Background Ensuring the survival of the distal end of a random flap during hypoperfusion (ischaemia) is difficult in clinical practice. Effective prevention of programmed cell death is a potential strategy for inhibiting ischaemic flap necrosis. The activation of stimulator of interferon genes (STING) pathway promotes inflammation and leads to cell death. The epidermal growth factor family member neuregulin-1 (NRG1) reduces cell death by activating the protein kinase B (AKT) signalling pathway. Moreover, AKT signalling negatively regulates STING activity. We aimed to verify the efficacy of NRG1 injection in protecting against flap necrosis. Additionally, we investigated whether NRG1 effectively enhances ischemic flap survival by inhibiting pyroptosis and necroptosis through STING suppression. Methods A random-pattern skin flap model was generated on the backs of C57BL/6 mice. The skin flap survival area was determined. The blood supply and vascular network of the flap was assessed by laser Doppler blood flow analysis. Cluster of differentiation 34 immunohistochemistry (IHC) and haematoxylin and eosin (H&E) staining of the flap sections revealed microvessels. Transcriptome sequencing analysis revealed the mechanism by which NRG1 promotes the survival of ischaemic flaps. The levels of angiogenesis, oxidative stress, necroptosis, pyroptosis and indicators associated with signalling pathways in flaps were examined by IHC, immunofluorescence and Western blotting. Packaging adeno-associated virus (AAV) was used to activate STING in flaps. Results NRG1 promoted the survival of ischaemic flaps. An increased subcutaneous vascular network and neovascularization were found in ischaemic flaps after the application of NRG1. Transcriptomic gene ontology enrichment analysis and protein level detection indicated that necroptosis, pyroptosis and STING activity were reduced in the NRG1 group. The phosphorylation of AKT and forkhead box O3a (FOXO3a) were increased after NRG1 treatment. The increased expression of STING in flaps induced by AAV reversed the therapeutic effect of NRG1. The ability of NRG1 to phosphorylate AKT-FOXO3a, inhibit STING and promote flap survival was abolished after the application of the AKT inhibitor MK2206. Conclusions NRG1 inhibits pyroptosis and necroptosis by activating the AKT-FOXO3a signalling pathway to suppress STING activation and promote ischaemic flap survival.

中文翻译：

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Neuregulin-1 是表皮生长因子家族的成员，可减轻 STING 介导的缺血皮瓣焦亡和坏死性凋亡


背景 在临床实践中，确保随机皮瓣远端在低灌注（缺血）期间存活是很困难的。有效预防程序性细胞死亡是抑制缺血性皮瓣坏死的潜在策略。干扰素基因刺激物 (STING) 通路的激活会促进炎症并导致细胞死亡。表皮生长因子家族成员神经调节蛋白-1 (NRG1) 通过激活蛋白激酶 B (AKT) 信号通路来减少细胞死亡。此外，AKT 信号传导负向调节 STING 活性。我们的目的是验证 NRG1 注射在防止皮瓣坏死方面的功效。此外，我们还研究了 NRG1 是否通过 STING 抑制来抑制焦亡和坏死性凋亡，从而有效增强缺血皮瓣的存活率。方法在C57BL/6小鼠背部制作随机图案皮瓣模型。确定皮瓣存活面积。通过激光多普勒血流分析评估皮瓣的血液供应和血管网络。皮瓣切片的分化簇 34 免疫组织化学 (IHC) 和苏木精和伊红 (H&E) 染色显示微血管。转录组测序分析揭示了NRG1促进缺血皮瓣存活的机制。通过免疫组化、免疫荧光和蛋白质印迹法检测皮瓣中血管生成、氧化应激、坏死性凋亡、细胞焦亡以及信号通路相关指标的水平。包装腺相关病毒 (AAV) 用于激活皮瓣中的 STING。结果NRG1促进缺血皮瓣的成活。应用NRG1后，缺血皮瓣中皮下血管网络增加，新生血管增多。 转录组基因本体富集分析和蛋白水平检测表明NRG1组的坏死性凋亡、焦亡和STING活性降低。 NRG1 处理后 AKT 和叉头盒 O3a (FOXO3a) 的磷酸化增加。 AAV 诱导的皮瓣中 STING 表达增加逆转了 NRG1 的治疗效果。应用 AKT 抑制剂 MK2206 后，NRG1 磷酸化 AKT-FOXO3a、抑制 STING 和促进皮瓣存活的能力被消除。结论 NRG1通过激活AKT-FOXO3a信号通路抑制焦亡和坏死性凋亡，抑制STING激活，促进缺血皮瓣存活。