, Nrf 2, HO-1, and GSH activity were increased significantly (P<0.01), but there were no significant changes in Fe 2+content, ACSL 4 and GPX 4 expression levels (P>0.05). Compared with the nalmefene group, in the nalmefene+EX527 group, the nalmefene+ML385 group and the nalmefene+Fe group, wet/dry weight ratio, lung tissue damage score, ACSL 4 expression level, TNF-α, IL-6 and MDA content were significantly increased (P<0.01), the expression level of GPX 4 and GSH activity were significantly decreased (P<0.01). The expression levels of Sirt 1, Nrf 2, HO-1 messenger RNA and protein were significantly decreased in the nalmefene+EX527 group (P<0.01). The expression levels of Nrf 2, HO-1 messenger RNA and protein decreased significantly in the namemefene+ML385 group (P<0.01), but there was no significant change in Sirt 1 messenger RNA and protein expression level (P>0.05). Sirt 1, Nrf 2, HO-1 messenger RNA-protein expression levels did not change significantly in the nalmefene+Fe group (P>0.05). Conclusion: Post-ischemic treatment with nalmefene hydrochloride may alleviate pulmonary ischemia-reperfusion injury by inhibiting ferroptosis through activation of the Sirt 1/Nrf 2/HO-1 axis.

Emodin alleviates lung ischemia-reperfusion injury by suppressing gasdermin D-mediated pyroptosis in rats.

Pyroptosis refers to programmed cell death associated with inflammation. Emodin has been reported to alleviate lung injuries caused by various pathological processes and attenuate ischemia-reperfusion (I/R) injuries in diverse tissues. Lewis rats were assigned into the sham, the I/R, and the I/R + emodin groups. Emodin and phosphate-buffered saline were intraperitoneally injected into rats of the emodin group and I/R group for 30 min, respectively. These rats were then subjected to left thoracotomy followed by 90-min clamping of the left hilum and 120-min reperfusion. Sham-operated rats underwent 210-min ventilation. Lung functions, histological changes, lung edema, and cytokine levels were assessed. Protein levels were measured by western blotting. Immunofluorescence staining was conducted to evaluate pyroptosis. Emodin alleviated the I/R-induced lung dysfunction, lung damages, and inflammation. Protective effects of emodin against I/R-mediated endothelial pyroptosis was observed in vivo and in vitro. Mechanistically, emodin inactivated the TLR4/MyD88/NF-κB/NLRP3 pathway. Emodin attenuates lung ischemia-reperfusion injury by inhibiting GSDMD-mediated pyroptosis in rats.

Inhibition of DNA methylation attenuates lung ischemia–reperfusion injury after lung transplantation

DNA methylation plays an important role in inflammation and oxidative stress. This study aimed to investigate the effect of inhibiting DNA methylation on lung ischemia-reperfusion injury (LIRI). We adopted a completely random design for our study. Thirty-two rats were randomized into the sham, LIRI, azathioprine (AZA), and pluripotin (SC1) groups. The rats in the LIRI, AZA, and SC1 groups received left lung transplantation and intravenous injection of saline, AZA, and SC1, respectively. After 24 hours of reperfusion, histological injury, the arterial oxygen partial pressure to fractional inspired oxygen ratio, the wet/dry weight ratio, protein and cytokine concentrations in lung tissue, and DNA methylation in lung tissue were evaluated. The pulmonary endothelium that underwent hypoxemia and reoxygenation was treated with AZA or SC1. Endothelial apoptosis, chemokines, reactive oxygen species, nuclear factor-κB, and apoptotic proteins in the endothelium were studied. Inhibition of DNA methylation by AZA attenuated lung injury, inflammation, and the oxidative stress response, but SC1 aggravated LIRI injury. AZA significantly improved endothelial function, suppressed apoptosis and necrosis, reduced chemokines, and inhibited nuclear factor-κB. Inhibition of DNA methylation ameliorates LIRI and apoptosis and improves pulmonary function via the regulation of inflammation and oxidative stress.

Resolution of post-lung transplant ischemia-reperfusion injury is modulated via Resolvin D1-FPR2 and Maresin 1-LGR6 signaling

Dysregulation of inflammation-resolution pathways leads to postlung transplant (LTx) ischemia-reperfusion (IR) injury and allograft dysfunction. Our hypothesis is that combined treatment with specialized pro-resolving lipid mediators, that is, Resolvin D1 (RvD1) and Maresin-1 (MaR1), enhances inflammation-resolution of lung IR injury. Expression of RvD1 and MaR1 was analyzed in bronchoalveolar lavage (BAL) fluid of patients on days 0, 1, and 7 post-LTx. Lung IR injury was evaluated in C57BL/6 (WT), FPR2-/-, and LGR6 siRNA treated mice using a hilar-ligation model with or without administration with RvD1 and/or MaR1. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by RvD1 and MaR1 against lung IR injury. In vitro studies analyzed alveolar macrophages and type II epithelial cell activation after treatment with RvD1 or MaR1. RvD1 and MaR1 expressions in BAL from post-LTx patients was significantly increased on day 7 compared to days 0 and 1. Concomitant RvD1 and MaR1 treatment significantly mitigated early pulmonary inflammation and lung IR injury in WT mice, which was regulated via FPR2 and LGR6 receptors. In the murine orthotopic donation after cardiac death LTx model, RvD1 and MaR1 treatments significantly attenuated lung IR injury and increased PaO2 levels compared to saline-treated controls. Mechanistically, RvD1/FPR2 signaling on alveolar macrophages attenuated HMGB1 and TNF-α secretion and upregulated uptake of macrophage-dependent apoptotic neutrophils (efferocytosis), whereas MaR1/LGR6 signaling mitigated CXCL1 secretion by epithelial cells. Bioactive proresolving lipid mediator-dependent signaling that is, RvD1/FPR2 and MaR1/LGR6- offers a novel therapeutic strategy in post-LTx injury.

Protease-Activated Receptor-1 Antagonist Protects Against Lung Ischemia/Reperfusion Injury.

Protease-activated receptor (PAR)-1 is a thrombin-activated receptor that plays an essential role in ischemia/reperfusion (IR)-induced acute inflammation. PAR-1 antagonists have been shown to alleviate injuries in various IR models. However, the effect of PAR-1 antagonists on IR-induced acute lung injury (ALI) has not yet been elucidated. This study aimed to investigate whether PAR-1 inhibition could attenuate lung IR injury. Lung IR was induced in an isolated perfused rat lung model. Male rats were treated with the specific PAR-1 antagonist SCH530348 (vorapaxar) or vehicle, followed by ischemia for 40 min and reperfusion for 60 min. To examine the role of PAR-1 and the mechanism of SCH530348 in lung IR injury, western blotting and immunohistochemical analysis of lung tissue were performed. In vitro, mouse lung epithelial cells (MLE-12) were treated with SCH530348 or vehicle and subjected to hypoxia-reoxygenation (HR). We found that SCH530348 decreased lung edema and neutrophil infiltration, attenuated thrombin production, reduced inflammatory factors, including cytokine-induced neutrophil chemoattractant-1, interleukin-6 and tumor necrosis factor-α, mitigated lung cell apoptosis, and downregulated the phosphoinositide 3-kinase (PI3K), nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in IR-injured lungs. In addition, SCH530348 prevented HR-induced NF-κB activation and inflammatory chemokine production in MLE12 cells. Our results demonstrate that SCH530348 exerts protective effects by blocking PAR-1 expression and modulating the downstream PI3K, NF-κB and MAPK pathways. These findings indicate that the PAR-1 antagonist protects against IR-induced ALI and is a potential therapeutic candidate for lung protection following IR injury.

Protective effect of necrosulfonamide on rat pulmonary ischemia-reperfusion injury via inhibition of necroptosis

BackgroundNecroptosis plays an important role in cell death during pulmonary ischemia-reperfusion injury (IRI). We hypothesized that therapy with necrosulfonamide (NSA), a mixed-lineage kinase domain-like protein inhibitor, would attenuate lung IRI. MethodsRats were assigned at random into the sham operation group (n = 6), vehicle group (n = 8), or NSA group (n = 8). In the NSA and vehicle groups, the animals were heparinized and underwent left thoracotomy, and the left hilum was clamped for 90 minutes, followed by reperfusion for 120 minutes. NSA (0.5 mg/body) and a solvent were administered i.p. in the NSA group and the vehicle group, respectively. The sham group underwent 210 minutes of perfusion without ischemia. After reperfusion, arterial blood gas analysis, physiologic data, lung wet-to-dry weight ratio, histologic changes, and cytokine levels were assessed. Fluorescence double immunostaining was performed to evaluate necroptosis and apoptosis. ResultsArterial partial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2) was better, dynamic compliance was higher, and mean airway pressure and lung edema were lower in the NSA group compared with the vehicle group. Moreover, in the NSA group, lung injury was significantly alleviated, and the mean number of necroptotic cells (55.3 ± 4.06 vs 78.2 ± 6.87; P = .024), but not of apoptotic cells (P = .084), was significantly reduced compared with the vehicle group. Interleukin (IL)-1β and IL-6 levels were significantly lower with NSA administration. ConclusionsIn a rat model, our results suggest that NSA may have a potential protective role in lung IRI through the inhibition of necroptosis.

Necrostatin-1 Synergizes the Pan Caspase Inhibitor to Attenuate Lung Injury Induced by Ischemia Reperfusion in Rats.

Background Both apoptosis and necroptosis have been recognized to be involved in ischemia reperfusion-induced lung injury. We aimed to compare the efficacies of therapies targeting necroptosis and apoptosis and to determine if there is a synergistic effect between the two therapies in reducing lung ischemia reperfusion injury. Methods Forty Sprague-Dawley rats were randomized into 5 groups: sham (SM) group, ischemia reperfusion (IR) group, necrostatin-1+ischemia reperfusion (NI) group, carbobenzoxy-Val-Ala-Asp-fluoromethylketone+ischemia reperfusion (ZI) group, and necrostatin-1+carbobenzoxy-Val-Ala-Asp-fluoromethylketone+ischemia reperfusion (NZ) group. The left lung hilum was exposed without being clamped in rats from the SM group, whereas the rats were subjected to lung ischemia reperfusion by clamping the left lung hilum for 1 hour, followed by reperfusion for 3 hours in the IR group. 1 mg/kg necrostatin-1 (Nec-1: a specific necroptosis inhibitor) and 3 mg/kg carbobenzoxy-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk: a pan caspase inhibitor) were intraperitoneally administrated prior to ischemia in NI and ZI groups, respectively, and the rats received combined administration of Nec-1 and z-VAD-fmk in the NZ group. Upon reperfusion, expressions of receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), and caspase-8 were measured, and the flow cytometry analysis was used to assess the cell death patterns in the lung tissue. Moreover, inflammatory marker levels in the bronchoalveolar lavage fluid and pulmonary edema were evaluated. Results Both Nec-1 and z-VAD-fmk, either alone or in combination, significantly reduced morphological damage, inflammatory markers, and edema in lung tissues following reperfusion, and cotreatment of z-VAD-fmk with Nec-1 produced the optimal effect. The rats treated with Nec-1 had lower levels of inflammatory markers in the bronchoalveolar lavage fluid than those receiving z-VAD-fmk alone (P < 0.05). Interestingly, the z-VAD-fmk administration upregulated RIP1 and RIP3 expressions in the lung tissue from the ZI group compared to those in the IR group (P < 0.05). Reperfusion significantly increased the percentages of necrotic and apoptotic cells in lung tissue single-cell suspension, which could be decreased by Nec-1 and z-VAD-fmk, respectively (P < 0.05). Conclusions Nec-1 synergizes the pan caspase inhibitor to attenuate lung ischemia reperfusion injury in rats. Our data support the potential use of Nec-1 in lung transplantation-related disorders.

Hydrogen Sulfide Ameliorates Lung Ischemia-Reperfusion Injury Through SIRT1 Signaling Pathway in Type 2 Diabetic Rats.

Lung ischemia-reperfusion (IR) injury remains a significant factor for the early mortality of lung transplantations. Diabetes mellitus (DM) is an independent risk factor for 5-year mortality following lung transplantation. Our previous study showed that DM aggravated lung IR injury and that oxidative stress played a key role in this process. Previously, we demonstrated that hydrogen sulfide (H2S) protected against diabetic lung IR injury by suppressing oxidative damage. This study aimed to examine the mechanism by which H2S affects diabetic lung IR injury. High-fat-diet-fed streptozotocin-induced type 2 diabetic rats were exposed to GYY4137, a slow-releasing H2S donor with or without administration of EX527 (a SIRT1 inhibitor), and then subjected to a surgical model of IR injury of the lung. Lung function, oxidative stress, cell apoptosis, and inflammation were assessed. We found that impairment of lung SIRT1 signaling under type 2 diabetic conditions was further exacerbated by IR injury. GYY4137 treatment markedly activated SIRT1 signaling and ameliorated lung IR injury in type 2 DM animals by improving lung functional recovery, diminishing oxidative damage, reducing inflammation, and suppressing cell apoptosis. However, these effects were largely compromised by EX527. Additionally, treatment with GYY4137 significantly activated the Nrf2/HO-1 antioxidant signaling pathway and increased eNOS phosphorylation. However, these effects were largely abolished by EX527. Together, our results indicate that GYY4137 treatment effectively attenuated lung IR injury under type 2 diabetic conditions via activation of lung SIRT1 signaling. SIRT1 activation upregulated Nrf2/HO-1 and activated the eNOS-mediated antioxidant signaling pathway, thus reducing cell apoptosis and inflammation and eventually preserving lung function.

Extracellular Vesicle-Mediated Delivery of microRNA-206 Antagomir Attenuates Lung Ischemia-Reperfusion Injury via Targeting Type II Epithelial Cell-Secreted CXCL1

Our hypothesis is that human adipose mesenchymal stem cell (MSC)-derived extracellular vesicle (EV) can be used as a delivery platform for microRNAs to effectively attenuate lung ischemia-reperfusion injury (IRI). C57Bl/6 (WT) mice underwent sham or lung IRI (1hr of ischemia followed by 2hr reperfusion using left lung hilar-ligation model) with or without treatment (1hr prior to ischemia) with EVs or antagomiR-206 enriched EVs (3x106 EVs given intratracheally; n=5/group). MSCs were transfected with antagomiR-206 using Lipofectamine reagent for enrichment of purified EVs. Lung tissue was used for Affymetrix GeneChip microRNA microarray hybridization. Cytokine expression in BAL fluid by Luminex array, and lung injury by measuring neutrophil infiltration (immunohistochemistry) and activation (myeloperoxidase levels), was evaluated. Murine type II epithelial (MLE12 cells) were co-cultured with EVs and exposed to hypoxia/reoxygenation (3hr/1hr) followed by analysis of supernatants by ELISA. Groups were compared using ANOVA and data is shown as mean±S.E. miRNA microarray analysis of lung tissue demonstrated a significant upregulation of miR-206 (∼50-fold) after lung IRI compared to sham. Treatment with antagomiR-206-enriched EVs displayed significantly better lung function [decreased airway resistance (0.6±0.03 vs. 1.1±0.05 cm H2O/μl/sec; p<0.05), pulmonary artery pressure (7.3±0.3 vs. 9.7±0.3 cm H2O; p<0.05) and increased pulmonary compliance (5.8±0.2 vs. 3.7±0.3 μl/cmH2O; p<0.05)] and mitigation of lung injury (neutrophil infiltration and myeloperoxidase levels) compared to treatment with EVs alone, respectively, after lung IRI. Treatment with EVs or antagomiR-206-enriched EVs significantly decreased proinflammatory cytokines (IL-17, TNF-α, MCP-1, HMGB1 and CXCL1) compared to IR alone. AntagomiR-206 enriched EVs displayed a significant decrease in CXCL1 expression, a hallmark of lung epithelial activation, compared to EVs alone. Co-culture of MLE12 cells with antagomiR-206 enriched EVs showed a significant attenuation of HR-induced CXCL1 expression compared to co-culture with EVs. MSC-derived EVs can be used as biomimetic nanovehicles for protective immunomodulation against lung IRI by enriching them with antagomiR-206.

Hydrogen sulfide attenuates lung ischemia–reperfusion injury through SIRT3-dependent regulation of mitochondrial function in type 2 diabetic rats

BackgroundLung ischemia–reperfusion injury is a complex pathophysiologic process associated with high morbidity and mortality. We have demonstrated elsewhere that diabetes mellitus aggravated ischemia-induced lung injury. Oxidative stress and mitochondrial dysfunction are drivers of diabetic lung ischemia-reperfusion injury; however, the pathways that mediate these events are unexplored. In this study using a high-fat diet–fed model of streptozotocin-induced type 2 diabetes in rats, we determined the effect of hydrogen sulfide on lung ischemia-reperfusion injury with a focus on Sirtuin3 signaling. MethodsRats with type 2 diabetes were exposed to GYY4137, a slow release donor of hydrogen sulfide with or without administration of the Sirtuin3 short hairpin ribonucleic acid plasmid, and then subjected to a surgical model of ischemia–reperfusion injury of the lung (n = 8). Lung function, oxidative stress, inflammation, cell apoptosis, and mitochondrial function were measured. ResultsCompared with nondiabetic rats, animals with type 2 diabetes at baseline exhibited significantly decreased Sirtuin3 signaling in lung tissue and increased oxidative stress, apoptosis, inflammation, and mitochondrial dysfunction (P < .05 each). In addition, further impairment in Sirtuin3 signaling was found in diabetic rats subjected to this model of lung ischemia–reperfusion. Simultaneously, the indexes showed further aggravation. Treatment with hydrogen sulfide restored Sirtuin3 expression and decreased lung ischemia–reperfusion injury in animals with type 2 diabetes mellitus by improving lung functional recovery, decreasing oxidative damage, suppressing inflammation, ameliorating cell apoptosis, and preserving mitochondrial function (P < .05). Conversely, these protective effects were largely reversed in Sirtuin3 knockdown rats. ConclusionImpaired lung Sirtuin3 signaling associated with type 2 diabetic conditions was further attenuated by an ischemia-reperfusion insult. Hydrogen sulfide ameliorated reperfusion-induced oxidative stress and mitochondrial dysfunction via activation of Sirtuin3 signaling, thereby decreasing lung ischemia-reperfusion damage in rats with a model of type II diabetes.

Dexmedetomidine alleviates lung ischemia-reperfusion injury in rats by activating PI3K/Akt pathway.

This research aims to investigate the role and mechanism of PI3K/Akt pathway in the pathological process of lung ischemia-reperfusion injury in dexmedetomidine-treated rats. Forty-five healthy male Sprague-Dawley rats were divided into three groups: sham operation group, lung ischemia-reperfusion group (IR group) and dexmedetomidine pretreatment group (Dex group). Rats in the sham operation group did not receive other procedures except for opening left chest. The left lung hilar of rats in the IR group was clamped with non-invasive vascular clamp after anesthesia to establish an ischemic model. After 1 hour, the vascular clamp was released and the rats were reperfused for 2 hours. As for rats in the Dex group, 3 μg/kg of dexmedetomidine (pumping time of 10 min) was pumped through the tail vein before releasing the left hilar clamp. After the experiment, blood samples and lung tissues were collected. Serum levels of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), IL-10, and IL-1 in rats were examined. Activities of malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD) and catalase (CAT) in rat lung tissues were also detected. Besides, the expressions of hypoxia-inducible factor-la (HIF-la), p-Akt, Caspase-3, and Caspase-9 in lung tissues were detected by Western blot. The mRNA expression levels of HIF-1a, p-Akt, Caspase-3, and Caspase-9 in lung tissues were evaluated by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Lung ischemia-reperfusion markedly increased the levels of IL-6, TNF-α, IL-10, and IL-1 in the IR group. In contrast, dexmedetomidine pretreatment decreased the expression levels of IL-6, TNF-α, IL-10, and IL-1in the Dex group. Also, the activities of MDA and MPO in lung tissues of rats in the IR group significantly increased after lung ischemia-reperfusion injury, whereas dexmedetomidine pretreatment reversed the elevated activities of MDA and MPO in the Dex group. Furthermore, dexmedetomidine pretreatment also improved the activities of SOD and CAT in rat lung tissues compared with rats with lung ischemia-reperfusion injury. In addition, dexmedetomidine pretreatment increased the expression levels of HIF-1α, p-Akt and HIF- in the Dex group when compared to those in the IR group. The mRNA expressions of HIF-1a, p-Akt, Caspase-3, and Caspase-9 in lung tissue of rats was significantly reduced after dexmedetomidine pretreatment. Rat lung ischemia-reperfusion can induce severe lung injury. Dexmedetomidine treatment can attenuate lung ischemia-reperfusion injury by activating the PI3K/Akt signaling pathway at the transcriptional level.

Low-intensity pulsed ultrasound pretreatment inhibits HMGB1 expression and attenuates lung ischemia-reperfusion injury in rats via the cholinergic anti-inflammatory pathway

To observe the effects of low-intensity pulsed ultrasound (LIPUS) pretreatment on pulmonary expression of high mobility group box-1 (HMGB1) in a rat model of lung ischemia-reperfusion (IR). Thirty-two male SpragueDawley rats weighing 250-300 g were randomly divided (n=8) into sham-operated group, lung IR group, LIPUS pretreatment group and pretreatment with α7-nicotinic cholinergic receptor (α7nAChR) antagonist group. In the sham-operated group, the left pulmonary hilum was dissociated without occlusion; in the other 3 groups, the left pulmonary hilum was occluded for 45 min followed by reperfusion for 180 min; LIPUS pretreatment for 30 min and intraperitoneal injection of methyllycaconitine (2 mg/kg), an α7nAChR antagonist, were administered before the operation. The wet/dry weight ratio (W/D) and pulmonary permeability index (LPI) of the lung tissue were measured, and the lung histopathology was observed and scored. The contents of interleukin-1 (IL-1) and IL-6 in the lung tissues were measured using ELISA, and the pulmonary expression of HMGB1 protein was detected using immunofluorescence assay and Western blotting. Compared with those in the sham-operated group, the W/D of the lung tissue, LPI, pathological scores, IL-1 and IL-6 contents in the lung tissue, and pulmonary HMGB1 expression all significantly increased in the other 3 groups (P &lt; 0.05). LIPUS preconditioning significantly lowered the W/D values, LPI, pathological score, IL-1 and IL-6 contents and HMGB1 expression in the lung tissues following lung IR, and these effects were significantly inhibited by administration of methyllycaconitine. LIPUS preconditioning can reduce lung IR injury possibly by activating α7nAChR-dependent cholinergic anti-inflammatory pathway to reduce lung tissue HMGB1 expression.

Induction of Tolerance to Fully Allogeneic Pulmonary Allograft by Adenosine A2A Receptor Agonist in MHC-defined CLAWN Miniature Swine

Introduction Adenosine has been found on immune cells such as neutrophils, macrophages, T-cells as well as on endothelial cells and the agonist of the adenosine A2A receptor (A2AR), one of four subtypes of the adenosine receptor family is known to potently attenuate lung ischemia-reperfusion injury (IRI). However, the long-term effects of this agonist on lung allograft survival remain unclear especially in large animals. The aim of this study is to investigate whether A2AR agonist has beneficial effects on pulmonary allograft survival using MHC-defined CLAWN miniature swine. Methods Twelve swine received fully MHC-mismatched lungs with 12 days of FK506 (35-45 ng/ml). In Group 1, six recipients received FK506 alone. In Group 2, three recipients were transplanted with lungs from brain-dead (BD) donors induced by subdural balloon inflation for 6 hours before organ procurement in order to examine whether BD affects the graft survival. In Group 3, three recipients were transplanted with lungs from BD donors treated with adenosine A2AR agonist (CGS21680: 5 μg/kg/min) starting 3 hours after BD induction. All recipients in group 3 additionally received the same dose of CGS21680 for 3 hours after reperfusion of the graft. Graft function was assessed by blood gases from the graft pulmonary vein (PV) and serial lung biopsies. The expression of mRNA of proinflammatory cytokines of the grafts (IL-1β and IL-6) were quantified by real-time RT-PCR. Results All grafts in Group 1 was rejected by day 63 with diffuse mononuclear cell infiltrates associated with intra-alveolar hemorrhage and capillary congestion. All three animals in Group 2 completely rejected the grafts by day 35, indicating rejection of grafts was accelerated by donor BD. In Group 3, CGS21680 was effective on early graft function. PO2 of graft PV in group 3 was higher than that of Group 2 (552 ± 22 vs. 414 ± 106 mmHg at 2 hour; 553 ± 19 vs. 349 ± 28 mmHg at day 2) associated with fewer inflammatory cell infiltrates both at 2-hour and day 2 biopsies. Up-regulation of proinflammatory cytokines in group 3 was markedly reduced at the time of organ procurement as well as at 2 hour and day 2 compared with Group 2. Although we lost one animal due to severe pneumonia at day 21, other two animals accepted the grafts over 150 days even after cessation of 12 days of FK506. Anti-donor T-cell responses assessed by MLR and anti-donor IgG development assessed by FACS was significantly reduced in Group3. Moreover, higher percentage of FoxP3-positive infiltrating cells were detected in Group 3, suggesting adenosine-mediated production of regulatory T cells. Conclusions Not only short-term effects, treatment of adenosine A2AR agonist with 12 days of FK506 facilitates induction of tolerance to fully allogeneic lung. This is the first evidence of successful induction of tolerance with short course of FK506 with adenosine A2AR agonist in a clinically relevant large animal model.

Mesenchymal stromal cell-derived extracellular vesicles attenuate lung ischemia-reperfusion injury and enhance reconditioning of donor lungs after circulatory death

BackgroundLung ischemia-reperfusion (IR) injury after transplantation as well as acute shortage of suitable donor lungs are two critical issues impacting lung transplant patients. This study investigates the anti-inflammatory and immunomodulatory role of human mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) to attenuate lung IR injury and improve of ex-vivo lung perfusion (EVLP)-mediated rehabilitation in donation after circulatory death (DCD) lungs.MethodsC57BL/6 wild-type (WT) mice underwent sham surgery or lung IR using an in vivo hilar-ligation model with or without MSCs or EVs. In vitro studies used primary iNKT cells and macrophages (MH-S cells) were exposed to hypoxia/reoxygenation with/without co-cultures with MSCs or EVs. Also, separate groups of WT mice underwent euthanasia and 1 h of warm ischemia and stored at 4 °C for 1 h followed by 1 h of normothermic EVLP using Steen solution or Steen solution containing MSCs or EVs.ResultsLungs from MSCs or EV-treated mice had significant attenuation of lung dysfunction and injury (decreased edema, neutrophil infiltration and myeloperoxidase levels) compared to IR alone. A significant decrease in proinflammatory cytokines (IL-17, TNF-α, CXCL1 and HMGB1) and upregulation of keratinocyte growth factor, prostaglandin E2 and IL-10 occurred in the BAL fluid from MSC or EV-treated mice after IR compared to IR alone. Furthermore, MSCs or EVs significantly downregulated iNKT cell-produced IL-17 and macrophage-produced HMGB1 and TNF-α after hypoxia/reoxygenation. Finally, EVLP of DCD lungs with Steen solution including MSCs or EVs provided significantly enhanced protection versus Steen solution alone. Co-cultures of MSCs or EVs with lung endothelial cells prevents neutrophil transendothelial migration after exposure to hypoxia/reoxygenation and TNF-α/HMGB1 cytomix.ConclusionsThese results suggest that MSC-derived EVs can attenuate lung inflammation and injury after IR as well as enhance EVLP-mediated reconditioning of donor lungs. The therapeutic benefits of EVs are in part mediated through anti-inflammatory promoting mechanisms via attenuation of immune cell activation as well as prevention of endothelial barrier integrity to prevent lung edema. Therefore, MSC-derived EVs offer a potential therapeutic strategy to treat post-transplant IR injury as well as rehabilitation of DCD lungs.

Lung inflation with hydrogen sulfide during the warm ischemia phase ameliorates injury in rat donor lungs via metabolic inhibition after cardiac death

Hydrogen sulfide attenuates lung ischemia-reperfusion injury when inhaled or administered intraperitoneally. This study investigated the effects of lung inflation with H2S during the warm ischemia phase on lung grafts from rat donors after cardiac death. One hour after cardiac death, donor lungs were inflated in situ for 2h with either O2 or H2S (O2 or H2S group) during the warm ischemia phase or were deflated as a control procedure (n=8). After 3h of cold preservation, lung transplantation was performed. During the warm ischemia phase, the metabolism and mitochondrial structures of donor lungs were analyzed. Arterial blood gas analysis was performed on the recipients. Protein expression in the graft of nuclear factor E2-related factor (Nrf)2 and nuclear factor kappa B (NF-κB) was analyzed by Western blotting, and static compliance, inflammation,oxidative stress, and cell apoptosis were assessed after 3h of reperfusion. When the O2 and H2S groups were compared with the control group, the mitochondrial structures were improved, and lactic acid levels, inflammation, oxidative stress, and cell apoptosis were significantly decreased; and glucose levels, as well as graft oxygenation and static compliance were increased. Simultaneously, the above indices showed further improvements, and the Nrf2 protein expression was significantly greater, and NF-κB protein expression was less in the H2S group than the O2 group. Lung inflation with H2S during the warm ischemia phase inhibited metabolism in donor lungs via mitochondrial protection, attenuated graft ischemic-reperfusion injury, and improved graft function through NF-κB-dependent anti-inflammatory and Nrf2-dependent antioxidative and antiapoptotic effects.

Alda-1 Attenuates Lung Ischemia-Reperfusion Injury by Reducing 4-Hydroxy-2-Nonenal in Alveolar Epithelial Cells.

Excessive oxidative stress is a main cause of lung ischemia-reperfusion injury, which often results in respiratory insufficiency after open-heart surgery for a cardiopulmonary bypass. Previous studies demonstrate that the activation of aldehyde dehydrogenase-2 could significantly reduce the oxidative stress mediated by toxic aldehydes and attenuate cardiac and cerebral ischemia-reperfusion injury. However, both the involvement of aldehydes and the protective effect of the aldehyde dehydrogenase-2 agonist, Alda-1, in lung ischemia-reperfusion injury remain unknown. Prospective laboratory and animal investigation were conducted. State Key Laboratory of Cardiovascular Disease. Primary human pulmonary alveolar epithelial cells, human pulmonary microvascular endothelial cells, and Sprague-Dawley rats. A hypoxia/reoxygenation cell-culture model of human pulmonary alveolar epithelial cell, human pulmonary microvascular endothelial cell, and an isolated-perfused lung model were applied to mimic lung ischemia-reperfusion injury. We evaluated the effects of Alda-1 on aldehyde dehydrogenase-2 quantity and activity, on aldehyde levels and pulmonary protection. We have demonstrated that ischemia-reperfusion-induced pulmonary injury concomitantly induced aldehydes accumulation in human pulmonary alveolar epithelial cells and lung tissues, but not in human pulmonary microvascular endothelial cells. Moreover, Alda-1 pretreatment significantly elevated aldehyde dehydrogenase-2 activity, increased surfactant-associated protein C, and attenuated elevation of 4-hydroxy-2-nonenal, apoptosis, intercellular adhesion molecule-1, inflammatory response, and the permeability of pulmonary alveolar capillary barrier, thus alleviated injury. Our study indicates that the accumulation of 4-hydroxy-2-nonenal plays an important role in lung ischemia-reperfusion injury. Alda-1 pretreatment can attenuate lung ischemia-reperfusion injury, possibly through the activation of aldehyde dehydrogenase-2, which in turn removes 4-hydroxy-2-nonenal in human pulmonary alveolar epithelial cells. Alda-1 pretreatment has clinical implications to protect lungs during cardiopulmonary bypass.

NOX2 Activation of Natural Killer T Cells Is Blocked by the Adenosine A2A Receptor to Inhibit Lung Ischemia-Reperfusion Injury.

Ischemia-reperfusion (IR) injury after lung transplantation, which affects both short- and long-term allograft survival, involves activation of NADPH oxidase 2 (NOX2) and activation of invariant natural killer T (iNKT) cells to produce IL-17. Adenosine A2A receptor (A2AR) agonists are known to potently attenuate lung IR injury and IL-17 production. However, mechanisms for iNKT cell activation after IR and A2AR agonist-mediated protection remain unclear. We tested the hypothesis that NOX2 mediates IL-17 production by iNKT cells after IR and that A2AR agonism prevents IR injury by blocking NOX2 activation in iNKT cells. An in vivo murine hilar ligation model of IR injury was used, in which left lungs underwent 1 hour of ischemia and 2 hours of reperfusion. Adoptive transfer of iNKT cells from p47(phox-/-) or NOX2(-/-) mice to Jα18(-/-) (iNKT cell-deficient) mice significantly attenuated lung IR injury and IL-17 production. Treatment with an A2AR agonist attenuated IR injury and IL-17 production in wild-type (WT) mice and in Jα18(-/-) mice reconstituted with WT, but not A2AR(-/-), iNKT cells. Furthermore, the A2AR agonist prevented IL-17 production by murine and human iNKT cells after acute hypoxia-reoxygenation by blocking p47(phox) phosphorylation, a critical step for NOX2 activation. NOX2 plays a key role in inducing iNKT cell-mediated IL-17 production and subsequent lung injury after IR. A primary mechanism for A2AR agonist-mediated protection entails inhibition of NOX2 in iNKT cells. Therefore, agonism of A2ARs on iNKT cells may be a novel therapeutic strategy to prevent primary graft dysfunction after lung transplantation.