Page 50 - 《中国药房》2021年23期

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with 250 μg/mL DOT. Gene ontology（GO）function enrichment analysis and KEGG pathway enrichment 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-β 3，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.
KEYWORDS Yam protein； Diabetes-induced erectile dysfunction； RNA-Seq technology； Fibrosis； Oxidative stress；
Differentially expressed genes

糖尿病性勃起功能障碍（diabetic erectile dysfunc- 护作用机制，从改善内皮功能角度为DOT防治DIED的
tion，DIED）是糖尿病的常见并发症，发病率高达50％～新治疗靶点研究提供参考。
75％，严重影响了患者的生活质量 [1－3] 。高血糖引起的 1 材料
机体氧化应激、纤维化是致使内皮功能障碍，进而诱发 1.1 主要仪器
DIED的重要原因。然而，由于内皮功能障碍患者的内 AL204 型电子天平、FE20 型 pH 计均购自德国
[4]
源性一氧化氮（NO）生成率较低，致使治疗勃起功能障 Mettler-Toledo公司；Mini Protean型蛋白电泳仪、CFX型
碍的“黄金药物”——5 型磷酸二酯酶抑制剂对 DIED 的实时荧光定量聚合酶链式反应（qPCR）仪购自美国 Bio-
疗效较差 [5－7] 。因此，研发可有效防治DIED的新型药物 Rad 公司；iBrightTMFL1000 型凝胶成像系统、ACCU-
具有重要意义。 CHEK型血糖仪均购自上海罗氏制药有限公司；3111型
山药是薯蓣科植物薯蓣 Dioscorea opposita Thunb. CO2细胞培养箱购自美国Thermo Fisher Scientific公司；
的根茎，入肾经，是《金匮要略》《圣济总录》等医学经典 EVOS 型数码倒置显微镜购自美国 AMG 公司；Infnite
著作中治疗阳痿、消渴等病症的临床常用药 [8－10] 。本课 200 PRO 型酶标仪购自美国 Life Sciences 公司；HiSeq
题组前期研究发现，山药总蛋白能够通过改善肾阳虚模 2500 型基因测序仪购自美国 Illumina 公司；EVOS FL
型大鼠睾丸组织纤维化和氧化应激，发挥治疗肾阳虚型 Auto型全自动荧光显微镜购自美国Life Technologies公
[11]
勃起功能障碍的作用。然而，目前关于山药蛋白防治司；WP-UO-YJ-40型超纯水机购自四川沃特尔水处理设
DIED、改善内皮功能的研究几近空白，且发挥作用的有备有限公司。
效物质基础尚不明确。在以往的研究中，多数是以人脐 1.2 主要药品与试剂
静脉内皮细胞作为研究高糖所致内皮功能损伤的体外 DOT（批号CM202010003，纯度99.9％）由长春中医
模型，但其并不能准确地模拟海绵体内皮细胞的微血管药大学中医药大健康创新中心制备；乙二胺四乙酸
环境。因此，本研究以从山药总蛋白中分离、纯化得到（EDTA，批号 20191221）、磷酸盐缓冲液（PBS，批号
的高纯度山药蛋白——DOT为研究对象，通过建立大鼠 20200428）均购自北京索莱宝科技有限公司；一氧化氮
DIED 模型，考察 DOT 对模型大鼠的干预作用，以及合酶（eNOS）、环磷酸鸟苷（cGMP）、胰岛素酶联免疫吸
DOT 对模型大鼠内皮功能的影响；在此基础上，采用附检测（ELISA）试剂盒（批号均为 202104）均购自上海
Matrigel 3D 技术分离、培养小鼠海绵体内皮细胞优选生物科技有限公司；MCECs 完全培养基（批号
（MCECs），然后利用高糖诱导致MCECs损伤，在体外模 WH01112005XP）、Hank’s 平衡盐溶液（批号 WH0111-
拟糖尿病诱导的内皮功能障碍模型，探讨DOT对高糖损 2005XP）均购自武汉普诺赛生命科技有限公司；CCK-8
伤 MCECs 的恢复作用；然后，进一步采用转录组测序检测试剂盒（批号 I02020141G14）购自美国 Invigentech
（RNA-Seq）技术，从分子水平分析DOT对内皮功能的保公司；M199培养基（批号RNBJ1753）、青霉素-链霉素双

·2860 · China Pharmacy 2021 Vol. 32 No. 23 中国药房 2021年第32卷第23期

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