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. 2026 Sep 29;50(10):e70216. doi: 10.1002/cbin.70216

Fisetin Attenuates Human Macrophage Polarization and Pro‐Inflammatory Responses via NLRP3 and NF‐κB/MAPK Suppression

Nattaya Onpan 1, Chanchao Lorthongpanich 1,2,✉, Pakpoom Kheolamai 3,4, Pimjai Chingsuwanrote 1, Napachai Rodboon 1, Sudjit Luanpitpong 1, Chonticha Saisawang 5, Surapol Issaragrisil 1,6,7
PMCID: PMC13621920  PMID: 42808783

ABSTRACT

Natural compounds with anti‐inflammatory properties are increasingly explored as therapeutic agents due to their lower risk of side effects compared with conventional drugs. Fisetin, a dietary bioflavonol abundant in fruits and vegetables, exhibits anti‐inflammatory activity in several cell types, including murine macrophages. However, its effects on human macrophages remain unclear. In this study, human pro‐inflammatory M1 macrophages were generated from THP‐1 monocytes using PMA, LPS, and IFN‐γ. The effects of fisetin on cytokine and chemokine secretion, reactive oxygen species (ROS) production, apoptosis, and phagocytic activity were evaluated. In addition, the NF‐κB, MAPK, and NLRP3 inflammasome pathways were analyzed. This study shows that fisetin pre‐treatment significantly suppressed LPS/IFN‐γ‐induced secretion of pro‐inflammatory cytokines (IL‐6, TNF‐α, IL‐1β, IL‐8) and chemokines (MCP‐1, CCL5, CXCL9, CXCL10). It also reduced ROS generation, NLRP3 inflammasome activation, and phagocytic activity. Mechanistically, fisetin inhibited NF‐κB activation as well as JNK/MAPK and p38/MAPK signaling, indicating that its anti‐inflammatory actions are mediated through multiple pathways. Importantly, fisetin did not affect the viability or proliferation of THP‐1‐derived macrophages, suggesting that reduced cytokine release was primarily due to attenuation of macrophage polarization rather than cytotoxicity. Fisetin attenuates inflammatory responses in human M1 macrophages through suppression of NF‐κB, MAPK, and NLRP3 inflammasome signaling. By limiting cytokine release, ROS production, and phagocytic activity without impairing cell survival, fisetin emerges as a promising natural candidate for managing chronic inflammatory disorders, including atherosclerosis, neurodegenerative diseases, and inflammatory bowel disease.

Keywords: anti‐inflammation, Fisetin, macrophages, MAPK pathway, NF‐κB, NLRP3 inflammasome, THP‐1

Abbreviations

CCK‐8

cell counting kit 8

CCL

CC motif chemokine ligand

CXCL

CXC motif chemokine ligand

DMSO

dimethyl sulfoxide

Fi

fisetin

IFN‐γ

interferon‐gamma

IL‐1β

interleukin‐1β

IL‐6

interleukin‐6

IL‐8

interleukin‐8

LPS

lypopolysaccharide

MCP‐1

monocyte chemoattractant protein‐1

MTT

3‐[4,5‐dimethylthiazol‐2‐yl]−2,5 diphenyl tetrazolium bromide

NF‐κB

nuclear factor‐kappa B

NLRP3

NOD‐like receptor protein 3, ROS

Reactive oxygen species

TGF‐β

transforming growth factor‐beta

TNF‐α

tumor necrosis factor‐alpha

1. Introduction

Macrophages play a vital role in host innate immune responses by defending against many pathogens and cancers. The macrophages were heterogenous and could be polarized into pro‐inflammatory M1 macrophages and anti‐inflammatory M2 macrophages depending on the environmental signals (Chen et al. 2023a; Viola et al. 2019). M1 macrophages produces pro‐inflammatory cytokines such as interleukin (IL)−6 (Tanaka et al. 2014; Gabay 2006), tumor necrosis factor (TNF)‐α (Tracey, M.D and Cerami, Ph.D 1994), IL‐1β (Dinarello 1996), and IL‐8 (Apostolopoulos et al. 1996), and chemokines such as C‐C motif chemokine ligand (CCL)−5 (Jin et al. 2024; Keophiphath et al. 2010), C‐X‐C motif chemokine ligand (CXCL)−9 (Callahan et al. 2021), CXCL10 (Callahan et al. 2021; Song et al. 2021), and monocyte chemoattractant protein‐1 (MCP‐1/CCL2) (Deshmane et al. 2009; Hada et al. 2021). M1 macrophages also produce high levels of reactive oxygen species (ROS) (Canton et al. 2021; Tan et al. 2016) and nitric oxide (Hada et al. 2021), which are important for pathogen clearance. The M1 phenotype could be induced by LPS and IFN‐γ (Genin et al. 2015; Meng and Lowell 1997). LPS binds to Toll‐like receptor 4 (TLR4) and sends a signaling cascade through the nuclear factor kappa B (NF‐κB) and MAPK kinase pathways (Chen et al. 2023a; Javaid and Choi 2020; Xia et al. 2021). In contrast, M2 macrophage, which produces anti‐inflammatory cytokines such as IL‐10 and TGF‐β (Zhang et al. 2017), is induced by IL‐4 and IL‐13 (Abdelaziz et al. 2020). Overreactive pro‐inflammatory responses from M1 macrophages cause chronic debilitating diseases, such as atherosclerosis (Hou et al. 2023; Wu et al. 2023), inflammatory bowel diseases (Hegarty et al. 2023) and sepsis (Wang and Wang 2023; Chen et al. 2021). Therefore, finding anti‐inflammatory agents that could be used to inhibit overactive M1 macrophages with low adverse effects could have great therapeutic potential.

Natural compounds with anti‐inflammatory property are gaining more popularity as potential therapeutic agents (Zhou et al. 2023), due to their lower adverse effects compared to other conventional anti‐inflammatory agents, such as steroids and non‐steroidal anti‐inflammatory (NSAIDs) drugs, especially in long‐term treatment. Among these natural compounds, fisetin (3,7,3’,4’‐tetrahydroxyflavone), a natural flavonoid mostly found in grapes, apples, strawberries (Khan et al. 2013), has been shown to be an effective anti‐inflammatory (Wu et al. 2022), antioxidant (Ahmad et al. 2019) and anti‐cancer agent (Zhou et al. 2023; Khan et al. 2013; Syed et al. 2013; Kubina et al. 2022) and has been recently used as food supplement for health benefits (Khan et al. 2013). Fisetin has been shown to have anti‐inflammatory activity in several cell types, such as mouse bronchial epithelial BEAS‐2B cells (Wu et al. 2022), mouse kidney inflammation (Ren et al. 2020), mouse microglia (Ahmad et al. 2019; Chuang et al. 2014), human primary pulmonary epithelial cell (Sim et al. 2020), and human gingival fibroblasts (Gutiérrez‐Venegas et al. 2014). Fisetin has also been shown to inhibit LPS‐induced pro‐inflammatory responses in mouse macrophages RAW264.7 cells (Kim et al. 2012; Molagoda et al. 2021a) by inhibiting the production of nitric oxide, IL‐6 and TNF‐α secretion through the Syk/Src/NF‐κB, JNK/MAPK (Kim et al. 2012, 2015a) and PIE3K/AKT/mTOR signaling pathways (Sun et al. 2021; Molagoda et al. 2021a). Moreover, fisetin also reduced the production of inflammation mediators, IL‐1β, MCP‐1 and nitric oxide in LPS and IFN‐γ induced pro‐inflammatory peritoneal by suppressing p‐ERK and p‐JNK pathways (Hada et al. 2021).

Although there are many studies show the anti‐inflammatory effect of fisetin in mouse macrophages, its effect on human macrophages has not yet been comprehensively investigated.

In this study, we study the effect of fisetin in human monocyte‐derived macrophages focusing on its anti‐inflammatory responses and the underlying mechanisms. Knowledges gained from this study would provide a better understanding of the role of fisetin in inhibiting inflammatory responses in human pro‐inflammatory M1 macrophages that could be beneficial for the development of new therapeutic strategies for chronic inflammatory diseases such as atherosclerosis, neurodegeneration, and inflammatory bowel disease in the future.

2. Materials and Methods

2.1. Cell Culture

THP‐1, a human monocytic leukemia cell line (JCRB0112, JCRB, Japan; CVCL_0006) was cultured in Roswell Park Memorial Institute medium (RPMI) 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat‐inactivated FBS (Gibco) in a 5% CO2 incubator at 37°C.

The THP‐1 cell line used in this study was subjected to short tandem repeat (STR) analysis to confirm its 100% authentication and tested negative for mycoplasma contamination prior to use in the experiments. The cell line has not been previously reported as misidentified or contaminated.

2.2. Derivation of Human M1 Macrophage From THP‐1 Cells

THP‐1 cells, a density of 5 × 105 cells/well, were seeded in a 6‐well in 2.5 ml of RPMI 1640 medium supplemented with 10% FBS and 20 nM phorbol 12‐myristate 13‐acetate (PMA) (P8139, Sigma‐Aldrich Corporation St. Louis, MO, USA) for 2 days to induce their differentiation to macrophages. The THP‐1‐derived macrophages were then polarized to pro‐inflammatory M1 macrophages by adding fresh media supplemented with 10 ng/ml LPS (L8274, Sigma‐Aldrich) and 20 ng/ml IFN‐γ (Miltenyi Biotec) (Supporting Figure 1) (Genin et al. 2015; Meli et al. 2020).

2.3. Fisetin and Inhibitor Treatment

Fisetin (Sigma‐Aldrich) was dissolved in dimethyl sulfoxide (DMSO) (Sigma‐Aldrich) as described previously (Lorthongpanich et al. 2021, 2024). THP‐1 cells were pretreated with fisetin across a concentration range of 10–60 µM for 1 h before activation into M1 macrophages by treatment with 10 ng/mL LPS and 20 ng/mL IFN‐γ. THP‐1 cells pretreated with DMSO serve as a control. JSH‐23 (Cat no. HY‐113982, MedChem Express) was used as a p‐65 NF‐kB inhibitor.

2.4. MTT Cell Cytotoxicity Assay

Cell cytotoxicity was determined using the MTT assay. THP‐1 cells were seeded in a 96‐well culture plate at a density of 5 × 104/well. Cells were treated with 0−60 µM fisetin for 48 h. The MTT solution was added to the cell culture at a final concentration of 0.5 mg/mL for 3 h. The reaction was stopped by adding the MTT solubilizer containing 10% sodium dodecyl sulfate (SDS) to solubilize formazan. Cell cytotoxicity was determined using a microplate reader (Synergy H1, BioTek Instruments Inc., Winooski, VT, USA) at an absorbance of 570 nm. After the test, concentration of 10, 20 and 40 μM were used for further experiments.

2.5. Enzyme‐Linked Immunosorbent Assays (ELISA)

The cell culture supernatant was collected, centrifuged at 2000 rpm for 5 min to remove cell debris. The levels of secreted pro‐inflammatory cytokines: IL‐6, TNF‐α and IL‐1β were determined by an ELISA (IL‐6 #430504, TNF‐α #430204 and IL‐1β #437004, Biolegend) according to the manufacturer's instructions.

2.6. Reactive Oxygen Species (ROS) Production Assay

Cells were seeded in a 96‐well plate at a density of 2.5 × 104 cells/well and treated with various concentrations of fisetin for 1 h before treating with 10 ng/mL LPS and 20 ng/mL IFN‐γ for 24 h. ROS production levels were determined by incubating cells with the 2’, 7’‐dichlorofluorescein diacetate (DCFDA) (ab113851, Abcam) for 45 min at 37°C in the dark according to the manufacturer's instruction and analyzed using flow cytometry.

2.7. Cell‐Counting Kit (CCK) 8 Cell Proliferation Assay

Cells were seeded in a 96‐well plate at a density of 2.5 × 104 cells/well, pretreated with various concentrations of fisetin for 1 h, and then treated with 10 ng/mL LPS and 20 ng/mL IFN‐γ for 24 h. Subsequently, 10 µL of CCK‐8 solution (CK04, Dojindo Laboratories) was added to each well and incubated for 2 h according to the manufacturer's instructions. Absorbance was measured at 450 nm using a microplate reader (Synergy H1).

2.8. Apoptosis Assay

Cells were harvested and resuspended in 100 µL of 1 × binding buffer containing 2 µL of 7‐AAD (BD Bioscience, San Jose, CA, USA), and incubated for 15 min at room temperature in the dark. The binding buffer was then added to a final volume of 300 µL, and the cells were analyzed by flow cytometry.

2.9. FITC‐Dextran Uptake Assay

Cells were incubated with 1 mg/mL FITC‐dextran (53379, Sigma‐Aldrich) in culture medium for 20 min at 37°C. Cells were harvested by incubating with trypsin/EDTA (Gibco) for 5 min, washed with 2% FBS in PBS, and then analyzed using flowcytometry (FACS Canto (BD Bioscience) and photographed using Amnis Imaging flow cytometers (Luminex, Texas, USA) (Luanpitpong et al. 2022).

2.10. Quantitative Real‐Time Reverse Transcription‐Polymerase Chain Reaction (qRT‐PCR)

RNA was isolated using TRIzol reagent (Molecular Research Center, Cincinnati, OH, USA). A total of 500 ng RNA, quantified by Nanodrop 2000 spectrophotometer (Thermo Fisher Science), was reverse transcribed using a ReverseAid First Strand cDNA synthesis Kit (Thermo Fisher Scientific). qRT‐PCR was performed using a CFX356 Real‐Time PCR Detection System (Bio‐ Rad Laboratories) with SYBR select master mix (Thermo Fisher Scientific). Data were normalized to GAPDH. The list of primers is provided in Table S1.

2.11. Western Blot Analysis

Cells were collected, washed with PBS, and lysed by incubation with radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) containing proteinase K (Thermo Fisher Scientific) and phosphatase inhibitors (Sigma‐Aldrich) on ice for 30 min. Protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific). Protein was separated by 10%–15% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a 0.45 µM polyvinylidene fluoride (PDVF) membrane (Merck Millipore).

The membrane was then blocked with 5% skim milk (Merck Millipore) in Tris‐buffered saline containing 0.1% Tween‐20 for 1 h at room temperature and incubated with the appropriate primary antibodies at 4°C overnight. After incubation with primary antibodies, the membrane was incubated with HRP‐conjugated secondary antibody at room temperature for 2 h and then incubated with 1:10,000 HRP‐conjugated anti‐human β‐actin for 30 min. The list of antibodies is provided in Table S2. Finally, the membrane was incubated with enhanced chemiluminescence (ECL) (Bio‐Rad Laboratories, Hercules, CA, USA), and SuperSignal west atto ultimate sensitivity chemiluminescent substrate (A38555, Thermo Scientific) and visualized using a biomolecular imager (ImageQuant LAS4000; GE Healthcare, Chicago, IL, USA).

2.12. Statistical Analysis

The results are presented as mean ± standard error of the mean (SEM) of at least three independent experiments. The significant difference was determined using Student's t‐test when comparing between two groups or by one‐way analysis of variance (ANOVA) when comparing more than two groups. Analysis was performed using GraphPad Prism software version 10 (GraphPad Software, San Diego, CA, USA). A p < 0.05 was considered statistically significant.

3. Results

3.1. Derivation of Human Pro‐Inflammatory M1 Macrophages From THP‐1 Cells

To derive M1 macrophages for investigating the anti‐inflammatory effect of fisetin, human monocytic cells, the THP‐1 cells were first induced to become non‐activated macrophages (M0) by treatment with 20 nM PMA for 2 days. The resulting M0 macrophages were then activated to become pro‐inflammatory macrophages (M1) by treating with LPS and IFN‐γ as described in Figure 1A. After PMA treatment, most THP‐1 cells attach to the culture surface and become macrophage‐like cells. There is no significant difference between the morphology of M0 and M1 macrophages derived from THP‐1 cells (Figure 1B). As expected, the THP‐1 derived M1 macrophages expressed much higher levels of pro‐inflammatory cytokines: IL‐6, TNF‐α and IL‐1β, compared to their M0 counterparts and the non‐differentiated THP‐1 cells (mono) (Figure 1C). Consistent with gene expression, THP‐1 derived M1 macrophages also released significantly higher levels of IL‐6, TNF‐α and IL‐1β protein in conditioned medium compared to their M0 counterparts and non‐differentiated THP‐1 cells (Figure 1D). These results confirm that our culture procedure successfully derived pro‐inflammatory M1 macrophages from THP‐1 cells.

Figure 1.

Figure 1

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Derivation of THP‐1 monocytic cell line to THP‐1 M1 like macrophages. (A) Schematic of derivation of M1 macrophage from THP‐1 monocytic cell line. (B) Cell morphology of THP‐1 derived M0 and M1 macrophages. (C) Relative gene expression of pro‐inflammatory genes: IL‐6, TNF‐α and IL‐1β normalized with GAPDH of THP‐1 derived M1 macrophage compared to THP‐1 monocyte and THP‐1 M0 macrophage. (D) Pro‐inflammatory cytokine secretion of THP‐1 derived M1 macrophage compared to THP‐1 monocyte and THP‐1 M0 macrophage analyze by ELISA. Mono=THP‐1 monocyte, mac = THP‐1 derived macrophage after PMA treatment for 2 days, M0 = THP‐1 derived M0 macrophage without LPS and IFN‐γ stimulation and M1 = THP‐1 derived M1 macrophage with LPS and IFN‐γ stimulation. Data represent the mean ± standard error of the mean (SEM), *p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar = 100 μM.

3.2. Fisetin Inhibits the Production and Secretion of Pro‐Inflammatory Cytokines in THP‐1‐Derived M1 Macrophages

The cytotoxicity assay showed that 10, 20 and 40 µM fisetin did not cause significant cell death in THP‐1 cells (Figure 2A, Figure S2), and therefore these concentrations were chosen for subsequent experiments. We first evaluate the effect of fisetin on M0 activation in pro‐inflammatory M1 macrophages by pre‐treating M0 macrophages with various concentrations of fisetin for 1 h, followed by stimulation with LPS and IFN‐γ (Figure 2B). The results showed that fisetin significantly reduced the expression levels of pro‐inflammatory cytokine genes: IL‐6, TNF‐α, IL‐1β, IL‐8 in THP‐1 derived macrophage, in a dose‐dependent manner compared to the untreated control, but did not significantly affect the expression levels of M2 anti‐inflammatory cytokine genes: TGF‐β1 and IL‐10 (Figure 2C). Consistent with its effect on gene expression, fisetin significantly reduced the levels of IL‐6, TNF‐α and IL‐1β protein released from the THP‐1 derived macrophages, in a dose‐dependent manner compared to the untreated control (Figure 2D). Furthermore, fisetin also significantly reduced NOD‐like receptor protein 3 (NLRP3) levels, an important component of the inflammasome complex that regulates IL‐1β secretion (Kelley et al. 2019; Olona et al. 2022), in THP‐1 derived macrophages, in a dose‐dependent manner compared to the untreated control (Figure 3A). Consistency with IL‐1β protein levels (Figure 3B). These findings demonstrate that fisetin effectively inhibits the activation of M0 macrophages into pro‐inflammatory M1 macrophages by reducing pro‐inflammatory cytokine expression and secretion without promoting a shift toward the anti‐inflammatory M2 phenotype.

Figure 2.

Figure 2

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Fisetin inhibited LPS and IFN‐γ induced pro‐inflammatory cytokine production and secretion in THP‐1 derived M1 macrophages. (A) Cytotoxicity of fisetin to THP‐1 monocyte analyzed by MTT assay. (B) Schematic of fisetin treatment to THP‐1 derived macrophages. (C) Relative gene expression of M1 macrophage related genes: IL‐6, TNF‐α, IL‐1β and IL‐8 and M2 macrophage related genes: TGF‐β1 and IL‐10 normalized with GAPDH of THP‐1 derived M1 macrophage after fisetin treatment for 24 h. (D) Pro‐inflammatory cytokine secretion of THP‐1 derived M1 macrophage after fisetin treatment analyze by ELISA at 24 and 48 h. Data represent the mean ± standard error of the mean (SEM), *p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3.

Figure 3

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Fisetin inhibited IL‐1β and NLRP3 inflammasome expression in THP‐1 derived M1 macrophages. (A) Expression of NLRP3 inflammasome after 0‐40 μM of fisetin treatment at 24 and 48 h and fold intensity changes analyzed by Image J. (B) Expression of IL‐1β after 0–40 μM of fisetin treatment at 24 and 48 h and fold intensity changes analyzed by Image J. Data represent the mean ± standard error of the mean (SEM), *p < 0.5, **p < 0.01. Full‐length, uncropped Western blot gels corresponding to Figure 3A,B are provided in the supplementary western blot file. The selected band for each protein is highlighted with a green box.

3.3. Fisetin Inhibits the Production of Chemokines and Reactive Oxygen Species (ROS) in THP‐1 Derived M1 Macrophages

Next, we investigated the effect of fisetin on the production of chemokines, including CCL‐5, MCP‐1, CXCL‐9, and CXCL‐10 which play key roles in leukocytes recruitment and enhancing of inflammatory signals (Dong et al. 2003; Singh et al. 2021), in THP‐1 derived M1 macrophages. Similarly, to its suppressive effect on pro‐inflammatory cytokine production, pre‐treatment with fisetin significantly reduced the expression levels of the CCL‐5, MCP‐1, CXCL‐9, and CXCL‐10 genes in THP‐1 derived macrophages, in a dose‐dependent manner compared to the untreated control (Figure 4A). In addition, fisetin significantly decreased reactive oxygen species (ROS) production, which is known to enhance inflammatory responses (Tran and Mills 2024; Herb and Schramm 2021), also in a dose‐dependent manner (Figure 4B–C). These findings further support the anti‐inflammatory effect of fisetin, as evidenced by its ability to reduce chemokine expression and ROS production in THP‐1‐derived M1 macrophages, both of which contribute to leukocyte recruitment and the amplification of inflammatory responses.

Figure 4.

Figure 4

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Fisetin reduced chemokines and reactive oxygen species (ROS) production in THP‐1 derived M1 macrophages. (A) Relative gene expression of chemokines: CCL5, MCP‐1, CXCL9 and CXCL10 of THP‐1 derived M1 macrophage after fisetin treatment for 24 h. (B) Flow cytometry analysis of ROS production of THP‐1 derived M1 macrophage after fisetin treatment. (C) Percentage of ROS positive cells after fisetin treatment for 24 h. Data represent the mean ± standard error of the mean (SEM), *p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3.4. Fisetin Inhibits the Phagocytosis Activity of THP‐1 Derived M1 Macrophages

In addition to its effect on gene and protein expression, we next investigated the effect of fisetin on phagocytic activity, which is an important function of pro‐inflammatory M1 macrophages, in the THP‐1 derived macrophage using the phagocytic FITC‐dextran beads assay (Figure 5A). As expected, the THP‐1 derived M1 macrophage exhibited a significantly higher level of phagocytosis activity than its M0 counterparts (90% vs. 70%, p < 0.05) analyzed after 24 h of M1 induction (Figure 5B). The result showed that treatment with 10‐20 μM fisetin slightly suppress phagocytic activity by 20%–30% (Figure 5C). However, at 40 μM fisetin significantly reduced the phagocytic activity of THP‐1 derived M1 macrophages compared to the untreated control (Figure 5C–F). This result highlights the effect of fisetin on a key pro‐inflammatory macrophage function.

Figure 5.

Figure 5

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Fisetin reduced phagocytosis activity of THP‐1 derived M1 macrophages. (A) Schematic of phagocytosis assay by FITC‐dextran uptake. (B) Comparison of percent phagocytosis cells of THP‐1 derived M1 macrophage (+LPS/IFN‐γ) and THP‐1 derived M0 macrophage (‐LPS/IFN‐γ). (C) Flow cytometry histogram of phagocytose FITC‐dextran cells after fisetin treatment for 24 h. (D) Percentage of phagocytose FITC‐dextran cells after fisetin treatment. (E) Mean fluorescence intensity of cells after fisetin treatment. (F) Representative image of phagocytose FITC‐dextran cells.

3.5. Fisetin Does Not Affect the Viability and Proliferative Capacity of THP‐1 Derived M1 Macrophages

We then determined whether the suppressive effect of fisetin on the THP‐1 derived macrophage was caused by a decrease in viability or the proliferative capacity of these cells by performing a 7‐AAD apoptosis assay and a CCK8 cell proliferation assay. The results showed that fisetin treatment did not affect the viability of the THP‐1‐derived macrophage compared to its untreated counterpart (Figure 6A). Consistent with this, the results of the western blot also showed that fisetin did not alter the levels of Caspase 3 and cleaved caspase 3 proteins compared to the untreated control (Figure 6B). These results indicated that fisetin at concentrations 10, 20 and 40 μM did not significantly induce cell death. Similar to its effect on the viability of THP‐1‐derived macrophages, the CCK8 cell proliferation assay showed that fisetin did not affect THP‐1‐derived M1 macrophage proliferation compared to their untreated control (Figure 6C). Therefore, the reduction in M1‐specific cytokine release could be attributed to fisetin's attenuation of macrophage polarization rather than to effects on viability or proliferation.

Figure 6.

Figure 6

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Fisetin did not significant induced cell dead or inhibited cell proliferation in THP‐1 derived M1 macrophages. (A). Percentages of cell dead stained with 7‐AAD after fisetin treatment at various concentration for 48 h. (B) Caspase 3 and cleaved caspase 3 staining of THP‐1 derived M1 macrophages after 24 h of fisetin treatment. (C) Cell proliferation assay analyzed by CCK8 assay kit of THP‐1 derived M1 macrophages after 48 h of fisetin treatment. Data represent the mean ± standard error of the mean (SEM), ns = not significant different. Full‐length, uncropped Western blot gels corresponding to Figure 6B are provided in the supplementary western blot file. The selected band for each protein is highlighted with a green box.

3.6. Fisetin Inhibits the NF‐κB, JNK/MAPK and p38/MAPK Signaling Pathways in THP‐1‐Derived M1 Macrophages

To investigate the mechanism, underline the anti‐inflammatory effect of fisetin, we determine its effect on the nuclear factor‐kappa B (NF‐κB) and mitogen‐activated protein kinase (MAPK) pathway (Ren et al. 2020) which have been known to play important roles in the regulation of many pro‐inflammatory cytokines, including IL‐6, and TNF‐α (Lawrence and Fong 2010; Tornatore et al. 2012). The results showed that 40 μM fisetin significantly decrease the level of phosphorylated p‐65 (NF‐κB) (phos‐p‐65) protein but not the total p65 proteins in THP‐1‐derived M1 macrophages (Figure 7A). Then we used a NF‐κB inhibitors, JSH‐23 which inhibits NF‐κB transcriptional activity and inhibits nuclear translocation of NF‐κB p‐65. Fisetin co‐treatment with JSH synergistic reduced the secretion of pro‐inflammatory cytokines and phos‐p‐65 levels (Supporting Figure 3). Similarly, to its effect on NF‐κB signaling, 20 and 40 μM fisetin significantly decrease the level of phosphorylated JNK (p‐JNK) and phosphorylated p38 (p‐p38) proteins in THP‐1‐derived M1 macrophages compared to the untreated control (Figure 7B,C). However, fisetin did not alter the level of phosphorylated ERK (p‐ERK) protein in these cells (Figure 7D). These findings suggest that fisetin inhibits the polarization of THP‐1‐derived macrophages toward the pro‐inflammatory M1 phenotype, at least in part, by suppressing the NF‐κB, JNK, and p38 MAPK signaling pathways.

Figure 7.

Figure 7

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Fisetin inhibited pro‐inflammation via NF‐κB pathway and JNK and p38/MAPK pathway in THP‐1 derived M1 macrophages. (A) Western blot analysis of phos‐p‐65 and (total) p‐65 expression after various concentration of fisetin treatment (left) and the band intensity of phos‐p‐65 (middle) and p‐65 (right) analyzed by Image J. (B‐D) Western blot analysis of JNK, p38 and ERK/MAPK pathway (top) and band intensity analyzed by Image J (bottom), respectively. Data represent the mean ± standard error of the mean (SEM), *p < 0.5, **p < 0.01, ***p < 0.001. Full‐length, uncropped Western blot gels corresponding to Figure 7A–D are provided in the supplementary western blot file. The selected band for each protein is highlighted with a green box.

4. Discussion

In this study, we showed that fisetin is an effective anti‐inflammatory agent that suppresses the production of pro‐inflammatory cytokines, chemokines, NLRP3 inflammasome components, ROS, and phagocytic activity in human THP‐1‐derived M1 macrophages. These findings are consistent with previous studies demonstrating the anti‐inflammatory effects of fisetin in mouse macrophages (Hada et al. 2021; Kim et al. 2012; Sun et al. 2021; Molagoda et al. 2021a; Kim et al. 2015b). In this study, we chose macrophages as a model because macrophages play a critical role in regulating inflammation and tissue repair. While previous studies have focused primarily on hepatic injury and liver transplantation (Peng et al. 2026; Li et al. 2025), our study specifically aimed to investigate the anti‐inflammatory effects of fisetin and its regulation of macrophage polarization (Peng et al. 2026; Li et al. 2025). Our study specifically aimed to investigate the anti‐inflammatory effects of fisetin and its regulation of macrophage polarization.

Our results showed that fisetin inhibited the activation of M0 to M1 macrophages by LPS and IFN‐γ treatment partly by reducing the expression levels of pro‐inflammatory cytokines IL‐6, TNF‐α, IL‐1β, and IL‐8 which play a pivotal role in acute and chronic inflammatory diseases, including septic shock syndrome (Gabay 2006; Tracey, M.D and Cerami, Ph.D 1994) and rheumatoid arthritis (Kay 2004). Furthermore, fisetin also significantly reduced NOD‐like receptor protein 3 (NLRP3) levels in THP‐1‐derived M1 macrophages. The NLRP3 inflammasome, a protein complex that regulates IL‐1β and IL‐18 secretion by activating caspase‐1 activity (Blevins et al. 2022), have been shown to play an importance role in inflammatory‐related diseases (Chen et al. 2023b). Consistent with the previous studies showing that fisetin inhibit NLRP3 inflammasome expression in both mouse BV2 microglia cells and a vascular dementia mouse model (Cordaro et al. 2022; Molagoda et al. 2021b). Fisetin has also been reported to attenuate hepatic ischemia‐reperfusion injury through regulation of the GSK3β/AMPK/NLRP3 inflammasome pathway (Pu et al. 2021). In that study, the anti‐inflammatory effects of fisetin were associated with modulation of the GSK3β/AMPK pathway and subsequent inhibition of NLRP3 inflammasome activation. In contrast, our study provides evidence that the anti‐inflammatory effects of fisetin in human THP‐1‐derived macrophages are also associated with inhibition of the NF‐κB signaling pathway. In particular, treatment with the NF‐κB inhibitor JSH‐23 reduced the production of IL‐6, TNF‐α, and IL‐1β, while co‐treatment with JSH‐23 and fisetin further reduced these pro‐inflammatory cytokines. These findings suggest that, although both studies support the anti‐inflammatory activity of fisetin and its effects on NLRP3‐related inflammation, the signaling pathways investigated are distinct, with the present study highlighting NF‐κB signaling as an important mechanism in fisetin‐mediated regulation of macrophage inflammatory responses.

Our results also suggest that fisetin inhibited the activation of THP‐1 derived M1 macrophages toward the pro‐inflammatory M1 phenotype, at least in parts, by inhibiting NF‐κB, a well‐known transcription factor that positively regulated the expression of IL‐6 and TNF‐α (Lawrence and Fong 2010; Tornatore et al. 2012), and MAPK signaling pathway, which also plays important role in inflammatory responses by interacting with NF‐κB pathway (Guo et al. 2024). These findings are consistent with previous studies showing that fisetin inhibits NF‐κB activation in RAW264.7 mouse macrophages (Kim et al. 2015a; Molagoda et al. 2021a), and inhibits MAPK signaling in primary mouse macrophages (Hada et al. 2021) and human lung epithelial cells (Peng et al. 2018). These are also in line with previous in vivo studies showing that fisetin alleviates colitis (Sahu et al. 2016), airway inflammation (Huang et al. 2018), and kidney inflammation (Ren et al. 2020) in mouse models. There are many signaling pathways involved in M1/M2 macrophage polarization, such as the ADH4/NRF2‐CSF1 axis (Yan et al. 2026) and angiopoietin‐like 4 (ANGPTL4) (Huang et al. 2025). ANGPTL4 inhibits pro‐inflammatory cytokines such as TNF‐α and IL‐1β and promotes M2 polarization in Kuffer cell (Huang et al. 2025).

In addition to cytokine and inflammasome suppression, our study also revealed that fisetin significantly decreased ROS production and phagocytic activity in M1 macrophages. Excessive ROS is known to amplify inflammatory signaling and cause tissue damage, while increased phagocytosis is a hallmark of activated M1 macrophages. Therefore, the ability of fisetin to attenuate these functional aspects further underscores its role in diminish inflammatory macrophage responses.

Taken together, our findings provide new evidence that fisetin inhibits M1 macrophage activation by suppressing inflammatory mediator production, inflammasome activation, oxidative stress, and phagocytic function. By targeting key signaling pathways such as NF‐κB and MAPK, fisetin interferes with core regulatory mechanisms underlying inflammation. Given the central role of M1 macrophages in the pathogenesis of chronic inflammatory diseases, including cardiovascular disease, neurodegeneration, metabolic disorders, and autoimmunity. Our results support further investigation into the therapeutic potential of fisetin. Future studies using human primary cells or in vivo models are warranted to evaluate its efficacy, safety, and translational relevance in human inflammatory conditions.

5. Conclusions

Our study reveals that fisetin is a potent anti‐inflammatory compound that effectively inhibits pro‐inflammatory cytokine and chemokine production, ROS generation, and phagocytic activity in human M1 macrophages. Mechanistically, fisetin suppresses the polarization of M0 macrophages to the pro‐inflammatory M1 phenotype without promoting a shift toward the M2 phenotype. These findings support the potential of fisetin as a therapeutic candidate for managing chronic inflammatory diseases and for future applications in immunotherapy.

Author Contributions

Nattaya Onpan: conceptualization, formal analysis, investigation, methodology, visualization, writing – original draft, editing and revised the manuscript. Chanchao Lorthongpanich: conceptualization, formal analysis, supervision, methodology, resources, writing – review, funding acquisition and editing the manuscript. Pakpoom Kheolamai: conceptualization, formal analysis, supervision, methodology, writing – review, and editing. Pimjai Chingsuwanrote: investigation, methodology, data analysis. Napachai Rodboon: investigation, methodology, data analysis. Sudjit Luanpitpong: investigation, methodology, data analysis. Chonticha Saisawang: methodology, resources, data analysis. Surapol Issaragrisil: conceptualization, resources, supervision, writing – review, and editing.

Ethics Statement

The experiments in this study were conducted exclusively using a human monocytic leukemia, THP‐1 cell line purchased from JCRB, Japan. No primary human tissues were used; hence, institutional ethical approval was not applicable.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1

CBIN-50-0-s002.docx (13.3MB, docx)

Supporting File 2

CBIN-50-0-s001.docx (16.5KB, docx)

Supporting File 3

CBIN-50-0-s003.pptx (249.6MB, pptx)

Acknowledgments

We thank Ms. Ruechaphorn Buasamrit for her assistance with laboratory management, and administrations. Figures 1 and 4 were created in part with BioRender.com. This research project was supported by Siriraj research development fund, Grant number (IO) R016833021, Faculty of Medicine Siriraj Hospital and National Research Council of Thailand (NRCT) and Mahidol University (Grant no. N42A680269).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting File 1

CBIN-50-0-s002.docx (13.3MB, docx)

Supporting File 2

CBIN-50-0-s001.docx (16.5KB, docx)

Supporting File 3

CBIN-50-0-s003.pptx (249.6MB, pptx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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