通过海绵体内注射间充质干细胞（MSC）是治疗糖尿病引起的勃起功能障碍（DMED）的潜在治疗方法。然而，肺栓塞和致瘤性是致命的不良事件，限制了MSC的临床应用。在这项研究中，我们检查了 MSC 衍生的细胞外囊泡 (MSC-EV) 的治疗功效和潜在机制。在这项研究中，使用了 40 只 8 周龄的雄性 SpragueDawley (SD) 大鼠。对照组10只大鼠腹腔注射PBS。其余大鼠腹腔注射STZ（60mg/kg）建立糖尿病（DM）模型。随后将糖尿病大鼠随机分为三组：DM组（海绵体内注射PBS）、EVs组（海绵体内注射MSC-EVs）、EVs-200a组（海绵体内注射miR-200a-3p） -丰富的细胞外囊泡）。通过实时测量海绵体内压力并利用海绵体神经的电刺激来确定勃起功能。通过使用免疫荧光染色、马森三色染色和安乐死后蛋白质印迹对阴茎组织进行研究来评估平滑肌含量。通过 ELISA 测量海绵体中的超氧化物歧化酶 (SOD)、丙二醛 (MDA) 和谷胱甘肽 (GSH) 水平。在体外，过氧化氢（H2O2）用于诱导氧化应激。使用 CCK8 测定法测量有或没有 H2O2 孵育的海绵体平滑肌细胞 (ccSMC) 的活力。使用流式细胞术评估 ccSMC 中的活性氧 (ROS) 水平和细胞凋亡。此外，进行双荧光素酶报告基因测定以验证 miR-200a-3p 和 Keap1 之间的关系。在 EVs 组中观察到勃起功能的逆转，尤其是在 EVs-200a 组中。 DM 增加了 MDA 水平，降低了 SOD 和 GSH 水平。 DM组中α平滑肌肌动蛋白（α-SMA）和平滑肌22α（SM22α）的表达减少，骨桥蛋白（OPN）的表达增加。蛋白质印迹显示海绵体组织中 Nrf2、HO-1 和 Bcl2 表达减少，Keap1、Bax 和 cleaved caspase3 表达增加。富含miR-200a-3p的细胞外囊泡（EVs-200a）逆转了这些变化并抑制平滑肌含量的损失和海绵体纤维化。在体外，H2O2 诱导 ccSMC 中高 ROS 水平并增加细胞凋亡，而这些效应可被 EVs-200a 逆转。 H2O2 降低了 Nrf2、HO-1 和 Bcl2 的表达，并增加了 Keap1、Bax 和 cleaved caspase-3 的表达，这些效应被 MSC-EVs（尤其是 EVs-200a）逆转。双荧光素酶报告基因检测结果表明，miR-200a-3p以阴性方式直接靶向Keap1。MSC-EVs，尤其是EVs-200a，通过调节表型转换、细胞凋亡和纤维化来减轻糖尿病大鼠的勃起功能障碍。从机制上讲，miR-200a-3p 靶向 Keap1/Nrf2 通路以减轻糖尿病大鼠的氧化应激。版权所有 © 2024 作者。由 Elsevier Masson SAS 出版。保留所有权利。

The administration of mesenchymal stem cells (MSCs) through intracavernous injection is a potential therapeutic approach for managing diabetes mellitus-induced erectile dysfunction (DMED). However, pulmonary embolism and tumorigenicity are fatal adverse events that limit the clinical application of MSCs. In this study, we examined the therapeutic efficacy and potential mechanism of MSC-derived extracellular vesicles (MSC-EVs).In this study, forty 8-week-old male SpragueDawley (SD) rats were utilised. In the control group, ten rats were administered an intraperitoneal injection of PBS. STZ (60 mg/kg) was intraperitoneally injected into the remaining rats to establish a diabetes mellitus (DM) model. Afterwards, the diabetic rats were divided into three groups at random: the DM group (intracavernosal injection of PBS), the EVs group (intracavernosal injection of MSC-EVs), and the EVs-200a group (intracavernosal injection of miR-200a-3p-enriched extracellular vesicles). Erectile function was determined by measuring intracavernous pressure in real time and utilising electrical stimulation of the cavernous nerves. The smooth muscle content was evaluated through the investigation of penile tissue using immunofluorescence staining, Masson's trichrome staining, and western blotting after euthanasia. Superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) levels in the corpus cavernosum were measured via ELISA. In vitro, hydrogen peroxide (H2O2) was used to induce oxidative stress. The viability of corpus cavernosum smooth muscle cells (ccSMCs) incubated with or without H2O2 was measured using a CCK8 assay. Flow cytometry was used to assess the levels of reactive oxygen species (ROS) and apoptosis in ccSMCs. Furthermore, a dual-luciferase reporter assay was performed to validate the relationship between miR-200a-3p and Keap1.Reversal of erectile function was observed in the EVs groups, especially in the EVs-200a group. DM increased the MDA level and decreased the SOD and GSH levels. In the DM group, the expression of alpha-smooth muscle actin (α-SMA) and smooth muscle 22 alpha (SM22α) was decreased, and the expression of osteopontin (OPN) was increased. Western blotting revealed decreased Nrf2, HO-1, and Bcl2 expression and increased Keap1, Bax and cleaved caspase3 expression in the cavernous tissue. miR-200a-3p-enriched extracellular vesicles (EVs-200a) reversed these changes and inhibited the loss of smooth muscle content and cavernous fibrosis. In vitro, H2O2 induced high ROS levels in ccSMCs and increased apoptosis, and these effects reversed by EVs-200a. H2O2 reduced Nrf2, HO-1, and Bcl2 expression and increased Keap1, Bax and cleaved caspase-3 expression, and these effects were reversed by MSC-EVs, especially EVs-200a. The of dual-luciferase reporter assay results indicated that miR-200a-3p directly targeted Keap1 in a negative manner.MSC-EVs, especially EVs-200a, alleviated erectile dysfunction in diabetic rats through the regulation of phenotypic switching, apoptosis and fibrosis. Mechanistically, miR-200a-3p targeted the Keap1/Nrf2 pathway to attenuate oxidative stress in diabetic rats.Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.