Myricetin reduces neutrophil extracellular trap release in a rat model of rheumatoid arthritis,which is associated with a decrease in disease severity

Abstract

Rheumatoid arthritis (RA) is a chronic disease characterized by joint inflammation and severe disability. However, there is a lack of safe and effective drugs for treating RA. In our previous study, we discovered that myricetin (MC) and celecoxib have a synergistic effect in the treatment of RA. We conducted in vitro and in vivo experiments to further investigate the effects and mechanisms of action of MC. Our findings demonstrated that MC treatment effectively reduced the release of neutrophil extracellular traps (NETs) and alleviated the inflammatory response in RA. Mechanistic studies showed that MC prevents the entry of PADI4 and MPO into the cell nucleus, thereby protecting DNA from decondensation. In a rat arthritis model, MC improved histological changes in ankle joints and suppressed NET-related signaling factors. In conclusion, MC protects the ankle joints against arthritis by inhibiting MPO and PADI4, thereby reducing NET release. The pharmacological mechanism of MC in RA involves the inhibition of NET release.

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by erosive arthritis, synovial hyperplasia, and joint destruction. The clinical manifestation of RA primarily involves multiple, symmetrical joint inflammation, which has a detrimental impact on the physical and mental well-being as well as the overall quality of life of patients. ¹ The precise pathogenesis of RA has not yet been fully elucidated; however, it is widely acknowledged that the immune system plays a crucial role in its onset and progression. The immune system functions as a key mediator throughout the process of RA, serving as the connecting link between pathogenic factors and phenotypic expression of the disease. ² Neutrophils, regarded as the “frontline warriors” of the immune response, are pivotal in the body's immunological reactions to pathogenic and autoimmune disorders. ³

In recent years, a novel mechanism called neutrophil extracellular traps (NETs) has been identified. It involves rupturing the nuclear membrane in neutrophils upon stimulation, forming a chromatin fiber network. This process is called NETosis. ⁴ During NETosis, the synovial fluid of patients with RA shows high concentrations of granular proteins from neutrophils, such as myeloperoxidase (MPO) and neutrophil elastase (NE), which are believed to contribute to joint damage. ⁵ Furthermore, NETs have been identified as a significant source of citrullinated self-antigens that trigger the autoimmunity associated with RA. NETosis has been increasingly detected in the circulation and synovial fluid of RA patients. ⁶ Extensive research suggests that NETs promote autoimmunity and exacerbate tissue damage.^5–7 As a result, modulating the release of NETs has become a critical target for treating RA. Although significant progress has been made in the management and treatment of RA, with mainstream therapies, including nonsteroidal anti-inflammatory drugs and biologics, their long-term use is limited due to potentially life-threatening side effects. ⁷ Thus, there is an urgent need to explore new and more effective treatment options for RA to complement existing therapeutic strategies.

Many researchers are looking toward natural products as a potential solution to this challenge. ⁸ Our previous findings showed that total flavones of Pterocarya rhoifolia, found in flavonoid compounds of natural plant products, can effectively intervene in RA by inhibiting the migration and invasion of synovial fibroblasts. ⁹ Moreover, they can modulate the functional effects of neutrophils stimulated by immune complexes in healthy individuals. ¹⁰ Among these compounds, myricetin (MC), a major component of the total flavones in Pterocarya rhoifolia, ¹¹ stands notable for its remarkable therapeutic effects. ¹² It possesses various pharmacological properties, including anti-aging of stem cells, ¹³ anti-hyperlipidemia, ¹⁴ anti-diabetic, ¹⁵ and anti-inflammatory effects. ¹⁶ Myricetin has been shown to significantly reduce inflammation in RA through different mechanisms, including modulation of IL-21 and histone K activity and targeting AIM2 to inhibit fibroblast-like synoviocyte-mediated rheumatoid synovial inflammation and joint destruction.^17–19

To further refine the mechanism of action of MC intervention in RA, we conducted a network pharmacology analysis based on existing databases to identify common target proteins between MC and RA. We focused on the involvement of PADI4 and MPO proteins, both of which are closely related to NET formation. Using AutoDock, we attempted molecular docking and found a strong binding activity, suggesting that MC may directly interact with MPO and PADI4. Subsequently, we focused on PADI4, which can deaminate or citrullinate histones, causing DNA to unravel and subsequently dissociate, leading to the binding of MPO to decondensed chromatin and release of NETs into the extracellular space. ²⁰ NETs are a source of citrullinated antigens in patients with RA, and excessive formation of NETs can induce tissue damage and promote key immune events within the synovium, including the production of anti-citrullinated protein antibodies (ACPAs), which further exacerbate the condition of RA patients. ⁶

Based on these findings, we propose that MC may alleviate RA by regulating NETs mediated by MPO and PADI4. Thus, in this study, we systematically investigated the potential mechanisms of MC in the release of NETs by neutrophils and a rat collagen-induced arthritis (CIA) model, providing new insights into the potential application of MC in immune-related diseases.

Materials and methods

Chemical reagents

Myricetin (M111176-10 mg) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd, China. Bovine collagen and Fuchs’ complete adjuvant were sourced from Sigma, USA. The Rat Peripheral Blood Neutrophil Isolate Kit was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd, China. PADI4 antibody (FNab06105) was purchased from Fine Test, USA. Cit-H3 antibody (ab281584) and NE antibody (ab131260) were purchased from Abcam, USA. Goat anti-rabbit IgG (A16098) and goat anti-mouse IgG (31160) were both purchased from Proteintech, China. MPO/DNA ELISA (YJ903251), tumor necrosis factor (TNF)-α ELISA (ml002859), and IL-1β ELISA Kits (ml037361) were obtained from Shanghai Enzyme-linked Biotechnology Co., Ltd, China. Phorbol-12-myristate-13-acetate (PMA) (S1819-5 mg) and Nucleoplasmic Isolation Kit (P0028) and MPO antibody (AF7494) were purchased from Beyotime, China. The CCK-8 kit (KR0009) was purchased from Wuhan Kerui Biotechnology Co.China.

Animals

Male Sprague Dawley (SD) rats, aged 6–8 weeks, with specific pathogen free animal (SPF) grade, were purchased from the Experimental Animal Center of Xiangxi University with license number laboratory animal production license (Hunan) 2020-0018. Before the start of the experiment, the study was approved by the Animal Ethics Committee of Hubei Enshi College. Throughout the research process, the rats were maintained under SPF conditions, and all experimental procedures and operations were conducted following the requirements and principles of the Animal Ethics Committee of Hubei Enshi College.

Effect of MC on CIA rats

A total of 25 rats were randomly divided into five groups: control group (Control), model group (CIA), MC 1 mg/kg group (MC (1 mg/kg)), MC 5 mg/kg group (MC (5 mg/kg)), and positive medication group using triptolide (TPT)(Chikin Hip Lik Pharmaceutical Co.14002046838, Hunan, China) (10 mg/kg). Bovine type II collagen (Sigma,234184-M, USA) emulsion and an equal volume of complete Freund's adjuvant (Sigma, AR001, USA) were prepared to establish the CIA rat model. Except for the control group, the remaining rats received subcutaneous injections of 0.2 US emulsion at the base of the tail and the pad of the right hind paw on day 1 for initial immunization. To obtain secondary immunization, on day 14, except for the control group, all rats were injected with 0.2 ml of bovine type II collagen mixed with incomplete Freund's adjuvant (Sigma, AR002, USA). Myricetin was dissolved in sodium carboxymethyl cellulose and administered orally starting on day 14 at 2 ml per rat once daily. The control and CIA groups received equal volumes of sodium carboxymethyl cellulose. To evaluate ankle joint swelling, body weight, joint volume (using the drainage method with a three-way stopcock), and paw thickness (measured with a digital caliper [Mitutoyo, Kawasaki, Japan]) were measured twice weekly. The severity of arthritis was assessed using a scoring scale ranging from 0 (normal) to 4 (severe swelling and disability), with scores of 1–3 indicating varying degrees of swelling and limited mobility. From the sixth week onward, blood samples were collected from the tail vein of the rats using blood collection tubes with or without anticoagulant. In the seventh week, the rats were euthanized under anesthesia, and blood samples were collected from the abdominal aorta in blood collection tubes with or without anticoagulant. Blood samples with anticoagulant were used to isolate neutrophils, while blood samples without anticoagulant were used for subsequent serological testing. The rats were then sacrificed, and ankle joints were collected and fixed with 4% paraformaldehyde for histopathological analysis.

Isolation and culture of rat peripheral blood neutrophils

Strictly following the Rat Neutrophil Isolation Kit instructions, 1 ml of neutrophil isolation solution was transferred to a centrifuge tube. Briefly, rat peripheral blood was added to the tube under negative pressure using a pipette and centrifuged at 600×g for 25 min at 21°C. After centrifugation, the supernatant was carefully aspirated into a separate tube, leaving isolated neutrophils in the original tube. The cells were then washed with phosphate buffered saline at 21°C and 1000 t 2 for 5 min. Residual red blood cells were removed using red blood cell lysis buffer. The isolated neutrophils were resuspended in 1 ml of complete Roswell park memorial institute (RPMI) 1640 culture medium, and their identity as neutrophils was confirmed using the Diff-Quick Stain Kit (Supplement 1). The cells were cultured in a humidified incubator at 37°C and 5% CO₂.

CCK8 experiment

Neutrophils were seeded in 96-well plates at a density of 104 cells per well and supplemented with RPMI 1640 medium. The plates were then placed in an incubator at 37°C with 5% CO₂. Cells (5 × 106) were added to each well. Subsequently, various concentrations of MC (0, 12.5, 25, 50, 100, and 200 μmol/l) were administered to neutrophils and incubated for 4 h. After treatment, 10 μL of the Cell Counting Kit 8 (CCK-8) solution was added to each well and incubated for 1 h. Absorbance at 450 nm was measured using a multifunctional enzyme marker (Thermo Fisher Scientific). This experiment was performed 3–4 times to ensure the reliability and reproducibility of the results.

MC treatment induces NET release in PMA-stimulated neutrophils

Isolated and purified neutrophils were seeded into six-well plates at a concentration of 1 × 106 cells/ml. The cells were incubated with or without MC (100 µM) or PMA (25 nM) for 4 h. The PMA group served as a model group to simulate the release of the NET network. In contrast, in the MC group and the intervention group, along with subsequent cellular experiments, the cells were treated with MC.

Cellular thermal shift assay (CETSA) for target protein identification

Following the extraction and isolation of purified neutrophils, they were allocated into 12 groups: six control groups treated with 25 nM PMA and six experimental groups treated with an additional 100 μmol/l MC. Subsequently, the cells were cultured for 1 h at 37°C in an incubator with 5% CO2. After incubation, temperature gradients ranging from 40°C to 65°C were applied. The cells were subjected to heat treatment for 3 ter using a polymerase chain reaction instrument, followed by rapid cooling on ice. The cooled cells were then centrifuged at 150 g for 5 min at 4°C. The supernatant was carefully discarded, and the cells were collected. The collected cells were subsequently subjected to western blotting to identify and analyze the target proteins of interest.

Western blotting (WB) analysis

Total neutrophil protein was extracted using a protein extraction solution composed of radio immunoprecipitation assay buffer and phenylmethylsulfonyl fluoride at a ratio of 100:1. The resulting mixture was centrifuged at 3000 rpm for 5 min at a chilling temperature of 4°C. Subsequently, the supernatant was carefully discarded, and a Protein Sampling Buffer was introduced. To ensure optimal cell dispersion, gentle vortexing or tapping of the tube bottom was performed. The lysate was added at 150–250 µL per cell per well for effective cell lysis in a 6-well plate. Thorough cell lysis was achieved by gently scraping the tube bottom. The absence of observable cell precipitates indicated complete lysis. To finalize the preparation, eppendorf micro test tubes containing the lysate were subjected to a 10-min boiling process in water heated to its boiling point. Thereafter, the samples were stored at −20°C. For electrophoresis analysis, a consistent amount of protein (30–50 r) was maintained for each sample. The protein samples were separated on lauryl sodium sulfate-PAGE gels with varying concentrations (12% and 15%) and subsequently electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were immersed in 5% skimmed milk solution for 1 e to block non-specific binding. Following this, the membranes were incubated with specific primary antibodies targeting PADI4 (1:1000), MPO (1:1000), Cit-H3 (1:1000), NE (1:1000), β-actin (1: 10,000), and Histone H3 (1:1000) for 12–16 h. Afterwards, the membranes were washed three times, and a suitable secondary antibody (1: 10,000) was applied, followed by incubation at 37°C for 2 h. The bands were developed using diaminobenzidine (DAB), and the resulting images were captured and stored using ImageJ software. The band intensities were quantitatively analyzed. To ensure accuracy, the data were normalized to the endogenous controls, β-actin, and histone H3, and the entire experiment was replicated three times.

Enzyme-linked immunosorbent assay (ELISA)

Serum samples and cell suspensions were prepared in strict accordance with the instructions provided by the ELISA Kit. A washing solution was prepared by diluting double distilled water at a ratio of 1:20. The MPO-DNA standard was prepared at concentrations of 1,000, 500, 250, 125, 62.5, and 31.25 pg/ml. Duplicate wells were set up for each concentration, and blank wells were included. Standards and test samples were added to the plate at a volume of 0.1 ml per well. The plates were incubated at room temperature for 2 e. Subsequently, the liquid in the wells was discarded, and the plate was washed three times with the provided Wash Solution and blotted dry. The mixture was again incubated at room temperature for 1 h. The plate was washed three times with Wash Solution and blotted dry. Next, 0.1 ml of the pre-concentrated Color Developer was added to each well, and the plate was incubated for 30 min in the dark at room temperature. Stop Solution (0.1 ml per well) was added and thoroughly mixed, and the enzyme marker was used to measure the absorbance (A) at 450 nm. Serum levels of MPO/DNA, TNF-α, and IL-1β were calculated using a standard curve. The sample grouping was used as the horizontal coordinate, whereas the standard concentration was the vertical coordinate for the analysis.

Observation of hematoxylin and eosin (HE) staining

The ankle joints of the euthanized rats were removed, skinned, and immersed in formalin. Dehydration and transparency were achieved using alcohol and xylene, respectively. The transparent tissues were embedded in paraffin and sectioned. The sections were then stained with hematoxylin and eosin and observed under a microscope. The fixed joint tissues were subjected to a series of procedures: deparaffinization using xylene, dehydration using ethanol, staining with hematoxylin, differentiation with differentiation solution, rinsing with running water, dehydration using an ethanol gradient, staining with eosin, further dehydration using an ethanol gradient, transparency using xylene, and sealing with a neutral mountant. Photomicrographs were taken under a microscope to observe pathological changes in rat joint tissues.

Immunohistochemical (IHC) analysis

Immunohistochemistry was performed using an Immunohistochemistry Application Solution Kit (Bioss). To remove the paraffin, paraffin blocks of the ankle joints were heated at 60°C and then immersed in xylene, ethanol with gradient concentrations, and ultrapure water. After blocking with 10% bovine serum albumin, the sections were incubated overnight with rabbit anti-NE monoclonal antibody (Abcam) or rabbit anti-Cit-H3 monoclonal antibody (Abcam) diluted 1:400, followed by incubation with the secondary antibody for 1 h. The tissue sections were then treated with a DAB working solution and observed under a 200-fold magnification.

Statistical analysis

The data presented in the previous sections are reported as the mean ± standard deviation (SD). Statistical analyses were conducted using the GraphPad Prism software (version 8.0). One-way analysis of variance was used, followed by the Bonferroni multiple comparison post-hoc test. Statistical significance was set at P < 0.05.

Results

Bioinformatics analysis to investigate the mechanism of action of MC intervention in rheumatoid arthritis

Our previous investigation revealed a potential synergistic effect of MC and celecoxib on anti-inflammatory activity, as shown by the random forest model [12]. Based on the anti-inflammatory properties of MC, we employed network pharmacology methods to predict the target genes of MC in RA, resulting in the identification of 6721 target genes for RA and 239 target genes for MC (Figure 1(A),B). Notably, we found a significant overlap of 115 common targets (Figure 1(C)). To focus on the primary targets, a protein-protein interaction (PPI) network was constructed using Cytoscape and further validated using the STRING database (Figure 1(B)). Using Degree scoring, we identified 17 key genes: MMP12, PADI4, MPO, MMP2, MMP3, MMP9, MMP13, TTR, TNF, STAT3, ALOX5, AKT1, MAPK3, MAPK1, PIK3CG, ADPRA3, and spleen tyrosine kinase. Interestingly, we observed that the major targets of MC, such as AKT1 and TNF-α, were associated with the critical proteins PADI4 and MPO involved in NET development (Figure 1(D)). Consequently, we performed molecular docking studies to explore the potential mechanisms of MC in RA by evaluating the interaction between MC and PADI4, as well as between MC and MPO. The results indicated possible binding of MC with PADI4 and MPO (Figure 1(E),F), suggesting that the inhibitory effect of MC on RA may be linked to the activation of PADI4 and MPO in neutrophils.

Figure 1.

Network pharmacological study of MC for rheumatoid arthritis (RA).

The CETSA revealed a direct interaction of MC with MPO and PADI4 proteins in neutrophils

To investigate the direct interaction between MC, MPO, and PADI4 proteins in neutrophils, we employed the CETSA method. Initially, a CCK-8 assay was conducted to assess the impact of different concentrations of MC on neutrophil activity. The results indicated no significant difference in neutrophil activity between the groups treated with 100 μM MC and the control group. However, neutrophil activity decreased following treatment with 200 μM MC (P < 0.01, Figure 2(A)). Next, we applied the CETSA, which directly detects intracellular drug binding to target proteins. Neutrophils were treated with both PMA and MC at various temperatures, and total protein was extracted for western blot analysis (Figure 2(B), (C)). Interestingly, a positive shift in the melting temperature of PADI4 and MPO proteins was observed in neutrophils treated with MC combined with PMA (Figure 2(D), (E)). This finding suggests that in neutrophils, the presence of MC leads to enhanced stability in the protein structure of MPO and PADI4 after heating to 45°C, compared to the group treated with PMA alone. In conclusion, our experimental findings and previous research indicate that MC directly interacts with MPO and PADI4 proteins in neutrophils, thereby influencing subsequent cellular phenotypic changes. The CETSA provides further evidence supporting a direct interaction between MC and these target proteins.

Figure 2.

MC can directly interact with both MPO and PADI4 proteins.

In neutrophils, MC inhibits nuclear entry of PADI4 and MPO and inhibits NETosis

NETs, which are released from neutrophils during inflammation through a process called NETosis, play a crucial role in the pathogenesis of RA. The MPO/DNA complex serves as a reliable marker of NETosis. To assess the effect of MC treatment on NET release from neutrophil traps, we initially examined the levels of myeloperoxidase (MPO)/DNA complexes in neutrophil suspensions induced by the addition of 25 nM PMA, followed by the addition of 100 μm MC, using ELISA (Figure 3(A)).

Figure 3.

In neutrophils, MC inhibits NETosis by inhibiting the entry of PADI4 and MPO into the nucleus.

NE and Cit-H3 are typical biomarkers of NETs. During NETosis, histone 3 (H3) in the nucleus undergoes modification, forming citrullinated histone 3 (Cit-H3). This modification leads to decondensation of chromatin and its release into the extracellular space. Additionally, intracytoplasmic granule-derived proteins, including enzymes and antimicrobial peptides, attach to chromatin scaffolds to form a network-like complex that releases NE from the nucleus. The expression of NE and Cit-H3 was analyzed via WB, and both were found to be significantly upregulated in the PMA group but downregulated in the MC group (Figure 3(B), (C)). This indicates that MC inhibits PMA-induced upregulation of Cit-H3 and NE in neutrophils, consequently suppressing the release of NETs (Figure 3(D), (E)).

Furthermore, NET formation involves central mechanisms such as anti-myeloperoxidase antibody (MPO), histone modification, and chromatin degradation. Histone guanylation by peptidylarginine deiminase 4 (PADI4) and nucleosome degradation are key steps in NET formation in vivo. Both MPO and PADI4 need to be transported into the neutrophil nucleus to fulfill their role in releasing NETs. To examine whether MC blocks the nuclear transportation of PADI4 and MPO, we performed separate WB analyses of the nuclear and cytoplasmic fractions of cells using the Nucleus Plasmid Separation Kit. The results showed a significant increase in PADI4 and MPO expression in the chromatin fraction compared to that in the cytoplasm in the PMA group (Figure 3(D), (F)). Conversely, in the MC group, both PADI4 and MPO proteins were significantly more highly expressed in the cytoplasm than in the chromatin fraction (Figure 3(D), (E)). This indicates that MC inhibits PMA-induced upregulation of PADI4 and MPO in the nucleus, thereby successfully impeding their entry into the nucleus and preventing NET formation.

In conclusion, MC binds to PADI4 and MPO with abnormally elevated levels in the cytoplasm of neutrophils and inhibits their nuclear entry, thus exerting suppressive effects on undesired behaviors and abnormal NET production.

In a CIA rat model, MC administration effectively suppressed joint inflammation

Based on the aforementioned in vitro experiments, we sought to investigate the potential of MC to ameliorate arthritis (CIA) induced by bovine type II collagen and complete Freund's adjuvant in rats (Figure 4(A)). Remarkably, our findings demonstrated a significant reduction in joint swelling in CIA rats after MC treatment (Figure 4(B)). Ankle joint swelling was assessed by measuring ankle joint drainage volume, ankle joint thickness, and arthritis scores at regular intervals from week 0 to week 7. In control rats without significant paw joint swelling, the bipedal injection of bovine type II collagen and complete Freund's adjuvant into the metatarsal and caudal roots resulted in increased ankle joint diameter from 2.9 A on day 0 to 5.3 mm at week 3 after induction (Figure 4(C)). This was accompanied by a corresponding increase in arthritis scores from 0 at week 0 to 4.0 at week 3 (Figure 4(D)). Additionally, ankle drainage volume increased from 1.75 ml at week 0 to 2.65 ml at the end of week 3 (Figure 4(E)). Notably, compared to the CIA rat model group, MC treatment significantly reduced ankle joint thickness (Figure 4(C)), arthritis scores (Figure 4(D)), and ankle joint drainage (Figure 4(E)), particularly in the high-dose MC group (5 mg/ml) (P < 0.05). Moreover, the reduction in plantar foot swelling was more pronounced in the high-dose MC (5 mg/ml) group than in the TPT group (P < 0.05).

Figure 4.

MC treatment ameliorates arthritis in rats with collagen-induced arthritis (CIA).

Furthermore, MC treatment effectively attenuated the upregulation in the expression of proinflammatory cytokines (TNF-α and IL-1β) in the serum of CIA rats (Figure 4(F),(G)). Histological evaluation of ankle joint sections using HE staining also confirmed the ameliorative effect of MC in CIA rats. Importantly, compared to the control group, MC treatment significantly reduced the infiltration of inflammatory cells, synovial hyperplasia, and cartilage erosion observed in the ankle joints of the CIA group (Figure 4H). Taken together, these findings strongly suggest that MC treatment significantly reduces both local and systemic inflammatory responses in the ankle joints.

In a CIA rat model, MC treatment reduces the release of NETs

To investigate the effect of MC treatment on the release of NETs, we examined the expression levels of Cit-H3 and NE, well-established markers of NETs, in joint tissue sections from rats with CIA using immunohistochemistry techniques.

Figures 5(A) and (B) present the results, demonstrating a significant upregulation in the expression of Cit-H3 and NE in the RA group compared to the control group. However, in the MC group, there was a notable downregulation of Cit-H3 and NE expression (Figure 5(A), (B), (C)). Furthermore, to gain a more comprehensive understanding of this phenomenon, we assessed the expression of another characteristic NET marker, the MPO/DNA complex, in serum samples using ELISA. The findings revealed a significant upregulation in the expression of the MPO/DNA complex in the RA group, whereas the MC group exhibited a substantially decreased expression compared to the CIA group (Figure 5(D)). Importantly, the expression in the MC group differed significantly from that in the CIA group (P < 0.05).

Figure 5.

MC inhibits NE expression and histone 3 citrullination in CIA rats.

Collectively, these findings demonstrate that MC treatment effectively inhibits the upregulation in the expression of markers associated with NET release, indicating a reduction in NET release in CIA rats.

Discussion

NET release is a newly recognized form of programmed cell death, characterized by releasing a meshwork composed of decondensed chromatin and intracellular granule proteins into the extracellular space. Morphologically, this process involves the dissolution of nuclear and granular membranes, contact between chromatin and cytoplasmic granules, rupture of the cell membrane, and subsequent formation of a network structure in the extracellular space. Inhibiting NET release has emerged as a promising intervention for the treatment of RA and is considered a strategy for cancer prevention. ²¹ However, most NET-targeted therapies for RA, primarily confined to animal models, are currently at the research stage. ²² This study aimed to investigate the therapeutic mechanisms of NETs in RA by combining an animal model with a cellular model constructed using primary cells from animals.

As a natural product, MC has been shown to have a synergistic effect with rhein, fisetin, and celecoxib in RA intervention based on a random forest model. ¹² Although recent progress has been made in understanding the anti-inflammatory effects of MC, the immunological mechanisms involved remain unclear. Therefore, to explore the potential targets of MC in RA intervention, we used network pharmacology to identify 115 potential targets shared between MC and RA. Among the top 17 main targets with the most significant network crosstalk, AKT1 and TNF-α, which may be related to cellular autophagy, have been studied extensively. ²³ Subsequently, targets such as MPK3 and STAT3, which also show significant phenomena, have been identified, ²⁴ while other targets such as MPO and PADI4, despite their significant phenomenon, seem to have received less attention from researchers. However, because of the limitations of network pharmacology predictions that rely solely on the intersection of two sets, it has not yet been confirmed that MPO and PADI4 are the targets of MC intervention in RA. Therefore, we conducted molecular docking between MC and MPO as well as PADI4 to further verify them as targets of MC and explore their direct interactions. MPO and PADI4 are crucial targets induced by NETs, and after binding with MPO and PADI4, MC can influence the release of NETs.^25,26 The discovery of these results validates our initial intention to find new targets for natural drug treatment of RA, and the finding that the binding energy between MC and the targets is less than or equal to −7 kcal/mol also lays the foundation for exploring the potential mechanisms of MC intervention in RA.

To explore the mechanism of MC intervention in RA more accurately, we further demonstrated through CETSA technology that MC can influence the release of NETs after binding with MPO and PADI4. CETSA is a biophysical technique that directly obtains protein small-molecule drug complexes by subjecting them to different temperature treatments. The use of CETSA technology to investigate the target proteins of taxane drugs in resistant cells has provided valuable assistance in the preclinical evaluation of the efficacy of taxane drugs. ²⁷ In this study, using CETSA in combination with WB, we observed a forward shift in the protein melting temperature, indicating the binding of myricetin to MPO and PADI4 proteins, suggesting that MPO and PADI4 may be candidate targets for RA intervention. This method involves harsh conditions such as protein denaturation and temperature control, and it is considered a technically challenging approach. However, the results obtained by assessing binding stability through a stability shift can provide important insights into the interactions between small-molecule compounds and proteins. ²⁸ Furthermore, we found that MC-induced PMA-treated neutrophils showed more stable binding between MC and induced PADI4 at 50°C, indicating that the optimal binding temperature of the PADI4 protein is approximately 50°C. Therefore, the results obtained from WB experiments were more pronounced. Based on this, we speculated whether this temperature-controlled operation can be applied to general WB experiments to determine whether finding the optimal temperature for the target protein can improve the efficiency and accuracy of the experiments, which may be promising news for researchers.

Furthermore, we investigated the role of MC in inhibiting PMA-induced NETosis. NETosis is a novel form of programmed cell death in neutrophils and is characterized by the extracellular release of fibrous DNA networks combined with granular and nuclear proteins. In recent years, researchers have confirmed that excessive release of NETosis plays an important role in the pathogenesis of RA. ²⁹ In RA, the production of ACPAs is closely associated with NETs. NETs serve as an important source of autoantigens that stimulate ACPA production, and excessive release of NETs easily induces an immune response. ACPAs also promote the release of peptide-arginine deiminases from neutrophils, which convert l-arginine to citrulline, creating a vicious cycle. ³⁰ In addition to autoantibody production, NETs also induce the production of inflammatory cytokines, thereby amplifying the inflammatory response. Reports suggest that NET release can be used to assess the therapeutic effect in patients with RA. ³¹ Furthermore, NE and Cit-H3 are major components of NETs and are considered biomarkers of NETosis. ²⁰ In this study, we demonstrated that MC binds to MPO and PADI4 in neutrophils, reducing their entry into the nucleus. This significantly inhibits the expression of NET-associated NE, Cit-H3, and MPO/DNA complexes, thereby preventing NET formation. These findings provide new potential mechanisms for the pharmacological effects of MC in RA intervention and broaden our understanding of its immunological mechanisms.

Finally, our research findings suggest that MC treatment significantly intervenes in joint swelling and the degree of inflammatory cell infiltration in a rat CIA model. We also observed that MC treatment reduced the levels of proinflammatory cytokines (TNF-α and IL-1β) in the plasma of CIA rats. Additionally, the high-dose MC group (5 mg/ml) exhibited more significant intervention effects in the CIA rat model than the positive control group treated with Tripterygium wilfordii polyglycoside tablets, which are widely recognized in clinical applications with documented safety and efficacy. ³² These results indicate the potential of MC as a new therapeutic candidate, providing a novel approach for RA intervention in basic medicine.

These data demonstrate the systemic anti-inflammatory effects of MC in CIA rats. This may be attributed to the significant inhibition of NET release, thereby disrupting the vicious cycle. Our study further confirmed that MC significantly reduced the expression of the biological markers NE and Cit-H3 as well as the MPO/DNA complex in the ankle joints of the CIA rat model. Additionally, this effect may be attributed to its binding interaction with PADI4, which reduces arginine citrullination. Acknowledging the pivotal role of NETs in the host defense against viral and inflammatory diseases is crucial. ³³ However, it has been observed that neutrophil bactericidal activity is not compromised in PADI4-deficient animals^{, 34} suggesting that MC targeting PADI4 could be a potential therapeutic approach to prevent inflammation-related diseases without impairing neutrophil antibacterial capabilities caused by the reduction of neutrophils through targeted antibodies.

Our current data indicate that MC affects inflammation caused by NETs by targeting the functionality of PADI4 and MPO, ultimately halting the malignant progression of RA. We aimed to enrich the scientific understanding of the intervention of RA with Pterocaryarhoifolia total flavonoids by studying the mechanisms of MC intervention in RA, providing experimental and theoretical foundations for the utilization and development of total flavonoids for the treatment of RA.^11,18 This will better serve the clinical setting and benefit patients by offering solutions for preventing and treating RA-related conditions (Figure 6). Furthermore, we will continue our research and exploration in subsequent experiments to investigate whether MC achieves its intervention effects in RA through other pathways and whether there are any differences in expression in neutrophils extracted from human blood samples.

Figure 6.

Mechanisms associated with the inhibition of NET release in rheumatoid arthritis by myricetin.

Conclusion

This ground-breaking study unveils the protective effects of myricetin in intervening in the progression of RA by suppressing the neutrophil trap network mediated by MPO and PADI4 enzymes. In this study, we deepened our understanding of the anti-inflammatory characteristics of myricetins and shed light on their potential therapeutic applications in immune disorders. These findings contribute to the current scientific knowledge and establish a robust basis for future research and translation of myricetins into clinical practice.

Supplemental Material

sj-png-1-ini-10.1177_17534259241255439 - Supplemental material for Myricetin reduces neutrophil extracellular trap release in a rat model of rheumatoid arthritis, which is associated with a decrease in disease severity

Supplemental material, sj-png-1-ini-10.1177_17534259241255439 for Myricetin reduces neutrophil extracellular trap release in a rat model of rheumatoid arthritis, which is associated with a decrease in disease severity by Yiqin Shu, Rui Yang, Huijie Wen, Qiannan Dong, Zhiqi Chen, Yang Xiang and Hao Wu in Innate Immunity