Probucol enhances the therapeutic efficiency of mesenchymal stem cells in the treatment of erectile dysfunction in diabetic rats by prolonging their survival time via Nrf2 pathway

Abstract

Background Intracavernous injection of mesenchymal stem cells (MSCs) is a promising method for Diabetic mellitus-induced erectile dysfunction (DMED), but short survival time of MSCs in cavernous is fatal defect for therapy. This study investigated therapeutic efficiency and potential mechanism of Probucol combined with MSCs. Methods In vivo study, total forty-eight 10-week-old male Sprague-Dawley (SD) rats were used in this study. Twelve rats received intraperitoneal injection of PBS as Sham Group; the rest received intraperitoneal injection of 60mg/kg streptozotocin to establish DM models. DM rats were randomly divided into three groups: received intracavernosal (IC) injection of either PBS (DM Group), MSCs (M Group), and administrated Probucol after intracavernosal injection of MSCs (P+M Group). Erectile function was assessed by electrical stimulation of the cavernous nerves with real-time intracavernous pressure measurement. After euthanasia, penile tissue was investigated for histologic examination and Western blotting. In vitro experiment, H 2 O 2 was used to create oxidative stress environment to detect changes in cell viability. CCK8 was used to measure cell viability of MSCs treated with or without Probucol. Intracellular ROS changes were detected by flow cytometry. Autophagy and apoptosis were detected by Western blotting and confocal microscopy. Results Recovery of erectile function was observed in the P+M Group. The combination therapy decreased fibrosis and increased endothelial function compared with MSCs therapy alone. Western blotting results confirmed the increased expression of Nrf2 and HO-1 in cavernous body. Vitro experiment indicated H 2 O 2 -induced high oxidative stress reduced cell viability, which was gradually reversed with increased concentration of Probucol. H 2 O 2 reduced Nrf2 expression, which was reversed by Probucol's intervention. Furthmore, the expression of Bax, Caspase3 and Cleaved-Caspase3 decreased, and the expression of Bcl-2 increased in a dose-dependent manner because of Probucol's intervention. In addition, Beclin1 and LC3II both increased in a dose-dependent manner. Meanwhile, the expression of P62 decreased. In study of autophagy flux, we found Probucol didn’t block it. Conclusion Probucol enhanced therapeutic efficiency of MSCs in DMED by prolonging their survival time, which mediated through improving the transplanted microenvironment of MSCs, increasing self-antioxidant ability of MSCs, strengthening protective autophagy and inhibiting apoptosis of MSCs via Nrf2 pathway.

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“…Another previous study also showed that intra-cavernous transplantation of VEGF gene-modified bone marrow-derived MSCs resulted in improved erectile function in diabetic rats ( 20 ). The mechanism of this improvement may be via differentiating into endothelial cells and smooth muscle cells, and the secretion by these cells of many kinds of cytokines and other effects ( 21 , 22 ). In addition, another study also demonstrated that MSCs engineered to express stromal cell-derived factor 1 (SDF-1) have been shown to reduce erectile dysfunction in streptozotocin-induced diabetic rats by increasing levels of neuronal nitric oxide synthase (nNOS), VEGF, and growth factors ( 23 ).…”