Supplementary Material for: Sub-chronic restraint stress suppresses sexual potency and erection efficiency by targeting the hypothalamic-pituitary-testicular axis and the NO/cGMP/PDE5α pathway in adult rats.

Introduction: Male sexual potency and vigor are a complex neuroendocrine process and an important component of well-being. Psychological stress is one of the leading causes of male impotence worldwide. Therefore, to better understand the effects of psychological stress on male sexual potency, vigor, and the physiology of erection, we used the rat restraint stress (RS) model, which can most aptly simulate psychological stress. Methods: Adult male SD rats were exposed to RS for 1.5 or 3 hours/day for 30 days. Neuromodulators and hormones of sexual potency and penile erection were quantified using ELISA kit. The histoarchitecture of the penis was examined using Masson trichrome staining. Immunoblotting and immunofluorescence were used to assess the expression and immunolocalization patterns of penile erection markers. To assess sexual potency and vigor, a non-contact erection and a copulatory test were performed. Results: RS exposure decreased the circulatory levels of gonadotropins and testosterone while increasing the serum corticosterone level. RS exposure altered the histomorphology of the penis by decreasing the smooth muscle/collagen ratio and increasing oxidative stress in penile tissue. Furthermore, RS adversely affected NO availability for penile erection by decreasing the neurotransmitter acetylcholine and other erection facilitatory markers such as p-Akt, nNOS, eNOS, and cGMP, while increasing the inhibitory marker PDE5α in the penis. RS exposure significantly reduced the frequencies of mount, intromission and ejaculation, whereas it prolonged sexual exhaustion by increasing latencies of postejaculatory mount, intromission, and ejaculation. Conclusion: The current findings suggest that psychological stressors, such as restraint stress, cause erectile dysfunction in adult male rats by modulating the hypothalamic-pituitary-testicular axis, oxidative balance, penile fibrosis, and the NO/cGMP/PDE5α pathway of penile erection.

引言：男性性能力与性活力是一项复杂的神经内分泌过程，亦是机体健康福祉的重要组成部分。心理应激是全球范围内男性勃起功能障碍的主要诱因之一。为此，为深入阐明心理应激对男性性能力、性活力及阴茎勃起生理的影响，本研究采用了最能模拟心理应激状态的大鼠束缚应激（restraint stress, RS）模型。

方法：将成年雄性SD大鼠每日施以1.5小时或3小时的束缚应激，持续30天。采用酶联免疫吸附试验（ELISA）试剂盒对与性能力及阴茎勃起相关的神经调节因子与激素进行定量检测。通过马松三色染色法观察阴茎的组织学结构。采用免疫印迹与免疫荧光技术，评估阴茎勃起相关标志物的表达水平与免疫定位模式。为评估性能力与性活力，本研究开展了非接触式勃起实验与交配行为实验。

结果：束缚应激处理可降低循环系统中的促性腺激素与睾酮水平，同时升高血清皮质酮浓度。束缚应激通过降低平滑肌/胶原蛋白比值、升高阴茎组织氧化应激水平，改变了阴茎的组织形态学特征。此外，束缚应激通过下调神经递质乙酰胆碱及其他勃起促进标志物（如p-Akt、nNOS、eNOS与cGMP）的表达，同时上调阴茎内的抑制性标志物PDE5α的水平，对阴茎勃起所需的一氧化氮（NO）生物利用度产生不利影响。束缚应激显著降低了雄鼠的爬跨、插入与射精频率，同时通过延长射精后爬跨、插入及射精的潜伏期，延长了性耗竭时长。

结论：本研究结果表明，束缚应激这类心理应激源可通过调控下丘脑-垂体-睾丸轴、氧化平衡、阴茎纤维化及阴茎勃起的NO/cGMP/PDE5α通路，诱发成年雄性大鼠的勃起功能障碍。