| Title | 淫羊藿次苷Ⅱ对糖尿病环境下人阴茎海绵体血管内皮细胞中miR-181c及其靶基因KLF6,KLF9,KLF10和KLF15的表达影响研究 |
| Other Titles | Effect of icarisideⅡ on miR-181c and its targeting genes KLF6, KLF9, KLF10 and KLF15 in a ;diabetic-like human cavernous endothelial cells |
| Authors | 关瑞礼 雷洪恩 唐渊 方冬 郑卫 辛钟成 |
| Affiliation | 100034,开云app体育 第一医院男科中心 |
| Keywords | 淫羊藿次苷Ⅱ 内皮细胞功能障碍 miR-181c 晚期糖基化终产物 KLF TXNIP IcarisideⅡ Endothelial dysfunction miR-181c Advanced glycationend products KLF TXNIP |
| Issue Date | 2016 |
| Publisher | 中华临床医师杂志（电子版） |
| Citation | 中华临床医师杂志（电子版）.2016,(1),56-61. |
| Abstract | 目的：检测淫羊藿次苷Ⅱ（ICAⅡ）对糖尿病环境下人阴茎海绵体血管内皮细胞（HCECs）中miR-181c及其靶基因KLF6、KLF9、KLF10和KLF15的表达影响。方法经分离鉴定的原代HCECs，随机分为三组：正常＋BSA组（NC组）、AGE-BSA＋高糖组（DM组），ICAⅡ治疗组（DM＋ICAⅡ组）。蛋白印迹实验检测eNOS和RAGE蛋白水平；实时定量PCR检测miR-181c在模型中的表达差异；生物学信息网站预测miR-181c的靶基因；实时定量PCR检测对应靶基因在模型中的变化；蛋白印迹实验验证靶基因KLF6、KLF9及KLF6下游TXNIP蛋白表达水平差异。结果与NC组相比，蛋白印迹实验的DM组在AGE-BSA联合高糖刺激下，eNOS蛋白表达水平明显降低，而RAGE蛋白表达水平明显升高，表明成功构建用于模拟体内的糖尿病模型；而ICAⅡ干预后能够有效恢复eNOS和RAGE蛋白表达（P＜0.05）。与NC组相比，实时定量PCR检测结果发现糖尿病环境下miR-181c表达水平明显降低，而ICAⅡ治疗后可显著提高其表达水平（P＜0.05）；miR-181c的靶基因KLF6和KLF9在DM组明显升高（P＜0.05），而KLF10和KLF15的mRNA水平无显著差异（P＞0.05）；ICAⅡ干预后有效降低其KLF6和KLF9的mRNA表达水平（P＜0.05）。蛋白印迹实验结果进一步验证了KLF6和KLF9的蛋白水平变化，与上述的KLF6和KLF9的mRNA表达变化结果一致。另外，KLF6作用的下游TXNIP蛋白水平变化在模型下呈现和其一样的变化趋势。结论 AGE-BSA联合高糖刺激下miR-181c及其靶基因的显著表达差异揭示其参与内皮细胞损伤的发生发展，而ICAⅡ能够有效逆转上述变化，为进一步研究ICAⅡ与miR-181c信号通路具体的作用机制提供前期依据。 Objective To investigate the effect of icarisideⅡ(ICAⅡ) on miR-181c and its targeting genes including KLF6, KLF9, KLF10, and KLF15 in a diabetic-like human cavernous endothelial cells (HCECs). Methods Purified HCECs were randomly divided into three groups:normal group+BSA (NC group), AGE-BSA+Glucose group (DM group) and DM group treated by ICAⅡ intervention (ICAⅡgroup). Using Western blot to detect the protein expressions of eNOS and RAGE;Real-time PCR to detect miR-181 expression and its predicted targeting genes mRNA levels; Western blot to further verify the protein levels of relevant target genes and relative downstream TXNIP gene. Results Compared with NC group, protein expressions of eNOS and RAGE significantly decreased and increased separately with the stimulation of AGE-BSA plus high glucose, while ICAⅡ intervention could reverse this trend (P<0.05). Real-time PCR showed that miR-181c expression was lower in DM group than that in NC group, but its expression was recovered by ICAⅡ treatment (P<0.05). The mRNA levels of KLF6 and KLF9 but not KLF10 or KLF15 significantly increased in DM group;Western blot analysis further verified the protein levels of KLF6 and KLF9 with an increase in DM group, whereas ICAⅡ supplement could reverse this trend (P<0.05). Protein expression of TXNIP showed the similar change trend with KLF6 under the model. Conclusion Differentially expressed changes of miR-181c and its target genes under AGE-BSA combined with glucose simulation with or without ICAⅡ intervention suggest miR-181c may be involved in the endothelial dysfunction under the diabetic-like environment and ICAⅡ may be as a new compound for treating the impairment of endothelial dysfunction by regulating the miR-181c pathway. The detailed mechanism needs further more investigation. |
| URI | http://hdl.handle.net/20.500.11897/448590 |
| ISSN | 1674-0785 |
| DOI | 10.3877/cma.j.issn.1674-0785.2016.01.013 |
| Appears in Collections: | 第一医院 |
