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Icariin alleviates ferroptosis-related atherosclerosis by promoting autophagy in xo-LDL-induced vascular endothelial cell injury and atherosclerotic mice
淫羊藿苷通过促进 xo-LDL 诱导的血管内皮细胞损伤和动脉粥样硬化小鼠的自噬减轻铁死亡相关的动脉粥样硬化

Xindong Wang

Corresponding Author

Xindong Wang

Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, China

Correspondence

Xindong Wang, Cardiovascular Department, Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, No. 100 Shizi Street, Hongshan Road, Qixia District, Nanjing City 210028, Jiangsu Province, China.

Email: wangxindong@jsatcm.com

Xiaoxia Qi, Science and Technology Department, Nanjing University of Chinese Medicine, Nanjing 210026, Jiangsu, China.

Email: 150058@njucm.edu.cn

Contribution: Conceptualization

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Meng Zhang

Meng Zhang

The Third Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, China

Contribution: Supervision, Validation

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Chenhan Mao

Chenhan Mao

The Third Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, China

Contribution: Methodology, Project administration, Software, Supervision

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Chengbo Zhang

Chengbo Zhang

The Third Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, China

Contribution: Supervision

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Wenqi Ma

Wenqi Ma

The Third Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, China

Contribution: Visualization

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Jiahui Tang

Jiahui Tang

The Third Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, China

Contribution: Visualization, Writing - original draft, Writing - review & editing

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Dongyang Xiang

Dongyang Xiang

Guizhou University of Traditional Chinese Medicine, Guiyang, China

Contribution: Formal analysis

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Xiaoxia Qi

Corresponding Author

Xiaoxia Qi

Science and Technology Department, Nanjing University of Chinese Medicine, Nanjing, China

Correspondence

Xindong Wang, Cardiovascular Department, Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, No. 100 Shizi Street, Hongshan Road, Qixia District, Nanjing City 210028, Jiangsu Province, China.

Email: wangxindong@jsatcm.com

Xiaoxia Qi, Science and Technology Department, Nanjing University of Chinese Medicine, Nanjing 210026, Jiangsu, China.

Email: 150058@njucm.edu.cn

Contribution: Writing - original draft, Writing - review & editing

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First published: 21 June 2023
Citations: 1
UBC eLink

Xindong Wang and Meng Zhang were contributed equally to this work.

Abstract  抽象的

Vascular endothelial cells (VECs) are located between the blood plasma and the vascular tissue, and the ferroptosis (iron-dependent programmed cell death) of VECs can lead to a range of cardiovascular diseases. Icariin is the main active ingredient of Epimedium brevicornum Maxim., which can improve endothelial cell dysfunction. In the present study, the protective effects of icariin on oxidised low-density lipoprotein (ox-LDL)-treated VECs and high-fat diet-fed Apolipoprotein E-deficient mice were investigated. Inflammatory fibrosis in tissues and inflammatory factors in serum and cell supernatants were detected, and mitochondrial membrane potential and the expression levels of ferroptosis-associated proteins were also detected. The results revealed that icariin reduced the endothelial atherosclerotic plaque area and collagen fibres in aortic sinus tissue, and increased the viability and mitochondrial membrane potential, whereas it reduced the reactive oxygen species levels of VECs. The nucleation of transcription factor EB (TFEB) and subsequent autophagy were negatively associated with ferroptosis in endothelial cells, and the more prominent the autophagy, the lower the levels of ferroptosis. Furthermore, by co-treating the cells with icariin and the two autophagy inhibitors, Bafilomycin A1 (blocking autophagosome and lysosome fusion) and 3-methyladenine (blocking autophagosome formation), respectively, the promoting effects of icariin on autophagy were found to be mediated through the process of autophagosome–lysosome fusion. In in vivo experiments, icariin reduced ferroptosis, alleviated atherosclerotic lesions and increased the rate of TFEB nucleation. Additionally, it was found that ARG304, THR308 and GLN311 were the optimal binding sites for the interaction between icariin and TFEB. Taken together, these results suggest that the fusion of autophagosomes and lysosomes promoted by icarrin enhances autophagy and thus reduces ferroptosis. Therefore, icariin may be a potential candidate for the prevention of ferroptosis of VECs and, thus, for the treatment of cardiovascular diseases.
血管内皮细胞(VEC)位于血浆和血管组织之间,VEC的铁死亡(铁依赖性程序性细胞死亡)可导致一系列心血管疾病。淫羊藿苷是淫羊藿的主要活性成分,可改善内皮细胞功能障碍。在本研究中,研究了淫羊藿苷对氧化低密度脂蛋白(ox-LDL)处理的 VEC 和高脂饮食喂养的载脂蛋白 E 缺陷小鼠的保护作用。检测组织炎症纤维化情况以及血清和细胞上清液中炎症因子,并检测线粒体膜电位和铁死亡相关蛋白的表达水平。结果显示,淫羊藿苷减少了主动脉窦组织中的内皮动脉粥样硬化斑块面积和胶原纤维,增加了活力和线粒体膜电位,同时降低了VECs的活性氧水平。转录因子EB(TFEB)的成核和随后的自噬与内皮细胞的铁死亡呈负相关,并且自噬越显着,铁死亡水平越低。此外,通过分别用淫羊藿苷和两种自噬抑制剂巴弗洛霉素A1(阻断自噬体和溶酶体融合)和3-甲基腺嘌呤(阻断自噬体形成)共同处理细胞,发现淫羊藿苷对自噬的促进作用是通过介导的自噬体-溶酶体融合的过程。在体内实验中,淫羊藿苷可减少铁死亡,减轻动脉粥样硬化病变并增加 TFEB 成核率。 此外,还发现ARG304、THR308和GLN311是淫羊藿苷与TFEB相互作用的最佳结合位点。综上所述,这些结果表明淫羊藿素促进自噬体和溶酶体的融合增强自噬,从而减少铁死亡。因此,淫羊藿苷可能是预防 VEC 铁死亡的潜在候选者,从而可用于治疗心血管疾病。

Abbreviations  缩写

  • 3-MA
  • 3-methyladenine  3-甲基腺嘌呤
  • Baf A1  巴夫A1
  • bafilomycin C1  巴弗洛霉素C1
  • BCA
  • bicinchoninic acid  二辛可宁酸
  • DCFH-DA
  • 2,7-dichlorodihydrofluorescein diacetate
    2,7-二氯二氢荧光素二乙酸酯
  • FTH1  光纤到户1
  • ferritin heavy chain 1  铁蛋白重链1
  • H&E  
  • hematoxylin and eosin  苏木精和伊红
  • HUVECs  人脐静脉内皮细胞
  • human umbilical vein endothelial cells
    人脐静脉内皮细胞
  • MMP  基质金属蛋白酶
  • mitochondrial membrane potential
    线粒体膜电位
  • ROS  活性氧
  • reactive oxygen species  活性氧
  • S.D.  标清
  • standard deviation  标准差
  • TC
  • total cholesterol  总胆固醇
  • TFEB  铁氟龙
  • transcription factor EB  转录因子EB
  • TfR1  转铁蛋白R1
  • transferrin receptor1  转铁蛋白受体1
  • TG
  • triglycerides  甘油三酯
  • TRP
  • transient receptor potential
    瞬时受体电位
  • VECs  血管内皮细胞
  • vascular endothelial cells
    血管内皮细胞
  • 1 INTRODUCTION  1 简介

    Atherosclerosis is the most common disease of the cardiovascular system worldwide; however, its exact cause remains unknown. Endothelial cells, also known as vascular endothelial cells (VECs), are a continuous flat layer of cells lining the blood vessels and vascular smooth muscle. These cells play a vital role in maintaining vascular permeability, transmitting vascular information and secreting vasoactive substances (Aird, 2003). The impairment of VEC function is closely related to hypertension, diabetes, hyperlipidaemia and hypoxia, and can induce atherosclerosis, from the basic to the clinical level (Brown et al., 2017; Gimbrone & García-Cardena, 2016; Wils et al., 2017).
    动脉粥样硬化是全世界最常见的心血管系统疾病;然而,其确切原因仍不清楚。内皮细胞,也称为血管内皮细胞(VEC),是衬在血管和血管平滑肌上的连续平坦细胞层。这些细胞在维持血管通透性、传递血管信息和分泌血管活性物质方面发挥着至关重要的作用(Aird, 2003 )。 VEC功能受损与高血压、糖尿病、高脂血症和缺氧密切相关,可诱发动脉粥样硬化,从基础到临床水平(Brown et al., 2017 ; Gimbrone & García-Cardena, 2016 ; Wils et al., 2017 )。

    During the early stages of atherosclerosis, large amounts of low-density lipoprotein (LDL) leak into the intima to be oxidised by free radicals, such as reactive oxygen species (ROS), to produce oxidised LDL (ox-LDL). The main source of endogenous ROS is the mitochondria, which are close to lysosomes (Li et al., 2015). In damaged mitochondria, the depolarisation of mitochondrial membrane potential (MMP) occurs and a depolarisation in MMP leads to the opening of the permeability transition pore and the release of large amounts of ROS. In addition, the excessive accumulation of ROS can lead to the ferroptosis (iron-dependent programmed cell death) of VECs (Bai et al., 2020; Redza-Dutordoir & Averill-Bates, 2016). Ferroptosis is a novel form of cell death triggered by lipid peroxidation in an iron-dependent manner, and has been a new hotspot for cardiovascular diseases, including endothelial cell injury, ischemic organ reperfusion injury and cardiovascular diseases (He et al., 2021; Jiang et al., 2021; Kobayashi et al., 2018). There are two main protein biomarkers for ferroptosis. One is transferrin receptor1 (TfR1), located on the cell membrane and is responsible for controlling Fe3+ transport across the cell membrane. The other is ferritin heavy chain 1 (FTH1), which is responsible for the storage of Fe2+; they are both considered to be ferroptosis markers (Feng et al., 2020; Gao et al., 2015). Recently, it has been reported that the inhibition of ferroptosis-induced lipid peroxidation and dysfunction in endothelial cells alleviated atherosclerosis (Bai et al., 2020; He et al., 2021).
    在动脉粥样硬化早期,大量低密度脂蛋白(LDL)渗入内膜,被活性氧(ROS)等自由基氧化,产生氧化型低密度脂蛋白(ox-LDL)。内源性ROS的主要来源是线粒体,它靠近溶酶体(Li et al., 2015 )。在受损的线粒体中,线粒体膜电位(MMP)发生去极化,MMP的去极化导致通透性转换孔打开并释放大量ROS。此外,ROS的过度积累会导致VEC的铁死亡(铁依赖性程序性细胞死亡)(Bai et al., 2020 ;Redza-Dutordoir & Averill-Bates, 2016 )。铁死亡是一种由铁依赖性脂质过氧化引发的新型细胞死亡形式,已成为心血管疾病的新热点,包括内皮细胞损伤、缺血性器官再灌注损伤和心血管疾病(He et al., 2021 ; Jiang等人, 2021 ;小林等人, 2018 )。铁死亡有两种主要的蛋白质生物标志物。其中之一是转铁蛋白受体1 (TfR1),位于细胞膜上,负责控制Fe 3+跨细胞膜的转运。另一个是铁蛋白重链1(FTH1),负责储存Fe 2+ ;它们都被认为是铁死亡标记物(Feng et al., 2020 ;Gao et al., 2015 )。 最近有报道称,抑制铁死亡诱导的内皮细胞脂质过氧化和功能障碍可减轻动脉粥样硬化(Bai et al., 2020 ;He et al., 2021 )。

    Clinically, 3-atorvastatinstatins are used to prevent and treat atherosclerosis caused by the apoptosis of VECs; however, the long-term use of statins may lead to numerous severe adverse effects, such as liver damage, muscle toxicity and gastrointestinal irritation (Singh & Bedi, 2013). In addition, aspirin has also been used to reduce blood clots in patients with atherosclerosis, due to its properties of inhibiting platelet aggregation, although the reduction in blood clotting capacity may cause major bleeding (Baigent et al., 2009). Therefore, attempts have been made to identify safer and more effective therapeutic agents, and there are numerous reports of natural drug extracts or monomers that improve VEC dysfunction and inhibit endothelial cell apoptosis (Duan et al., 2021; Tang et al., 2009; Zhang et al., 2012). Icariin, a natural plant extract and traditional herbal monomer, is the main active ingredient in Epimedium brevicornum Maxim., and previous studies have demonstrated its safety and stability (Fan et al., 2019; Qin et al., 2020). A variety of pharmacological effects have been discovered in icarrin. It has been found that the use of 40 mg/kg icarrin improves cardiomyocyte apoptosis in spontaneously hypertensive rats (Qian, Zhu, Li, Li, et al., 2021). Icariin has also been shown to exert hepatoprotective effects on rodents with liver fibrosis induced by carbon tetrachloride (Ye et al., 2020). Of note, it is known to increase the number of cardiomyocytes and improve endothelial dysfunction, and it can also correct endothelial dysfunction by inhibiting the proliferation and migration of smooth muscle cells to prevent atherosclerosis (Duan et al., 2021; Fang & Zhang, 2017; Wang et al., 2016; Zhu et al., 2005).
    临床上,3-阿托伐他汀用于预防和治疗VEC凋亡引起的动脉粥样硬化;然而,长期使用他汀类药物可能会导致许多严重的副作用,例如肝损伤、肌肉毒性和胃肠道刺激(Singh & Bedi, 2013 )。此外,阿司匹林还被用于减少动脉粥样硬化患者的血栓,因为它具有抑制血小板聚集的特性,尽管凝血能力的降低可能会导致大出血(Baigent et al., 2009 )。因此,人们尝试寻找更安全、更有效的治疗药物,并且有大量关于改善VEC功能障碍和抑制内皮细胞凋亡的天然药物提取物或单体的报道(Duan等, 2021 ;Tang等, 2009 ;张等人, 2012 )。淫羊藿苷是一种天然植物提取物和传统草药单体,是淫羊藿中的主要活性成分,前期研究已证明其安全性和稳定性(Fan et al., 2019 ;Qin et al., 2020 )。淫羊藿苷具有多种药理作用。研究发现,使用 40 mg/kg 淫羊藿苷可改善自发性高血压大鼠的心肌细胞凋亡(Qian, Zhu, Li, Li, et al., 2021 )。淫羊藿苷还被证明对四氯化碳诱导的肝纤维化啮齿动物具有保肝作用(Ye et al., 2020 )。 值得注意的是,已知它可以增加心肌细胞数量并改善内皮功能障碍,还可以通过抑制平滑肌细胞的增殖和迁移来纠正内皮功能障碍,从而预防动脉粥样硬化(Duan et al., 2021 ; Fang & Zhu, 2017) ;王等人, 2016 ;朱等人, 2005 )。

    In the present study, to investigate the role of icarrin in alleviating atherosclerosis, an in vitro model of VEC treated with ox-LDL and an in vivo model of Apolipoprotein E-deficient (ApoE−/−) mice fed a high-fat diet (HFD) were established. By investigating the protective effects of icariin on VECs and the possible mechanisms by measuring cell viability, Fe2+ content, ROS levels, autophagy in mitochondria and lysosomes, as well as the expression levels of related proteins, the present study aimed to provide a theoretical basis for exploring new VEC dysfunctions.
    在本研究中,为了研究淫羊藿苷在缓解动脉粥样硬化中的作用,使用ox-LDL治疗的VEC体外模型和喂食高脂饮食的载脂蛋白E缺陷(ApoE -/− )小鼠的体内模型( HFD)成立。本研究通过测定细胞活力、Fe 2+含量、ROS水平、线粒体和溶酶体自噬以及相关蛋白的表达水平,探讨淫羊藿苷对VEC的保护作用及其可能的机制,旨在提供理论依据。探索新的 VEC 功能障碍的基础。

    2 METHODS AND MATERIALS  2 方法和材料

    2.1 Reagents and antibodies
    2.1 试剂和抗体

    Icariin (purity ≥98%), purchased from Beijing Solarbio Science & Technology Co., Ltd., was dissolved in dimethyl sulphoxide (DMSO, aprotic polar solvents). The following antibodies used in the present study, such as anti-LAMP1 (cat. no. ab24170), anti-TRPML1 (cat. no. ab272608), anti-TfR1 (cat. no. ab214039), anti-glutathione peroxidase 4 (GPX4; cat. no. ab125066), anti-ferritin heavy chain (FTH1; cat. no. ab75972), anti-LC3B antibody (cat. no. ab192890), anti-GAPDH (cat. no. ab8245) and goat anti-rabbit IgG H&L (HRP) (cat. no. ab6721) antibodies were purchased from Abcam. pCMV-GFP-LC3B (D2815) was purchased from Beyotime Institute of Biotechnology. The fusion protein of GFP-LC3 can be efficiently expressed in the target cells after transfection, exhibiting bright green fluorescence, which can be used for the study of autophagy. 3-Methyladenine (3-MA; HY-19312, MedChemExpress) and bafilomycin C1 (Baf A1, HY-130173, MedChemExpress) were both autophagy inhibitors. Ox-LDL was purchased from MilliporeSigma.
    淫羊藿苷(纯度≥98%)购自北京索拉宝科技有限公司,溶解于二甲亚砜(DMSO,非质子极性溶剂)中。本研究中使用的抗体包括抗LAMP1(目录号ab24170)、抗TRPML1(目录号ab272608)、抗TfR1(目录号ab214039)、抗谷胱甘肽过氧化物酶4( GPX4;目录号 ab125066),抗铁蛋白重链(FTH1;目录号。抗 LC3B 抗体(目录号 ab75972)、抗 LC3B 抗体(目录号 ab192890)、抗 GAPDH(目录号 ab8245)和山羊抗兔 IgG H&L(HRP)(目录号 ab6721)抗体购自 Abcam。 pCMV-GFP-LC3B(D2815)购自碧阳天生物技术研究所。 GFP-LC3融合蛋白转染后可在靶细胞中高效表达,呈现亮绿色荧光,可用于自噬的研究。 3-甲基腺嘌呤(3-MA;HY-19312,MedChemExpress)和巴弗洛霉素 C1(Baf A1,HY-130173,MedChemExpress)都是自噬抑制剂。 Ox-LDL 购自 MilliporeSigma。

    2.2 Cells and cell culture
    2.2 细胞和细胞培养

    Human umbilical vein endothelial cells (HUVECs) obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (3rd generation) were placed in Dulbecco's modified Eagle's medium containing penicillin–streptomycin–amphotericin B solution (Beyotime Institute of Biotechnology) with 10% fetal bovine serum and cultured in a 5% CO2 humidified incubator at 37°C. Passages were performed when the confluence rate exceeded 80%.
    取自中国科学院典型培养物保藏中心细胞库(第3代)的人脐静脉内皮细胞(HUVEC)置于含有青霉素-链霉素-两性霉素B溶液的Dulbecco改良Eagle培养基(碧瑶天生物技术研究所)中,加入10 %胎牛血清并在5%CO 2湿润培养箱中于37℃培养。当汇合率超过80%时进行传代。

    2.3 Cell viability assay  2.3 细胞活力测定

    Cell viability was detected using a Cell Viability Assay Kit (CCK-8, Elabscience). In the presence of electron-coupled reagents, it can be oxidised by intracellular dehydrogenases to produce a water-soluble orange formazan, and the amount of formazan produced is proportional to the number of living cells. The cells were placed in 96-well plates with 100 μL cells per well and pre-incubated in a 5% CO2 incubator for 4 h at 37°C. The cells were treated with the drug for 4 h, and were then treated with 10 μL CCK-8 solution per well for 2 h. The absorbance at 450 nm was then measured.
    使用细胞活力测定试剂盒(CCK-8,Elabscience)检测细胞活力。在电子耦合试剂存在下,可被细胞内脱氢酶氧化,产生水溶性橙色甲臜,产生的甲臜量与活细胞数量成正比。将细胞置于96孔板中,每孔100μL细胞,并在5%CO 2培养箱中37℃预孵育4小时。将细胞用药物处理4小时,然后每孔用10μL CCK-8溶液处理2小时。然后测量 450 nm 处的吸光度。

    2.4 Experimental protocol
    2.4 实验方案

    For the first experiment, the cells were divided into five groups as follows: The control group (negative control), ox-LDL group (positive control), 12.5-Icariin group (12.5 μg/mL icariin), 25-Icariin group (25 μg/mL icariin) and 50-Icariin group (50 μg/mL icariin). The cells were plated in six-well plates at 6 × 104 cells/well and incubated for 24 h. The cells were pre-treated with 12.5, 25 and 50 μg/mL icariin for 2 h, and the incubation was continued for 24 h with the replacement of each drug culture medium containing 20 μg/mL ox-LDL solution. The cells in the control group grew normally without treatment. The cells in the ox-LDL group were treated with 20 μg/mL ox-LDL and without icariin.
    第一个实验将细胞分为五组:对照组(阴性对照)、ox-LDL组(阳性对照)、12.5-淫羊藿苷组(12.5 μg/mL淫羊藿苷)、25-淫羊藿苷组(25 μg/mL 淫羊藿苷)和 50-淫羊藿苷组(50 μg/mL 淫羊藿苷)。将细胞以6×10 4 个细胞/孔铺在六孔板中并孵育24小时。细胞用12.5、25和50 μg/mL淫羊藿苷预处理2 h,继续培养24 h,更换含20 μg/mL ox-LDL溶液的每种药物培养基。对照组的细胞在未经处理的情况下正常生长。 ox-LDL 组的细胞用 20 μg/mL ox-LDL 处理,不含淫羊藿苷。

    Furthermore, a lentiviral vector (TRPML1-siRNA) was constructed for RNA interference against the TRPML1 gene and its negative control. A total of 1 × 105 cells, infected with TRPML1-siRNA as described above, were seeded on six-well plates for 24 h and then exposed to 6 μg/mL polyethylene glycol and lentiviral vector. After 4 h, the medium was replaced with fresh normal medium and the cells were then treated with icariin.
    此外,构建了慢病毒载体(TRPML1-siRNA),用于针对TRPML1基因及其阴性对照的RNA干扰。将如上所述用TRPML1-siRNA感染的总共1×10 5 个细胞接种在六孔板上24小时,然后暴露于6μg/mL聚乙二醇和慢病毒载体。 4小时后,用新鲜的正常培养基更换培养基,然后用淫羊藿苷处理细胞。

    2.5 JC-1 flow detection of mitochondrial membrane potential

    In damaged mitochondria, the depolarisation of MMP occurs and the reduction in MMP causes a series of regulatory cascade responses, such as the opening of the permeability transition pore and the release of large amounts of ROS. JC-1 (J6004, UE, China) is an ideal fluorescent probe for the detection of the mitochondrial membrane potential Δ Ψm. A decrease in MMP is a marker of early apoptosis and, therefore, the transition from red to green fluorescence of JC-1 can be used as an indicator of early apoptotic cell death. Following incubation at 37°C for 15 min, the supernatant was removed by centrifugation and the procedure is repeated, followed by re-election in phosphate buffer solution and assaying on the machine. For normal cells, JC-1 red aggregates in mitochondria can be detected in PE or PI channels; for apoptotic cells, JC-1 green monomers can be detected in FITC channels.
    在受损的线粒体中,MMP发生去极化,MMP的减少引起一系列调节级联反应,例如通透性转换孔的开放和大量ROS的释放。 JC-1(J6004,UE,中国)是一种理想的线粒体膜电位ΔΨm检测荧光探针。 MMP 的减少是早期凋亡的标志,因此,JC-1 从红色荧光到绿色荧光的转变可以用作早期凋亡细胞死亡的指标。 37℃孵育15分钟后,离心去除上清液,重复该过程,然后在磷酸盐缓冲溶液中重选并在机器上进行测定。对于正常细胞,PE或PI通道可检测到线粒体内的JC-1红色聚集体;对于凋亡细胞,可以在 FITC 通道中检测到 JC-1 绿色单体。

    2.6 Relative level of ROS detected by DCF probe
    2.6 DCF探针检测的ROS相对水平

    The level of intracellular ROS was measured using the ROS fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, No. S0033M, Beyotime Institute of Biotechnology), which can freely cross the cell membrane and be hydrolysed to DCFH, which is then oxidised by intracellular ROS to the green fluorescent substance DCF. The cells were incubated overnight in 96-well plates (1.5 × 105 cells/well) and then incubated with a final concentration of 25 μM DCFH-DA for 30 min, followed by treatment with icariin. The fluorescence intensity of DCF was detected using a fluorescence microscope and images were obtained, and the green fluorescence intensity was analysed using ImageJ software (National Institutes of Health), which can indicate the relative amount of ROS in the cells.
    使用ROS荧光探针2,7-二氯二氢荧光素二乙酸酯(DCFH-DA,编号S0033M,碧云天生物技术研究所)测量细胞内ROS水平,该探针可以自由穿过细胞膜并水解为DCFH,然后DCFH被氧化通过细胞内ROS转化为绿色荧光物质DCF。将细胞在96孔板中孵育过夜(1.5×10 5 个细胞/孔),然后与终浓度25μM DCFH-DA一起孵育30分钟,然后用淫羊藿苷处理。使用荧光显微镜检测DCF的荧光强度并获得图像,使用ImageJ软件(National Institutes of Health)分析绿色荧光强度,可以指示细胞中ROS的相对量。

    2.7 Staining of lysosomes and mitochondria
    2.7 溶酶体和线粒体染色

    The cells were fixed in paraformaldehyde and treated with 0.5% Triton X-100 for 20 min at room temperature and then sealed with serum for 30 min. The cells were then incubated with Lyso-Tracker Red (lysosome, C1046, Beyotime Institute of Biotechnology) for 60 min at 37°C, and incubated with YF®488 Click-iT EdU Imaging Kits (mitochondrion, C6015, UE) for 40 min protected from light. They were then observed using a fluorescence microscope and representative images were obtained.
    将细胞固定在多聚甲醛中,并在室温下用0.5%Triton X-100处理20分钟,然后用血清密封30分钟。然后将细胞与 Lyso-Tracker Red(溶酶体,C1046,Beyotime Institute of Biotechnology)在 37°C 孵育 60 分钟,并与 YF®488 Click-iT EdU 成像试剂盒(线粒体,C6015,UE)孵育 40 分钟避光。然后使用荧光显微镜观察它们并获得代表性图像。

    2.8 Immunofluorescence detection of TFEB activation in the nucleus
    2.8 免疫荧光检测细胞核内TFEB活化情况

    The cells were treated as aforementioned in section 2.7 and then incubated with TFEB antibody overnight at 4°C, followed by 1 h of incubation with secondary antibody at 37°C. The cytoskeleton and nuclei were stained using Actin-Tracker Red-Rhodamine (C2207S, Beyotime Institute of Biotechnology) and DAPI (C1002, Beyotime Institute of Biotechnology), respectively.
    按照第 2.7 节中的上述方法处理细胞,然后与 TFEB 抗体在 4°C 下孵育过夜,然后与二抗在 37°C 下孵育 1 小时。细胞骨架和细胞核分别使用Actin-Tracker Red-Rhodamine(C2207S,Beyotime Institute of Biotechnology)和DAPI(C1002,Beyotime Institute of Biotechnology)染色。

    2.9 pCMV-GFP-LC3 transfection and autophagy monitoring
    2.9 pCMV-GFP-LC3转染和自噬监测

    The cells were seeded in 24-well plates and cultured to 90% confluency. They were then transfected with pCMV-GFP-LC3 plasmid using transfection reagent according to the manufacturer's instructions. The cells were treated as aforementioned in section 2.4 and then observed and photographed using a fluorescence microscope. In the case of non-autophagy, GFP-LC3 is present in the cytoplasm in a diffused form under a fluorescence microscope; however, in the case of autophagy, GFP-LC3 accumulates on the autophagosomal membrane and is expressed as dots or punctae.
    将细胞接种到24孔板中并培养至90%汇合。然后根据制造商的说明使用转染试剂用pCMV-GFP-LC3质粒转染它们。按照2.4节所述处理细胞,然后使用荧光显微镜观察和拍照。非自噬情况下,荧光显微镜下GFP-LC3以弥散形式存在于细胞质中;然而,在自噬的情况下,GFP-LC3 积聚在自噬体膜上并表达为点或点。

    2.10 Animals and experimental protocol
    2.10 动物和实验方案

    For the present study, 8-week-old (weighing 25 ± 2 g) ApoE−/− male mice with C57BL/6 mice were obtained from Jiangsu ALF Biotechnology Co., Ltd. and housed in a specific pathogen-free environment. The animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing University of Chinese Medicine. The model of atherosclerosis was constructed using ApoE−/− mice fed a HFD (0.15% cholesterol +25% fat; Fuzhou Junke Biological Technology Co., Ltd.) for 12 weeks (Bai et al., 2020). The ApoE−/− mice were randomly divided into four groups as follows: (a) The model group: ApoE−/− mice fed a HFD and received 0.5% CMC-Na by gavage; (b) 40-Icariin group: ApoE−/− mice fed a HFD and gavaged with 40 mg/kg icariin (0.5% CMC-Na as a solvent); (c) 80-Icariin group: ApoE−/− mice fed a HFD and gavaged with 80 mg/kg icariin (0.5% CMC-Na as a solvent); (d) 3-atorvastatin group: ApoE−/− mice fed a HFD and gavaged with 3 mg/kg atorvastatin (0.5% CMC-Na as a solvent) (Lv et al., 2021). The C57BL/6 mice in the control group were fed with normal feed and gavaged with 0.5% CMC-Na. Treatment with icariin and atorvastatin began at week 4 and continued for 8 weeks. At the end of the experiment, the mice were euthanised by cervical dislocation. The mice were grouped using a random method as follows: Mice were numbered sequentially from 1 to 48, and 48 numbers were arbitrarily transcribed, starting from a certain number in a row of the random number table; each number was divided by 4 and the remainder 1, 2, 3 and 4 represented A, B, C and D. Mice were assigned to the four groups and then adjusted to ensure that there were 12 mice in each group.
    本研究中,8周龄(体重25±2g)ApoE −/−雄性小鼠与C57BL/6小鼠均购自江苏艾尔福生物科技有限公司,饲养在特定的无病原体环境中。动物实验经南京中医药大学机构动物护理与使用委员会批准。采用HFD(0.15%胆固醇+25%脂肪;福州君科生物科技有限公司)喂养12周的ApoE −/−小鼠构建动脉粥样硬化模型(Bai et al., 2020 )。将ApoE −/−小鼠随机分为四组:(a)模型组:ApoE −/−小鼠高脂饮食饲喂,0.5%CMC-Na灌胃; (b) 40-淫羊藿苷组:ApoE −/−小鼠喂食 HFD 并灌胃 40 mg/kg 淫羊藿苷(0.5% CMC-Na 作为溶剂); (c) 80-淫羊藿苷组:ApoE -/−小鼠喂食 HFD 并灌胃 80 mg/kg 淫羊藿苷(0.5% CMC-Na 作为溶剂); (d) 3-阿托伐他汀组:ApoE -/−小鼠喂食 HFD 并灌胃 3 mg/kg 阿托伐他汀(0.5% CMC-Na 作为溶剂)(Lv 等人, 2021 )。对照组C57BL/6小鼠正常饲料喂养,并用0.5%CMC-Na灌胃。淫羊藿苷和阿托伐他汀治疗从第 4 周开始并持续 8 周。实验结束时,通过颈椎脱位法处死小鼠。采用随机方法对小鼠进行分组:将小鼠从1到48依次编号,从随机数字表中某一行的某一数字开始任意转录48个数字;每个数字除以 4,余数 1、2、3 和 4 分别代表 A、B、C 和 D。 将小鼠分为四组,然后进行调整以确保每组有 12 只小鼠。

    2.11 Histopathological analysis
    2.11 组织病理学分析

    The aortic arch was fixed in 4% paraformaldehyde and cut into 4-μm-thick sections, and then stained with hoematoxylin and eosin (H&E) and Masson's trichrome stain for histopathological analysis.
    将主动脉弓固定在4%多聚甲醛中并切成4μm厚的切片,然后用苏木精和伊红(H&E)和Masson三色染色进行组织病理学分析。

    2.12 Western blot analysis
    2.12 蛋白质印迹分析

    The cells were lysed with cell lysis buffer for western blot analysis and IP (P0013, Beyotime Institute of Biotechnology) on ice and collected as protein extracts by centrifugation at 12,000 × g for 30 min at 4°C. The protein concentrations were determined using a bicinchoninic acid (BCA) Protein Assay kit (P0012S, Beyotime Institute of Biotechnology) and equal amounts of protein extracts were separated by 10% SDS–PAGE and then transferred to nitrocellulose membranes. The membranes were blocked in 5% skimmed milk for 2 h followed by overnight incubation with primary antibodies at 4°C and then incubated with corresponding secondary antibody for 2 h at room temperature. The gray values of the protein bands were analysed using ImageJ software (National Institutes of Health) and GAPDH was used as a loading control.
    用细胞裂解缓冲液裂解细胞,用于冰上蛋白质印迹分析和 IP(P0013,Beyotime 生物技术研究所),并通过在 4°C 下以 12,000 × g 离心 30 分钟收集蛋白质提取物。使用二辛可宁酸 (BCA) 蛋白质测定试剂盒(P0012S,Beyotime 生物技术研究所)测定蛋白质浓度,并通过 10% SDS-PAGE 分离等量的蛋白质提取物,然后转移到硝酸纤维素膜上。将膜在 5% 脱脂牛奶中封闭 2 小时,然后与一抗在 4℃ 下孵育过夜,然后与相应的二抗在室温下孵育 2 小时。使用ImageJ软件(美国国立卫生研究院)分析蛋白质条带的灰度值,并使用GAPDH作为上样对照。

    2.13 Molecular docking analyses of TFEB
    2.13 TFEB的分子对接分析

    In order to elucidate the association between icariin (CAS: #489-32-7) and TFEB (AF-P19484-F1, AlphaFold database), protein molecules and drug small molecules were processed and saved as PDB QT files using Autodock software. The processed ligand molecules (icariin) and receptor molecules (TFEB) were analysed to generate docked.dlg files, and the lowest binding conformation was selected based on binding energy, etc. and saved as PDBQT files. The molecular conformations were Cartoon displayed and images were generated using PyMOL software.
    为了阐明淫羊藿苷(CAS:#489-32-7)和 TFEB(AF-P19484-F1,AlphaFold 数据库)之间的关联,使用 Autodock 软件处理蛋白质分子和药物小分子并保存为 PDB QT 文件。对处理后的配体分子(淫羊藿苷)和受体分子(TFEB)进行分析,生成docked.dlg文件,并根据结合能等选择最低结合构象,保存为PDBQT文件。分子构象以卡通方式显示并使用 PyMOL 软件生成图像。

    2.14 Microscale thermophoresis
    2.14 微尺度热泳

    The TFEB protein was commixed with a Cy5-labelled double-stranded DNA probe in a binding buffer and incubated for 5 min. All liquids were added to the capillary tube and various parameters of the interaction between these two molecules were obtained by testing the impact of assorted concentrations of binding molecules on the distribution of the dynamic equilibrium state of the fluorescent molecule by thermophoresis, using Affinity Analysis 2.3 for software data analysis and curve fitting.
    将 TFEB 蛋白与 Cy5 标记的双链 DNA 探针在结合缓冲液中混合并孵育 5 分钟。将所有液体加入毛细管中,通过热泳测试不同浓度的结合分子对荧光分子动态平衡态分布的影响,使用Affinity Analysis 2.3进行分析,得到这两个分子之间相互作用的各种参数。软件数据分析和曲线拟合。

    2.15 Statistical analysis
    2.15 统计分析

    The data in the present study were analysed using Graphpad Prism 8.02 software and expressed as the mean ± standard deviation (S.D.). Differences between two groups were compared using an unpaired Student's t-test and differences between multiple groups were compared using a one-way ANOVA and Tukey's post hoc test.
    本研究中的数据使用Graphpad Prism 8.02软件进行分析并表示为平均值±标准差(SD)。使用未配对的学生 t 检验比较两组之间的差异,使用单向方差分析和 Tukey 事后检验比较多组之间的差异。

    3 RESULTS  3 个结果

    3.1 Icariin improves the viability and MMP
    3.1淫羊藿苷提高活力和MMP

    As shown in Figure 1a, the viability of the VECs decreased significantly when the concentration of icariin reached 100 μg/mL, and the concentrations set for the experiments were 12.5, 25 and 50 μg/mL. Furthermore, the modelling concentration of ox-LDL for was chosen to be 20 μg/mL (Figure 1b). The viability of the VECs in the ox-LDL group was significantly lower than that in the control group; however, various concentrations of icariin increased cell viability compared with the ox-LDL group (p < 0.05, Figure 1c). As shown in Figure 1d, the Fe2+ content in the ox-LDL group was increased significantly compared with that of the control group, and icariin treatment decreased the content of Fe2+ in a concentration-dependent manner. The results of JC-1 staining revealed that ox-LDL treatment reduced the aggregate/monomer ratio; however, icariin increased the aggregate/monomer ratio, which indicated that icariin attenuated the reduction in MMP induced by ox-LDL (Figure 1e). In addition, the levels of ROS were also detected. The level of ROS was markedly higher in ox-LDL group compared with the control group, and icariin decreased the levels of ROS in a concentration-dependent manner (Figure 1f).
    如图1a所示,当淫羊藿苷浓度达到100μg/mL时,VEC的活力显着下降,实验设定的浓度为12.5、25和50μg/mL。此外,ox-LDL 的建模浓度选择为 20 μg/mL(图1b )。 ox-LDL组VEC活力显着低于对照组;然而,与 ox-LDL 组相比,不同浓度的淫羊藿苷增加了细胞活力( p < 0.05,图1c )。如图1d所示,ox-LDL组Fe 2+含量较对照组显着升高,且淫羊藿苷处理呈浓度依赖性降低Fe 2+含量。 JC-1染色结果显示ox-LDL处理降低了聚集体/单体比;然而,淫羊藿苷增加了聚集体/单体比率,这表明淫羊藿苷减弱了ox-LDL引起的MMP的减少(图1e )。此外,还检测了ROS的水平。与对照组相比,ox-LDL组ROS水平明显升高,淫羊藿苷以浓度依赖性方式降低ROS水平(图1f )。

    Details are in the caption following the image
    Icariin improves cell viability and mitochondrial membrane potential. (a) The screening of icariin for use in experiments (n = 5). (b) The screening of ox-LDL for modeling (n = 5). (c) The viability of VECs in the different groups (n = 5). (d) The content of Fe2+ in the different groups (n = 3). (e) The aggregate:monomer (red/green) ratio in the different groups (n = 3). (f) The levels of ROS in the different groups (n = 3). “*” indicates a statistically significant difference (*p < 0.05 and **p < 0.01). Similar results were observed in another two dependent experiments. ox-LDL, oxidised low-density lipoprotein; VECs, vascular endothelial cells; ROS, reactive oxygen species.
    淫羊藿苷可提高细胞活力和线粒体膜电位。 (a) 筛选用于实验的淫羊藿苷 ( n = 5)。 (b) 用于建模的 ox-LDL 筛选( n = 5)。 (c) 不同组中 VEC 的活力 ( n = 5)。 (d) 不同组中Fe 2+的含量( n = 3)。 (e) 不同组中的聚集体:单体(红色/绿色)比率( n = 3)。 (f) 不同组中的 ROS 水平( n = 3)。 “*”表示统计学上显着的差异(* p < 0.05 和 ** p < 0.01)。在另外两个相关实验中也观察到了类似的结果。 ox-LDL,氧化低密度脂蛋白; VEC,血管内皮细胞; ROS,活性氧。

    3.2 Icariin increases mitophagy in ox-LDL-treated cells
    3.2 淫羊藿苷增加ox-LDL处理细胞的线粒体自噬

    Mitochondrial depolarisation or ROS production is known to cause autophagy, and mitophagy occurs if there is a large amount of damaged mitochondria (Wang et al., 2012). In the present study, in order to investigate the role of icariin in the mitophagy, autophagic induction was measured in VECs stably expressing GFP-LC3. Exposure to ox-LDL led to an increase in the numbers of green punctae, and this increase was promoted by icariin, which indicated that icariin could further increase autophagy (Figure 2a,b). In addition, for VECs exposed to ox-LDL, there was a minimal initiation of mitophagy. Accompanied by the increasing concentrations of icariin, the yellow fluorescence became more prominent (Figure 2c,d), suggesting that ox-LDL-induced mitophagy was further enhanced by icariin.
    已知线粒体去极化或 ROS 产生会引起自噬,如果线粒体大量受损,就会发生线粒体自噬 (Wang et al., 2012 )。在本研究中,为了研究淫羊藿苷在线粒体自噬中的作用,在稳定表达GFP-LC3的VEC中测量了自噬诱导。暴露于ox-LDL导致绿色点的数量增加,并且这种增加是由淫羊藿苷促进的,这表明淫羊藿苷可以进一步增加自噬(图2a ,b)。此外,对于暴露于 ox-LDL 的 VEC,线粒体自噬的启动程度极低。随着淫羊藿苷浓度的增加,黄色荧光变得更加明显(图2c,d ),表明淫羊藿苷进一步增强了ox-LDL诱导的线粒体自噬。

    Details are in the caption following the image
    Icariin increases the reduction in mitophagy due to ox-LDL. (a) In VECs stably expressing GFP-LC3B, icariin increased the green fluorescence puncta (n = 3). (b) Data of GFP-LC3B in each group (n = 3). (c) The co-localisation (yellow) of the mitochondrion (green) with lysosome (red) puncta was enhanced by icariin (n = 3). (d) Data of mitochondria co-localised with lysosomes in each group (n = 3). “*” indicates a statistically significant difference (*p < 0.05 and **p < 0.01). Similar results were observed in another two dependent experiments. ox-LDL, oxidised low-density lipoprotein; VECs, vascular endothelial cells.
    淫羊藿苷可增加 ox-LDL 导致的线粒体自噬的减少。 (a) 在稳定表达 GFP-LC3B 的 VEC 中,淫羊藿苷增加绿色荧光点( n = 3)。 (b) 每组中 GFP-LC3B 的数据 ( n = 3)。 (c) 淫羊藿苷增强了线粒体(绿色)与溶酶体(红色)斑点的共定位(黄色)( n = 3)。 (d) 每组中线粒体与溶酶体共定位的数据 ( n = 3)。 “*”表示统计学上显着的差异(* p < 0.05 和 ** p < 0.01)。在另外两个相关实验中也观察到了类似的结果。 ox-LDL,氧化低密度脂蛋白; VEC,血管内皮细胞。

    3.3 Icariin increases TFEB activation in the nucleus and autophagy
    3.3 淫羊藿苷增加细胞核中TFEB的激活和自噬

    The hypothesis is that ROS-induced mitophagy is designed to scavenge excess ROS and prevent further oxidative damage (Narendra et al., 2008). The present study then investigated the possible mechanisms responsible for the enhanced autophagy and mitophagy induced by icariin. Previous studies have demonstrated that TFEB regulates the biogenesis of both autophagosomes and lysosomes (Settembre et al., 2011; Yamamoto & Mizushima, 2021). As shown in Figure 3a, the location of the nucleus and cytoskeleton was determined by staining with DAPI and phalloidin; the fluorescence staining of TFEB revealed a significantly higher rate of nucleation in the icariin-treated cells (Figure 3b). Lamin B1 (nuclear fibrillar protein) is a structural component of the nuclear membrane. The increase rate of TFEB entry into the nucleus was further determined by measuring the ratio of nuclear TFEB/Lamin B1, which was significantly increased (Figure 3c). Subsequently, the expression of related proteins was examined (Figure 3d). The expression levels of Lamp1, TRPML1 and LC3 II/I increased in the ox-LDL group compared with the control group, and further increased following treatment with icariin, whereas the expression levels of GPX4 and FTH1 decreased significantly in the ox-LDL-treated group and increased with icariin treatment. Lamp1 is a lysosomal-associated membrane protein and TRPML1 is a lysosomal ion channel protein (Zhang et al., 2016). Following icariin treatment, the increase in the levels of these two proteins was accompanied by an increase in the LC3 II/I levels, indicating an increase in autophagy, while the increase in the GPX4 and FTH1 and a decrease in the TfR1 levels indicated a decrease in ferroptosis.
    假设ROS诱导的线粒体自噬旨在清除过量的ROS并防止进一步的氧化损伤(Narendra等, 2008 )。本研究随后研究了淫羊藿苷诱导增强自噬和线粒体自噬的可能机制。先前的研究表明,TFEB 调节自噬体和溶酶体的生物合成(Settembre 等人, 2011 ;Yamamoto 和 Mizushima, 2021 )。如图3a所示,通过DAPI和鬼笔环肽染色确定细胞核和细胞骨架的位置; TFEB 的荧光染色显示淫羊藿苷处理的细胞中成核率明显较高(图3b )。核纤层蛋白 B1(核纤维蛋白)是核膜的结构成分。通过测量核TFEB/Lamin B1的比率进一步确定TFEB进入细胞核的增加率,该比率显着增加(图3c )。随后,检查相关蛋白的表达(图3d )。 ox-LDL组中Lamp1、TRPML1和LC3 II/I的表达水平较对照组升高,并且在淫羊藿苷治疗后进一步升高,而ox-LDL治疗组中GPX4和FTH1的表达水平显着降低组并随着淫羊藿苷治疗而增加。 Lamp1是一种溶酶体相关膜蛋白,TRPML1是一种溶酶体离子通道蛋白(Zhang et al., 2016 )。 淫羊藿苷处理后,这两种蛋白水平的增加伴随着LC3 II/I水平的增加,表明自噬增加,而GPX4和FTH1的增加以及TfR1水平的降低表明自噬减少。在铁死亡中。

    Details are in the caption following the image
    Icariin increases TFEB activation in the nucleus and autophagy. (a) The co-localisation of the cytoskeleton (phalloidin, red), nucleus (DAPI, blue) and TFEB (green) (n = 3). (b) TFEB nuc/cyt ratio in each group (n = 3). (c) icariin increased nuclear TFEB expression (n = 3). (d) Western blot analysis of Lamp1, TfR1, TRPML-1, GPX4, FTH1 and LC3 II/I (n = 3). “*” indicates a statistically significant difference (*p < 0.05 and **p < 0.01). Similar results were observed in another two dependent experiments. TFEB, transcription factor EB; TfR1, transferrin receptor 1; GPX4, glutathione peroxidase 4; FTH1, ferritin heavy chain 1.
    淫羊藿苷可增加细胞核中 TFEB 的激活和自噬。 (a) 细胞骨架(鬼笔环肽,红色)、细胞核(DAPI,蓝色)和 TFEB(绿色)的共定位( n = 3)。 (b) 每组中的 TFEB nuc/cyt 比率 ( n = 3)。 (c) 淫羊藿苷增加核 TFEB 表达 ( n = 3)。 (d) Lamp1、TfR1、TRPML-1、GPX4、FTH1 和 LC3 II/I ( n = 3) 的蛋白质印迹分析。 “*”表示统计学上显着的差异(* p < 0.05 和 ** p < 0.01)。在另外两个相关实验中也观察到了类似的结果。 TFEB,转录因子EB; TfR1,转铁蛋白受体1; GPX4、谷胱甘肽过氧化物酶4; FTH1,铁蛋白重链1。

    3.4 Icariin promotes autophagy by promoting the lysosome–autophagosome fusion
    3.4 淫羊藿苷通过促进溶酶体-自噬体融合促进自噬

    The process of autophagy is divided into four parts, namely the formation of the phagophore, the formation of autophagosomes, the formation of lysosomes, and the fusion and degradation of the autophagosome–lysosome complex (Yamamoto & Mizushima, 2021). As demonstrated in Figure 4a, icariin notably promoted the expression levels of TFEB/Lamin B1 and LC3II/I in the cells exposed to xo-LDL. In the cells treated with the 3-MA, the expression levels of LC3II/I and TFEB/Lamin B1 were significantly reduced, and the addition of icariin did not result in a significant difference in LC3II/I expression, whereas TFEB/Lamin B1 expression was significantly increased. Lamin B1 is a housekeeping gene in the nucleus, and the significant increase in TFEB/Lamin B1 levels with the addition of icariin indicated that blocking autophagosome formation did not affect TFEB entry into the nucleus. In the cells treated with BafA1, the levels of LC3II/I and TFEB were significantly reduced. Co-treatment with icariin was found to have a significant effect on LC3 II/I levels, but not on TFEB/Lamin B1 levels, suggesting that icariin affects TFEB nucleation via the lysosomal autophagosome pathway. As shown in Figure 4b, the nucleation rate of TFEB was not significantly affected by BafA1, while 3-MA had a significant effect on the nucleation rate of TFEB. Furthermore, with TRPML1 expression was interfered with using siRNA-TRPML1 and it was found that TRPML1 knockdown significantly attenuated icariin-mediated autophagy and nuclear TFEB expression compared with the siRNA-ctrl.
    自噬过程分为四个部分,即吞噬泡的形成、自噬体的形成、溶酶体的形成以及自噬体-溶酶体复合物的融合和降解(Yamamoto & Mizushima, 2021 )。如图4a所示,淫羊藿苷显着促进暴露于xo-LDL的细胞中TFEB/Lamin B1和LC3II/I的表达水平。 3-MA处理的细胞中,LC3II/I和TFEB/Lamin B1的表达量显着降低,淫羊藿苷的添加并没有导致LC3II/I表达量的显着差异,而TFEB/Lamin B1的表达量则显着降低。显着增加。 Lamin B1是细胞核内的管家基因,添加淫羊藿苷后TFEB/Lamin B1水平显着增加,表明阻断自噬体形成并不影响TFEB进入细胞核。在用 BafA1 处理的细胞中,LC3II/I 和 TFEB 的水平显着降低。研究发现淫羊藿苷联合治疗对 LC3 II/I 水平有显着影响,但对 TFEB/Lamin B1 水平没有显着影响,表明淫羊藿苷通过溶酶体自噬体途径影响 TFEB 成核。如图4b所示,BafA1对TFEB的成核率没有显着影响,而3-MA对TFEB的成核率有显着影响。此外,使用siRNA-TRPML1干扰TRPML1表达,发现与siRNA-ctrl相比,TRPML1敲低显着减弱淫羊藿苷介导的自噬和核TFEB表达。

    Details are in the caption following the image
    Icariin suppresses autophagy by inhibiting the autophagosome–lysosome fusion. (a) Ratio of TFEB nuc/cyt and expression of LC3 II/I in each group (n = 3). (b) The co-localisation of the cytoskeleton (phalloidin, red), nucleus (DAPI, blue) and TFEB (green) (n = 3). (c) Western blot analysis of TRPML1 protein levels in siRNA-ctrl and siRNA-TRPML1 cells, and the expression of LC3II/I and TFEB in the presence of TRPML1 interference (n = 3). “*” indicates a statistically significant difference (*p < 0.05 and **p < 0.01). Similar results were observed in another two dependent experiments. TFEB, transcription factor EB.
    淫羊藿苷通过抑制自噬体-溶酶体融合来抑制自噬。 (a) 每组中 TFEB nuc/cyt 的比率和 LC3 II/I 的表达 ( n = 3)。 (b) 细胞骨架(鬼笔环肽,红色)、细胞核(DAPI,蓝色)和 TFEB(绿色)的共定位( n = 3)。 (c) siRNA-ctrl 和 siRNA-TRPML1 细胞中 TRPML1 蛋白水平的蛋白质印迹分析,以及 TRPML1 干扰存在下 LC3II/I 和 TFEB 的表达 ( n = 3)。 “*”表示统计学上显着的差异(* p < 0.05 和 ** p < 0.01)。在另外两个相关实验中也观察到了类似的结果。 TFEB,转录因子EB。

    3.5 Icariin alleviates atherosclerotic lesions in mice
    3.5 淫羊藿苷减轻小鼠动脉粥样硬化病变

    As shown in Figure 5a,c, the endothelial structure of the aortic sinus tissue in the control group of mice was intact, smooth and uninterrupted, with no obvious abnormalities; however, the mice in the model group had large subendothelial deposits of lipids, and treatment with icariin reduced the endothelial atherosclerotic plaque area of the aortic sinus tissue in mice. Similarly, the collagen fibre area of the mice in the model group was markedly increased compared with that of the control group, while the use of icariin reduced the collagen fibre area in the mice (Figure 5b,d).
    如图5a、c所示,对照组小鼠主动脉窦组织内皮结构完整、光滑、不间断,未见明显异常;然而,模型组的小鼠有大量的内皮下脂质沉积,淫羊藿苷治疗减少了小鼠主动脉窦组织的内皮动脉粥样硬化斑块面积。同样,模型组小鼠的胶原纤维面积较对照组明显增加,而淫羊藿苷的使用则减少了小鼠的胶原纤维面积(图5b,d )。

    Details are in the caption following the image
    Icariin alleviates atherosclerotic lesions in mice. (a) Representative images of histological sections were stained with haematoxylin and eosin, and black double arrows indicate the thickness of the lipid plaques (n = 6). (b) Representative images of histological sections were stained with Masson's trichrome staining (n = 6). (c) Percentage of the atherosclerotic plaque area of mice in each group (n = 6). (d) Percentage of the collagen fibre area of mice in each group (n = 6). “*” indicates a statistically significant difference (*p < 0.05 and **p < 0.01). Similar results were observed in another two dependent experiments.
    淫羊藿苷可减轻小鼠动脉粥样硬化病变。 (a) 组织学切片的代表性图像用苏木精和伊红染色,黑色双箭头表示脂质斑块的厚度 ( n = 6)。 (b) 组织学切片的代表性图像采用马森三色染色 ( n = 6)。 (c) 每组小鼠动脉粥样硬化斑块面积的百分比 ( n = 6)。 (d) 每组小鼠胶原纤维面积的百分比 ( n = 6)。 “*”表示统计学上显着的差异(* p < 0.05 和 ** p < 0.01)。在另外两个相关实验中也观察到了类似的结果。

    3.6 Icariin inhibits pro-inflammatory factors and promotes autophagy in vivo
    3.6 淫羊藿苷抑制体内促炎因子并促进自噬

    Total cholesterol (TC) and triglycerides (TG) are critical indicators of atherosclerosis. As shown in Figure 6a,b, the values of TC and TG were significantly increased in the model group compared with the control group, whereas treatment with icariin decreased the values of TC and TG. Furthermore, the levels of the pro-inflammatory factors, IL-6 and TNF-α, also markedly increased in the model group compared with the control group; however, this trend was reversed by icariin (Figure 6c,d). The in vivo results of western blot analysis also revealed that icariin reduced the increase in ferroptosis (Figure 6e). As shown in Figure 6f, the expression of nuclear TFEB was increased by icariin, which as consistent with the results of the in vitro experiment.
    总胆固醇(TC)和甘油三酯(TG)是动脉粥样硬化的重要指标。如图6a、b所示,与对照组相比,模型组TC和TG值显着升高,而淫羊藿苷治疗则降低了TC和TG值。此外,与对照组相比,模型组促炎因子IL-6和TNF-α的水平也明显升高;然而,淫羊藿苷逆转了这一趋势(图6c,d )。体内蛋白质印迹分析结果也显示淫羊藿苷减少了铁死亡的增加(图6e )。如图6f所示,淫羊藿苷增加了核TFEB的表达,这与体外实验的结果一致。

    Details are in the caption following the image
    Icariin inhibits pro-inflammatory factors and autophagy. (a) The serum total cholesterol (TC) of mice in each group (n = 6). (b) The serum TG levels of mice in each group (n = 6). (c) The serum IL-6 levels of mice in each group (n = 6). (d) The serum TNF-α levels of mice in each group (n = 6). (e) The expression levels of ferroptosis related proteins in each group (n = 3). (f) The expression levels of nucleus TFEB in each group (n = 3). “*” indicates a statistically significant difference (*p < 0.05 and **p < 0.01). Similar results were observed in another two dependent experiments. TG, triglycerides; TFEB, transcription factor EB.
    淫羊藿苷抑制促炎因子和自噬。 (a)各组小鼠的血清总胆固醇(TC)( n =6)。 (b)各组小鼠血清TG水平( n =6)。 (c)各组小鼠血清IL-6水平( n =6)。 (d)各组小鼠血清TNF-α水平( n =6)。 (e)每组中铁死亡相关蛋白的表达水平( n = 3)。 (f)各组中细胞核TFEB的表达水平( n =3)。 “*”表示统计学上显着的差异(* p < 0.05 和 ** p < 0.01)。在另外两个相关实验中也观察到了类似的结果。 TG、甘油三酯; TFEB,转录因子EB。

    3.7 Icariin interacts with TFEB via ARG304, THR308, GLN311
    3.7 淫羊藿苷通过ARG304、THR308、GLN311与TFEB相互作用

    A computational model was applied to predict the potential binding site of icariin at TFEB. Molecular dynamics simulations revealed that icarrin interacted with TFEB at ARG304, THR308 and GLN311 to build hydrogen bonds (Figure 7a,b). Additionally, the microscale thermophoresis experiments denoted that the mutation of all three interacting amino acids to alanine revealed a significant reduction in the binding affinity of icariin to TFEB. Therefore, icariin may interact with TFEB via these three amino acids, and the target of icariin action is TFEB (Figure 7c,d).
    应用计算模型来预测淫羊藿苷在 TFEB 上的潜在结合位点。分子动力学模拟表明,淫羊藿苷与 TFEB 在 ARG304、THR308 和 GLN311 处相互作用,形成氢键(图7a、b )。此外,微尺度热泳实验表明,所有三个相互作用的氨基酸突变为丙氨酸,表明淫羊藿苷与 TFEB 的结合亲和力显着降低。因此,淫羊藿苷可能通过这三个氨基酸与TFEB相互作用,淫羊藿苷作用的靶点是TFEB(图7c,d )。

    Details are in the caption following the image
    Icariin interacts with TFEB via ARG304, THR308, GLN311. (a) The structure of icariin binding to the TFEB protein in a three-dimensional model and a (b) two-dimensional ligand interaction diagram. Microscale thermophoresis binding curves of the Cy5-labelled DNA probe and cell lysates that were pre-treated with icariin-TFEB; (c) the curve of icariin and TFEB (Kd = 0.00936 μM); and (d) the curve of icariin and mutation-TFEB (Kd = 30.17 μM). TFEB, transcription factor EB.
    淫羊藿苷通过 ARG304、THR308、GLN311 与 TFEB 相互作用。 (a) 三维模型中淫羊藿苷与 TFEB 蛋白结合的结构和 (b) 二维配体相互作用图。 Cy5标记的DNA探针与淫羊藿苷-TFEB预处理的细胞裂解液的微量热泳结合曲线; (c) 淫羊藿苷和TFEB的曲线(Kd = 0.00936 μM); (d) 淫羊藿苷与突变-TFEB (Kd = 30.17 μM) 的曲线。 TFEB,转录因子EB。

    4 DISCUSSION  4 讨论

    Atherosclerosis is the leading cause of cardiovascular disease worldwide. The exact etiology remains unknown, and the risk factors include hyperlipidaemia. The basic lesions of atherosclerosis are lipid deposition in the intima of the artery, focal fibrosis of the intima forming atherosclerotic plaques resulting in lumen stenosis and ischemic changes of the corresponding organs. The ApoE−/− mouse model is a widely accepted animal model of atherosclerosis, where the ApoE gene is knocked out and cholesterol is unable to be bound by cell surface lipoprotein receptors for degradation and accumulation occurs, leading to atherosclerosis (Lee et al., 2017). A previous study demonstrated that icarrin inhibits the progression of atherosclerosis in ApoE−/− mice and analysed the possible role of Icarrin in downregulating CX3CR1; however, it did not explore the protective mechanisms of icarrin in atherosclerosis (Wang et al., 2016).
    动脉粥样硬化是全世界心血管疾病的主要原因。确切的病因尚不清楚,危险因素包括高脂血症。动脉粥样硬化的基本病变是动脉内膜的脂质沉积,内膜局灶性纤维化形成动脉粥样硬化斑块,导致管腔狭窄和相应器官的缺血性改变。 ApoE −/−小鼠模型是广泛接受的动脉粥样硬化动物模型,其中ApoE基因被敲除,胆固醇无法与细胞表面脂蛋白受体结合而发生降解和积累,导致动脉粥样硬化(Lee等, 2017 )。先前的一项研究表明,淫羊藿苷可抑制 ApoE −/−小鼠动脉粥样硬化的进展,并分析了淫羊藿苷在下调 CX3CR1 中的可能作用;但并未探讨淫羊藿苷对动脉粥样硬化的保护机制(Wang et al., 2016 )。

    Based on the inhibitory effects of icarrin on ferroptosis and the critical role of ferroptosis in atherosclerosis, the present study demonstrated that icarrin attenuated atherosclerosis by inhibiting ferroptosis. In the present study, it was found that icarrin treatment significantly reduced the development of atherosclerotic plaques and collagen thickness in the aorta of the atherosclerotic mouse model (Figure 5), and improved dyslipidaemia in mice, specifically reducing the serum levels of TC and TG (Figure 6a,b). However, in previous research, although icarrin was shown to alleviate atherosclerosis, it did not reduce blood lipids; this may be related to the different study animals used (Zhang et al., 2013). Herein, combined icarrin treatment reduced the oxidative stress levels (Figure 1e,f) and ferroptosis (Figure 1d), thus increasing mitophagy (Figure 2) in HUVECs. Another study demonstrated the safety of icarrin in in vivo experiments (Yuan et al., 2022); the results of that study, in combination with the present study, provides new experimental evidence suggesting the possible use of icarrin in clinical practice for alleviating atherosclerosis.
    基于淫羊藿苷对铁死亡的抑制作用以及铁死亡在动​​脉粥样硬化中的关键作用,本研究证明淫羊藿苷通过抑制铁死亡来减轻动脉粥样硬化。本研究发现,淫羊藿苷治疗可显着减少动脉粥样硬化小鼠模型主动脉内动脉粥样硬化斑块的发展和胶原蛋白厚度(图5 ),并改善小鼠的血脂异常,特别是降低血清TC和TG水平(图5)。图6a、b )。然而,在之前的研究中,虽然淫羊藿苷被证明可以缓解动脉粥样硬化,但它并不能降低血脂;这可能与使用的不同研究动物有关(Zhang et al., 2013 )。在此,联合淫羊藿苷治疗降低了氧化应激水平(图1e,f )和铁死亡(图1d ),从而增加了HUVEC中的线粒体自噬(图2 )。另一项研究在体内实验中证明了淫羊藿苷的安全性(Yuan et al., 2022 );该研究的结果与本研究相结合,提供了新的实验证据,表明淫羊藿苷在临床实践中可能用于缓解动脉粥样硬化。

    At present, the treatment of atherosclerosis is mainly aimed at regulating the traditional risk factors, and anti-inflammatory intervention may be a novel potential therapy. Ferroptosis is characterised by abnormal mitochondrial structure and increases in iron and lipid hydroxide levels (which produce large quantities of free radicals when the Fenton reaction is catalysed by iron ions) (Kurz et al., 2008; Latunde-Dada, 2017; Wang et al., 2020). In the present study, as was expected, the ox-LDL-treated VECs exhibited an increase in the Fe2+ and ROS levels, as well as an increase in TfR1 and a decrease in FTH levels, further suggesting that ferroptosis was promoted; however, icarrin treatment attenuated these effects.
    目前,动脉粥样硬化的治疗主要针对调节传统的危险因素,抗炎干预可能是一种新型的潜在疗法。铁死亡的特征是线粒体结构异常以及铁和脂质氢氧化物水平增加(当铁离子催化芬顿反应时,会产生大量自由基)(Kurz 等人, 2008 ;Latunde-Dada, 2017 ;Wang 等人) ., 2020 ).在本研究中,正如预期的那样,ox-LDL处理的VEC表现出Fe 2+和ROS水平的增加,以及TfR1的增加和FTH水平的降低,进一步表明铁死亡得到促进。然而,淫羊藿苷治疗减弱了这些影响。

    The present study also aimed to explore the mechanisms through which icarrin reduces ferroptosis. It is known that endogenous ROS are mainly derived from the mitochondria, and the release of ROS is related to lysosomes. As previously demonstrated, TRPML1 is a receptor of oxidative stress in lysosomes and promotes the release of Ca2+ from lysosomes into cells, facilitates TFEB dephosphorylation into the nucleus and regulates autophagic gene expression (Settembre et al., 2011). Thus, mitophagy is enhanced to promote the clearance of damaged mitochondria, thus lessening the mitochondrial production of free radicals, which is a negative feedback to ROS accumulation (Zhang et al., 2016). Notably, TRPML1 protein expression has been shown to be higher in both in vivo and in vitro models compared with the negative control group, which may be due to the body's own response to oxidative stress (Chodari et al., 2021). Furthermore, herein, the addition of icarrin significantly increased TRPML1 expression compared with the model group. Similarly, the increase in autophagy also increased with the addition of icarrin (Figure 2a,b).
    本研究还旨在探讨淫羊藿素减少铁死亡的机制。众所周知,内源性ROS主要来源于线粒体,ROS的释放与溶酶体有关。如之前所证明的,TRPML1是溶酶体中氧化应激的受体,促进Ca 2+从溶酶体释放到细胞中,促进TFEB去磷酸化进入细胞核并调节自噬基因表达(Settembre等, 2011 )。因此,线粒体自噬增强,促进受损线粒体的清除,从而减少线粒体产生自由基,这是ROS积累的负反馈(Zhang et al., 2016 )。值得注意的是,与阴性对照组相比,体内和体外模型中 TRPML1 蛋白表达均较高,这可能是由于人体自身对氧化应激的反应所致 (Chodari et al., 2021 )。此外,本文中,与模型组相比,淫羊藿苷的添加显着增加了TRPML1表达。同样,自噬的增加也随着淫羊藿苷的添加而增加(图2a,b )。

    The catabolic process of mitophagy is that the autophagy system targets the damaged mitochondria and delivers them to the lysosome for degradation, including the initiation of isolation film, the formation of the autophagosome, the production of the lysosome, and autophagosome–lysosome fusion and degradation (Murrow & Debnath, 2013; Shen & Mizushima, 2014). The present study also found that the overlap of mitochondria and lysosomes in the degree of autophagy was consistent (Figure 2c). In order to elucidate which stage of autophagy icariin acts, we treated VECs with two autophagy inhibitors, BafA1 (blocking the fusion of autophagosomes with lysosomes) and 3-MA (blocking the formation of autophagosomes). The results revealed that icariin still had a significant facilitatory effect on TFEB nucleation when autophagosome formation was inhibited, whereas the facilitatory effect of icariin on TFEB nucleation was blocked when autophagosome–lysosome fusion was inhibited, suggesting that icariin promotes autophagy via the mitochondrial lysosome fusion pathway.
    线粒体自噬的分解代谢过程是自噬系统针对受损的线粒体,将其递送至溶酶体进行降解,包括隔离膜的引发、自噬体的形成、溶酶体的产生以及自噬体-溶酶体的融合和降解。 Murrow 和 Debnath, 2013 ;Shen 和 Mizushima, 2014 )。本研究还发现,线粒体和溶酶体在自噬方面的重叠程度是一致的(图2c )。为了阐明自噬淫羊藿苷作用的哪个阶段,我们用两种自噬抑制剂BafA1(阻断自噬体与溶酶体的融合)和3-MA(阻断自噬体的形成)处理VEC。结果表明,当自噬体形成受到抑制时,淫羊藿苷对TFEB成核仍具有显着的促进作用,而当自噬体-溶酶体融合受到抑制时,淫羊藿苷对TFEB成核的促进作用被阻断,表明淫羊藿苷通过线粒体溶酶体融合途径促进自噬。

    The data presented herein suggest that icarrin may be a novel and promising candidate for use as a natural product that can effectively increase VEC survival and alleviate atherosclerosis, possibly by promoting mitochondria–lysosome fusion to promote autophagy and reduce ROS levels, thereby reducing ferroptosis. In addition, the present study analysed the possible interaction between icarrin and TFEB, and found ARG304, THR308 and GLN311 were the optimal binding sites for the interaction between icariin and TFEB. As the representative constituent of Epimedium brevicornu Maxim., icarrin has been long used in traditional medicine and is considered a safe and effective natural product with globally accessible resources (Eftekhari et al., 2021). Previous studies have also demonstrated the in vivo safety of icarrin, as for example, using H&E staining of sections of heart tissue from rats, and kidney tissue from mice (He, 2023; Qian, Zhu, & Li, 2021). In 2022, the icariin small molecule drug was launched in China as an indication treatment for hepatocellular carcinoma. In addition to its well-documented anticancer and anti-inflammatory activities, the present study reports a potential pleiotropic effect of icarrin, including lowering lipid levels and increasing VEC viability; the present study also supports a practical clinical application of icarrin in atherosclerosis. However, the selection of the optimal starting dose of a drug for experimental or clinical trials remains a matter of debate. Based on previous reports, the dose for adults should be 3.25 mg/kg for conversion from 40 mg/kg in mice, which was 195 mg in the first human clinical trial (Chodari et al., 2021). Of course, the exponential conversion of doses between animals and humans using body surface area (0.67) is not the gold standard, and estimates always require the careful consideration of differences in pharmacokinetics and pharmacodynamics between species. In addition, research on the combination of nanotechnology and drugs in recent years can improve bioavailability and markedly reduce the drug dose, which is also worthy of further exploration in the future (Liang et al., 2023).
    本文提供的数据表明,淫羊藿苷可能是一种新颖且有前途的天然产物候选物,可以有效增加 VEC 存活并减轻动脉粥样硬化,可能通过促进线粒体-溶酶体融合来促进自噬并降低 ROS 水平,从而减少铁死亡。此外,本研究分析了淫羊藿苷与TFEB之间可能的相互作用,发现ARG304、THR308和GLN311是淫羊藿苷与TFEB相互作用的最佳结合位点。作为淫羊藿的代表成分,淫羊藿苷在传统医学中长期使用,被认为是一种安全有效、资源丰富的天然产物(Eftekhari et al., 2021 )。先前的研究也证明了淫羊藿苷的体内安全性,例如,使用大鼠心脏组织切片和小鼠肾组织切片的 H&E 染色(He, 2023 ;Qian、Zhu 和 Li, 2021 )。 2022年,淫羊藿苷小分子药物在中国上市,作为肝细胞癌的适应症治疗。除了其有据可查的抗癌和抗炎活性外,本研究报告了淫羊藿苷潜在的多效作用,包括降低脂质水平和增加 VEC 活力;本研究还支持淫羊藿苷在动脉粥样硬化中的实际临床应用。然而,实验或临床试验中药物最佳起始剂量的选择仍然存在争议。根据之前的报道,成人的剂量应从小鼠的 40 mg/kg 换算为 3.25 mg/kg,第一次人体临床试验中的剂量为 195 mg(Chodari 等,2017)。, 2021 )。当然,使用体表面积(0.67)对动物和人类之间的剂量进行指数换算并不是黄金标准,估计总是需要仔细考虑物种之间药代动力学和药效学的差异。此外,近年来纳米技术与药物结合的研究可以提高生物利用度并显着降低药物剂量,这也值得未来进一步探索(Liang et al., 2023 )。

    The present study demonstrated that treatment with icarrin alleviated the progression of atherosclerosis in mouse and cellular models and inhibited oxidative stress and ferroptosis. However, the present study has some limitations that need to be addressed. Firstly, there may be other proteins or pathways other than TFEB that contribute to the inhibitory effects of icarrin on ferroptosis; thus further studies are required. Secondly, due to the absence of a positive control for icarrin in the cell assay, the promoting effects of icarrin cell viability and its suppressive effects on oxidative stress levels are not convincing. Thirdly, the toxicological analysis of icarrin was not included as a part of the in vivo experiments; the dose was selected by referring only to other studies, which may be insufficient. Therefore, in future research, the authors aim to carefully design experiments to address these questions.
    本研究表明,淫羊藿苷治疗可减轻小鼠和细胞模型中动脉粥样硬化的进展,并抑制氧化应激和铁死亡。然而,本研究存在一些需要解决的局限性。首先,除了TFEB之外,可能还有其他蛋白质或途径有助于淫羊藿素对铁死亡的抑制作用;因此需要进一步研究。其次,由于细胞测定中缺乏淫羊藿苷的阳性对照,淫羊藿苷对细胞活力的促进作用及其对氧化应激水平的抑制作用并不令人信服。第三,体内实验中没有对淫羊藿苷进行毒理学分析;仅参考其他研究来选择剂量,这可能是不够的。因此,在未来的研究中,作者的目标是仔细设计实验来解决这些问题。

    5 CONCLUSION  5 结论

    In conclusion, icariin reduced ROS levels by promoting the expression of TRPML1, thereby promoting autophagy and accelerating the clearance of damaged mitochondria in the cellular ferroptosis model. In addition, in a mouse model of atherosclerosis, icariin reduced ferroptosis and attenuated the development of atherosclerotic lesions. These findings firstly suggest that the natural drug, icarrin, may be an effective treatment option for atherosclerosis. Due to the high prevalence of atherosclerosis, icarrin may have broad application prospects in the medical field. However, a limitation of the present study is that icariin may reduce ferroptosis by promoting TFEB expression, although other possible influencing factors cannot be ruled out. The authors aim to further explore this matter in future studies.
    总之,在细胞铁死亡模型中,淫羊藿苷通过促进TRPML1的表达来降低ROS水平,从而促进自噬并加速受损线粒体的清除。此外,在动脉粥样硬化小鼠模型中,淫羊藿苷可减少铁死亡并减轻动脉粥样硬化病变的发展。这些发现首先表明天然药物淫羊藿苷可能是动脉粥样硬化的有效治疗选择。由于动脉粥样硬化的高患病率,淫羊藿苷在医学领域可能具有广阔的应用前景。然而,本研究的局限性在于淫羊藿苷可能通过促进 TFEB 表达来减少铁死亡,但不能排除其他可能的影响因素。作者旨在在未来的研究中进一步探讨这个问题。

    AUTHOR CONTRIBUTIONS  作者贡献

    Xindong Wang: Conceptualization. Meng Zhang: Supervision; validation. Chenhan Mao: Methodology; project administration; software; supervision. Chengbo Zhang: Supervision. Wenqi Ma: Visualization. Jiahui Tang: Visualization; writing – original draft; writing – review and editing. Dongyang Xiang: Formal analysis. Xiaoxia Qi: Writing – original draft; writing – review and editing.
    王新东:概念化。张萌:监督;验证。毛辰汉:方法论;项目管理;软件;监督。张成波:监督。马文琪:可视化。唐家辉:可视化;写作——原稿;写作——审查和编辑。向东阳:形式分析。齐晓霞:写作——初稿;写作——审查和编辑。

    ACKNOWLEDGMENTS  致谢

    This work was partially supported by grants from National Natural Science Foundation of China (No. 81973766, 81403386, 81873274), Natural Science Foundation of Jiangsu Province of China (No. BK20211387) and Science and Technology Planning Project of Chinese Medicine of Jiangsu Province of China (No. YB201922).
    该工作得到国家自然科学基金(No. 81973766、81403386、81873274)、江苏省自然科学基金(No. BK20211387)和江苏省中医药科技规划项目的部分资助中国(编号:YB201922)。

      CONFLICT OF INTEREST STATEMENT
      利益冲突声明

      The authors declare that they have no competing interest.
      作者声明他们没有竞争利益。

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