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基于转录组测序技术分析山药蛋白防治糖尿病性勃起功能障碍的作用机制
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| 篇名： | 基于转录组测序技术分析山药蛋白防治糖尿病性勃起功能障碍的作用机制 |
| TITLE： | Analysis of the Mechanism of Yam Protein in the Prevention and Treatment of Diabetes-induced Erectile Dysfunction Based on RNA-Seq Technology |
| 摘要： | 目的：研究山药蛋白（DOT）防治糖尿病性勃起功能障碍（DIED）的潜在作用机制。方法：采用高糖高脂饲料+腹腔注射链脲佐菌素（40mg/kg）的方法复制DIED大鼠模型。实验设置正常对照组（生理盐水），模型组（生理盐水），DOT低、中、高剂量组（0.3、0.6、0.9mg/kg）和西地那非组（阳性对照，4.4mg/kg），每组9只大鼠。在糖尿病模型已建立成功、开始诱导DIED发生的阶段，各组大鼠开始灌胃相应溶液，每天1次，连续11周。测定大鼠体质量、空腹血糖（FPG）值、勃起次数、勃起率、空腹胰岛素（FINS）、胰岛素抵抗指数（IR）以及阴茎海绵体组织中内皮型一氧化氮合酶（eNOS）、环磷酸鸟苷（cGMP）含量，以评价DOT对DIED模型大鼠的干预作用。选择30mmol/L葡萄糖诱导48h建立高糖损伤小鼠海绵体内皮细胞（MCECs）模型，并给予125、250、500μg/mLDOT进行干预，然后检测细胞活力，以评价DOT对高糖损伤MCECs的影响。继而采用转录组测序技术分别筛选正常MCECs与高糖损伤MCECs、高糖损伤MCECs与250μg/mLDOT处理的MCECs中的差异表达基因，对差异表达基因进行基因本体（GO）功能富集分析和京都基因与基因组百科全书（KEGG）通路富集分析；分析两组细胞间共同的差异表达基因，并选择6个关键基因在mRNA水平上进行验证。结果：不同剂量DOT对DIED模型大鼠体质量降低、FINS和IR升高、勃起次数减少、勃起率降低以及阴茎海绵体组织中eNOS、cGMP含量降低均有不同程度的逆转作用，其中高剂量DOT作用后DIED模型大鼠的上述指标变化均被显著逆转（P＜0.05或P＜0.01）。125、250、500μg/mLDOT均可显著提高高糖损伤MCECs的活力（P＜0.05或P＜0.01）。转录组测序结果表明，与正常MCECs比较，高糖损伤MCECs中共有48个差异表达基因；与高糖损伤MCECs比较，DOT处理的MCECs中共有779个差异表达基因；两组差异表达基因在生物学过程注释中均以细胞过程为主、在细胞组分注释中均以细胞部分为主、在分子功能注释中均以绑定分子功能为主，并主要富集在细胞外基质受体相互作用通路、错配修复通路、磷脂酰肌醇3激酶-蛋白激酶B（PI3K-Akt）信号通路等。在两组差异表达基因中，共得到Aldh1a1、Abcc5、Tac1等13个共同的差异表达基因，DOT均可显著回调高糖损伤MCECs中上述共同差异表达基因的表达。经验证，DOT可以显著逆转高糖损伤MCECs中关键基因TGF-β3、Txnip、Aldh1a1、Loxl1、Mt1、Mt2的mRNA表达。结论：DOT对DIED模型大鼠的症状具有改善作用；其作用机制可能与抑制纤维化、降低氧化应激等生物学途径，从而改善海绵体内皮功能有关。 |
| ABSTRACT： | OBJECTIVE：To study the potential mechanism of yam protein （DOT） in the prevention and treatment of diabetes-induced erectile dysfunction （DIED）. METHODS ：DIED model was induced by high-glucose and high-fat diet and intraperitoneal injection of streptozotocin （40 mg/kg）. The experiment was set up in the normal control group （normal saline ）， model group （normal saline ），DOT low-dose ，medium-dose and high-dose groups （0.3，0.6，0.9 mg/kg），sildenafil group （positive control ，4.4 mg/kg），with 9 rats in each group. In the stage of successful establishment of diabetes model and initiation of inducing DIED ，rats in each group were given relevant solution intragastrically ，once a day ，for consecutive 11 weeks. Body weight，fasting plasma glucose （FPG），the times and rate of penile erection ，fasting insulin （FINS），insulin resistance index （IR），the contents of endothelial nitric oxide synthase （eNOS）and cyclic guanosine monophosphate （cGMP）in penile cavernous tissue were determined so as to evaluate the intervention effects of DOT on DIED model rats. High-glucose damaged mice cavernous endothelial cells （MCECs）model was induced by 30 mmol/L glucose for 48 h，and then give DOT 125，250，500 μg/mL. The cell viability was detected so as to evaluate the effects of DOT on high-glucose damaged MCECs model. RNA-Seq mail：xingxin0902@163.com technology was adopted to screen the differentially expressed genes between normal MCECs and high-glucose damaged MCECs，high-glucose damaged MCECs and MCECs treated with 250 μg/mL DOT. Gene ontology（GO）function enrichment analysis and KEGG pathway enrichme nt analysis were performed for differentially expressed genes. The common differentially expressed genes between 2 groups were analyzed ，and mRNA expressions of six key genes were validated. RESULTS ：Different doses of DOT could reverse the reduction of body weight ，the increase of FINS and IR ，the reduction of the times and rate of penile erection ，the decrease of eNOS and cGMP contents in penile cavernous tissue of DIED model rats ；above indexes of DIED model rats were reversed significantly after treated with high-dose of DOT（P＜0.05 or P＜0.01）. 125，250，500 μg/L DOT could significantly improve the activity of high-glucose damaged MCECs （P＜0.05 or P＜0.01）. RNA-Seq technology showed that compared with normal MCECs ，a total of 48 differentially expressed genes were found in high-glucose damaged MCECs. Compared with high-glucose damaged MCECs ，a total of 779 differentially expressed genes were found in MCECs treated with DOT. The differentially expressed genes of 2 groups were mainly cellular process in biological process annotation ，cellular part in cell component annotation and binding molecular function in molecular function annotation ，which were mainly enriched in extracellular matrix receptor interaction pathway ，mismatch repair pathway ， phosphatidylinositol 3 kinase-protein kinase B （PI3K-Akt）signal pathway and so on. Among differentially expressed genes of 2 groups，13 common differentially expressed genes such as Aldh1a1，Abcc5，Tac1 were found. DOT could significantly reverse the expression of the above common differentially expressed genes in high-glucose damaged MCECs. After validation ，DOT could significantly reverse the mRNA expression of TGF-β3，Txnip，Aldh1a1，Loxl1，Mt1 and Mt2 in high-glucose damaged MCECs. CONCLUSIONS：DOT could improve the symptom of DIED model rats ，the mechanism of which may be related to biological pathway of inhibiting fibrosis and reducing oxidative stress ，so as to improve the endothelial function of cavernous body. |
| 期刊： | 2021年第32卷第23期 |
| 作者： | 邢欣，赵大庆，王思明，于士婷，李懿轩，刘美辰 |
| AUTHORS： | XING Xin，ZHAO Daqing ，WANG Siming ，YU Shiting ，LI Yixuan ，LIU Meichen |
| 关键字： | 山药蛋白；糖尿病性勃起功能障碍；转录组测序技术；纤维化；氧化应激；差异表达基因 |
| KEYWORDS： | Yam protein ； Diabetes-induced erectile dysfunction ； RNA-Seq technology ； Fibrosis； Oxidative stress ； |
| 阅读数： | 223 次 |
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