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 Sprague-Dawley (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.