背景:通过海绵体内注射给予间充质干细胞(MSC)是管理糖尿病诱导的勃起功能障碍(DMED)的潜在治疗方法。然而,肺栓塞和致瘤性是限制MSCs临床应用的致命性不良事件。在这项研究中,我们检查了MSC衍生的细胞外囊泡(MSC-EV)的疗效和潜在机制。
方法:在本研究中,使用40只8周龄雄性SpragueDawley(SD)大鼠。在对照组中,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。之后,将糖尿病大鼠随机分为3组:DM组(海绵体注射PBS),EV组(海绵体内注射MSC-EV),和EVs-200a组(海绵体内注射富含miR-200a-3p的细胞外囊泡)。通过实时测量海绵体内压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对阴茎组织进行调查来评估平滑肌含量,马森三色染色,安乐死后的西方印迹。超氧化物歧化酶(SOD),丙二醛(MDA),用ELISA法测定海绵体中谷胱甘肽(GSH)水平。体外,过氧化氢(H2O2)用于诱导氧化应激。使用CCK8测定法测量在有或没有H2O2的情况下孵育的海绵体平滑肌细胞(ccSMC)的活力。流式细胞术用于评估ccSMC中活性氧(ROS)和细胞凋亡的水平。此外,我们进行了双荧光素酶报告基因试验,以验证miR-200a-3p和Keap1之间的关系.
结果:在电动汽车组中观察到勃起功能逆转,特别是在EVs-200a组中。DM增加了MDA水平,降低了SOD和GSH水平。在DM组中,α-平滑肌肌动蛋白(α-SMA)和平滑肌22α(SM22α)的表达降低,骨桥蛋白(OPN)表达增加。Western印迹显示海绵状组织中Nrf2,HO-1和Bcl2的表达降低,Keap1,Bax和caspase3的表达增加。富含miR-200a-3p的细胞外囊泡(EVs-200a)逆转了这些变化,并抑制了平滑肌含量和海绵状纤维化的损失。体外,H2O2在ccSMCs中诱导高ROS水平并增加细胞凋亡,这些影响被EV-200a逆转。H2O2降低Nrf2,HO-1和Bcl2的表达,增加Keap1,Bax和裂解的caspase-3的表达,这些效应被MSC-EV逆转,尤其是EVS-200a。双荧光素酶报告基因测定结果表明miR-200a-3p以阴性方式直接靶向Keapl。
结论:MSC-EV,尤其是电动汽车-200a,通过调节表型转换减轻糖尿病大鼠的勃起功能障碍,细胞凋亡和纤维化。机械上,miR-200a-3p靶向Keap1/Nrf2途径以减轻糖尿病大鼠的氧化应激。
BACKGROUND: 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).
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.