Department of Otorhinolaryngology, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Respir Res. 2021 Oct 19;22(1):267. doi: 10.1186/s12931-021-01865-y.
The non-cancerous functions of Akt in the airway are understudied. In some tissues, Akt phosphorylates and activates endothelial nitric oxide synthase (eNOS) to produce nitric oxide (NO) that has anti-inflammatory effects. NO production has antibacterial and antiviral effects in the airway, and increasing NO may be a useful anti-pathogen strategy. Akt also stimulates the nuclear factor erythroid 2-related factor 2 (Nrf-2) transcription factor, which transcribes antioxidant genes. Therefore, we hypothesized that activation of the Akt/eNOS pathway, which also activates Nrf-2, may have protective effects in human airway cells against injury.
To directly test the effects of Akt signaling in the airway, we treated A549 and 16HBE cells as well as primary bronchial, nasal, and type II alveolar epithelial cells with small molecule Akt activator SC79. We examined the effects of SC79 on eNOS activation, NO production, Nrf-2 target levels, and interleukin-8 (IL-8) transcription during exposure to TNF-α or Pseudomonas flagellin (TLR5 agonist). Additionally, air-liquid interface bronchial cultures were treated with cadmium, an oxidative stressor that causes airway barrier breakdown.
SC79 induced a ~ twofold induction of p-eNOS and Nrf-2 protein levels blocked by PI3K inhibitor LY294002. Live cell imaging revealed SC79 increased acute NO production. Quantitative RT-PCR showed a ~ twofold increase in Nrf-2 target gene transcription. TNF-α or flagellin-induced IL-8 levels were also significantly reduced with SC79 treatment. Moreover, the transepithelial electrical resistance decrease observed with cadmium was ameliorated by SC79, likely by an acute increase in tight junction protein ZO-1 levels.
Together, the data presented here demonstrate SC79 activation of Akt induces potentially anti-pathogenic NO production, antioxidant gene transcription, reduces IL-8 transcription, and may protect against oxidative barrier dysfunction in a wide range of airway epithelial cells.
Akt 在气道中的非癌性功能尚未得到充分研究。在某些组织中，Akt 磷酸化并激活内皮型一氧化氮合酶 (eNOS) 以产生具有抗炎作用的一氧化氮 (NO)。NO 在气道中具有抗菌和抗病毒作用，增加 NO 可能是一种有用的抗病原体策略。Akt 还刺激核因子红细胞 2 相关因子 2 (Nrf-2) 转录因子，该转录因子转录抗氧化基因。因此，我们假设激活 Akt/eNOS 途径（也激活 Nrf-2）可能对人呼吸道细胞免受损伤具有保护作用。
为了直接测试气道中 Akt 信号的作用，我们用小分子 Akt 激活剂 SC79 处理 A549 和 16HBE 细胞以及原代支气管、鼻和 II 型肺泡上皮细胞。我们研究了 SC79 在 TNF-α 或假单胞菌鞭毛蛋白（TLR5 激动剂）暴露期间对 eNOS 激活、NO 产生、Nrf-2 靶水平和白细胞介素 8 (IL-8) 转录的影响。此外，用镉处理气液界面支气管培养物，镉是一种引起气道屏障破坏的氧化应激源。
SC79 诱导 p-eNOS 和 Nrf-2 蛋白水平升高约 2 倍，被 PI3K 抑制剂 LY294002 阻断。活细胞成像显示 SC79 增加了急性 NO 产生。定量 RT-PCR 显示 Nrf-2 靶基因转录增加约 2 倍。用 SC79 处理还显著降低了 TNF-α 或鞭毛蛋白诱导的 IL-8 水平。此外，SC79 改善了观察到的镉引起的跨上皮电阻降低，可能是通过紧密连接蛋白 ZO-1 水平的急性增加。
综上所述，这里呈现的数据表明，SC79 激活 Akt 诱导潜在的抗病原体 NO 产生、抗氧化基因转录、减少 IL-8 转录，并可能防止广泛的气道上皮细胞发生氧化屏障功能障碍。
