Zhang Chi, Luo Daosheng, Li Tingting, Yang Qiyun, Xie Yun, Chen Haicheng, Lv Linyan, Yao Jiahui, Deng Cuncan, Liang Xiaoyan, Wu Rongpei, Sun Xiangzhou, Zhang Yuanyuan, Deng Chunhua, Liu Guihua
Department of Andrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
Reproductive Medicine Research Center, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510000, China.
Stem Cells Int. 2019 Sep 9;2019:2168709. doi: 10.1155/2019/2168709. eCollection 2019.
Cavernosal endothelial dysfunction is one of the factors in developing diabetic erectile dysfunction (DED), but the mechanism of cavernosal endothelial dysfunction is unclear. The present study is aimed at determining the contribution of autophagy in cavernosal endothelial dysfunction of DED rats and explaining the therapeutic effect of urine-derived stem cells (USCs).
After rat corpus cavernosal vascular endothelial cells (CCECs) were isolated and cultured , CCECs were treated with advanced glycation end products (AGEs) to mimic the diabetic situation. Autophagy flux, proliferation, and apoptosis of CCECs were determined by mRFP-GFP-LC3 adenovirus infection combined with fluorescence observation and western blot analysis. USCs were isolated from the urine of six healthy male donors, and coculture systems of USCs and CCECs were developed to assess the protective effect of USCs for CCECs . The contribution of autophagy to the cellular damage in CCECs was evaluated by the autophagic inhibitor, 3-methyladenine (3-MA). Then, DED rats were induced by streptozotocin (50 mg/kg) and screened by apomorphine test (100 g/kg). In DED rats, USCs or PBS as vehicle was administrated by intracavernous injection ( = 15 per group), and another 15 normal rats served as normal controls. Four weeks after injection, erectile function was evaluated by measuring the intracavernosal pressure (ICP) and mean arterial pressure (MAP). Cavernosal endothelial function and autophagic activity were examined by western blot, immunofluorescence, and transmission electron microscopy.
, AGE-treated CCECs displayed fewer LC3 puncta formation and expressed less LC3-II, Beclin1, and PCNA but expressed more p62 and cleaved-caspase3 than controls ( < 0.05). Coculture of USCs with CCECs demonstrated that USCs were able to protect CCECs from AGE-induced autophagic dysfunction and cellular damage, which could be abolished by 3-MA ( < 0.05). DED rats showed lower ratio of ICP/MAP, reduced expression of endothelial markers, and fewer autophagic vacuoles in the cavernosal endothelium when compared with normal rats ( < 0.05). Intracavernous injection of USCs improved erectile function and cavernosal endothelial function of DED rats ( < 0.05). Most importantly, our data showed that the repaired erectile function and cavernosal endothelial function were the result of restored autophagic activity of the cavernosal endothelium in DED rats ( < 0.05).
Impaired autophagy is involved in the cavernosal endothelial dysfunction and erectile dysfunction of DED rats. Intracavernous injection of USCs upregulates autophagic activity in the cavernosal endothelium, contributing to ameliorating cavernosal endothelial dysfunction and finally improving the erectile dysfunction induced by diabetes.
海绵体内皮功能障碍是糖尿病性勃起功能障碍（DED）发生发展的因素之一，但海绵体内皮功能障碍的机制尚不清楚。本研究旨在确定自噬在DED大鼠海绵体内皮功能障碍中的作用，并解释尿液来源干细胞（USCs）的治疗效果。
分离培养大鼠海绵体血管内皮细胞（CCECs）后，用晚期糖基化终末产物（AGEs）处理CCECs以模拟糖尿病状态。通过mRFP-GFP-LC3腺病毒感染结合荧光观察和蛋白质印迹分析来测定CCECs的自噬通量、增殖和凋亡。从6名健康男性供体的尿液中分离出USCs，并建立USCs与CCECs的共培养体系，以评估USCs对CCECs的保护作用。通过自噬抑制剂3-甲基腺嘌呤（3-MA）评估自噬对CCECs细胞损伤的作用。然后，用链脲佐菌素（50mg/kg）诱导DED大鼠，并通过阿扑吗啡试验（100μg/kg）进行筛选。在DED大鼠中，通过海绵体内注射给予USCs或作为对照的PBS（每组n = 15），另外15只正常大鼠作为正常对照。注射4周后，通过测量海绵体内压（ICP）和平均动脉压（MAP）评估勃起功能。通过蛋白质印迹、免疫荧光和透射电子显微镜检查海绵体内皮功能和自噬活性。
与对照组相比，AGE处理的CCECs显示出较少的LC3斑点形成，LC3-II、Beclin1和PCNA表达较少，但p62和裂解的caspase3表达较多（P < 0.05）。USCs与CCECs共培养表明，USCs能够保护CCECs免受AGE诱导的自噬功能障碍和细胞损伤，而3-MA可消除这种保护作用（P < 0.05）。与正常大鼠相比，DED大鼠的ICP/MAP比值较低，内皮标志物表达减少，海绵体内皮中的自噬空泡较少（P < 0.05）。海绵体内注射USCs改善了DED大鼠的勃起功能和海绵体内皮功能（P < 0.05）。最重要的是，我们的数据表明，修复后的勃起功能和海绵体内皮功能是DED大鼠海绵体内皮自噬活性恢复的结果（P < 0.05）。
自噬受损参与了DED大鼠的海绵体内皮功能障碍和勃起功能障碍。海绵体内注射USCs上调了海绵体内皮中的自噬活性，有助于改善海绵体内皮功能障碍，最终改善糖尿病引起的勃起功能障碍。
