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 (). 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 (). 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 (). 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.

中文翻译：

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Necrostatin-1 协同泛半胱天冬酶抑制剂减轻大鼠缺血再灌注引起的肺损伤


背景。细胞凋亡和坏死性凋亡均被认为与缺血再灌注引起的肺损伤有关。我们的目的是比较针对坏死性凋亡和细胞凋亡的疗法的疗效，并确定两种疗法在减少肺缺血再灌注损伤方面是否存在协同作用。方法。将40只Sprague-Dawley大鼠随机分为5组：假手术组（SM）、缺血再灌注（IR）组、necrostatin-1+缺血再灌注（NI）组、苯甲酰基-Val-Ala-Asp-氟甲基酮+缺血再灌注（ZI）组组、necrostatin-1+苯甲酰基-Val-Ala-Asp-氟甲基酮+缺血再灌注（NZ）组。 SM组大鼠不夹闭左肺门，暴露左肺门；IR组大鼠夹闭左肺门1小时，再灌注3小时，进行肺缺血再灌注。在缺血前腹膜内给予 1 mg/kg necrostatin-1（Nec-1：一种特异性坏死性凋亡抑制剂）和 3 mg/kg 苯甲氧基-Val-Ala-Asp-氟甲基酮（z-VAD-fmk：一种泛半胱天冬酶抑制剂） NI组和ZI组分别给予Nec-1和z-VAD-fmk，NZ组大鼠联合给予Nec-1和z-VAD-fmk。再灌注后，测量受体相互作用蛋白1（RIP1）、受体相互作用蛋白3（RIP3）和caspase-8的表达，并使用流式细胞术分析评估肺组织中的细胞死亡模式。此外，还评估了支气管肺泡灌洗液和肺水肿中的炎症标志物水平。结果。 Nec-1 和 z-VAD-fmk 单独或联合使用均可显着减少再灌注后肺组织的形态损伤、炎症标志物和水肿，其中 z-VAD-fmk 与 Nec-1 联合治疗产生最佳效果。与单独接受 z-VAD-fmk 治疗的大鼠相比，接受 Nec-1 治疗的大鼠支气管肺泡灌洗液中炎症标志物的水平较低（ ）。有趣的是，与 IR 组相比，z-VAD-fmk 给药上调了 ZI 组肺组织中 RIP1 和 RIP3 的表达（ ）。再灌注显着增加了肺组织单细胞悬液中坏死和凋亡细胞的百分比，Nec-1和z-VAD-fmk可分别降低坏死和凋亡细胞的百分比（）。结论。 Nec-1 协同泛 caspase 抑制剂减轻大鼠肺缺血再灌注损伤。我们的数据支持 Nec-1 在肺移植相关疾病中的潜在用途。