尿源干细胞通过FGF2-PI3K/mTOR通路抑制内皮细胞过度自噬治疗糖尿病性勃起功能障碍的机制研究

The incidence of diabetic erectile dysfunction (DED) is getting higher, but the existing treatment is not effective. The core pathogenesis of DED is cavernous endothelial dysfunction. Our previous study verified that the excessive autophagy of cavernous endothelial cells (ECs) is an important mechanism of endothelial dysfunction of DED. Our previous study also demonstrated that transplantation of urine derived stem cells (USCs) could repair the endothelial function and erectile function in DED rats, the underlying mechanism is remained unknown. Our further study found that the fibroblast growth factor 2 (FGF2) secreted by USCs could inhibit the autophagy of ECs, which was associated with the activation of PI3K/Akt/mTOR pathway. Therefore, we hypothesis that USCs can activate the PI3K/Akt/mTOR pathway trough the paracrine of FGF2 to inhibit the excessive autophagy of ECs and thus repair the endothelial function and erectile function in DED rats. We intend to analyze the mechanism of FGF2 regulation of endothelial autophagy by cell co-culture, gene microarray, RPPA analysis and western blot et al. This program will obtain the direct evidence that FGF2 activates the PI3K/Akt/mTOR pathway to inhibit endothelial cell autophagy and repair the cavernous endothelial function and erection function, which will provide sufficient theoretical basis for the promotion of USCs as an effective treatment for diabetic erectile dysfunction.

糖尿病性勃起功能障碍（DED）发病率高且疗效不佳，其核心发病机制是阴茎海绵窦内皮功能障碍。我们前期研究证实细胞过度自噬是海绵窦内皮功能障碍的重要机制，并首次证明尿源干细胞（USCs）可改善DED大鼠的内皮功能和勃起功能，但具体机制未明。进一步预实验发现USCs旁分泌成纤维细胞生长因子2（FGF2）可抑制海绵窦内皮细胞自噬，可能与PI3K/Akt/mTOR通路有关。因此我们提出USCs通过旁分泌FGF2激活PI3K/Akt/mTOR通路抑制内皮细胞过度自噬，从而修复DED大鼠内皮功能和勃起功能的科学假设。本项目拟通过体外共培养、基因芯片和反向蛋白芯片等技术获得FGF2激活PI3K/Akt/mTOR通路抑制内皮细胞过度自噬的直接证据，明确其关键调控节点，从FGF2-PI3K/Akt/mTOR通路抑制内皮细胞过度自噬的角度揭示USCs治疗DED的分子机制，为USCs治疗DED提供更充分的科学依据

糖尿病性勃起功能障碍（DED）发病率高且疗效不佳，其核心发病机制是阴茎海绵窦内皮功能障碍。我们前期研究证实细胞自噬活性改变是海绵窦内皮功能障碍的重要机制，并首次证明尿源干细胞（USC）可改善DED大鼠的内皮功能和勃起功能，但具体机制未明。本项目通过构建DED大鼠模型并检测发现，其阴茎海绵窦内皮细胞自噬受损（自噬体数量减少，自噬标记物LC3II、Beclin1表达降低，自噬底物P62表达增多），可能是导致内皮功能障碍和勃起功能障碍的重要机制；通过USC移植治疗，DED大鼠阴茎海绵窦内皮细胞自噬功能得到修复，海绵体内皮功能改善（CD31、eNOS、p-eNOS、VEGF、VEGFR表达增多）、勃起功能得到提高（ICP和ICP/MAP提高），提示USC可能通过提高阴茎海绵窦内皮细胞自噬水平、修复细胞损伤，进而改善内皮功能和阴茎勃起功能，达到治疗作用。为验证上述作用机制，我们在体外培养大鼠阴茎海绵窦内皮细胞（CCVEC），使用晚期糖基化终产物（AGEs）处理CCVEC模拟糖尿病状态下内皮功能障碍；体外实验结果表明，AGEs处理后CCVEC自噬活性降低（LC3II/LC3I比例下降、Beclin1表达下降、p62含量增多，胞内自噬小体及自噬溶酶体数量下降）、增殖水平降低（PCNA表达减少）、凋亡水平增高（凋亡通路蛋白cleaved-caspase 3表达增多）；与USC共培养后，CCVEC自噬功能部分恢复、增殖水平较前提高、凋亡水平较前降低，提示USC可通过提高细胞自噬水平保护CCVEC；用3-MA抑制自噬活性后，USC对CCVEC的保护作用消失，进一步验证USC是通过调控自噬活性发挥保护作用。综合上述体外、体内实验结果，本项目证实USC可通过提高DED大鼠阴茎海绵窦内皮细胞自噬活性改善内皮功能和勃起功能，最终治疗DED。该研究结果为USC治疗DED提供了更加充分的科学依据。