药学学报

Hub target	Binding affinity/kcal·mol^-1
Nitric oxide synthase 3 (NOS3)	-6.03
Tumor necrosis factor (TNF)	-5.9
Mammalian target of rapamycin (MTOR)	-5.99
Cyclin D1 (CCND1)	-5.69
Prostaglandin-endoperoxide synthase 2 (PTGS2)	-4.83
Caspase-3 (CASP3)	-5.19
Heat shock protein 90 alpha family class A member 1 (HSP90AA1)	-6.44
G protein-coupled estrogen receptors (GPER)	-5.42

Hub target	Binding affinity/kcal·mol^-1
Nitric oxide synthase 3 (NOS3)	-6.03
Tumor necrosis factor (TNF)	-5.9
Mammalian target of rapamycin (MTOR)	-5.99
Cyclin D1 (CCND1)	-5.69
Prostaglandin-endoperoxide synthase 2 (PTGS2)	-4.83
Caspase-3 (CASP3)	-5.19
Heat shock protein 90 alpha family class A member 1 (HSP90AA1)	-6.44
G protein-coupled estrogen receptors (GPER)	-5.42

芒柄花黄素通过上调GPER激活PI3K/Akt/eNOS信号通路减轻心肌缺血再灌后无复流的作用及机制研究

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刘海瑞 , 叶麟晰 , 林佳美慧 , 刘倩 , 彭雅旋 , 陈婷 ^*

药学学报 | 专题报道: 基于整合药理学的中医药现代化研究 2023,58(6): 1496-1504

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药学学报 |专题报道: 基于整合药理学的中医药现代化研究 2023 , 58 (6) : 1496 -1504

芒柄花黄素通过上调GPER激活PI3K/Akt/eNOS信号通路减轻心肌缺血再灌后无复流的作用及机制研究

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刘海瑞, 叶麟晰, 林佳美慧, 刘倩, 彭雅旋, 陈婷^*

作者信息

湖南中医药大学, 中西医结合心脑疾病防治湖南省重点实验室, 湖南长沙 410208

通讯作者:

*陈婷, Tel: 86-731-88458862, E-mail: chenting520@hnucm.edu.cn

The effect and mechanism of formononetin on alleviating no-reflow after myocardial ischemia and reperfusion by up-regulating the PI3K/Akt/eNOS signal pathway activated by GPER

Hai-rui LIU, Lin-xi YE, Jia-mei-hui LIN, Qian LIU, Ya-xuan PENG, Ting CHEN^*

Affiliations

Key Laboratory of Hunan Province for Integrated Traditional Chinese and Western Medicine on Prevention and Treatment of Cardio-Cerebral Diseases, Hunan University of Chinese Medicine, Changsha 410208, China

出版时间: 2023-06-12 doi: 10.16438/j.0513-4870.2022-1442

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摘要

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基于整合药理学及实验验证探讨芒柄花黄素(formononetin, FMN) 对心肌缺血再灌后无复流(no-reflow, NR) 的心脏保护作用及其分子机制。首先, 采用人乳腺癌细胞(MCF-7) 和心肌NR大鼠证实FMN的雌激素活性和减轻NR的药效作用。将雄性Sprague-Dawley (SD) 大鼠分为Sham、NR、FMN (20 mg·kg^-1) 和硝普钠(sodium nitroprusside, SNP, 5.0 mg·kg^-1) 组, 给药1周, 每天1次, 本实验获得天津中医药大学伦理委员会批准(TCM-LAEC2019095)。再整合药理学分析和NR大鼠体内研究, 揭示FMN改善NR的作用机制。结果发现, FMN具有雌激素样作用, 且通过改善心脏结构和功能、减少无复流、缺血心肌面积和心肌细胞病理损伤, 起到减轻NR的作用。整合药理学预测FMN改善NR机制主要与磷脂酰肌醇-3-激酶-蛋白激酶B (phosphatidyinositol-3-kinase-protein kinase B, PI3K-Akt) 信号通路有关。植物雌激素主要通过上调G蛋白偶联雌激素受体(G protein-coupled estrogen receptor, GPER) 发挥保护心血管的作用, GPER也作为PI3K-Akt信号通路上游的重要调节因子, 本研究发现FMN能显著上调GPER、p-PI3K、p-Akt和磷酸化内皮型一氧化氮合酶(phospho-endothelial nitric oxide synthase, p-eNOS) 蛋白表达, 且与GPER及eNOS蛋白具有良好结合能力。本研究通过整合药理学和实验评估, 揭示FMN通过上调GPER激活PI3K/Akt/eNOS信号通路, 从而显著改善NR的机制。

关键词

心脏保护作用 / 分子机制 / 整合药理学 / 芒柄花黄素 / G蛋白偶联雌激素受体 / PI3K/Akt/eNOS

Abstract

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To investigate the cardioprotective effect of formononetin (FMN) on no-reflow (NR) after myocardial ischemia-reperfusion and its molecular mechanism based on integrated pharmacology and experimental verification, firstly, human breast cancer MCF-7 cells and myocardial NR rats were used to confirm the estrogenic activity and the effect of alleviating NR of FMN, respectively. Male SD rats were divided into Sham, NR, FMN (20 mg·kg^-1) and sodium nitroprusside (SNP, 5.0 mg·kg^-1) groups, which were administered once a day for one week, the experiment was approved by the Ethics Committee of Tianjin University of Traditional Chinese Medicine (TCM-LAEC2019095). The pharmacological analysis and in vivo study of NR rats were integrated to reveal the mechanism of FMN improving NR. The results showed that FMN had estrogenic effect and reduced NR by improving cardiac structure and function, reducing NR, ischemic myocardial area and pathological injury of cardiomyocytes. Integrated pharmacology predicts that the mechanism of FMN improving NR is mainly related to phosphatidyinositol-3-kinase-protein kinase B (PI3K-Akt) signal pathway. Phytoestrogens play a role in cardiovascular protection mainly by activating G protein-coupled estrogen receptor (GPER). GPER is also an important regulator in the upstream of PI3K-Akt signaling pathway. This study found that FMN can significantly activate GPER, p-PI3K, p-Akt and phospho-endothelial nitric oxide synthase (p-eNOS). It has good binding ability with GPER and eNOS protein. In this study, through the integration of pharmacology and experimental evaluation, it is revealed that FMN activates PI3K/Akt/eNOS signal pathway by activating GPER, thus significantly improving NR.

Key words

cardioprotective effect / molecular mechanism / network pharmacology / formononetin / G protein-coupled estrogen receptor / PI3K/Akt/eNOS

引用本文

刘海瑞, 叶麟晰, 林佳美慧, 刘倩, 彭雅旋, 陈婷. 芒柄花黄素通过上调GPER激活PI3K/Akt/eNOS信号通路减轻心肌缺血再灌后无复流的作用及机制研究. 药学学报, 2023 , 58 (6) : 1496 -1504 . DOI: 10.16438/j.0513-4870.2022-1442

Hai-rui LIU, Lin-xi YE, Jia-mei-hui LIN, Qian LIU, Ya-xuan PENG, Ting CHEN. The effect and mechanism of formononetin on alleviating no-reflow after myocardial ischemia and reperfusion by up-regulating the PI3K/Akt/eNOS signal pathway activated by GPER[J]. Acta Pharmaceutica Sinica, 2023 , 58 (6) : 1496 -1504 . DOI: 10.16438/j.0513-4870.2022-1442

正文

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无复流(no-reflow, NR) 是急性心肌梗死(AMI) 患者在经皮冠状动脉介入治疗(PCI) 后心肌组织再灌注并不完全, 甚至无再灌注的现象, 表现为冠脉血流减慢或无血流^[1]。临床上AMI患者PCI术后无复流发生率高达30%^{[2, 3]}。NR是一个复杂的病理过程, 它起始于缺血期, 恶化于再灌注期, 并以心肌微循环障碍为病理核心^[4]。现有研究表明, 其主要机制与炎症反应、钙超载、线粒体损伤、氧化应激和细胞凋亡等有关^{[5, 6]}。近年来, 虽在NR发病机制及病理变化等研究领域取得了显著进展, 然而在治疗上仍缺乏真正有效控制病情发展的药物。因此, 研究和探求治疗NR的新途径、新药物成为目前国内外学者研究重点。

雌激素能显著改善NR, 减少缺血再灌后心肌微血管损伤^[7], 其主要通过膜受体和下游级联磷脂酰肌醇-3-激酶/蛋白激酶B/内皮型一氧化氮合酶(phosphatidyinositol-3-kinase/protein kinase B/endothelial nitric oxide synthase, PI3K/Akt/eNOS) 激活非基因组机制产生一氧化氮(nitric oxide, NO), 从而促进血管舒张、调节血压、减少血管炎症和动脉粥样硬化并改善NR^{[8, 9]}。G蛋白偶联雌激素受体(G protein-coupled estrogen receptor, GPER) 可介导雌激素在体内快速反应, 其对于心血管系统的调节和保护作用已成为研究热点^[10]。前期研究也发现, GPER能激活PI3K/Akt/eNOS和环磷酸腺苷/蛋白激酶A (cyclic adenosine monophosphate/protein kinase A, cAMP/PKA) 途径, 活化eNOS产生NO, 产生舒张血管效应, 从而改善NR^[11]。

芒柄花黄素(formononetin, FMN) 是异黄酮家族的植物雌激素成员, 具有抗氧化、抗高血压、抗肿瘤和抗感染等药理作用^[12]。主要存在于黄芪和红三叶等豆科植物中, 富含FMN的芪苈强心胶囊、芪参益气丸、脑心通胶囊, 已在临床上广泛用于治疗高血压和心肌缺血再灌注损伤^[13]; FMN可以通过激活雌激素受体α (ERα) 增强ROCK-II信号通路产生促血管生成作用^[14]; FMN可以通过抑制ROS-TXNIP-NLRP3通路, 最终抑制NLRP3炎性小体的激活, 对心肌缺血再灌注损伤发挥有效的保护作用^[15]。值得关注的是, 富含FMN的通脉养心丸已被证实具有改善NR的作用^[11], 而FMN是否具有改善NR的作用及其作用机制尚不清楚。

网络药理学是通过构建“药物成分-药物作用靶点-疾病相关靶点”相互作用网络, 从系统生物学角度出发, 基于多组分、多靶点、多途径的整体观揭示中药复杂体系治疗疾病的相关机制, 近年来成为中药研究领域比较常用的研究技术^[16]。在本研究中, 通过整合药理学和实验验证来阐明FMN在心肌缺血再灌注后NR中的潜在机制。首先, 本研究采用人乳腺癌细胞(MCF-7) 和NR大鼠验证FMN激素样作用和改善NR的作用; 然后构建“关键靶点-通路”网络, 以预测FMN治疗疾病的潜在靶点和作用通路; 最后, 采用NR大鼠体内实验, 揭示FMN减轻NR的机制。

材料与方法

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FMN的作用靶点收集 将“Formononetin”输入中药系统药理数据库TCMSP (https://old.tcmsp-e.com/tcmsp.php) 检索框, 限定词为“chemical name”, 得出FMN的CAS号并记录, 将所得CAS号输入PubChem (https://pubchem.Ncbi.nlm.nih.gov) 数据库得出SMILE号。将SMILE号导入Swiss (Swiss target prediction.ch) 和Sea (https://sea.bkslab.org) 数据库得到FMN的作用预测靶点, 删去重复值, 获得FMN作用靶点, 将获得的靶点导入UniProt数据库(http://www.uniprot.org.) 转换成相应的基因名称。

NR相关靶点收集 以“no reflow after myocardial ischemia and reperfusion”为关键词, 在OMIM (http://www.omim.org/)、Gene Cards (https://www.genecards.org/)、Pharmgkb (https://www.pharmgkb.org/)、TTD (https://db.idrblab.net/ttd/) 及DisGeNET (https://www.disgenet.org) 数据库进行检索, 删去重复值, 获得NR疾病靶点。将获得的靶点导入UniProt数据库(http://www.uniprot.org.) 转换成相应的基因名称。

FMN-NR共同靶点筛选 在Venny2.1.0在线软件作图工具平台上输入FMN作用靶点与NR相关靶点, 进行交集处理后, 以韦恩图的形式展示FMN-NR共同靶点, 获得FMN治疗NR的潜在靶点^[17]。

FMN治疗NR核心靶点的筛选 蛋白质相互作用(protein-protein interaction, PPI) 是体现蛋白与蛋白之间相互关系的网络图, 将FMN-NR共同靶点上传至String10.0 (https://stringdb.org) 数据库, 选择multiple proteins功能, 设置蛋白种类为“Homo sapiens”, 相互作用阈值为0.4, 获取靶点相互作用的网络关系数据; 将分析得到的PPI结果保存为tsv文件格式, 进而导入Cytoscape 3.9.1软件选择网络拓扑分析功能进行可视化结果, 以节点的大小、颜色及深浅变化代表degree值的大小; degree值越大, 点越大, 说明这个靶点在FMN治疗NR方面具有更重要的潜在作用, 选取degree值前8位的靶点, 作为FMN治疗NR的核心靶点^[18]。

FMN治疗NR靶点的功能与通路富集分析 将FMN-NR共同靶点导入David (https://david.ncifcrf.gov) 数据库, 通过功能分析模块进行基因本体(GO) 和京都基因和基因组百科全书(KEGG) 富集分析。GO分析包括3个类别: 细胞组分(cellular component, CC)、分子功能(molecular function, MF)、生物过程(biological process, BP), 3组中选取具有统计学意义(P < 0.05) 的通路, KEGG结果选择具有统计学意义(P < 0.05) 的通路, 应用微生信基因富集分析在线工具数据平台(www.bioinformatics.com.cn) 绘制柱状图, 对上述所得FMN-NR共同靶点进行GO功能和KEGG通路富集分析^[19]。

化合物-核心靶点相互作用验证 核心蛋白的三维(3D) 结构从PDB数据库(https://www.rcsb.org/) 下载。使用AutoDockTools-1.5.6通过去除配体、除水和加氢来处理核心蛋白。候选化合物的三维结构可从PubChem下载。使用AutoDockTools将蛋白质受体和配体转换为PDBQT格式。最后, 使用AutoDock Vina软件进行分子对接, 并使用PyMOL和Discovery Studio 2016选择最低自由能模型进行可视化分析, 对接分数 < -5表示具有良好亲和力^[20]。

MCF-7细胞增殖实验 以雌激素依赖性的MCF-7乳腺癌细胞为对象, 采用CCK-8实验检测FMN对MCF-7细胞增殖的影响, 并筛选有效浓度。实验开始前3天改为在无酚红高糖培养基(Dulbecco's modified eagle medium, DMEM) (含5% CDT-FBS) 条件下培养, 以耗尽细胞内源性雌激素。选用对数生长期细胞, 0.05%胰酶消化后, 加入无酚红DMEM, 以3 000个/孔的浓度接种于96孔板内, 每孔200 µL。待细胞贴壁生长至60%~70%融合度后, 约24 h, 分别加入FMN (10、20和30 μmol·L^-1)、雌二醇(estradiol, E₂, 10^-8 mol·L^-1), 置于37 ℃、5% CO₂细胞培养箱内, 培养48 h。每孔加入20 µL 10% CCK-8, 继续孵育4 h后, 使用酶联免疫检测仪, 于450 nm下测定吸光度(A) 值, 计算平均A值和增殖率。其中, 增殖率%= (A_实验组-A_对照组)/A_对照组×100%。

实验动物 雄性Sprague-Dawley (SD) 大鼠(250 ± 10) g购自北京维通利华实验动物技术有限公司(证书编号: 11401300051612; SCXK20160006)。所有实验方案均按照天津中医药大学动物伦理委员会批准的指南进行(TCM-LAEC2019095)。动物标准饲养, 自由摄食饮水, 实验前12 h禁食, 不禁水。

大鼠NR模型的制备 适应性饲养大鼠1周, 术前禁食不禁水12 h, 实验组大鼠腹腔内注射0.3%戊巴比妥钠(1 mL/100 g) 进行麻醉, 麻醉后胸前手术备皮(用剃毛机将大鼠胸前区鼠毛脱去, 使手术视野充分暴露), 将大鼠用皮筋仰卧位固定四肢及头部于鼠板。在胸骨左缘0.5 cm处, 自胸骨左缘第3~4肋间开胸, 逐层钝性分离皮下组织、肌肉, 将心脏压出胸壁外。找到冠状动脉左前降支(LAD), 用5/0缝合线在左心耳根部下方2 mm处, 以深1.5~2 mm、宽2~3 mm穿过心肌表层, 打一松结, 松结内放入2/0棉线。而后将冠脉及棉线一起进行结扎, 第二结为滑结, 造成心肌缺血。结扎后, 迅速将心脏送回胸腔, 并挤出胸腔内空气, 用一把直头止血钳夹闭皮肤及肌肉层, 造成假关胸。阻断冠状动脉左前降支2 h后, 松开止血钳, 轻轻提起结扎的线头, 用一把显微镊顺势轻轻拔出棉线, 剪断胸腔外的结扎线, 恢复血流灌注并缝合, 其中假手术组只穿线不结扎。再灌注后2 h进行超声心动图检查, 射血分数(ejection fraction, EF) 值< 50%和明显的心肌NR^{[7, 21]}, 标志构造模型成功。

分组和给药 再灌注2 h后, 根据大鼠EF值(< 50%) 将大鼠随机分为NR组、FMN (20 mg·kg^-1) 和SNP (sodium nitroprusside, 5.0 mg·kg^-1, 阳性对照组) (n = 16)。假手术组(Sham) 和NR组大鼠灌胃相同体积的0.5%羧甲基纤维素(CMC-Na, 批号C8621, 北京索莱宝科技有限公司) 溶剂, FMN和SNP组大鼠是在NR模型基础上分别进行灌胃和腹腔注射。每日1次, 连续给药7天, 给药体积为1 mL/100 g, 大鼠每隔2天称重1次, 根据体重变化及时调整给药体积。第一次给药时间为造模后4 h。

无复流心肌面积及缺血心肌面积评估 给药7天后, 每组随机选择5只大鼠, 下腔静脉注入6%硫磺素S 1 mL·kg^-1, 硫磺素S经过的区域365 nm波长光源下发紫色荧光, 代表复流区范围, 无荧光区域为无复流区。硫磺素S注入完毕1 min后原位结扎LAD, 下腔静脉注入2% Evans Blue 1 mL·kg^-1[22], 1 min后立即取出心脏置于-40 ℃冰箱进行冷冻15 min, 再从结扎线下1 mm平行均匀将心脏切成5片, 荧光显微镜365 nm拍照保存用于检测无复流心肌面积。心肌切片再于1% TTC溶液37 ℃水浴锅中孵化15~30 min, 于普通光照下再次拍照保存用于检测缺血心肌面积。普通光照下, 蓝染的区域为非缺血心肌区, 浅红色区域为缺血未梗死心肌区, 灰白色区域为梗死心肌区, 用Image-Pro Plus 6.0软件测定各区面积。计算公式: 无复流心肌面积百分数=无复流心肌面积/心脏面积×100%; 缺血心肌面积百分数=缺血心肌面积/心脏面积×100%。

大鼠心脏血流动力学及结构的超声测定 使用小动物超声仪测量大鼠心脏的功能和结构^[23] (Vevo 2100, VisualSonics, 加拿大)。大鼠麻醉后胸前备皮(用剃毛机将大鼠胸前区鼠毛脱去, 使超声视野充分暴露), 将大鼠用皮筋仰卧位固定四肢及头部于测量板。探头频率设置为12 Mhz, 探头涂耦合剂并放置在胸骨左侧进行检查, 与胸骨中线形成30°角。M型样本线垂直于室间隔和左心室后壁, 以获得M型超声心动图。测量包括左心室射血分数（EF）、左室短轴缩窄率(FS)、左室质量(LV质量)、左室舒张末期容积(LV Vold)、左室收缩末期容积(LV Vols)、流出道峰值血流速度(LVOT peak) 和每搏输出量(LVSV)。所有测量值均为3个心脏周期的平均值。

HE染色 对大鼠实施安乐死, 并取出心脏。采用常规HE染色^[23], 于显微镜下对病理切片进行观察、选取合适视野拍照, 然后进行专业描述与分析。

Western blot (WB) 实验 如前所述, 提取心脏左心室总蛋白, 进行蛋白质印迹定量分析^[23]。使用的抗体包括兔单克隆GPER (1∶1 000稀释, bs-1380R, Bioss公司); 兔单克隆p-PI3K (Tyr 458) (1∶1 000稀释, PA5-17387, Thermo公司); 兔单克隆p-Akt (Thr308, 1∶1 000稀释, 13038)、兔单克隆p-eNOS (Ser1177, 1∶1 000稀释, 9570S)、兔单克隆GAPDH (1∶1 000稀释, 5174T) 和抗兔IgG (1∶5 000稀释, 7074P2) 均购自Cell Signaling Technology公司。

统计学分析 应用SPSS 21.0版进行数据统计分析, 所有数据以平均值±标准差表示, 数据符合正态分布时通过单因素方差分析(ANOVA) 进行分析, 数据不符合正态分布时使用非参数检验, 当P < 0.05时, 差异被认为具有统计学意义。

结果

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1 “作用靶点-关键靶点-通路”网络的构建

通过GeneCards、OMIM、PharmGkb、TTD、DisGeNET 5个数据库进行检索, 得到不重复的NR相关靶点1 622个, 从TCMSP、Swiss、Sea、UniProt数据库得到不重复的FMN作用靶点261个。绘制韦恩图后得到FMN-NR共同靶点93个。FMN-NR共同靶点的PPI图显示共得到84个节点, 505条边。为筛选关键靶点中作用较大的靶点, 利用Analyze Network功能对网络进行拓扑分析, 根据关键靶点的度值(degree) 进行排序, 排名前8位的靶点将用于后续的分子对接验证。93个靶点中筛选出8个核心靶点, 其对应基因为TNF、NOS3、MTOR、CCND1、PTGS2、PPARG、CASP3和HSP90AA1 (图 1)。

2 潜在靶点的GO富集分析和KEGG通路分析及分子对接验证

对交集靶点进行GO富集分析与KEGG通路分析, 得到GO条目共2 526条(P < 0.05), 其中包括生物过程(biological process, BP) 条目517条、细胞组成(cell composition, CC) 条目58条、分子功能(molecular function, MF) 条目109个。根据P值对条目排序, 将各类别前10名富集结果进行可视化分析, FMN-NR共同靶点主要涉及的生物学过程包括药物反应、凋亡过程的负调控、缺氧反应、蛋白质磷酸化的正向调控等, 主要涉及的细胞成分包括细胞质膜、膜筏、胞溶质等, 主要涉及的分子功能包括酶结合、相同蛋白结合、蛋白激酶活性等。FMN-NR共同靶点的KEGG富集通路有153条, 将富集结果按显著性排序取前30个进行展示, 其中主要包括PI3K-Akt信号通路、环磷酸鸟苷酸蛋白激酶通路[NO-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG), cGMP-PKG]、晚期糖基化终末产物-糖基化终末产物受体[the advanced glycation end product (AGE)-receptor of AGE (RAGE)]、表皮生长因子受体(epidermal growth factor receptor, EGFR)、血管内皮生长因子(vascular endothelial growth factor，VEGF) (图 2)。本文采用分子对接方法预测FMN与主要靶点的亲和力(图 3), FMN和主要靶点(TNF、NOS3、MTOR、CCND1、CASP3及HSP90AA1) 以及GPER构象表现出良好的结合作用, 结合能结果如表 1。

3 FMN的雌激素样活性

采用CCK-8实验检测FMN对MCF-7细胞增殖的影响。如图 4所示, FMN显著促进MCF-7细胞的生长(P < 0.05, P < 0.01, P < 0.001), 提示FMN具有雌激素样作用。

4 FMN对NR大鼠NR心肌面积和缺血心肌面积的影响

为了研究FMN是否具有减轻NR的作用, 本文采用硫黄素S、伊文思蓝和TTC染色观察无复流心肌面积和缺血心肌面积。如图 5A所示, 采用硫磺素S染色, 发现FMN明显降低心肌无复流面积(P < 0.05, P < 0.01), 伊文思蓝/TTC染色结果显示, FMN可以显著降低心肌缺血面积(P < 0.05, P < 0.01), 表明FMN具有减轻NR的作用。

5 FMN对NR大鼠心肌组织病理损伤的影响

为了进一步观察FMN对心肌组织病理损伤的影响, 本文进行了HE染色。Sham组呈现完整的心肌细胞, 心肌纤维排列整齐且紧密。NR组与Sham组相比, 心肌细胞紊乱, 核固缩溶解, 肌纤维肿胀, 大量炎性细胞浸润。然而, 与NR组相比, FMN组和SNP组心肌损伤程度明显减轻, 病变面积明显缩小, 水肿明显减轻, 可见少量炎性细胞浸润和间质水肿, 偶见空泡细胞, 心肌细胞较为完整, 心肌组织损伤程度明显改善(图 5B)。以上结果表明, FMN对于心肌组织具有较为显著的改善作用。

6 FMN对NR大鼠心脏结构和功能的影响

为了研究FMN是否改善NR大鼠心脏结构和功能, 本文采用小动物超声仪测量大鼠心脏的功能和结构。如图 6所示, 与Sham组相比, NR组EF、FS、LVOT峰值和LVSV均显著降低(P < 0.05, P < 0.001)。与NR组相比, FMN和SNP组的EF、FS和LVOT峰值显著高于NR组(P < 0.01)。超声结果显示, FMN能增加射血分数、左室短轴缩窄率和峰值血流速度, 有增加每搏输出量的趋势, 提示FMN有潜在改善心肌缺血再灌后无复流大鼠心脏结构和功能的作用。

7 FMN对NR大鼠心肌GPER、p-PI3K、p-Akt和p-eNOS蛋白表达的影响

如图 7所示, 与Sham组相比, NR组GPER、p-PI3K、p-Akt和p-eNOS蛋白表达显著降低(P < 0.05, P < 0.01, P < 0.001), 与NR组相比, FMN治疗组和SNP治疗组GPER、p-PI3K、p-Akt和p-eNOS蛋白表达水平增加(P < 0.05, P < 0.01, P < 0.001)。这些结果表明, FMN可以上调GPER蛋白的表达, 以激活PI3K/Akt/eNOS信号传导。

讨论

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NR是心肌梗死缺血后PCI治疗的一个主要临床表现, 由于微循环再灌注不足导致内皮细胞受损, 继发性缺血进而导致NR。临床上, NR有4种致病因素: 远端动脉粥样硬化血栓栓塞、缺血损伤、再灌注损伤和冠状动脉微循环障碍^[24]。目前对于NR的改善, 已有药物干预和器械治疗等, 但由于作用环节单一、操作复杂或费用昂贵等不足, 均未能获得理想效果^{[25, 26]}。流行病学和实验研究结果提示, 雌激素具有潜在心血管保护作用, 然而雌激素作为心脏保护剂的使用受到女性致癌作用和男性女性化作用的限制^{[27, 28]}。如果非致癌和非女性化的雌激素样化合物, 如天然植物雌激素(PE), 能够提供心脏保护, 这将为预防心血管疾病提供一种安全的方法。因此, 更多的研究转向作用更为温和的植物雌激素。

FMN是异黄酮家族的植物雌激素成员, 具有抗氧化、抗高血压、抗肿瘤和抗感染等药理作用^[12]。FMN也能够抑制炎症因子的分泌, 对心血管疾病有一定的保护作用, 其作用机制主要与抑制TLR4-NF/κB和JNK-NF/κB信号通路有关^[29]。FMN还能通过调控线粒体稳态有效地发挥心脏保护作用, 其机制主要通过激活SIRT1通路抑制线粒体氧化应激, 通过抑制RohA通路促进线粒体复合物的活性, 通过与雌激素受体的相互作用维持线粒体稳态, 促进NO产生^[30]。FMN亦可通过促进过氧化物酶体增殖物激活受体γ (PPAR-γ) 表达保护血管内皮细胞^[31]。富含FMN的通脉养心丸已被证实具有改善NR的作用^[11], 提示FMN可能具有改善NR的作用。

本研究通过整合药理学分析预测FMN改善NR作用主要与PI3K-Akt信号通路有关。研究报道, GPER介导的PI3K/Akt/eNOS和cAMP/PKA通路在改善心肌缺血再灌后无复流中发挥必不可少的作用^{[7, 32]}, 前期研究也发现GPER能激活PI3K/Akt/eNOS和cAMP/PKA途径, 活化eNOS产生NO, 产生舒张血管效应, 从而改善NR^{[11, 23]}。本研究发现, FMN可以促进MCF-7细胞的生长, 提示FMN具有雌激素样作用。体内实验显示, FMN能减轻NR, 其通过增加射血分数、左室短轴缩窄率和峰值血流速度改善心脏结构和功能的作用。炎症反应是心肌缺血/再灌注(myocardial ischemia/reperfusion, I/R) 损伤的重要组成部分, 已被认为与NR有关^[33]。病理损伤结果表明, FMN能改善炎性细胞浸润和间质水肿情况, 心肌损伤程度明显减轻。WB实验证实, FMN促进GPER、p-PI3K、p-Akt和p-eNOS蛋白表达, 通过分子对接验证FMN与GPER及eNOS具有良好结合能力, 提示FMN可能通过上调GPER激活PI3K/Akt/eNOS信号通路, 产生舒张血管效应, 从而改善NR。

综上, 本研究进行了整合药理学和实验验证, 揭示了FMN改善NR的作用及机制。结果证实, FMN具有改善NR的作用, 其作用可能是通过上调GPER激活PI3K/Akt/eNOS信号通路来实现的。后期本课题组会运用心肌细胞GPER基因敲除小鼠和缺氧/复氧微血管内皮细胞和心肌细胞模拟体内外NR模型实验进行深入的机制研究。本研究为NR的临床诊断及治疗提供理论依据和实验基础, 进一步为中药新药开发探索新的研究模式。

作者贡献: 陈婷提出整体研究思路, 负责论文撰写的指导与修改; 刘海瑞负责动物实验和体外实验, 以及文章书写; 刘海瑞、叶麟晰、林佳美慧、刘倩和彭雅旋负责网络药理学分析。

利益冲突: 所有作者均声明没有利益冲突。

基金

收起

湖南省自然科学基金资助项目-科药联合基金(2022JJ80112)
湖南省教育厅科学研究优秀青年项目(22B0367)

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2023年第58卷第6期

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doi: 10.16438/j.0513-4870.2022-1442

接收时间：2022-12-30
首发时间：2025-11-21
出版时间：2023-06-12

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收稿日期：2022-12-30
修回日期：2023-04-26

基金

湖南省自然科学基金资助项目-科药联合基金(2022JJ80112)

湖南省教育厅科学研究优秀青年项目(22B0367)

作者信息

湖南中医药大学, 中西医结合心脑疾病防治湖南省重点实验室, 湖南长沙 410208

通讯作者:

*陈婷, Tel: 86-731-88458862, E-mail: chenting520@hnucm.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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