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。

3.1 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 ）。

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。

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。

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。

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 ）。

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。

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 ）。