基于miRNA-21网络调控ADMA水平探讨藤茶二氢杨梅素对内皮损伤的保护机制研究

Ampelopsis grossedentata shows great cardiovascular protection effects. Multiple studies have proved that dihydromyricetin (DMY), the main active component from ampelopsis grossedentata, has potent activity of anti-atherosclerosis. But the mechanism is still not fully illustrated. Our previous study had found that DMY could reverse the endothelial dysfunction induced by Ox-ldl in primary human umbilical vein endothelial cell (HUVEC), decreased the expression of miR-21, and overexpression of miR-21 could eliminate DMY’s reversing effect. Since numerous studies have shown that asymmetric dimethylarginine (ADMA) can result in atherosclerosis, based on this, we found that the gene of DDAH1 coding the key hydrolytic enzyme of ADMA was miR-21’s target gene, and the gene of SLC7A2 coding ADMA’s major transporter was also a potential target for miR-21 as predicted by bioinformatics analysis. We further proved that the expression of miR-21 was significantly increased, and expression of DDAH1 and SLC7A1 were both significantly decreased in human atherosclerotic plaque tissue. Above all, our findings indicate that the effect of DMY reversing the endothelial dysfunction may be based on miRNA-21 net regulating ADMA concentration through the DDAH1/SLC7A1 pathway. Our study will use gene knockout mice and primary human endothelial cell models to explore the mechanisms of DMY reversing endothelial dysfunction by using RNA interference and reporter gene technologies. Our study will illustrate a new mechanism of DMY’s anti-atherosclerosis effect, and wil provide evidence of developing drugs from DMY and ampelopsis grossedentata to prevent and treat atherosclerosis.

藤茶具有心血管保护功效，国内外研究已证实二氢杨梅素（DMY）是其抗动脉粥样硬化（AS）的主要活性成分，但机制尚不明确。本项目预实验发现DMY可抵御Ox-ldl诱导的原代人脐静脉内皮细胞功能损伤，降低miR-21表达，过表达miR-21则取消DMY的保护作用。大量研究已证实非对称性二甲基精氨酸（ADMA）可导致AS，我们在此基础上已发现ADMA的水解酶DDAH1是miR-21的靶基因，预测其转运体SLC7A1也是miR-21的潜在靶点，进一步研究发现人AS斑块中miR-21表达显著升高，DDAH1和SLC7A1表达均降低。综上，我们推测DMY可能通过作用miR-21网络调控DDAH1/SLC7A1通路影响ADMA水平，抵御AS内皮损伤。本研究拟使用基因敲除小鼠和原代内皮细胞模型，通过RNA干扰和报告基因等技术，阐明DMY对内皮损伤保护的新机制，为DMY和藤茶作为AS的潜在防治药物提供依据。

藤茶具有心血管保护功效，国内外研究已证实二氢杨梅素（DMY）是其抗动脉粥样硬化（AS）的主要活性成分，但机制尚不明确。非对称二甲基精氨酸（ADMA）可通过竞争性结合一氧化氮合酶（NOS），抑制一氧化氮（NO）生成，进而引起内皮功能障碍。人体内ADMA主要由二甲基精氨酸二甲胺水解酶1（DDAH1）代谢，由SLC7A1转运至血浆。本项目组前期研究首次发现DDAH1是miR-21的靶基因，DMY可显著改善Ox-ldl诱导的HUVEC细胞内皮功能损伤，降低miR-21表达，升高DDAH1以及SLC7A1表达；过表达miR-21则取消DMY的保护作用。进一步研究发现人AS斑块中miR-21表达显著升高，DDAH1和SLC7A1表达均降低。但经过扩大样本量再验证发现SLC7A1在人正常组织和AS斑块间表达无显著差异；细胞实验发现，各种AS致病因子刺激后，可显著影响DDAH1的表达，而对SLC7A1并无显著影响。接着我们在APOE-/-基因敲除AS小鼠模型上验证了DMY和藤茶提取物的抗AS作用，DMY灌胃可显著抑制AS小鼠动脉粥样硬化病变的形成、促炎基因的表达以及肝脏炎症，同时增加小鼠体内NO生成并改善脂质代谢。然而，NOS抑制剂L-NAME与DMY合用取消DMY的抗AS作用。进一步机制研究发现，DMY降低AS小鼠体内miR-21表达，增加DDAH1表达，降低ADMA水平，增加eNOS的磷酸化和NO的产生。而在AS小鼠模型上经尾静脉注射miR-21模拟物或在HUVEC细胞模型中过表达miR-21均取消了DMY介导的内皮保护作用。因此，本项目证实了DMY通过抑制miR-21表达，网络调控DDAH1/ADMA/NO通路，保护血管内皮功能，进而抵御AS的发生发展。本项目有利于阐明藤茶二氢杨梅素抗AS作用新的分子机制，为其作为AS新的潜在防治药物提供理论依据，并为民族药物藤茶进一步开发提供理论支持。