摘要
目的探讨淫羊藿次苷Ⅱ(ICAⅡ)通过mi R-155/内皮型一氧化氮合酶(e NOS)通路改善糖尿病性人阴茎海绵体血管内皮细胞(HCECs)功能障碍的作用机制。方法经原代分离培养HCECs,随机分为三组:正常+BSA对照组(NC组),Age-BSA联合高糖糖尿病组(DM组),ICAⅡ治疗组(DM+ICAⅡ组,ICAⅡ0.1、1、10μmol/L)。Western blot检测e NOS、晚期糖基化终产物受体(RAGE)蛋白的表达;实时定量PCR检测e NOS及其潜在上游mi RNAs(mi R-155、mi R-543、mi R-31、mi R-429、mi R-200c、mi R-200b)的表达;DAF-FM DA荧光探针法和硝酸盐/亚硝酸盐还原法检测细胞一氧化氮含量;进一步mi R-155过表达慢病毒感染,观察ICAⅡ对HCECs中mi R-155、e NOS和一氧化氮表达的影响。结果 Age-BSA联合高糖刺激下,HCECs中e NOS和RAGE蛋白表达水平分别显著下调和上调;而不同浓度的ICAⅡ可逆转e NOS和RAGE蛋白的表达(P<0.05);且随着ICAⅡ浓度的提高其作用逐渐增强。当HCECs发生糖尿病性功能障碍时,可调控e NOS表达的潜在上游mi RNAs中mi R-155的表达变化最为明显(P<0.05)。且在ICAⅡ干预下,发现糖尿病性功能障碍HCECs中e NOS和一氧化氮的表达恢复上调与mi R-155表达受到抑制有关。进一步利用mi R-155过表达慢病毒感染HCECs后,发现mi R-155表达显著升高,而ICAⅡ可明显抑制其表达(P<0.05);相应地,HCECs过表达mi R-155后,观察到其下游e NOS及一氧化氮表达明显降低,而ICAⅡ干预可显著恢复其表达(P<0.05)。结论 mi R-155/e NOS通路可能在糖尿病性HCECs功能障碍的发生中发挥重要作用,而ICAⅡ可能通过调控mi R-155/e NOS信号通路而发挥改善糖尿病性内皮细胞功能的作用。
ObjectiveTo investigate the changes of miR-155/eNOS signal pathway under the stimulation of Age-BSA and glucose with or without icarisideⅡ （ICAⅡ） intervention in human cavernous endothelial cells （HCECs）.MethodsPurified HCECs were first divided into three groups randomly： normal group＋BSA （NC group）, Age-BSA＋glucose group （DM group）, ICAⅡ treatment group （DM＋ICAⅡ group with different concentrations at 0.1μmol/L, 1μmol/L, 10μmol/L）. Western blotwas used to detect the protein expression of eNOS and RAGE; Real time PCR to detect the expression of miR-155 and eNOS; DAF-FM DA fluorescent probes assay and NaNO3/NaNO2assay to detect the NO concentration. Lentivirus mediated miR-155 overexpression was constructed to observe the changes of eNOS and NO.ResultsThe eNOS and RAGE expression in DM group was significantly reduced and increased respectively compared with that of NC group （P〈0.05）, while ICAⅡ intervention could reverse this change effectively. 10μmol/L of ICAⅡ had the most powerful effect. MiR-155 had the highest fold changes among candidate miRNAs in diabetic like HCECs （P〈0.05）. MiR-155 increased and eNOSnbsp;decreased remarkably in DM group, while ICAⅡ intervention could inhibit the miR-155 expression, which led to the significantly higher eNOS expression and NO concentration （P〈0.05）. In lentivirus mediated miR-155 overexpression with or without ICAⅡ intervention model, we found the similar trend with the above diabetic model.ConclusionMiR-155/eNOS signal pathway may be involved in the process of diabetic HCECs dysfunction. ICAⅡ could promote the recovery of the endothelial dysfunction by regulating the miR-155/eNOS signal pathway.
出处
《中华临床医师杂志（电子版）》
CAS
2016年第6期826-832,共7页
Chinese Journal of Clinicians(Electronic Edition)
基金
国家自然科学基金(81401194
81270693)
