Department of Urology, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Department of Radiation Oncology, Department of Urology, University Medical Center Utrecht, Utrecht, The Netherlands.
Head and Neck Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Department of Otolaryngology Head and Neck Surgery, Rabin Medical Center, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
J Sex Med. 2019 Nov;16(11):1721-1733. doi: 10.1016/j.jsxm.2019.08.015. Epub 2019 Oct 1.
The etiology of radiation-induced erectile dysfunction (ED) is complex and multifactorial, and it appears to be mainly atherogenic.
To focus on vascular aspects of radiation-induced ED and to elucidate whether the protective effects of sildenafil are mediated by attenuation of oxidative stress and apoptosis in the endothelial cells.
Bovine aortic endothelial cells (BAECs), with or without pretreatment of sildenafil (5 μM at 5 minutes before radiation), were used to test endothelial dysfunction in response to external beam radiation at 10-15 Gy. Generation of reactive oxygen species (ROS) was studied. Extracellular hydrogen peroxide (HO) was measured using the Amplex Red assay and intracellular HO using a fluorescent sensor. In addition, ROS superoxide (O•-) was measured using a O•- chemiluminescence enhancer. Both HO and O2•- are known to reduce the bioavailability of nitric oxide, which is the most significant chemical mediator of penile erection. Generation of cellular peroxynitrite (ONOO) was measured using a chemiluminescence assay with the PNCL probe. Subsequently, we measured the activation of acid sphingomyelinase (ASMase) enzyme by radioenzymatic assay using [C-methylcholine] sphingomyelin as substrate, and the generation of the proapoptotic C-ceramide was assessed using the diacylglycerol kinase assay. Endothelial cells apoptosis was measured as a readout of these cells' dysfunction.
Single high-dose radiation therapy induced NADPH oxidases (NOXs) activation and ROS generation via the proapoptotic ASMase/ceramide pathway. The radio-protective effect of sildenafil on BAECs was due to inhibition of this pathway.
Here, we demonstrate for the first time that radiation activated NOXs and induced generation of ROS in BAECs. In addition, we showed that sildenafil significantly reduced radiation-induced O•- and as a result there was reduction in the generation of peroxynitrite in these cells. Subsequently, sildenafil protected the endothelial cells from radiation therapy-induced apoptosis.
This is the first study demonstrating that single high-dose radiation therapy induced NOXs activation, resulting in the generation of O•- and peroxynitrite in endothelial cells. Sildenafil reduced ROS generation by inhibiting the ASMase/ceramide pathway. These studies should be followed in an animal model of ED.
This study demonstrated that sildenafil protects BAECs from radiation-induced oxidative stress by reducing NOX-induced ROS generation, thus resulting in decreased endothelial dysfunction. Therefore, it provides a potential mechanism to better understand the atherogenic etiology of postradiation ED. Wortel RC, Mizrachi A, Li H, et al. Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress. J Sex Med 2019;16:1721-1733.
放射性勃起功能障碍（ED）的病因复杂且多因素，主要表现为动脉粥样硬化。
关注放射性 ED 的血管方面，并阐明西地那非的保护作用是否通过减轻内皮细胞的氧化应激和细胞凋亡来介导。
使用牛主动脉内皮细胞（BAEC），用或不用西地那非预处理（辐射前 5 分钟用 5 μM），以检测 10-15Gy 外照射后内皮功能障碍。研究活性氧（ROS）的产生。使用 Amplex Red 测定法测量细胞外过氧化氢（HO），使用荧光传感器测量细胞内 HO。此外，使用 O•-化学发光增强剂测量 ROS 超氧化物（O2•-）。HO 和 O2•-都已知会降低阴茎勃起的最重要化学介质一氧化氮的生物利用度。使用含有 PNCL 探针的化学发光测定法测量细胞内过氧亚硝酸盐（ONOO）的产生。随后，我们使用放射性酶测定法通过 [C-甲基胆碱] 鞘磷脂作为底物测量酸性鞘磷脂酶（ASMase）酶的激活，并且通过二酰基甘油激酶测定法评估促凋亡 C-神经酰胺的生成。将内皮细胞凋亡作为这些细胞功能障碍的指标进行测量。
单次高剂量放射治疗通过促凋亡 ASMase/神经酰胺途径诱导 NADPH 氧化酶（NOXs）的激活和 ROS 的产生。西地那非对 BAEC 的放射保护作用归因于该途径的抑制。
在这里，我们首次证明辐射激活了 BAECs 中的 NOXs 并诱导了 ROS 的产生。此外，我们表明西地那非可显著降低辐射诱导的 O•-，因此减少了这些细胞中过氧亚硝酸盐的产生。随后，西地那非可防止内皮细胞受到放射治疗诱导的凋亡。
这是第一项证明单次高剂量放射治疗可诱导 NOXs 激活，从而导致内皮细胞中 O•-和过氧亚硝酸盐产生的研究。西地那非通过抑制 ASMase/神经酰胺途径减少 ROS 的产生。这些研究应在 ED 的动物模型中进行。
本研究表明，西地那非通过减少 NOX 诱导的 ROS 生成来保护 BAEC 免受辐射诱导的氧化应激，从而导致内皮功能障碍降低。因此，它为更好地理解放射后 ED 的动脉粥样硬化病因提供了潜在的机制。Wortel RC，Mizrachi A，Li H，等。西地那非可防止内皮细胞免受辐射诱导的氧化应激。性医学杂志 2019；16：1721-1733。
