Lung Ischemia-reperfusion Research Articles

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.

Carnosol suppresses interleukin-6 production in mouse lungs injured by ischemia-reperfusion operation and in RAW264.7 macrophages treated with lipopolysaccharide.

Carnosol is a naturally occurring herbal compound, known for its antioxidative properties. We previously found that carnosol protected mouse lungs from ischemia-reperfusion injury in ex vivo cultures. To elucidate the molecular mechanisms underpinning carnosol-mediated lung protection, we analyzed modes of interleukin-6 (IL-6) gene expression, which is associated with lung ischemia-reperfusion injury. Microarray analysis of mouse lungs suggested that IL-6 mRNA levels were elevated in the mouse lungs subjected to clamp-reperfusion, which was associated with elevated levels of other inflammatory modulators, such as activating transcription factor 3 (ATF3). Carnosol pretreatment lowered the IL-6 protein levels in mouse lung homogenates prepared after the clamp-reperfusion. On the other hand, the ATF3 gene expression was negatively correlated with that of IL-6 in RAW264.7 cells. IL-6 mRNA levels and gene promoter activities were suppressed by carnosol in RAW264.7 cells, but rescued by ATF3 knockdown. When RAW264.7 cells were subjected to hypoxia-reoxygenation, carnosol treatment lowered oxygen consumption after reoxygenation, which was coupled with a correlation with a transient production of mitochondrial reactive oxygen species and following ATF3 gene expression. These results suggest that carnosol treatment could be a new strategy for protecting lungs from ischemia-reperfusion injury by modulating the ATF3-IL-6 axis.

α7 Nicotinic acetylcholine receptor contributes to the alleviation of lung ischemia–reperfusion injury by transient receptor potential vanilloid type 1 stimulation

BackgroundActivation of transient receptor potential vanilloid type 1 (TRPV1) decreases lung ischemia–reperfusion injury (LIRI) in rabbits and rats. Stimulation of α7 nicotinic acetylcholine receptors (α7nAChRs) protects against lung injury. Here we examined whether α7nAChRs contribute to TRPV1-mediated protection against LIRI. MethodsWild-type (WT) and TRPV1-knockout (KO) mice were subjected to 1-h lung ischemia by clamping left hilum, followed by 2-h reperfusion. WT or KO mice were pretreated with vehicle, TRPV1 agonist capsaicin, TRPV1 antagonist capsazepine, α7nAChR antagonist methyllycaconitine, or α7nAChR agonist PNU-282987. Arterial blood and lung tissues were obtained for blood gas, lung wet-to-dry weight ratio, interleukin (IL)1β, IL6, tumor necrosis factor-α (TNF-α), apoptosis-related proteins (caspases, Bax, Fas), and pathologic scoring. ResultsCapsaicin pretreatment reduced wet-to-dry ratio, pathologic score, alveolar-arterial oxygen gradient (A-aDO2), and IL1β, IL6, and TNFα levels in WT mice, with no effects in KO mice. This reduction was reversed by TRPV1 blockade. Furthermore, α7nAChR blockade before capsaicin exacerbated LIRI as evidenced by enhanced alveolar-arterial oxygen gradient, pathologic score, and IL1β, IL6, and TNFα levels, while α7nAChR agonist pretreatment under TRPV1 blockade showed opposite changes. Capsaicin also decreased cleaved caspase-3, caspase-3/9, and Bax protein expression, effects abolished by TRPV1 blockade. Similarly, α7nAChR blockade diminished capsaicin-induced downregulation of apoptotic proteins, and α7nAChR activation decreased expression levels even under TRPV1 blockade. ConclusionsTRPV1 activation alleviates LIRI, partially dependent on α7nAChR activity. The α7nAChR stimulation with or without existence of TRPV1 alleviates LIRI. Thus, α7nAChR is involved in the pathway of TRPV1-mediated protection against LIRI and the specific mechanism remains to be revealed.

Lazaroid (U-74389G) ameliorates lung injury due to lipid peroxidation and nitric oxide synthase-dependent reactive oxygen species generation caused by remote systematic ischemia-reperfusion following thoracoabdominal aortic occlusion

IntroductionLung ischemia-reperfusion injury after thoracoabdominal aortic occlusion represents a major complication, which increases morbidity and mortality. In the present study we hypothesized that lazaroid U-74389G intravenous administration protects from lung ischemia-reperfusion injury through lipid peroxidation inhibition. Materials and methodsA total of 24 pigs were randomized in three groups. Group I (n = 8) underwent sham operation, group II (n = 8) underwent thoracoabdominal aortic occlusion for 45min and received placebo and group III (n = 8) received 3 doses of lazaroid (3 mg/kg) 60 and 30min before thoracoabdominal aortic occlusion and at 30min during thoracoabdominal aortic occlusion (duration 45min). Aortic occlusion was performed with aortic balloon-catheters under fluoroscopic guidance. All animals were sacrificed at the 7 t h postoperative day and lung specimens were harvested for molecular analysis. ResultsmRNA levels of leukotrienes LB4 (LTB4R2), LC4 (LTC4S) and nitric oxide synthase (NOS) isoforms including iNOS, nNOS and eNOS were determined with real-time RT-qPCR. Nitric oxide can either induce (iNOS) or inhibit (nNOS and eNOS) lipid peroxidation based on its specific isoform origin. Group III showed significantly reduced mRNA levels of LTB4R2 (−63.7%), LTC4S (−35.9%) and iNOS (−60.2%) when compared with group II (P < 0.05, for all). The mRNA levels of nNOS was significantly increased (+37.4%), while eNOS was slightly increased (+2.1%) in group III when compared with group II (P < 0.05 and P = 0.467 respectively). ConclusionLazaroid U-74389G may represent an effective pharmacologic intervention in reducing lung ischemia-reperfusion injury following thoracoabdominal aortic occlusion.

Spleen-derived classical monocytes mediate lung ischemia-reperfusion injury through IL-1β.

Ischemia-reperfusion injury, a form of sterile inflammation, is the leading risk factor for both short-term mortality following pulmonary transplantation and chronic lung allograft dysfunction. While it is well recognized that neutrophils are critical mediators of acute lung injury, processes that guide their entry into pulmonary tissue are not well understood. Here, we found that CCR2+ classical monocytes are necessary and sufficient for mediating extravasation of neutrophils into pulmonary tissue during ischemia-reperfusion injury following hilar clamping or lung transplantation. The classical monocytes were mobilized from the host spleen, and splenectomy attenuated the recruitment of classical monocytes as well as the entry of neutrophils into injured lung tissue, which was associated with improved graft function. Neutrophil extravasation was mediated by MyD88-dependent IL-1β production by graft-infiltrating classical monocytes, which downregulated the expression of the tight junction-associated protein ZO-2 in pulmonary vascular endothelial cells. Thus, we have uncovered a crucial role for classical monocytes, mobilized from the spleen, in mediating neutrophil extravasation, with potential implications for targeting of recipient classical monocytes to ameliorate pulmonary ischemia-reperfusion injury in the clinic.

Pannexin-1 channels on endothelial cells mediate vascular inflammation during lung ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) injury (IRI), which involves inflammation, vascular permeability, and edema, remains a major challenge after lung transplantation. Pannexin-1 (Panx1) channels modulate cellular ATP release during inflammation. This study tests the hypothesis that endothelial Panx1 is a key mediator of vascular inflammation and edema after I/R and that IRI can be blocked by Panx1 antagonism. A murine hilar ligation model of IRI was used whereby left lungs underwent 1 h of ischemia and 2 h of reperfusion. Treatment of wild-type mice with Panx1 inhibitors (carbenoxolone or probenecid) significantly attenuated I/R-induced pulmonary dysfunction, edema, cytokine production, and neutrophil infiltration versus vehicle-treated mice. In addition, VE-Cad-CreERT2+/Panx1fl/fl mice (tamoxifen-inducible deletion of Panx1 in vascular endothelium) treated with tamoxifen were significantly protected from IRI (reduced dysfunction, endothelial permeability, edema, proinflammatory cytokines, and neutrophil infiltration) versus vehicle-treated mice. Furthermore, extracellular ATP levels in bronchoalveolar lavage fluid is Panx1-mediated after I/R as it was markedly attenuated by Panx1 antagonism in wild-type mice and by endothelial-specific Panx1 deficiency. Panx1 gene expression in lungs after I/R was also significantly elevated compared with sham. In vitro experiments demonstrated that TNF-α and/or hypoxia-reoxygenation induced ATP release from lung microvascular endothelial cells, which was attenuated by Panx1 inhibitors. This study is the first, to our knowledge, to demonstrate that endothelial Panx1 plays a key role in mediating vascular permeability, inflammation, edema, leukocyte infiltration, and lung dysfunction after I/R. Pharmacological antagonism of Panx1 activity may be a novel therapeutic strategy to prevent IRI and primary graft dysfunction after lung transplantation.

The effect of morphine delta receptor activity on ischemic postconditioning in lung ischemia reperfusion injury

OBJECTIVE: In the context of the physiopathology of lung damage due to ischemia and reperfusion injury, we aimed to reveal the effects of the addition of morphine sulfate to ischemic postconditioning (PC) protocol. METHODS: In the present study, 48 Wistar albino female rats were employed. Group 1 was accepted as the Sham group that underwent thoracotomy through the fifth left intercostal space. Ischemia-reperfusion (IR) group: Thoracotomy and IR period. IRPC group: thoracotomy, IR period and ischemic PC. In IRPC3 and IRPC30 groups, in addition to ischemic PC different doses of morphine sulfate (3 μmol and 30 μmol) was administered. Tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-10 levels were measured in the biochemical assessment of the lung tissue samples obtained. RESULTS: TNF-α and IL-1 (pro-inflammatory cytokines) have lower values, and IL-10 (anti-inflammatory cytokine) have higher values both in the groups which have been subject to PC and morphine. TNF-α and IL-1 levels in lung tissue were statistically significant between the IRPC3 group and the IR and IRPC groups. In addition, IL-10 level in lung tissue was statistically significant between the IRPC3 group and the IRPC group. CONCLUSION: In the present study conducted with experimental animals where morphine was also injected beside ischemic PC protocols, statistically significant differences were determined in the lung tissue analyses when we compared pro-inflammatory and anti-inflammatory cytokine values. We firmly believe that adding morphine to the lung transplantation protocols and PC will decrease IR damage.

Human Multilineage-differentiating Stress-Enduring Cells Exert Pleiotropic Effects to Ameliorate Acute Lung Ischemia–Reperfusion Injury in a Rat Model

Posttransplantation lung ischemia–reperfusion (IR) injuries affect both patient survival and graft function. In this study, we evaluated the protective effects of infused human multilineage-differentiating stress-enduring (Muse) cells, a novel, easily harvested type of nontumorigenic endogenous reparative stem cell, against acute IR lung injury in a rat model. After a 2-h warm IR injury induction in a left rat lung, human Muse cells, human mesenchymal stem cells (MSCs), and vehicle were injected via the left pulmonary artery after reperfusion. Functionality, histological findings, and protein expression were subsequently assessed in the injured lung. In vitro, we also compared human Muse cells with human MSCs in terms of migration abilities and the secretory properties of protective substances. The arterial oxygen partial pressure to fractional inspired oxygen ratio, alveolar-arterial oxygen gradient, left lung compliance, and histological injury score on hematoxylin–eosin sections were significantly better in the Muse group relative to the MSC and vehicle groups. Compared to MSCs, human Muse cells homed more efficiently to the injured lung, where they suppressed the apoptosis and stimulated proliferation of host alveolar cells. Human Muse cells also migrated to serum from lung-injured model rats and produced beneficial substances (keratinocyte growth factor [KGF], hepatocyte growth factor, angiopoietin-1, and prostaglandin E2) in vitro. Western blot of lung tissue confirmed high expression of KGF and their target molecules (interleukin-6, protein kinase B, and B-cell lymphoma-2) in the Muse group. Thus, Muse cells efficiently ameliorated lung IR injury via pleiotropic effects in a rat model. These findings support further investigation on the use of human Muse cells for lung IR injury.

A (+)-Larixol Congener with High Affinity and Subtype Selectivity toward TRPC6.

Natural products have many health benefits, and their application can improve the quality of life. Recently, the diterpene (+)-larixol and its acetylated congeners demonstrated selective inhibition of the second-messenger-gated cation channel transient receptor potential canonical 6 (TRPC6) over its close isoforms TRPC3 and TRPC7. Building on this knowledge, we expanded these findings by chemical diversification of (+)-larixol mostly at position C6. Implementing high-throughput Ca2+ FLIPR screening assays and electrophysiological patch-clamp recordings, we showcase larixyl N-methylcarbamate, termed SH045, as a compound with nanomolar affinity and 13-fold subtype selectivity over TRPC3 in stably expressing HEK293 cells. Expanding on this finding, TRPC6 inhibition was also observed in rat pulmonary smooth muscle cells. Furthermore, treatment of isolated perfused lung preparations with SH045 led to a decrease in lung ischemia-reperfusion edema (LIRE), a life-threatening condition associated with TRPC6 that may occur after organ transplantation. Taken together, and given the inexpensive, straightforward, and scalable preparation of SH045, we report a TRPC6 blocker that holds promise for the translational treatment of LIRE.

Inhibition of regulated necrosis attenuates receptor-interacting protein kinase 1-mediated ischemia-reperfusion injury after lung transplantation.

Increasing evidence indicates that regulated necrosis plays a critical role during cell death caused by ischemia-reperfusion (IR) injury. Necroptosis is one form of regulated necrosis. Necrostatin-1 (Nec-1), an inhibitor of receptor-interacting protein kinase 1 (RIPK1), is known to reduce necroptosis. We investigated the effect of Nec-1 treatment on IR-induced lung injury in a rat lung transplant model. Lewis rats were divided into 4 groups (n = 6 each): (1) Control (no treatment), (2) Donor treatment (D), (3) Recipient treatment (R), and (4) Donor plus Recipient treatment (D+R) groups. Donor lungs were flushed and preserved for 18 hours at 4ºC before transplantation. Recipient animals underwent a left single lung transplant. After 2 hours of reperfusion, we assessed the physiologic function, cytokine expression, pathway activation, and the extent of necrosis. Pulmonary gas exchange in D+R group was significantly better than in the other 3 groups (p = 0.003). Lung edema was significantly lower in the D+R group compared with the Control group (p = 0.006). The expression of interleukin-6 in lung tissue and plasma was significantly reduced in the D+R group compared with the Control group (p = 0.036). The percentage of necrotic cells in D+R group was significantly lower than in the Control and D groups (p = 0.01), indicating Nec-1inhibited regulated necrosis. The administration of Nec-1 to both donor and recipient improved graft function after lung transplantation through the reduction of necroptosis. The inhibition of regulated necrosis appears to be a promising strategy to attenuate IR lung injury after lung transplantation.

Role of Nrf2/ARE signaling pathway in propofol-induced reduction of lung ischemia-reperfusion injury in aged rats

Objective To evaluate the role of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway in propofol-induced reduction of lung ischemia-reperfusion (I/R) injury in aged rats. Methods Thirty-two clean healthy male Sprague-Dawley rats, aged 18-22 months, weighing 450-600, were divided into 4 groups (n=8 each) using a random number table: sham operation group (group S), I/R group (group I/R), I/R plus propofol group (group I/R+ P) and all-trans retinoic acid (ARTA) plus I/R plus propofol group (group ARTA+ I/R+ P). Lung I/R was induced by occlusion of the right hilum of lung for 60 min followed by 120 min of reperfusion in anesthetized rats.In group ATRA+ I/R+ P, Nrf2/ARE signaling pathway blocker ARTA 6 mg/kg was intraperitoneally injected once a day for 3 consecutive days, and the model of lung I/R injury was established at 2 h after the last administration.In group I/R+ P and group ARTA+ I/R+ P, while the model of lung I/R injury was established, propofol 30 mg·kg-1·h-1 was infused via the caudal vein until 120 min of reperfusion.The rats were sacrificed at 120 min of reperfusion and then the lungs were removed for examination of the pathological changes which were scored and for measurement of wet/dry weight ratio (W/D ratio), superoxide dismutase (SOD) activity (by xanthine oxidase method), malondialdehyde (MDA) content (using thiobarbituric acid method) and expression of Nrf2 and heme oxygenase-l (HO-1) protein (by Western blot). Results Compared with group S, the pathological scores, W/D ratio and MDA content were significantly increased, and the activity of SOD was decreased in I/R and ATRA+ I/R+ P groups, and the expression of Nrf-2 and HO-1 was significantly up-regulated in I/R, I/R+ P and ATRA+ I/R+ P groups (P 0.05). Compared with group I/R+ P, the pathological scores, W/D ratio and MDA content were significantly increased, the activity of SOD was decreased, and the expression of Nrf-2 and HO-1 was down-regulated in group ARTA+ I/R+ P (P<0.05). Conclusion Nrf2/ARE signaling pathway activation is involved in propofol-induced reduction of lung I/R injury in aged rats. Key words: Propofol; Lung; Reperfusion injury; Nuclear factor-E2 related factor 2; Response elements

Double-Negative αβ T cells: A Novel Player in Lung Ischemic-Reperfusion Injury

Lung ischemia-reperfusion injury (IRI) remains one of the most common complications following lung transplantation and often accompanied by renal insufficiency. Recent work has elucidated a novel subset of αβ T cell receptor positive CD4-CD8- (double-negative; DN) T cells in the kidneys, which have protective anti-inflammatory properties against IRI. We hypothesized that DN T cells are found in lung, are activated by IRI, and could be a new therapeutic target for the injured lung.

Adiponectin protects against lung ischemia-reperfusion injury in rats with type 2 diabetes mellitus.

Adiponectin (APN) has been associated with the pathogenesis of acute brain, liver and heart injury. However, the role of APN in lung ischemia-reperfusion injury (LIRI) in diabetes mellitus remains unclear. To investigate this, the present study evaluated the effects of APN on lung dysfunction and pathological alterations in rats with type 2 diabetes mellitus via lung ischemia/reperfusion (I/R). The lung-protective effects of APN globular domain (gAPN) in rats with type 2 diabetes mellitus were also investigated by measuring the oxygenation index, inflammatory cytokines, lung edema, histopathology, oxidative stress, apoptosis and the protein expression levels of phosphorylated 5′adenosine monophosphate-activated protein kinase (p-AMPK), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). The results of the present study demonstrated that the diabetes mellitus rats + I/R (DIR) group exhibited greater concentrations of tumor necrosis factor-α and interleukin-6, and increases in the wet-weight to dry-weight ratio, lung injury score, oxidative stress and cellular apoptosis. These effects were accompanied by lower pulmonary oxygenation compared with the normal rat + I/R (NIR) group (P<0.05). Additionally, all of these alterations were attenuated in the NIR + gAPN and DIR + gAPN groups compared with in the NIR and DIR groups, respectively. In the DIR group, the expression levels of p-AMPK/AMPK and eNOS were significantly downregulated, and the levels of iNOS were upregulated, compared with those of the NIR group. Treatment with APN activated AMPK, increased eNOS expression and attenuated iNOS expression. The results of the present study demonstrated that APN exerted protective effects against LIRI via its anti-inflammatory, antioxidative stress and anti-apoptotic activities. These protective effects of APN were eliminated in rats with type 2 diabetes mellitus, in which LIRI was exacerbated. The present study indicated that APN may be a potential therapeutic agent for LIRI in type 2 diabetes mellitus.

Effect of penehyclidine hydrochloride injection on pulmonary ischemia-reperfusion in aortic dissection surgery

Objective: To evaluate the effect of Penehyclidine Hydrochloride Injection on pulmonary ischemia-reperfusion in aortic dissection surgery. Methods: This study was a prospective randomized controlled trial. Between September 2015 and October 2017 in the Second Affiliated Hospital of Zhengzhou University, 60 patients with aortic dissection underwent total arch replacement surgery were randomly divided into penehyclidine hydrochloride group (group A 30 cases) and control group (group B 30 cases). Patients in group A were infused with penehyclidine hydrochloride 0.05 mg/kg after entering the operating room. Patients in group B were infused with the same dose of 0.9% normal saline. Serum tumor necrosis factor (TNF-α), interleukin-6 (IL-6), interleukin-1 (IL-1) and oxygenation index (OI) before anesthesia (T1), before cardiopulmonary bypass (T2), 1 h after cardiopulmonary bypass (T3), the end of cardiopulmonary bypass (T4), 4 h after surgery (T5) and 24 h after surgery (T6) were measured. The time of postoperative ventilator support and ICU staywas recorded. Results: At the time of T4, T5 and T6, the serum levels of TNF-α[(0.10±0.08), (0.13±0.02), (0.23±0.17) mg/L], IL-6[(0.23±0.08), (0.34±0.07), (0.54±0.17) mg/L]and IL-1[(0.62±0.14), (1.02±0.27), (1.44±0.40) mg/L]in group A were significantly lower than those in group B[(0.30±0.09), (0.51±0.19), (0.86±0.02) mg/L; (0.73±0.19), (1.33±0.13), (1.98±0.13) mg/L; (0.93±0.19), (1.43±0.66), (2.04±0.45) mg/L]. The OI was higher in group A[(446.7±267.0), (386.7±169.5), (391.7±227.9) mmHg]than that of group B[(341.2±145.2), (299.5±98.7), (275.0±127.3) mmHg](P<0.05). The time of ventilator support and ICU stay after operation was (3.6 ±1.2)d, (8.4 ± 2.0) d in group A, which was shorter than that in group B[(4.3 ± 1.8), (10.0 ± 2.2) d], and there were statistical differences between the two groups (all P<0.05). Conclusions: In total arch replacement, intravenous penehyclidine hydrochloride injection may decrease the release of serum TNF-α, IL-6, IL-1, improve oxygenation index, reduce lung ischemia-reperfusion injury, shorten the time of ventilator support and ICU stay after operation, and thus improve the prognosis of patients.

Effect of therapeutic hypercapnia preconditioning on lung ischemia-reperfusion injury in rats

Objective To evaluate the effect of therapeutic hypercapnia preconditioning on lung ischemia-reperfusion (I/R) injury in rats. Methods Forty healthy adult Sprague-Dawley rats of either sex, aged 2 months, weighing 250-300 g, were divided into 5 groups (n=8 each) using a random number table: sham operation group (group S), group I/R and preconditioning with therapeutic hypercapnia of different level groups (group THP1-3). Lung I/R injury was induced by clamping the left hilum of lung for 45 min followed by 120 min of reperfusion.In THP1-3 groups, the respiratory parameters were adjusted at 5 min of stability after isolating the left hilum of lung to make PETCO2 reach 55-65, 65-75 and 75-85 mmHg respetively and maintained at this level for 5 min, normal ventilation was then used to make PETCO2 restore the normal level, continuously repeating for 3 circles, and then the left hilum of lung was blocked for 45 min followed by 120 min of reperfusion.The bronchoalveolar lavage fluid (BALF) was collected at the end of reperfusion for determination of the total protein (TP) concentration using Coomassie brilliant blue staining.Lung tissues were obtained at the end of reperfusion for examination of pathological changes after haematoxylin and eosin staining (under a light microscope) and for determination of wet/dry weight ratio (W/D ratio), malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, interleukin-8 (IL-8) and IL-10 contents (by enzyme-linked immunosorbent assay), tumor necrosis factor-alpha (TNF-α) expression (by immunohistochemistry) and expression of TNF-α mRNA (by real-time polymerase chain reaction). Results Compared with group S, the TP concentration in BALF, W/D ratio and contents of MDA, IL-8 and IL-10 in lung tissues were significantly increased, the SOD activity was decreased, the expression of TNF-α mRNA was up-regulated (P 0.05), the staining range and intensity of TNF-α were decreased, and the pathological changes of lung tissues were significantly attenuated in THP1-3 groups. Conclusion Therapeutic hypercapnia preconditioning can reduce lung I/R injury in rats, and the mechanism is related to inhibiting inflammatory responses and oxidative stress responses. Key words: Hypercapnia; Reperfusion injury; Lung; Preconditioning

Effects of pretreatment with exogenous insulin-like growth factors-1 on lung injury in rats undergoing cardiopulmonary bypass

Objective To evaluate the effects of pretreatment with exogenous insulin-like growth factor-1 (IGF-1) on lung injury in rats undergoing cardiopulmonary bypass(CPB). Methods Seventy-two SPF healthy male Sprague-Dawley rats, weighing 350-500 g, were divided into 4 groups (n=18 each) using a random number table: sham operation group (group S), CPB group, CPB plus left lung ischemia-reperfusion group (group LI) and IGF-1 group.The chest was only opened, and the rats underwent no CPB in group S. Only the CPB model was established in group CPB.The model of left lung ischemia-reperfusion injury was established based on the CPB model in group LI.The model of CPB and left lung ischemia-reperfusion injury was established, and IGF-1 30 μg/kg was intravenously injected at 10 min before clamping the hilum of lung and immediately after opening the hilum of lung in group IGF-1.Six rats were selected before operation (T1), 10 min after opening the left hilum (T2) and at the end of operation (T3), and blood samples were collected from the femoral artery for blood gas analysis.The oxygenation index (OI) and respiratory index (RI) were calculated.Serum was obtained from blood, and the concentrations of interleukin-6 (IL-6), IL-1β and tumor necrosis factor-alpha (TNF-α) in serum were measured using enzyme-linked immunosorbent assay.The left upper lung tissues were removed for examination of the pathological changes which were scored with a light microscope. Results Compared with the baseline at T1, OI was significantly decreased, and RI, concentrations of IL-6, IL-1β and TNF-α in serum and pathological scores of lung tissues were increased at T3 in CPB, LI and IGF-1 groups (P<0.05). Compared with group S, OI was significantly decreased, and RI, concentrations of IL-6, IL-1β and TNF-α in serum and pathological scores of lung tissues were increased at T3 in CPB, LI and IGF-1 groups (P<0.05). Compared with group CPB, OI was significantly decreased, and RI, concentrations of IL-6, IL-1β and TNF-α in serum and pathological scores of lung tissues were increased at T3 in group LI (P<0.05). Compared with group LI, OI was significantly increased, RI, concentrations of IL-6 and IL-1β in serum and pathological scores of lung tissues were decreased at T3, and the concentration of serum TNF-α was increased at T3 in group IGF-1 (P<0.05). Conclusion IGF-1 pretreatment can reduce lung injury in rats undergoing CPB, and the mechanism is related to inhibiting inflammatory responses. Key words: Insulin-like growth factor Ⅰ; Cardiopulmonary bypass; Respiratory distress syndrome, adult

Effects of dexmedetomidine on inflammatory response and lipid peroxidation during lung ischemia-reperfusion in rats: an in vitro experiment

Objective To investigate the effects of dexmedetomidine on inflammatory response and lipid peroxidation during lung ischemia-reperfusion(I/R)in rats. Methods Thirty lungs, which were isolated from SPF adult male Sprageue-Dawley rats, in which the model of isolated lung perfusion was successfully established, were divided into 3 groups(n=10 each)using a random number table: sham operation group(S group), I/R group and dexmedetomidine group(D group). The lungs were subjected to 60 min of ischemia and apnea after 15-min ischemia followed by 75 min of reperfusion and ventilation to establish the model of isolated lung I/R injury.In group D, the lungs were perfused with K-H solution containing 10 nmol/L dexmedetomidine for 15 min and then subjected to 60 min of ischemia and apnea followed by ventilation and reperfusion with K-H sloution containing 10 nmol/L dexmedetomidine for 75 min.Airway resistance, lung compliance, perfusion flow and partial pressure of arterial oxygen(PaO2)were recorded at 10 min of perfusion(T0)and 15, 45 and 75 min after restoration of perfusion(T1-3). Lung tissues were obtained at 75 min after restoration of perfusion for determination of wet/dry weight ratio(W/D ratio)and for examination of pathological changes and ultrastructure of lungs.The activity of superoxide dismutase(SOD)and content of malondialdehyde(MDA)in lung tissues were measured by improved pyrogallol autoxidation method and thiobarbituric acid method, respectively.The expression of β-endorphin and interleukin-8(IL-8)in lung tissues was detected by Western blot. Results Compared with group S, the airway resistance was significantly increased, and the lung compliance, perfusion flow and PaO2 were decreased during the perfusion restoration period, the W/D ratio and content of MDA were increased, the activity of SOD was decreased, and the expression of β-endorphin and IL-8 was up-regulated in I/R and D groups(P<0.05). Compared with group I/R, the airway resistance was significantly decreased, and the lung compliance, perfusion flow and PaO2 were increased during the perfusion restoration period, the W/D ratio and content of MDA were decreased, the activity of SOD was increased, the expression of β-endorphin and IL-8 was down-regulated(P<0.05), and the pathological changes of lungs were significantly attenuated in group D. Conclusion The mechanism by which dexmedetomidine reduces lung I/R injury may be related to inhibiting lipid peroxidation and inflammatory response of lung tissues in rats. Key words: Dexmedetomidine; Inflammation; Lipid peroxidation; Reperfusion injury; Lung

Melatonin attenuates lung ischaemia-reperfusion injury via inhibition of oxidative stress and inflammation.

Lung ischaemia-reperfusion injury is a complex pathophysiological process due to the production of reactive oxygen species and the generation of inflammatory reaction. We investigated the protective effects and the corresponding mechanism of melatonin (MT), a potent free-radical scavenger, on lung injury induced by ischaemia-reperfusion in a mouse model. Adult male C57BL/6J mice (n = 30) were randomly and equally allocated into 5 groups: sham controls, IR, IR + 10 mg/kg MT, IR + 20 mg/kg MT and IR + 30 mg/kg MT. Lung ischaemia-reperfusion injury was induced by thoracotomy followed by clamping of the left hilum for 1 h and subsequent reperfusion for 2 h. Histological scoring analysis showed that lung parenchymal damage was ameliorated in the melatonin pretreatment groups when compared with the IR group, with the IR + 20 mg/kg MT group showing the strongest effect among the melatonin pretreatment groups. Wet-to-dry weight ratio, detection of malondialdehyde, protein expressions of inflammatory factors (tumour necrosis factor-α, interleukin-1β, NF-κB and IKK-γ) and apoptotic factors (cleaved caspase-3 and Bax/Bcl-2), as well as TUNEL assay showed changes similar to those of the lung injury scores in all groups. In contrast, the examination of superoxide dismutase showed a pattern contrary to that of the lung injury score in all groups. In addition, immunohistochemistry staining showed that the expressions of the antioxidants glutathione peroxidase and glutathione reductase were increased in the melatonin pretreatment groups. This study demonstrated that melatonin pretreatment attenuated lung ischaemia-reperfusion injury via inhibition of oxidative stress, inflammation and apoptosis.

Protection function of acidification pretreatment of respiratory acidosis on lung ischemia-reperfusion injury

Objective: This research was aimed to build ischemia-reperfusion model of rats in vivo , and observe the effects of different degrees of acidification pre-treatment of respiratory acidosis on lung ischemiareperfusion injury. Methods: 50 Sprague-Dawley rats were randomly divided into five groups: the sham operation S group, the ischemia-reperfusion group, pretreatment L group, pretreatment M group and pretreatment H group. Rats in all groups were killed in 2 h after reperfusion and ischemia. Samples of lung tissue were selected after experiments. Wet to dry weight ratio, levels of malondialdehyde, superoxide dismutase, interleukin-8 and interleukin-10 in lung tissue were detected. Pathological changes of lung tissue were observed. Results: Compared with the sham operation group, wet to dry weight ratio, levels of malondialdehyde, superoxide dismutase, interleukin-8 and interleukin-10 in lung tissue increased obviously. Acidification pretreatment of respiratory acidosis can inhibit interleukin-8 production after ischemia-reperfusion, and lower the levels of malondialdehyde and superoxide dismutase. Conclusion: Acidification pretreatment of respiratory acidosis can inhibit proinflammatory cytokine production after ischemia-reperfusion injury, lower inflammatory reaction level, which may protect ischemia-reperfusion injury.

Methylene Blue Protects the Isolated Rat Lungs from Ischemia-Reperfusion Injury by Attenuating Mitochondrial Oxidative Damage.

Impaired mitochondrial function is a key factor attributing to the lung ischemia reperfusion injury (LIRI). Methylene blue (MB) has been reported to attenuate brain and renal ischemia-reperfusion injury. We hypothesized that MB also could have a protective effect against LIRI by preventing mitochondrial oxidative damage. Isolated rat lungs were assigned to the following four groups (n=6): a sham group: perfusion for 105min without ischemia; I/R group: shutoff of perfusion and ventilation for 45min followed by reperfusion for 60min; and I/R+MB group and I/R+glutathione (GSH) group: 2mg/kgMB or 4μM glutathione were intraperitoneally administered for 2h, and followed by 45min of ischemia and 60min of reperfusion. MB lessened pulmonary dysfunction and severe histological injury induced by ischemia-reperfusion injury. MB reduced the production of reactive oxygen species and malondialdehyde and enhanced the activity of superoxide dismutase. MB also suppressed the opening of the mitochondrial permeability transition pore and partly preserved mitochondrial membrane potential. Moreover, MB inhibited the release of cytochrome c from the mitochondria into the cytosol and decreased apoptosis. Additionally, MB downregulated the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-18). MB protects the isolated rat lungs against ischemia-reperfusion injury by attenuating mitochondrial damage.