Background: Lung ischemia-reperfusion injury (LIRI) is a complex pathophysiological process that can lead to poor patient outcomes. Inflammasome-dependent macrophage pyroptosis contributes to organ damage caused by ischemia/reperfusion injury. Oxidative stress and antioxidant enzymes also play an important role in LIRI. In this study, we conducted experiments to investigate whether and how preconditioning with rHMGB1 could ameliorate LIRI in a mouse model. Methods: Adult male BALB/c mice were anesthetized, the left hilus pulmonis was clamped, and reperfusion was performed. rHMGB1 was administered via intraperitoneal injection before anesthesia, and brusatol was given intraperitoneally every other day before surgery. We measured pathohistological lung tissue damage, wet/dry mass ratios of pulmonary tissue, and levels of inflammatory mediators to assess the extent of lung injury. Alveolar macrophage pyroptosis was evaluated by measuring release of lactate dehydrogenase, caspase-1 expression was assessed using flow cytometry, and gasdermin-D expression was analyzed using immunofluorescent staining. Levels of oxidative stress markers and antioxidant enzymes were also analyzed. Results: Preconditioning with rHMGB1 significantly ameliorated lung injury induced by ischemia-reperfusion, based on measurements of morphology, wet/dry mass ratios, as well as expression of IL-1β, IL-6, NF-κB, and HMGB1 in lung tissues. It also alleviated alveolar macrophage pyroptosis, reduced oxidative stress and restored the activity of antioxidant enzymes. These beneficial effects were mediated at least in part by the Keap1/Nrf2/HO-1 pathway, since they were reversed by the pathway inhibitor brusatol. Conclusions: Preconditioning with rHMGB1 may protect against LIRI by suppressing alveolar macrophage pyroptosis. This appears to involve reduction of oxidative stress and promotion of antioxidant enzyme activity via the Keap1/ Nrf2/HO-1 pathway.

Background: Lung ischemia reperfusion injury (LIRI) is a complex pathophysiological process activated by lung transplantation and acute lung injury. The p38 mitogen-activated protein kinase (MAPK) is involved in breakdown of the endothelial barrier during LIRI, but the mechanism is still unclear. Therefore, we investigated the function of p38 MAPK in LIRI in vivo and in vitro.Methods: Sprague–Dawley rats were subjected to ischemia reperfusion with or without pretreatment with a p38 MAPK inhibitor. Lung injury was assessed using hematoxylin and eosin staining, and pulmonary blood–air barrier permeability was evaluated using Evans blue staining. A rat pulmonary microvascular endothelial cell line was infected with lentiviral expressing short hairpin (sh)RNA targeting p38 MAPK and then cells were subjected to oxygen/glucose deprivation and reoxygenation (OGD/R). Markers of endothelial destruction were measured by western blot and immunofluorescence.Results:In vivo LIRI models showed structural changes indicative of lung injury and hyperpermeability of the blood–air barrier. Inhibiting p38 MAPK mitigated these effects. Oxygen/glucose deprivation and reoxygenation promoted hyperpermeability of the endothelial barrier in vitro, but knockdown of p38 MAPK attenuated cell injury; maintained endothelial barrier integrity; and partially reversed injury-induced downregulation of permeability protein AQP1, endothelial protective protein eNOS, and junction proteins ZO-1 and VE-cadherin while downregulating ICAM-1, a protein involved in destroying the endothelial barrier, and ET-1, a protein involved in endothelial dysfunction.Conclusion: Inhibition of p38 MAPK alleviates LIRI by decreasing blood–air hyperpermeability. Blocking p38 MAPK may be an effective treatment against acute lung injury.

Polarization of alveolar macrophages (AMs) into the M1 phenotype contributes to inflammatory responses and tissue damage that occur during lung ischemia–reperfusion injury (LIRI). Programmed cell death factor-1 (PD-1) regulates polarization of macrophages, but its role in LIRI is unknown. We examined the role of PD-1 in AM polarization in models of LIRI in vivo and in vitro. Adult Sprague–Dawley rats were subjected to ischemia–reperfusion with or without pretreatment with a PD-1 inhibitor, SHP1/2 inhibitor, or Akt activator. Lung tissue damage and infiltration by M1-type AMs were assessed. As an in vitro complement to the animal studies, rat alveolar macrophages in culture were subjected to oxygen/glucose deprivation and reoxygenation. Levels of SHP1/2 and Akt proteins were evaluated using Western blots, while levels of pro-inflammatory cytokines were measured using enzyme-linked immunosorbent assays. Injury upregulated PD-1 both in vivo and in vitro. Inhibiting PD-1 reduced the number of M1-type AMs, expression of SHP1 and SHP2, and levels of inflammatory cytokines. At the same time, it partially restored Akt activation. Similar results were observed after inhibition of SHP1/2 or activation of the PI3K/Akt pathway. PD-1 promotes polarization of AMs to the M1 phenotype and inflammatory responses through the SHP1/2-PI3K/Akt axis. Inhibiting PD-1 may be an effective therapeutic strategy to limit LIRI.

Background: Lung ischemia-reperfusion injury (LIRI) is a common and complex pathophysiological process that can lead to poor patient outcomes. Inflammasome-dependent macrophage pyroptosis contributes to organ damage caused by ischemia-reperfusion (I/R). Oxidative stress reaction and antioxidant enzymes also play an important role in LIRI. This experiment was conducted to investigate whether preconditioning with rHMGB1 could ameliorate LIRI and explore the mechanisms of its protective effect in a lung I/R mice model. Methods: Adult male mice were anesthetized and the left hilus pulmonis was clamped for 60 min, followed by 120 min of reperfusion. rHMGB1 was performed by intraperitoneal injection at 2 h before anesthesia. Brusatol (Nrf2 antagonist) was given intraperitoneally every other day for a total of five times before surgery. Measurements of pathohistological lung tissue damage, pulmonary wet/dry (W/D) ratios and inflammatory mediators were performed to assess the extent of lung injury after I/R. Alveolar macrophages (AMs) pyroptosis were evaluated by LDH release, caspase-1 expression in flow cytometry, GSDMD expression in immunofluorescent staining. The products of oxidative Stress (ROS, MDA, 15-F2t-Isoprostane) and the antioxidant enzymes (SOD, GSH-PX, CAT) were detected.Results: Preconditioning with rHMGB1 significantly ameliorated I/R-induced lung injury through measuring the morphology, wet/dry weight ratio, the expressions of IL-1β, IL-6, NF-κB and HMGB1 in lung tissue. rHMGB1 preconditioning remarkably alleviated AMs pyroptosis induced by lung I/R. rHMGB1 preconditioning significantly reduced oxidative stress and restored the activity of antioxidative enzymes. In addition, rHMGB1 preconditioning mediated the activity of Keap1/Nrf2/HO-1 pathway in LIRI. Furthermore, inhibiting Keap1/Nrf2/HO-1 pathway through brusatol administration could aggravate lung tissue damage and inflammatory response after lung I/R. And these effects by brusatol administration could be alleviated by rHMGB1 preconditioning in LIRI .Conclusions : rHMGB1 preconditioning protects against LIRI through suppressing AMs pyroptosis. The molecular mechanism could be partially explained by inhibiting oxidative stress and improving the activity of antioxidative enzymes via Keap1/Nrf2/HO-1 pathway upon rHMGB1 preconditioning.