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Lung ischemia-reperfusion injury (LIRI) is associated with many diseases, including primary graft dysfunction after lung transplantation, and has no specific and effective therapies. Necroptosis contributes to the pathogenesis of ischemia-reperfusion injury. Necrostatin-1 (Nec-1), the necroptosis inhibitor targeting RIPK1, has been reported to alleviate ischemia-reperfusion injury in various organs. However, the underlying mechanism of Nec-1 in LIRI remains unclear. In this paper, an in vivo LIRI model was built up by left lung hilar clamping in mice, and an in vitro cold ischemia-reperfusion (CI/R) model using BEAS-2B cells was applied to mimic the lung transplantation setting. We found Nec-1 significantly alleviated ischemia-reperfusion-induced lung injury, cytokine releasing, and necroptosis of epithelial cells in mouse lungs. In vitro, Nec-1 also mitigated CI/R-induced cell death and inflammatory responses in BEAS-2B cells, and these protective effects were achieved by simultaneously inhibiting the formation of necrosome and RIPK1-dependent apoptosis. However, Nec-1 decreased the necrosome number but increased the apoptosis level in lung tissues after ischemia reperfusion. We further clarified that Nec-1 could also attenuate lung injury by promoting neutrophil apoptosis from flow cytometry. In conclusion, Nec-1 alleviated lung ischemia-reperfusion injury by inhibiting necroptosis and apoptosis of epithelial cells and promoting the apoptosis of neutrophils. Thus, Nec-1 could be a promising medication against primary graft dysfunction after lung transplantation.

The CysLT2 receptor is involved in myocardial ischemia/reperfusion injury, differentiation of colorectal cancers, bleomycin-induced pulmonary inflammation and fibrosis. However, the signal transduction of cysteinyl leukotriene receptor 2 (CysLT2) in inflammatory responses remains to be clarified. In HEK293 cells stably expressing hCysLT1, hCysLT2 and rGPR17, we determined the signaling pathways for interleukin-8 (IL-8) production after CysLT2 receptor activation. HEK293 cells were stably transfected with the recombinant plasmids of pcDNA3.1(+)-hCysLT1, pcDNA3.1(+)-hCysLT2 and pcDNA3.1-rGPR17. Leukotriene C4 (LTC4) and LTD4 were used as the agonists to induce IL-8 production and the related changes in signal molecules. We found that LTC4 and LTD4 significantly induced IL-8 promoter activation in the HEK293 cells stably expressing hCysLT2, but not in those expressing hCysLT1 and rGPR17. In hCysLT2-HEK293 cells, LTC4 induced elevation of intracellular calcium, ERK1/2 phosphorylation and Egr-1 expression, and stimulated IL-8 expression and release. These responses were blocked by the selective CysLT2 receptor antagonist HAMI3379. The ERK1/2 inhibitor U0126 inhibited Egr-1 and IL-8 expression as well as IL-8 release, but the JNK and p38 inhibitors did not have the inhibitory effects. Down-regulation of Egr-1 by RNA interference with its siRNA inhibited the LTC4-induced IL-8 expression and release. In conclusion, these findings indicate the ERK-Egr-1 pathway of CysLT2 receptors mediates IL-8 production induced by the pro-inflammatory mediators LTC4 and LTD4. [ABSTRACT FROM AUTHOR]