In our study, we utilized Human Umbilical Vein Endothelial Cells (HUVECs) sourced from our research group's cell bank. The culture method we employed was based on the protocol described by Li et al.29. We maintained HUVECs in a controlled environment at 37°C within a 5% CO₂ incubator, using Dulbecco’s Modified Eagle’s Medium (DMEM; C11885500BT, Gibco), enriched with 10% Fetal Bovine Serum (FBS; FSP500, ExCell) and 1% Penicillin-Streptomycin Solution (P1400, Solarbio).

For assessing cytotoxicity, we seeded HUVECs in 96-well plates and treated them with varying concentrations of kaempferol (ranging from 0.625 to 40 µM in DMSO) for 24 hours. To evaluate cell viability, we added 10 µl of the Cell Counting Kit-8 (CCK-8; 40203ES60, YEASEN) solution to each well and incubated them for 3 hours at 37°C in a 5% CO₂ environment. Subsequently, we measured the absorbance at 450 nm using a spectrophotometer.

For the cell survival experiment, HUVECs were plated in 96-well plates and treated either with the GSH peroxidase 4 inhibitor RSL3 (Y-100218A, MCE) or kaempferol. After incubation at 37°C in a 5% CO₂ incubator for 3 hours, we measured absorbance at 450 nm using a spectrophotometer for cell viability assessment.

To detect ROS production, we utilized the Superoxide Anion Probe Dihydroethidium (DHE) assay30. This involved culturing HUVECs and subsequently incubating them with 10 μM DHE (S0063, Beyotime) at 37°C for 30 minutes. After washing the cells twice with phosphate-buffered saline (PBS) and fixing them with 4% paraformaldehyde for 30 minutes, we applied an anti-fluorescence quenching agent containing 4',6-diamidino-2-phenylindole (DAPI) (ZLI-9556, ZSGB-BIO) for counter-staining. We then examined and photographed the cells under a confocal microscope.

Following the methodology of Mei et al.31, we detected lipid peroxidation using the C11 BODIPY 581/591 indicator. After pretreating HUVECs, we added C11 BODIPY 581/591 (D3861, Invitrogen) at a final concentration of 5 μM to the culture medium and co-incubated it for one hour at 37°C. We washed the cells twice with PBS, treated them with trypsin, resuspended them in PBS containing 5% FBS, and finally analyzed them using flow cytometry.

We analyzed the intracellular protein content using the Western Blot technique32. After pretreatment, HUVECs were lysed with RIPA buffer containing protease inhibitors on ice for 30 minutes. The proteins were then separated by SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies against SLC7A11 (ab175186, Abcam), GPX4 (A1933, ABclonal), and GAPDH (A19056, ABclonal) overnight at 4°C, using a 1:1000 dilution. The next day, we incubated the membranes with an HRP-conjugated Goat Anti-Rabbit secondary antibody (RGAR001, Proteintech) at a 1:10,000 dilution for one hour at room temperature. Detection was achieved using a chemiluminescent substrate. GAPDH served as a loading control33, 34, and the bands were quantitatively analyzed using ImageJ software (version 1.4.3.67).

We extracted total RNA using TRI Reagent (T9424, Sigma) and synthesized cDNA with TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311-02, Transgen). We performed qPCR using the QuantStudioTM 3 System and PerfectStart® Green qPCR SuperMix (+Universal Passive Reference Dye) (AQ602-01, Transgen), detecting fluorescence with SYBR Green. The amplification efficiency was calculated as E = 10^(-1/k)-1, with efficiencies for SLC7A11 and GPX4 at 99.8% and 102.7%, respectively. We normalized the expression levels of SLC7A11 and GPX4 mRNAs to β-actin mRNA using the 2^-ΔΔCt method and employed the following primers for qPCR:

- β-actin: - Forward: 5′-CCTGGCACCCAGCACAAT-3′ - Reverse: 5′-GGGCCGGACTCGTCATAC-3′- SLC7A11: - Forward: 5′-ATGCAGTGGCAGTGACCTTT-3′ - Reverse: 5′-CATGGAGCCAAAGCAGGAGA-3′- GPX4: - Forward: 5′-GAAGATCCAACCCAAGGGCA-3′ - Reverse: 5′-GACGGTGTCCAAACTTGGTG-3′

We meticulously analyzed all data to ensure a normal distribution and presented the results as the mean ± standard deviation (SD). Statistical significance was determined using Student's t-test or one-way ANOVA, followed by post-hoc testing. We utilized Pearson's product-moment correlation for correlation analyses. All statistical procedures were conducted using GraphPad Prism version 8.0.2, considering p-values of < 0.05 as statistically significant.

Figure 1

Figure 2

Figure 3

Figure 4

Ferroptosis was induced in HUVECs using different concentrations of RSL-3 (DMSO, 0.125, 0.25, 0.5, and 1 μM). A dose-dependent decrease in cell viability in HUVECs was detected. The LD50 of RSL-3 in cell viability was achieved at a concentration of 0.25 μM. Subsequent induction experiments were carried out using this concentration (Figure 1A). To screen for the most active flavonoids, HUVECs were treated with different flavonoid components and co-cultured with RSL-3 for 24 hours. Kaempferol demonstrated the strongest ability to rescue the RSL-3-induced ferroptosis (Figure 1B). Figure 1C shows the chemical structural formula of Kaempferol.

The toxicity of kaempferol on HUVECs was analyzed using the CCK-8 assay. HUVECs were treated with different concentrations of kaempferol (DMSO, 0.625, 1.25, 2.5, 5, 10, 20, and 40 μM) for 24 hours, and the cell viability was analyzed. Interestingly, kaempferol showed no toxicity, but a slight growth-promoting effect (Figure 2A). Moreover, kaempferol was shown to resist ferroptosis and effectively rescue HUVECs similar to the control group at a concentration of 5 μM. The subsequent experiments were performed with this concentration to rescue HUVEC ferroptosis (Figure 2B).

Ferroptosis is a ROS-dependent, non-apoptotic, lipid-peroxidation-induced cell death closely related to the intracellular ROS content35, 36. ROS are generated during normal physiological processes and are essential for cell signaling and tissue homeostasis37. The C11 BODIPY 581/591 and DHE assays were used to determine the lipid oxidation profiles and oxidative stress in HUVECs, respectively. The results showed that the fluorescence intensity of DHE increased significantly following RSL-3 treatment and C11 BODIPY 581/591 addition, whereas both DHE and C11 BODIPY 581/591 decreased significantly upon treatment with kaempferol, indicating the antioxidant capacity of kaempferol (Figure 3).

The protein expression of SLC7A11 and GPX4 was significantly downregulated in the RSL-3 group. However, treatment with kaempferol restored the protein levels to that of the control, suggesting that kaempferol inhibits ferroptosis in HUVECs (Figure 4 A, B, C). This correlation was also verified at the RNA level, with qPCR results being consistent with the western blot findings (Figure 4 D, E).

The pathology of vascular diseases often involves the dysregulation of endothelial cell death, making the study of endothelial cell death models crucial for the identification of effective treatments for such diseases. Human Umbilical Vein Endothelial Cells (HUVECs) are frequently utilized in research on cell biology within the contexts of angiogenesis, vascular diseases, and cardiovascular diseases38. Due to their superior proliferation and migration abilities, as well as their capacity to form in vitro tubular structures resembling angiogenesis, HUVECs are considered ideal models for exploring endothelial cell death38.

Ferroptosis, a form of non-apoptotic cell death characterized by iron-dependent lipid peroxidation, is distinguished by an accumulation of lipid peroxides leading to cell swelling and the subsequent rupture of the cell membrane8, 39. The process of lipid peroxidation is fundamental to ferroptosis40. Compounds such as RSL3 and erastin, known inducers of ferroptosis, are employed to develop cell models for this form of cell death. Erastin targets System Xc- activity to disrupt glutathione (GSH) synthesis, a pathway leading to ferroptosis41, whereas RSL3, by directly inhibiting GPX4, activates iron-dependent, nonapoptotic cell death in cells with RAS mutations42, 43 and in various cell types44, 45. Observations of morphological changes, including cell and mitochondrial shrinkage as well as cell membrane damage, in HUVECs treated with RSL3 confirm its efficacy in creating an ideal model for endothelial cell ferroptosis.

Recent decades have seen the identification of pharmacological and natural compounds capable of modulating ferroptosis46, 47. Among these, kaempferol, a compound found in abundance in fruits, vegetables, and herbs, stands out for its minimal toxicity and promising therapeutic potential48. Its mechanisms involve the promotion of free radical scavenging, enhancement of antioxidant enzyme activities against lipid peroxidation, and prevention of hemolysis49. Moreover, kaempferol acts protectively in ischemic stroke by activating specific signaling pathways (Nrf2/SLC7A11/GPX4) to mitigate oxygen-glucose deprivation/reperfusion-induced cellular damage and suppress ferroptosis initiation50. Additionally, it reverses adverse effects such as hepatic iron overload and oxidative stress induced by acetaminophen in mice, showcasing its ability to reduce intracellular ROS accumulation, trigger the Nrf2 pathway, upregulate GPX4, and prevent hepatocyte ferroptosis51. Through our research, using HUVECs as a model, we established kaempferol's efficacy in attenuating RSL3-induced cell death, highlighting its potential in the treatment of vascular diseases through ferroptosis inhibition.

In exploring ferroptosis further, we discovered it to be a reactive oxygen species (ROS)-dependent cell demise mechanism, exacerbating oxidative damage through excessive ROS generation via the Fenton reaction52, 53. Our investigation into the impact of kaempferol on lipid peroxidation, utilizing assays like DHE for intracellular ROS levels30 and C11-BODIPY for lipid peroxidation40, confirmed significant inhibition of RSL3-induced lipid peroxidation in HUVECs.

Multiple regulatory signals such as GPX4 and SLC7A11 are involved in the regulation of cell ferroptosis54. The GSH–GPX4 limits membrane lipid peroxidation via targeting System Xc¯ cystine/glutamate antiporter55, 56. SLC7A11 maintains the production of GSH, a major endogenous antioxidant, through a series of reactions involving the exchange of extracellular cysteine with intracellular glutamate46. Inhibiting the SLC7A11 pathway stands out as a critical upstream mechanism for inducing ferroptosis57. The expression of GPX4 and SLC7A11 at both protein and RNA levels was investigated in RSL3-treated HUEVCs. This study also provides evidence that kaempferol could significantly protect HUEVCs ferroptosis through the regulation of GPX4 and SLC7A11 expression.

The implications of endothelial cell ferroptosis extend to a variety of vascular-related conditions, including peripheral vascular disease58, stroke59, heart disease60, diabetes61, venous thrombosis62, tumor growth63, and metastasis64, making the targeting of endothelial cell ferroptosis a novel therapeutic strategy. Kaempferol's multi-faceted pharmacological effects, combined with its minimal toxicity, endow it with significant potential in both health food and pharmaceutical sectors51.

In previous studies, kaempferol has shown potential effectiveness in the treatment of diseases such as Alzheimer's disease65 and colon cancer. It exhibits various effects such as antioxidant, anti-inflammatory, anti-tumor, and promotion of glucose metabolism by regulating multiple signaling pathways such as Nrf2/SLC7A11/GPX4, Toll-like receptor 4 (TLR4)/ nuclear factor kappa-B (NF-κB), immunoglobulin-regulated enhancer 1 (IRE1)/ c-Jun N-terminal kinase (JNK)/ C/EBP homology protein (CHOP), and mitogen-activated　protein　kinases (MAPKs). In addition, compared with some chemotherapeutic agents, kaempferol is not toxic to normal cells66 and appears to be relatively safe at certain doses67. However, clinical trials of kaempferol on humans are still scarce and remain controversial, as most studies are based on animal models or in vitro experiments. More studies are still needed to determine its safety, pharmacokinetics and potential adverse effects in humans. In addition, although kaempferol has shown potential therapeutic effects in vitro and in vivo models, there is a lack of clinical trial validation, and therefore more human studies are needed to confirm its efficacy and safety in clinical applications.

The present study had a limited experimental model and did not elucidate the molecular mechanisms of kaempferol in depth. Future studies could further explore the molecular mechanisms and interactions of kaempferol in regulating signaling pathways and inhibiting iron death, as well as the targets and biological effects of kaempferol. This will contribute to a better understanding of the mechanism of action of kaempferol and provide a more scientific basis for its future clinical applications.

We uncovered the protective role of kaempferol in safeguarding human umbilical vein endothelial cells (HUVECs) from ferroptosis, an iron-dependent form of cell death. This protective mechanism functions through the modulation of GPX4 and SLC7A11, crucial elements in the cell's defense against ferroptosis. These insights broaden our comprehension of ferroptosis mechanisms and position kaempferol as a potential therapeutic candidate for drug development.

ALOXs - Arachidonic Acid Lipoxygenases, ANOVA - Analysis of Variance, ACSL4 - Acyl-CoA Synthetase Long-Chain Family Member 4, CCK-8 - Cell Counting Kit-8, DAPI - 4',6-diamidino-2-phenylindole, DMEM - Dulbecco’s Modified Eagle’s Medium, DHE - Dihydroethidium, FBS - Fetal Bovine Serum, GSH - Glutathione, GPX4 - Glutathione Peroxidase 4, HO-1 - Heme Oxygenase-1, HRP - Horseradish Peroxidase, HUVECs - Human Umbilical Vein Endothelial Cells, IRF - Interferon Regulatory Factor, LPCAT3 - Lysophosphatidylcholine Acyltransferase 3, Nrf2 - Nuclear Factor Erythroid-Derived 2-Like 2, PBS - Phosphate-Buffered Saline, PCs - Procyanidins, PVDF - Polyvinylidene Difluoride, qPCR - Real-Time Fluorescence Quantitative Polymerase Chain Reaction, RCD - Regulated Cell Death, RIPA - Radioimmunoprecipitation Assay Buffer, ROS - Reactive Oxygen Species, SD - Standard Deviation, SDS-PAGE - Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SLC7A11 - Solute Carrier Family 7 Member 11

We would like to thank Editage (www.editage.cn) for English language editing.

All authors significantly contributed to this work, read and approved the final manuscript.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.