NJ025-1001

Kaempferol Mitigates H2O2-Induced Oxidative Stress in RAW 264.7 Macrophages and Zebrafish  

Muhammad Haroon1 and Sun Chul Kang1,2* 

1Department of Biotechnology, Daegu University, Gyeongsan, 38453, Republic of Korea, 2Dr. Kang Bio Co., Ltd., Gyeongsan, 38428, Republic of Korea. 

Correspondence to: Sun Chul Kang, sckang@daegu.ac.kr

Received: October 2, 2025; Revised: November 24, 2025; Accepted: November 26, 2025; Published: November 29, 2025 

NATPRO J. 2025, 2, 35-38 

https://doi.org/10.23177/NJ025.1001 

Copyright ©  The Asian Society of Natural Products

Abstract

Oxidative stress is a key mediator in numerous inflammatory diseases. This study investigated the protective effects of kaempferol against H₂O₂-induced oxidative damage in RAW 264.7 macrophages and a zebrafish model. In macrophages, kaempferol pre-treatment significantly attenuated H₂O₂-induced cytotoxicity and lactate dehydrogenase (LDH) release. It also reduced intracellular reactive oxygen species (ROS) and suppressed the secretion of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. Mechanistically, this protection was mediated by the activation of the Nrf2 antioxidant pathway and its downstream targets SOD1, catalase, and HO-1. Furthermore, in zebrafish, kaempferol improved survival and hatching rates, reduced ROS levels and activated the Nrf2 pathway. Collectively, our findings demonstrate that kaempferol exerts potent antioxidant and anti-inflammatory effects both in vitro and in vivo, suggesting its therapeutic potential against oxidative stress-related pathologies.

Keywords

kaempferol; oxidative stress; cytotoxicity; RAW 264.7; zebrafish

Introduction

Oxidative stress, characterized by an imbalance between ROS production and antioxidant defenses, contributes to cellular damage and various diseases, including inflammation, cancer, and cardiovascular disorders. Hydrogen peroxide (H2O2), a common ROS, is widely used to induce oxidative injury in cellular models. In this study, RAW 264.7 macrophages were utilized, a cell line widely used to study inflammation and redox responses due to their role in immune activation and cytokine production. To complement the in vitro findings, the zebrafish model was used as an ethical, rapid vertebrate system for assessing developmental toxicity and oxidative stress, offering insights into whole-organism effects such as survival, hatching, and ROS accumulation [1, 2].

Kaempferol, a natural flavonoid abundant in fruits and vegetables, exhibits potent antioxidant properties, including the ability to scavenge ROS, modulate the Nrf2/HO-1 pathway, and reduce inflammation. Previous studies have demonstrated its protective effects against H2O2-induced damage in various cell lines; however, an integrated evaluation in macrophages and the zebrafish model remains limited [3, 4]. This study aimed to investigate the protective role of kaempferol against H2O2-induced oxidative stress and toxicity using RAW 264.7 macrophages (in vitro cytotoxicity, ROS, cytokines, and antioxidant enzymes expression) and a zebrafish model (survival, hatching, ROS, and antioxidant enzymes expression). This combined strategy bridges cellular and organismal responses, highlighting kaempferol's therapeutic potential for oxidative stress-related conditions and supporting its development as a natural protective agent [5].

Materials and methods

Cell culture and treatments

RAW 264.7 macrophages (ATCC) were cultured in DMEM (10% FBS, 1% penicillin/streptomycin) at 37°C, 5% CO2. Cells were pre-treated with various concentrations of kaempferol (5-70 μM) for 6 h, followed by 300 μM H2O2 for 24 h in serum-free DMEM. Controls included untreated, H2O2 alone, and kaempferol alone groups.

Cell viability and cytotoxicity assays

Cell viability was assessed using the MTT assay. Following treatments, the cells were incubated with 0.5 mg/mL MTT for 4 h. The formazon crystals were dissolved with DMSO, and the absorbance was measured at 570 nm. The cytotoxicity was determined with LDH release assay by using a commercial kit according to the manufacturer’s instructions, with absorbance measured at 490 nm. Data were normalized to controls.

H2DCFDA assay

Intracellular ROS was measured using the fluorescent probe H2DCFDA. Treated cells were incubated with 10 μM H2DCFDA (30 min at 37°C), and fluorescence was quantified using an inverted fluorescent microscope (excitation 485 nm, emission 535 nm).

Cytokine measurements

Supernatants from treated cells were analyzed for TNF-α, IL-1β, and IL-6 using ELISA kits (R&D Systems). Absorbance was measured at 450 nm, and concentrations (pg/mL) were calculated from standard curves.

In vivo experiments

All experiments were performed using wild-type AB zebrafish (Danio rerio) embryos and larvae. Adult fish were maintained in a recirculating aquaculture system under standard husbandry conditions: a constant water temperature of 28.5 °C and a 14/10 h light/dark cycle. Embryos were raised in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄) in a Petri dish. Zebrafish embryos (4-6 hours post-fertilization; hpf) were exposed to 2 mM H2O2 ± kaempferol pre-treatment (30-50 μM) in E3 medium. The experiment was performed in three independent biological replicates (N=3). For survival, hatching rate and H2DCFDA assay, 30 embryos were allocated to each experimental group (n = 30 per group per replicate). For western blot analysis, approximately 200 embryos were used per group. Survival and hatching rates were recorded under a microscope at 24, 48, 72, and 96 hpf. ROS was detected in 96 hpf larvae using 20 μM H2DCFDA, imaged via fluorescence microscopy, and quantified with ImageJ software.

Western blot analysis

Protein expression of Nrf2, SOD1, catalase, and HO-1 was analyzed in both RAW 264.7 cells and zebrafish larvae. For cells, lysates were prepared directly in RIPA buffer. For zebrafish, pools of larvae (approximately 200 per group) were homogenized in ice-cold RIPA lysis buffer, and the homogenates were centrifuged at 12,000 × g for 15 min at 4°C to collect the supernatant. The protein concentration of all lysates was determined using the Bradford assay. Equal amounts of protein (100 µg) were separated by 10–12% SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against Nrf2, SOD1, catalase, HO-1, and β-actin (Cell Signaling Technology, 1:1000). After incubation with HRP-conjugated secondary antibodies, protein bands were visualized using an enhanced chemiluminescence (ECL) substrate and quantified by densitometry using ImageJ software.

Statistical analysis

All data are presented as the mean ± standard deviation (SD). Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons in GraphPad Prism software. A p-value of < 0.05 was considered statistically significant.

Results and Discussion

In RAW 264.7 macrophages, H2O2 (25-1000 μM) reduced cell viability dose-dependently, with 300 μM inducing ~50% cell death after 24 h (Figure 1A), consistent with H2O2's induction of oxidative stress and TNF-α production in macrophages, which highlights its role in triggering ROS-mediated cellular damage [6]. Kaempferol (5-70 μM) alone showed no cytotoxicity (Figure 1B), confirming its safety as a flavonoid antioxidant that supports its non-toxic profile in various cell lines [7-9]. Pre-treatment with kaempferol (5-50 μM for 6 h) significantly protected against 300 μM H2O2-induced cytotoxicity; specifically, 50 μM kaempferol increased cell survival by ~40% and reduced LDH release (Figure 1C and 1D). These results indicated the cytoprotection by attenuating membrane damage from oxidative stress, as reported in similar studies [10]. 

The H2DCFDA assay showed that kaempferol (30-50 μM) reduced H2O2-induced ROS by ~50% (Figure 2A), reflecting its potent scavenging activity, which aligns with previous reports on flavonoid-mediated ROS reduction in stressed cells [9]. Pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) decreased by up to 60% (Figure 2B), consistent with kaempferol's inhibition of cytokine release in macrophages, which may involve downregulation of inflammatory pathways under oxidative conditions [11]. Western blot analysis revealed a 1.5-3-fold upregulation of Nrf2, SOD1, catalase, and HO-1 (Figure 2C), suggesting enhanced antioxidant defenses via Nrf2 activation, which mirrors kaempferol's role in boosting enzymatic responses to oxidative stress [12]. We next evaluated whether these protective effects translated to an in vivo model. In zebrafish embryos, exposure to 2 mM H2O2 reduced survival to ~45% and hatching to ~40% by 96 hpf, reflecting H2O2's disruption of development via ROS accumulation as observed in models of oxidative toxicity. Kaempferol pre-treatment (30-50 μM) markedly improved these phenotypic outcomes, increasing survival and hatching by 30-50% and decreasing ROS by ~45% in surviving larvae (Figure 3A-3C). We also analyzed the protein levels of key components of the Nrf2 pathway in zebrafish larvae by western blot. Critically, western blot analysis of zebrafish larvae confirmed that the protective mechanism was conserved in vivo, showing that kaempferol significantly increased the protein expression of Nrf2 and its downstream targets, SOD1, catalase, and HO-1 (Figure 3D), demonstrating its protective capacity in vivo by alleviating oxidative damage in zebrafish, similar to flavonoid interventions in stress models [4, 13, 14]. 

Conclusion

In summary, our findings demonstrate that kaempferol confers significant protection against H₂O₂-induced oxidative damage across both cellular and whole-organism models. In macrophages, kaempferol not only attenuated cytotoxicity and ROS but also suppressed pro-inflammatory cytokine secretion. Mechanistically, we confirmed that this protection is mediated through the activation of the Nrf2 pathway, leading to the upregulation of key antioxidant enzymes (SOD1, catalase, and HO-1). The translational relevance of this mechanism was confirmed in vivo, where kaempferol improved survival, hatching rates, and reduced ROS in zebrafish, effects that were similarly associated with Nrf2 pathway activation. This integrated approach not only validates the zebrafish as a powerful model for oxidative stress research but, more importantly, strongly underscores kaempferol's potential as a therapeutic candidate for mitigating oxidative stress-related pathologies. Future studies should focus on elucidating the precise molecular triggers of Nrf2 activation by kaempferol and evaluating its efficacy in more complex mammalian disease models.

Ethical approval

The Daegu University Ethical Committee (LMO No. LML-16-1134) approved the study, and the experimental procedures were conducted under the animal care guidelines.

Conflict of Interest

The submission has been read and approved by all the authors indicated above. Additionally, they state that they have no conflicting financial or non-financial interests.

Funding

This research was supported by “National Research Foundation of Korea” grant, NRF RS-2023-00253438.

Authors' contributions

M. Haroon performed experiments and analysis. M. Haroon prepared the manuscript. S.C. Kang verified the results and approved the study. All authors have approved the submission of the manuscript.

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