【摘要】 目的 探究人脐带间充质干细胞(hUCMSCs)对2型糖尿病性勃起功能障碍(T2DMED)模型大鼠的改善作用及潜在机制。方法 取36只性成熟的雄性SD大鼠，采用高脂饲料联合腹腔注射40 mg/kg链脲佐菌素(STZ)建立2型糖尿病(T2DM)模型，经阿扑吗啡(APO)阴茎勃起实验及性行为学实验筛选出T2DMED大鼠。将建模成功的大鼠按随机数字表法分为模型组(MC组，尾静脉注射等量0.9%生理盐水)、二甲双胍组(MTF组，150 mg/kg MTF溶液灌胃)、hUCMSCs组(尾静脉注射1×10~6 cells/kg P5代hUCMSCs细胞悬液)。另取12只同批次未建模雄性SD大鼠为空白对照组(NC组),每周1次尾静脉注射等量0.9%生理盐水，连续4周。分别于第0、10及14周采用电子秤测定大鼠体重，血糖仪检测尾静脉血糖；采用生物机能实验系统测定大鼠最大阴茎海绵体内压(ICPmax)与平均动脉压(MAP),计算ICPmax/MAP比值评价勃起功能；腹主动脉取血离心制备血清，采用酶联免疫吸附测定(ELISA)试剂盒检测血清中白细胞介素-1β(IL-1β)、白细胞介素-4(IL-4)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)及肿瘤坏死因子α(TNF-α)水平。HE染色观察海绵体窦腔结构、平滑肌束形态排列，Masson染色评估阴茎海绵体纤维化程度；采用实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测阴茎组织中磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(AKT)、内皮型一氧化氮合酶(eNOS)的mRNA水平；采用蛋白质免疫印迹法(Western blot)检测阴茎组织中PI3K、p-PI3K、AKT、p-AKT、eNOS、p-eNOS及凋亡相关蛋白Bax、Bcl-2、Caspase-3、Cleaved Caspase-3的表达水平。结果 与NC组相比，MC组血糖、勃起潜伏期、爬背潜伏期、射精潜伏期、促炎因子水平、Cleaved Caspase-3/Caspase-3比值升高(P<0.001);勃起次数、爬背次数、射精次数、ICPmax/MAP比值、抗炎因子水平、阴茎海绵体平滑肌/胶原纤维比值、PI3K、AKT、eNOS的mRNA水平(P<0.01)及磷酸化蛋白水平、Bcl-2/Bax比值降低(P<0.001)。与MC组相比，MTF组和hUCMSCs组血糖、促炎因子水平降低(P<0.001);ICPmax/MAP比值、抗炎因子水平、eNOS磷酸化蛋白水平、Bcl-2/Bax比值升高(P<0.05)。与MTF组相比，hUCMSCs组勃起次数、爬背次数、射精次数、ICPmax/MAP比值(P<0.001)、抗炎因子水平、阴茎海绵体平滑肌/胶原纤维比值(P<0.01)、PI3K、AKT、eNOS的mRNA水平(P<0.05)及磷酸化蛋白水平、Bcl-2/Bax比值(P<0.001)升高；勃起潜伏期、爬背潜伏期、射精潜伏期、促炎因子水平(P<0.05)、Cleaved Caspase-3/Caspase-3比值(P<0.001)降低。结论 hUCMSCs能够激活PI3K-AKT-eNOS通路，通过降糖、抗炎、抗凋亡及抗纤维化等系统性调控作用改善T2DMED大鼠勃起功能，为临床应用转化提供了实验基础。更多还原

【Abstract】 Objective To investigate the multitarget regulatory effects and potential mechanisms of human umbilical cord mesenchymal stem cells（hUCMSCs） on rats in improving type 2 diabetes mellitus-induced erectile dysfunction（T2DMED）. Methods A total of 36 male SD rats were used. The T2DM model was established by high-fat and high-sugar diet combined with intraperitoneal injection of streptozocin（STZ, 40 mg/kg）. Rats with T2DMED were further screened via apomorphine（APO） test and mating test. The successfully modeled 36 rats were randomly divided into three groups: model control group（MC group, tail vein injection of equal volume of 0.9% normal saline）, metformin group（MTF group, intragastric administration of 150 mg/kg metformin solution）, and hUCMSCs group（tail vein injection of P5 generation hUCMSCs suspension at a dose of 1×10⁶ cells/kg）.Another 12 healthy male SD rats were set as the normal control group（NC group）, which received equal volume of 0.9% normal saline via tail vein injection once a week for consecutive 4 weeks. During intervention, body weight and tail vein blood glucose were detected at week 0, 10 and 14. In APO test, erectile latency and erection frequency within 1 800 s were recorded. In mating test, mount latency, mount frequency, ejaculation latency and ejaculation frequency within 1800 s were observed. After intervention, maximum intracavernous pressure（ICPmax） and mean arterial pressure（MAP） were measured by biological functional experimental system, and the ICPmax/MAP ratio was calculated to evaluate erectile function.Abdominal aortic blood was collected to separate serum. Enzyme-linked immunosorbent assay（ELISA） kits were used to detect serum levels of interleukin-1β（IL-1β）, IL-4, IL-6, IL-10 and tumor necrosis factor-α（TNF-α）. Penile corpus cavernosum tissues were collected to prepare paraffin sections. HE staining was performed to observe the structure of cavernous sinus and arrangement of smooth muscle bundles; Masson staining combined with Image J software was used to calculate the area ratio of smooth muscle to collagen fiber for fibrosis assessment.Quantitative real-time reverse transcription polymerase chain reaction（qRT-PCR） was adopted to detect mRNA expressions of PI3K, AKT and eNOS in penile tissues, and the relative expression was calculated by 2^{（-△△Ct）} method. Western blot was used to determine the protein expressions of PI3K, p-PI3K, AKT, p-AKT, eNOS, p-eNOS, as well as apoptosis-related proteins Bax, Bcl-2, Caspase-3 and Cleaved Caspase-3. The relative protein expression was analyzed by grayscale scanning with Image J software. Results Compared with the NC group, the MC group showed significantly increased blood glucose, erectile latency, mount latency, ejaculation latency, pro-inflammatory factor levels and Cleaved Caspase-3/Caspase-3 ratio（P<0.001）. Meanwhile, erection frequency, mount frequency, ejaculation frequency, ICPmax/MAP ratio, anti-inflammatory factor levels, smooth muscle/collagen fiber ratio of corpus cavernosum, mRNA and phosphorylated protein levels of PI3K, AKT and eNOS, as well as Bcl-2/Bax ratio were markedly decreased（P<0.01 or P<0.001）. Compared with the MC group, both MTF group and hUCMSCs group had decreased blood glucose and pro-inflammatory factor levels（P<0.001）, while ICPmax/MAP ratio, anti-inflammatory factor levels, phosphorylated eNOS level and Bcl-2/Bax ratio were increased（P<0.05）. Compared with the MTF group, the hUCMSCs group presented higher erection frequency, mount frequency, ejaculation frequency, ICPmax/MAP ratio（P<0.001）, anti-inflammatory factor levels, smooth muscle/collagen fiber ratio of corpus cavernosum（P<0.01）, mRNA and phosphorylated protein levels of PI3K, AKT and eNOS, as well as Bcl-2/Bax ratio（P<0.001）. By contrast, erectile latency, mount latency, ejaculation latency, pro-inflammatory factor levels（P<0.05） and Cleaved Caspase-3/Caspase-3 ratio（P<0.001） were reduced. Conclusion hUCMSCs can effectively improve erectile function in T2DMED rats through systemic regulatory mechanisms, including hypoglycemic, anti-inflammatory, anti-apoptotic, and anti-fibrotic effects. Its activation of the PI3K-AKT-eNOS pathway is a preliminary finding, which provides an experimental basis for clinical transformation.更多还原