Myricetin attenuates experimental autoimmune prostatitis and inflammatory and oxidative injury in LPS-induced RWPE-1 cells potentially through modulation of MAPK/STAT3/NF-κB/Nrf2 signaling pathway

Abstract

Background

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a prevalent urological condition marked by ongoing pelvic pain, inflammation, and a reduced quality of life. Increasing evidence indicates that dysregulated immune responses and oxidative stress contribute to CP/CPPS pathogenesis. Myricetin (MYR), a natural flavonoid, has been reported to exert anti-inflammatory and antioxidant effects in multiple disease models. However, its role in CP/CPPS remains unclear.

Methods

The protective effects of MYR were evaluated in an experimental autoimmune prostatitis (EAP) mouse model and in lipopolysaccharide (LPS)-induced RWPE-1 prostate epithelial cells. Prostatic histopathology, pelvic pain behavior, inflammatory cytokine levels, immune cell infiltration, and oxidative stress markers were assessed in vivo. In vitro, inflammatory mediator expression, reactive oxygen species (ROS) production, oxidative stress status, mitochondrial function, and signaling pathway activation were examined using qRT-PCR, Western blotting, immunofluorescence, and biochemical assays. Network pharmacology analysis was used to predict potential targets and pathways associated with the effects of MYR. The involvement of Nrf2 signaling was further examined using the Nrf2 inhibitor ML385.

Results

MYR markedly alleviated prostatic inflammation and pelvic pain in EAP mice, accompanied by reduced serum levels of pro-inflammatory cytokines and chemokines, decreased infiltration of CD4-positive T cells and CD68-positive macrophages, and improved oxidative stress status. In LPS-induced RWPE-1 cells, MYR suppressed the expression of inflammatory mediators, reduced ROS accumulation, restored redox balance, and attenuated mitochondrial dysfunction. Mechanistically, MYR inhibited the phosphorylation of ERK, JNK, p38, and STAT3, suppressed NF-κB activation, and promoted Nrf2 signaling. Pharmacological inhibition of Nrf2 largely abolished the anti-inflammatory and antioxidant effects of MYR.

Conclusions

MYR attenuated inflammation, oxidative stress, and pain-related changes in experimental prostatitis, and these effects were associated with coordinated modulation of MAPK, STAT3, NF-κB, and Nrf2 signaling pathways. These findings suggest that MYR may represent a potential candidate for further preclinical investigation in CP/CPPS.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13062-026-00840-1.

Introduction

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common urological condition in men, representing over 90% of all prostatitis cases [1]. Its clinical features mainly include persistent and refractory pelvic pain or discomfort, often accompanied by lower urinary tract symptoms and sexual dysfunction, all of which significantly affect patients’ quality of life [2].

The etiology of CP/CPPS is extremely complex and has not yet been fully elucidated. It is currently widely accepted that the pathogenesis is not attributable to a single pathogenic infection but rather involves a multifactorial interplay of immune dysregulation, neuroendocrine dysfunction, oxidative stress injury, and psychological factors. Consequently, the development of novel intervention strategies with multi-target and multi-pathway regulatory capabilities is of great significance. Excessive immune inflammatory responses and oxidative stress play central roles in the initiation and perpetuation of the disease [3–5]. Key signaling pathways, such as mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription (STAT), nuclear factor erythroid 2-related factor 2 (Nrf2), and nuclear factor kappa B (NF-κB), play a crucial role in the onset and progression of CP/CPPS [6–10]. Autoimmune dysfunction causes immune cells, including T lymphocytes and macrophages, to infiltrate prostatic tissue, where they release inflammatory cytokines (e.g., IL-1β, IL-17 A, IFN-γ) and chemokines (e.g., CCL2), creating a complex inflammatory network. This process intensifies both peripheral and central neural pathways, contributing to chronic pain. Disruption of the oxidative-antioxidative balance inside cells triggers oxidative stress, leading to the excessive accumulation of reactive oxygen species (ROS) [11]. In CP/CPPS, excessive ROS accumulation can lead to oxidative damage of intracellular proteins, DNA, organelles, and membrane structures through lipid peroxidation, resulting in a range of pathological changes in the prostate, such as tissue edema, congestion, epithelial hyperplasia, and fibrosis, which in turn accelerate disease progression [12, 13]. However, current clinical therapeutic approaches for CP/CPPS, such as antibiotics, α-blockers, and nonsteroidal anti-inflammatory drugs, often yield limited efficacy or provide only partial symptomatic relief, making optimal therapeutic outcomes difficult to achieve [14]. Thus, further exploration of the pathogenesis of CP/CPPS and the development of safe and effective therapeutic strategies are of considerable clinical importance.

Myricetin (MYR) is a flavonoid widely found in various plants, exhibiting a range of biological activities, such as anti-inflammatory, antioxidant, immunomodulatory, and antitumor effects [15, 16]. Previous studies have demonstrated that MYR can modulate the MAPK, STAT3, NF-κB, and Nrf2 signaling pathways, thereby exerting immunoregulatory and protective functions in multiple inflammatory and oxidative stress-related diseases [17–22]. However, the exact role of MYR in CP/CPPS and the mechanisms underlying its effects remain largely unclear.

In this study, we employed an integrated approach combining network pharmacology, animal experiments, and in vitro cell-based assays to assess the protective effects of MYR in experimental models of CP/CPPS and to further examine the potential roles of the MAPK, STAT3, NF-κB, and Nrf2 signaling pathways.

Materials and methods

Experimental animals

Male C57BL/6J mice (6–8 weeks old) and male Sprague-Dawley (SD) rats were purchased from Beijing Sibeifu Biotechnology Co., Ltd. All animal procedures were approved by the Animal Ethics Committee of the Second Hospital of Hebei Medical University (Approval No. 2024-AE244) and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Establishment of the experimental autoimmune prostatitis (EAP) model and drug treatment

The EAP mouse model was established as previously reported [23]. Briefly, prostate homogenates were obtained from SD rats, and the resulting supernatants were collected as prostate antigens (PAgs). PAgs or normal saline were then emulsified with an equal volume of complete Freund’s adjuvant (CFA; MCE). On days 0 and 28, mice received intradermal injections of the emulsion containing 300 µg PAgs per mouse at multiple sites, including the shoulders, tail base, and bilateral hind footpads. Control mice were injected with an equivalent volume of normal saline emulsified with CFA. All mice were euthanized on day 42 for tissue harvesting. Mice were randomly divided into four groups (n = 6 per group): control, MYR, EAP, and EAP + MYR. According to a previous report, mice in the EAP + MYR group were administered MYR by oral gavage at a dose of 200 mg/kg starting 2 days prior to the second immunization, the MYR dose was selected based on previous in vivo studies reporting protective anti-inflammatory and antioxidant effects of MYR at comparable doses in mouse models, together with preliminary tolerability considerations [24, 25]. Mice in the control group received an equivalent volume of vehicle, whereas mice in the MYR group were treated with MYR alone. Because MYR administration was initiated before the second immunization, the in vivo experiment mainly evaluated the preventive effect of MYR during disease progression rather than a definitive therapeutic effect on fully established disease.

Behavioral testing

Prior to euthanasia on day 42, mechanical allodynia in the suprapubic pelvic region was assessed using von Frey filaments with bending forces of 0.04, 0.16, 0.4, 1.0, and 4.0 g. Each mouse was tested ten times. A positive nociceptive response was defined as any of the following behaviors: (a) jumping; (b) immediate licking or scratching of the stimulated area; or (c) rapid abdominal retraction. The response rate was calculated as the number of positive responses divided by the total number of stimulations (10).

Enzyme-linked immunosorbent assay (ELISA)

Mouse blood samples were collected and centrifuged at 2000 × g for 10 min to separate the serum. Serum levels of TNF-α, IL-1β, IL-17 A, IFN-γ, and CCL2 were measured using ELISA kits (Feiyue Biotechnology, China) according to the manufacturer’s instructions.

Hematoxylin and eosin (HE) staining

Mouse prostate tissues were collected, fixed, dehydrated, and paraffin-embedded. The embedded tissues were sectioned into 4-µm slices and baked at 65 °C for 2 h. The sections were then deparaffinized in xylene for 10 min, rehydrated through a graded ethanol series (100%, 95%, and 75%) for 5 min each, and rinsed with distilled water. HE staining was subsequently performed to assess the inflammatory status of the prostate tissue. The severity of prostatic inflammation was scored according to established criteria [26], as summarized in Supplementary Table S1.

Immunohistochemistry (IHC) staining

Prostate tissue sections were prepared using the same procedure as that for H&E staining. After deparaffinization, rehydration, and antigen retrieval, the sections were incubated with 3% hydrogen peroxide for 25 min at room temperature, washed three times with phosphate-buffered saline (PBS), and blocked with goat serum. The sections were then incubated overnight at 4 °C with primary antibodies against CD4 (1:500, GB15064, Servicebio, China) or CD68 (1:400, GB113109, Servicebio, China). Following PBS washes, the sections were incubated with horseradish peroxidase (HRP)-linked secondary antibodies for 2 h at room temperature. Immunoreactivity was visualized using 3,3′-diaminobenzidine (DAB), followed by hematoxylin counterstaining. Images were acquired under a light microscope.

For histological and immunostaining analyses, prostate tissues from 6 mice per group were analyzed. For each animal, 3 non-overlapping sections were selected, and 5 randomly chosen fields per section were captured under identical microscope settings. Histological scoring and immunostaining quantification were performed by an investigator blinded to the experimental groups. The percentage of positively stained area was quantified using ImageJ software. The average value from all analyzed fields was used as the representative value for each animal before statistical analysis.

Immunofluorescence (IF) staining

Tissue sections were prepared as described for immunohistochemistry. After fixation and permeabilization, the sections were incubated overnight at 4 °C with an anti-CCL2 primary antibody (1:50, ab214819, Abcam, UK). The following day, the sections were incubated with a fluorescent secondary antibody for 2 h, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescence images were acquired using a laser scanning confocal microscope (Zeiss, Germany).

For immunofluorescence analysis, RWPE-1 cells grown on coverslips were fixed with paraformaldehyde and permeabilized with Triton X-100. After a 1-h blocking step, the cells were incubated overnight at 4 °C with anti-Nrf2 (1:200, 16396-1-AP, Proteintech, China) and anti-p65 (1:200, 10745-1-AP, Proteintech, China) primary antibodies. The next day, fluorescent secondary antibodies were applied for 1 h at room temperature. Nuclei were stained with DAPI, and the coverslips were mounted prior to fluorescence microscopic examination.

Cell culture and treatment

RWPE-1, a human prostate epithelial cell line, was obtained from the Chinese Academy of Sciences and cultured in keratinocyte serum-free medium (K-SFM, Gibco, USA) supplemented with keratinocyte growth supplement and 1% penicillin–streptomycin at 37 °C in a humidified incubator with 5% CO₂. To establish an in vitro inflammatory model, RWPE-1 cells were stimulated with 2.5 µg/mL LPS as previously described [27]. Cells were pretreated with MYR (10 µM) and/or ML385 (5 µM) for 2 h prior to LPS stimulation.

Cell viability assay

Cell viability was assessed using the CCK-8 assay kit (Seven Sea, China). Cells were seeded at a specified density in 96-well plates. At the indicated time points, 10 µL of CCK-8 solution was added to each well containing 100 µL of culture medium and incubated at 37 °C for 1–2 h. Absorbance was measured at 450 nm using a microplate reader. Each experimental condition was performed in triplicate, and independent experiments were repeated three times.

Measurement of intracellular reactive oxygen species (ROS) levels

Intracellular ROS levels were determined using a ROS assay kit (Solarbio, China) following the manufacturer’s protocol. DCFH-DA was diluted 1:1000 in serum-free medium to a final concentration of 10 µmol/L. After the culture medium was removed, the cells were washed three times with serum-free medium and incubated with the diluted probe at 37 °C for 30 min. Fluorescence signals were then immediately observed and recorded under a fluorescence microscope.

Measurement of antioxidant enzyme activities

Mouse serum samples and cell lysates were collected, and superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels were measured using the corresponding assay kits (Solarbio, China) in accordance with the manufacturer’s instructions. Absorbance was measured using a microplate reader, and the data were analyzed quantitatively.

Measurement of mitochondrial membrane potential (JC-1 Staining)

Mitochondrial membrane potential was evaluated using a JC-1 mitochondrial membrane potential assay kit (Solarbio, China). After incubation with JC-1 working solution at 37 °C for 20 min, the cells were washed three times with PBS and immediately imaged using a fluorescence microscope.

Nuclear and cytoplasmic protein extraction

A nuclear and cytoplasmic protein extraction kit (Beyotime, China) was used in accordance with the manufacturer’s instructions to minimize cross-contamination between nuclear and cytoplasmic proteins. Correspondingly treated RWPE-1 cells were collected, trypsinized, washed with PBS, and incubated with nuclear and cytoplasmic extraction reagents. After lysis on ice and centrifugation, the cytoplasmic and nuclear protein fractions were separated and subsequently subjected to Western blot analysis.

Western blot analysis

Following treatment, cells were washed three times with 1× PBS and lysed using whole-protein lysis buffer. After 15 min, cell lysates were collected by repeated scraping of the culture plates and centrifuged to obtain protein extracts. Protein concentrations were quantified, and equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h and then incubated with the indicated primary antibodies. The primary antibodies used were as follows: cyclooxygenase-2 (COX-2) (1:2000, 12375-1-AP), inducible nitric oxide synthase (iNOS) (1:1000, 22226-1-AP), IL-1β (1:1000, 16806-1-AP), ERK (1:8000, 11257-1-AP), p-ERK (1:3000, 28733-1-AP), JNK (1:10000, 66210-1-Ig), p-JNK (1:2000, 80024-1-RR), p38 (1:1000, 14064-1-AP), p-p38 (1:2000, 28796-1-AP), Nrf2 (1:10000, 16396-1-AP), HO-1 (1:3000, 10701-1-AP), p65 (1:1000, 10745-1-AP), p-p65 (1:5000, 82335-1-RR), IκBα (1:10000, 10268-1-AP), p-IκBα (1:2000, 82349-1-RR), Lamin B (1:20000, 12987-1-AP), and β-actin (1:30000, 20536-1-AP). Protein bands were detected using enhanced chemiluminescence and imaged with a Fusion FX imaging system (Vilber Lourmat, France). Band intensities were quantified using ImageJ software. All experiments were independently repeated at least three times.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using an RNA extraction kit (Seven Sea, China), and RNA concentration was measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). First-strand cDNA was synthesized using the M-MLV First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). qRT-PCR analysis was conducted using an ABI 7500 FAST system (Applied Biosystems, USA) with Platinum SYBR Green qPCR SuperMix-UDG (Thermo Fisher Scientific, USA). Relative gene expression levels were determined by the 2^−ΔΔCt method. Primer sequences are listed in Supplementary Table S2.

Network pharmacology analysis

Potential targets of MYR were predicted using SwissTargetPrediction. CP/CPPS-related targets were retrieved from the GeneCards, DisGeNET, and OMIM databases. After removal of duplicate entries, overlapping targets between MYR and CP/CPPS were identified and subjected to subsequent enrichment analyses. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using the clusterProfiler package (version 4.0) in R (version 4.3). A P value < 0.05 was considered statistically significant. Enrichment results were visualized as bubble plots using the ggplot2 package in R.

Statistical analysis

All quantitative data are presented as mean ± standard deviation (SD) from at least three independent experiments (n ≥ 3). Data distribution was assessed using the Shapiro-Wilk normality test. For datasets that met the normality assumption, comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey’s multiple comparisons test to adjust for multiple pairwise comparisons. For datasets that did not meet the normality assumption, the Kruskal-Wallis test followed by Dunn’s multiple comparisons test was applied. Statistical analyses were performed using GraphPad software (version 8.3) and R (version 4.3). Corrected p-values were reported. A p-value < 0.05 was considered statistically significant.

Results

MYR significantly alleviates prostatic inflammation and pelvic pain in EAP mice

The MYR treatment protocol and experimental design are shown in Fig. 1A. HE staining demonstrated that, compared with the control group, prostate tissues from EAP mice exhibited obvious perivascular distortion and extensive inflammatory cell infiltration. MYR treatment markedly alleviated these histopathological inflammatory changes (Fig. 1B) and significantly lowered the inflammation score (Fig. 1C). In addition, the response rate to pelvic von Frey filament stimulation was significantly higher in EAP mice than in control mice, whereas MYR administration substantially attenuated this abnormal nociceptive response (Fig. 1D). ELISA showed that the serum levels of TNF-α, IL-1β, IL-17 A, IFN-γ, and CCL2 were significantly elevated in the EAP group, while MYR treatment effectively reduced the levels of these inflammatory mediators (Fig. 1E). CD4 and CD68 were used as markers of CD4⁺ T cells and macrophages, respectively. Immunohistochemical staining revealed prominent infiltration of CD4⁺ T cells and CD68⁺ macrophages in the prostate tissues of EAP mice, which was significantly attenuated by MYR treatment (Fig. 1F). Consistently, immunofluorescence analysis showed that MYR markedly suppressed CCL2 expression in prostate tissues from EAP mice (Fig. 1G). To assess oxidative stress, serum oxidative stress-related indices were measured. Compared with control mice, EAP mice exhibited reduced SOD activity and increased MDA levels, both of which were significantly reversed by MYR treatment (Fig. 1H). IHC analysis further showed that Nrf2 expression was decreased, whereas p65 expression was increased, in the prostate tissues of EAP mice relative to controls. MYR treatment markedly upregulated Nrf2 expression and downregulated p65 expression (Fig. 1I). Taken together, these findings suggest that MYR alleviates pathological injury and inflammatory responses in EAP mice, accompanied by reduced immune cell infiltration and attenuation of oxidative stress.

Fig. 1.

MYR significantly alleviates prostatic inflammation and pelvic pain in EAP mice. (A) Flowchart of the experimental design. (B) HE staining of prostate tissues from mice in the control, MYR, EAP and the EAP + MYR treatment groups (scale bar: 100 μm). (C) Prostate inflammation scores of mice in the control, MYR, EAP and the EAP + MYR treatment groups. (D) Tactile response frequencies of mice in the control, MYR, EAP and the EAP + MYR treatment groups. (E) Serum levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-17 A, IFN-γ, CCL2) in the control, MYR, EAP and the EAP + MYR treatment groups. (F) IHC staining images of CD4 and CD68 in prostate tissues of mice in the control, MYR, EAP and the EAP + MYR treatment groups (scale bar: 100 μm). (G) IF staining images of CCL2 in prostate tissues of mice from the control, MYR, EAP and the EAP + MYR treatment groups (scale bar: 100 μm). (H) Serum levels of SOD and MDA in mice from the control, MYR, EAP and the EAP + MYR treatment groups. (I) IF staining images of Nrf2 and P65 in prostate tissues of mice from the control, MYR, EAP and the EAP + MYR treatment groups (scale bar: 100 μm). Data are presented as mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, “ns” P > 0.05

Network pharmacology analysis

To investigate the potential mechanisms by which MYR exerts its effects in CP/CPPS, network pharmacology analysis was performed to predict its putative targets. Gene Ontology (GO) enrichment analysis showed significant enrichment in biological processes related to inflammatory response, oxidative stress, cytokine-mediated signaling, response to lipopolysaccharide, and apoptosis, suggesting that inflammation and oxidative stress may represent major processes regulated by MYR (Fig. 2A). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified 174 significantly enriched pathways associated with the actions of MYR in CP/CPPS. The top 20 enriched KEGG pathways were further visualized as a bubble plot (Fig. 2B). These targets were mainly enriched in the MAPK, JAK-STAT, NF-κB, TNF, IL-17, and Toll-like receptor signaling pathways.

Fig. 2.

Network pharmacology analysis. (A) GO enrichment analysis. (B) A bubble map illustrating the KEGG enrichment analysis of potential targets

MYR suppresses LPS-induced inflammatory response and oxidative stress in RWPE-1 cells

To evaluate the protective effect of MYR in vitro, RWPE-1 cells were stimulated with LPS. As shown in Fig. 3A, the chemical structure of MYR was presented. Cell viability analysis showed that MYR at concentrations of 0.625–10 µM had no obvious cytotoxic effect on RWPE-1 cells, whereas 20 µM MYR significantly reduced cell viability; therefore, 10 µM MYR was selected for subsequent experiments (Fig. 3B). RT-qPCR results showed that LPS markedly increased the mRNA expression of inflammatory mediators, including TNF-α, IL-1β, IL-17 A, IFN-γ, and CCL2. MYR treatment significantly reversed these changes (Fig. 3C). Consistently, western blot analysis showed that LPS upregulated the protein expression of COX-2, iNOS, and IL-1β, while MYR treatment markedly reduced their expression levels (Fig. 3D, E). We next assessed oxidative stress and mitochondrial function. LPS stimulation decreased SOD activity and increased MDA levels, whereas MYR treatment restored SOD activity and reduced MDA accumulation (Fig. 3F). In addition, ROS staining and quantitative analysis showed that MYR significantly suppressed LPS-induced ROS accumulation (Fig. 3G). JC-1 staining further demonstrated that LPS reduced the mitochondrial membrane potential, as reflected by a decreased JC-1 red/green fluorescence ratio, while MYR treatment significantly restored mitochondrial membrane potential (Fig. 3H). Collectively, these results indicate that MYR suppresses LPS-induced inflammatory response and oxidative stress in RWPE-1 cells.

Fig. 3.

MYR suppresses LPS-induced inflammatory response and oxidative stress in RWPE-1 cells. (A) Chemical structure of MYR. (B) CCK8 assay was performed to analyze the effect of different concentrations of MYR on cells. (C) qRT-PCR was used to detect the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-17 A, IFN-γ, CCL2) in the control group, MYR group, LPS group, and LPS + MYR group. (D) Western blot was used to detect the expression levels of COX-2, iNOS, IL-1β in the control group, MYR group, LPS group, and LPS + MYR group. (E) Quantitative analysis of Western blot bands. (F) Levels of SOD and MDA in the control group, MYR group, LPS group, and LPS + MYR group. (G) Intracellular ROS levels in the control, MYR, LPS, and LPS + MYR groups were detected using DCFH probe and quantitative analysis of intracellular ROS intensity (scale bar: 100 μm). (H) Mitochondrial membrane potential levels in the control, MYR, LPS, and LPS + MYR groups were measured by JC-1 probe and quantitative analysis of mitochondrial membrane potential (red/green fluorescence ratio) (scale bar: 100 μm). Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01

MYR suppresses LPS-induced activation of MAPK and STAT3 pathways in RWPE-1 cells

To investigate the mechanisms by which MYR alleviates CP/CPPS, Western blot analysis was performed to assess whether MYR regulates MAPK and STAT3 signaling in LPS-induced RWPE-1 cells. Compared with the control group, LPS stimulation increased the phosphorylation levels of JNK, p38, and ERK. MYR treatment significantly inhibited the phosphorylation of these proteins (Fig. 4A, B). Similarly, phosphorylated STAT3 was activated upon LPS treatment, and MYR administration markedly reduced the levels of activated phosphorylated STAT3 protein (Fig. 4C, D). These findings suggest that MYR is associated with reduced activation of MAPK and STAT3 signaling in LPS-induced RWPE-1 cells.

Fig. 4.

MYR suppresses inhibits LPS-induced activation of MAPK and STAT3 pathways in RWPE-1 cells. (A) Western blot was used to detect the expression levels of ERK, p-ERK, JNK, p-JNK, p38, p-p38 in the control group, MYR group, LPS group, and LPS + MYR group. (B) Quantitative analysis of Western blot bands. (C) Western blot was used to detect the expression levels of STAT3, p-STAT3 in the control group, MYR group, LPS group, and LPS + MYR group. (D) Quantitative analysis of Western blot bands. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01

MYR enhances Nrf2-associated antioxidant signaling and suppresses NF-κB activation in LPS-induced RWPE-1 cells

To further investigate the antioxidant and anti-inflammatory mechanisms of MYR, we examined the NF-κB and Nrf2/HO-1 signaling pathways in LPS-induced RWPE-1 cells. Western blot analysis showed that LPS stimulation reduced both total and nuclear Nrf2 protein levels in RWPE-1 cells, accompanied by downregulation of its downstream target HO-1. MYR treatment markedly reversed these changes (Fig. 5A, B). Immunofluorescence staining further demonstrated that LPS exposure decreased Nrf2 fluorescence intensity and impaired its nuclear translocation. In contrast, MYR treatment not only increased overall Nrf2 fluorescence intensity but also facilitated its translocation from the cytoplasm to the nucleus (Fig. 5C). In addition, LPS stimulation significantly enhanced the phosphorylation of IκBα and p65 and promoted p65 nuclear translocation (Fig. 5D, E). Consistently, immunofluorescence analysis revealed increased numbers of p65-positive cells and obvious nuclear localization in the LPS-treated group, whereas MYR intervention markedly suppressed p65 nuclear translocation (Fig. 5F). Collectively, these results indicate that MYR augments Nrf2-mediated antioxidant defense while inhibiting NF-κB activation in LPS-induced RWPE-1 cells.

Fig. 5.

MYR enhances Nrf2-associated antioxidant signaling and suppresses NF-κB activation in LPS-induced RWPE-1 cells. (A) Expression levels of Nucl-Nrf2, Total-Nrf2 and HO-1 in cells from the control group, MYR group, LPS group, and LPS + MYR group were detected by Western blot. (B) Quantitative analysis of Western blot bands. (C) Expression levels of p-IκBα, IκBα, Nucl-p65, p-p65, and Total-p65 in cells from the control group, MYR group, LPS group, and LPS + MYR group were detected by Western blot. (D) Quantitative analysis of Western blot bands. (E) IF images and localization of Nrf2 in cells from the control group, MYR group, LPS group, and LPS + MYR group (scale bar: 100 μm). (F) IF images and localization of p65 in cells from the control group, MYR group, LPS group, and LPS + MYR group (scale bar: 100 μm). Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01

Nrf2 inhibition weakens the protective effects of MYR in LPS-induced RWPE-1 cells

To further clarify the role of Nrf2 in MYR-mediated protection, validation experiments were performed using ML385, a pharmacological inhibitor of Nrf2. Western blot analysis showed that ML385 markedly abolished the MYR-induced increase in Nrf2 nuclear translocation and HO-1 expression (Fig. 6A, B). In parallel, the inhibitory effect of MYR on NF-κB activation was attenuated by ML385, as evidenced by the restored phosphorylation of IκBα and p65, as well as enhanced p65 nuclear translocation (Fig. 6C, D). Immunofluorescence analysis further confirmed that ML385 largely reversed the regulatory effects of MYR on the subcellular localization of both Nrf2 and p65 (Fig. 6E, F). In addition, MYR significantly suppressed the LPS-induced upregulation of TNF-α, IL-1β, IL-17 A, IFN-γ, and CCL2 mRNA expression, whereas these inhibitory effects were largely weakened in the presence of ML385 (Fig. 6G). Consistently, MYR reduced MDA levels, restored SOD activity, and improved mitochondrial membrane potential in LPS-induced RWPE-1 cells, while ML385 markedly attenuated these effects (Fig. 6H, I). These findings indicate that inhibition of Nrf2 weakens the protective effects of MYR against LPS-induced inflammatory and oxidative injury in RWPE-1 cells. As summarized in Fig. 7, the protective effects of MYR were associated with coordinated inhibition of MAPK/STAT3/NF-κB signaling and activation of Nrf2-dependent antioxidant signaling.

Fig. 6.

Nrf2 inhibition weakens the protective effects of MYR in LPS-induced RWPE-1 cells. (A, B) Western blot analysis was performed to detect the expression levels of Nucl-Nrf2, Total-Nrf2 and HO-1 in the Control, LPS, LPS + MYR, and LPS + MYR+ML385 groups, with subsequent quantitative analysis of the bands. (C, D) Western blot was used to examine the expression levels of p-IκBα, IκBα, Nucl-p65, p-p65, and Total-p65 in the Control, LPS, LPS + MYR, and LPS + MYR+ML385 groups, followed by quantitative analysis of the bands. (E) Fluorescent expression and localization of Nrf2 in Control, LPS, LPS + MYR, and LPS + MYR+ML385 groups (Scale bar: 100 μm). (F) Fluorescent expression and localization of p65 in Control, LPS, LPS + MYR, and LPS + MYR+ML385 groups (Scale bar: 100 μm). (G) qRT-PCR was performed to detect the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-17 A, IFN-γ, CCL2) in cells from the control group, LPS group, LPS + MYR group, and LPS + MYR+ML385 group. (H) Levels of SOD and MDA in cells from the control group, LPS group, LPS + MYR group, and LPS + MYR +ML385 group. (I) Measurement of mitochondrial membrane potential in cells from the control group, LPS group, LPS + MYR group, and LPS + MYR +ML385 group using JC-1 probe (scale bar: 100 μm). Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01,“ns” P > 0.05

Fig. 7.

Proposed schematic model illustrating the mechanisms by which MYR attenuates experimental prostatitis-associated inflammatory and oxidative injury through coordinated regulation of MAPK, STAT3, NF-κB, and Nrf2 signaling pathways

Discussion

In the present study, we combined in vivo experiments, network pharmacology analysis, and in vitro mechanistic validation to investigate the protective effects of MYR in experimental models relevant to CP/CPPS. Our data showed that MYR markedly alleviated prostatic inflammation and pelvic pain-related responses in EAP mice, accompanied by reduced serum levels of pro-inflammatory cytokines and chemokines, decreased infiltration of CD4-positive T cells and CD68-positive macrophages, and improved oxidative stress status. In LPS-induced RWPE-1 cells, MYR suppressed inflammatory mediator expression, reduced ROS accumulation, restored redox balance, and attenuated mitochondrial dysfunction. Mechanistically, these protective effects were accompanied by reduced activation of MAPK, STAT3, and NF-κB signaling, together with enhanced Nrf2-associated antioxidant responses. Pharmacological inhibition of Nrf2 markedly weakened the anti-inflammatory and antioxidant effects of MYR.

The pathological hallmarks of CP/CPPS include chronic inflammation of the prostate tissue, immune cell infiltration, and persistent pelvic pain [28]. In this study, we utilized the EAP mouse model, which is induced by immunization and recapitulates the pathological processes of human CP/CPPS. This model is widely recognized as an ideal animal model for investigating this disease [29]. Current evidence indicates that autoimmune dysfunction is a central pathogenic mechanism of CP/CPPS, and aberrant activation and infiltration of immune cells not only contribute to disease progression but are also closely associated with clinical symptoms such as pain [30, 31]. Our study revealed that MYR significantly reduces the infiltration of CD4⁺ T cells and CD68⁺ macrophages in the prostate tissue, along with a concomitant decrease in the expression of the chemokine CCL2. CCL2 is a key chemokine responsible for recruiting monocytes/macrophages, and its expression is markedly elevated in the prostatic fluid of CP/CPPS patients, positively correlating with pain severity [32]. The downregulation of CCL2 by MYR may represent a crucial mechanism by which it reduces immune cell infiltration and alleviates local inflammation. Experimental studies have shown that EAP mice exhibit elevated serum levels of IL-1β, IFN-γ, IL-17 A, and CCL2, accompanied by immune cell infiltration in the prostatic parenchyma and increased prostatic CCL2 expression [28]. Consistent with these observations, our results demonstrated that MYR markedly alleviated histopathological injury in the prostate, reduced pelvic tactile allodynia, attenuated immune cell infiltration, and lowered the serum levels of major inflammatory and pain-related mediators, including IL-1β, IFN-γ, IL-17 A, and CCL2.

To investigate the potential mechanisms by which MYR alleviates CP/CPPS, network pharmacology analysis was conducted in this study. The results revealed multiple common targets shared by MYR and CP/CPPS, which were predominantly enriched in inflammation-related pathways such as NF-κB, MAPK, JAK-STAT, T cell receptor signaling, and Th17 cell differentiation. Furthermore, GO functional analysis indicated that “regulation of reactive oxygen species metabolic process” is a potentially critical process involved in the therapeutic effects of MYR. These predictive findings provided a clear direction for subsequent in vitro mechanistic investigations, focusing on key inflammatory signaling pathways including MAPK, JAK-STAT, and NF-κB, as well as the Nrf2-mediated antioxidant pathway.

To further clarify the mechanisms underlying the effects of MYR, we employed an LPS-induced inflammatory model in RWPE-1 cells. The results showed that MYR pretreatment significantly suppressed the LPS-induced expression of inflammatory mediators, including IL-1β, IL-17 A, IFN-γ, and CCL2, and also reduced the levels of COX-2 and iNOS, in agreement with the in vivo findings. Concurrently, MYR effectively suppressed LPS-induced intracellular ROS production and reversed the oxidative stress state characterized by decreased SOD activity and elevated MDA levels, further validating the antioxidant properties of MYR.

Mechanistically, this study focused on four pivotal signaling pathways: MAPK, STAT3, NF-κB, and Nrf2. These pathways play crucial roles in inflammation, and their aberrant activation can exacerbate and sustain the inflammatory response [33, 34], which can be verified by detecting the phosphorylation levels of relevant proteins. The MAPK family, including JNK, p38, and ERK, constitutes an important signal transduction module mediating inflammatory responses. Upon phosphorylation, MAPKs activate downstream transcription factors, promoting the expression of pro-inflammatory cytokines [35]. NF-κB is a central transcription factor orchestrating immune and inflammatory responses [36]. Under basal conditions, NF-κB remains inactive in the cytoplasm through its association with IκBα. In response to pro-inflammatory stimulation, IκBα is phosphorylated and subsequently degraded, thereby releasing NF-κB (p65), which then translocates into the nucleus to drive the transcription of downstream inflammatory genes. Previous studies have shown that the NF-κB and MAPK signaling pathways are activated in EAP mice, and such activation promotes the expression of multiple cytokines, thereby facilitating disease progression [37]. Our findings reveal that LPS stimulation significantly increases the phosphorylation levels of JNK, p38, ERK, IκBα, and p65, whereas MYR intervention inhibits p65 nuclear translocation and effectively suppresses these changes. The STAT3 signaling pathway plays a critical role in Th17 cell differentiation and IL-17 A production, which are closely linked to the pathogenesis of CP/CPPS [38]. The inhibitory effect of MYR on STAT3 phosphorylation may be associated with the reduction in IL-17 A levels. Additionally, studies have indicated that the STAT3 pathway is involved in the regulation of autophagy in CP/CPPS, thereby influencing disease onset and progression [39]. Our study demonstrated that MYR markedly attenuated LPS-induced STAT3 phosphorylation. Previous studies have shown that activation of the JNK/MAPK and NF-κB pathways promotes the expression of inflammatory cytokines and the pain-related chemokine CCL2 [40], which is consistent with our findings.

Nrf2 is the master transcription factor governing the cellular antioxidant defense system. Increased intracellular ROS levels stimulate the nuclear translocation of Nrf2, which subsequently binds to antioxidant response elements (AREs) in DNA and induces the transcription of phase II detoxification enzymes, including superoxide dismutase (SOD) and catalase (CAT), as well as antioxidant proteins such as HO-1 and NAD(P)H quinone oxidoreductase 1 (NQO1), thereby contributing to the maintenance of redox homeostasis [41, 42]. Accumulating evidence suggests that oxidative stress participates in the pathogenesis of CP/CPPS [3, 4]. Studies have shown that EAP models exhibit dysregulated levels of oxidative stress-associated biomarkers, such as glutathione (GSH), MDA, SOD, NADPH, and ROS [43, 44]. Furthermore, lycopene has been shown to activate the Nrf2 signaling pathway, upregulating the expression of CAT, glutathione peroxidase (GSH-Px), and SOD, while downregulating MDA expression, thereby attenuating CP/CPPS-associated oxidative stress [45]. Consistent with these findings, our results show that MYR reverses the decrease in SOD activity and the increase in MDA levels in the serum of EAP mice, improving the oxidative stress status. In vitro, LPS stimulation led to reduced Nrf2 protein levels and downregulation of its downstream target HO-1, whereas MYR pretreatment promoted Nrf2 nuclear translocation and effectively reversed these alterations. Moreover, MYR-induced upregulation of Nrf2 was accompanied by significant inhibition of the phosphorylation of key components of the NF-κB pathway, namely p65 and IκBα. The observed reciprocal regulation between Nrf2 and NF-κB by MYR may be attributed to the ability of Nrf2-induced HO-1 to suppress IκBα phosphorylation, or to the direct protein-protein interaction between Nrf2 and p65 that limits its transcriptional activity. Notably, the anti-inflammatory and antioxidant effects of MYR were largely abolished in the presence of the Nrf2 inhibitor ML385. These findings indicate that Nrf2 signaling makes an important contribution to the protective effects of MYR, although the current data do not fully establish the hierarchical relationship between Nrf2 and the other signaling pathways examined. However, the current findings do not exclude the contribution of additional upstream or parallel regulatory mechanisms. These results suggest that MYR not only suppresses pro-inflammatory signaling pathways but also enhances cellular antioxidant capacity, thereby exerting a dual synergistic effect of “inflammation suppression and antioxidant defense.”

Although MYR also inhibited MAPK, STAT3, and NF-κB signaling, we focused on Nrf2 inhibition in the mechanistic rescue experiments because oxidative stress and mitochondrial dysfunction were central pathological features in both the EAP model and LPS-induced RWPE-1 cells. Nrf2 is a key regulator of cellular antioxidant defense and controls downstream cytoprotective enzymes, including HO-1 and NQO1. Previous studies have also implicated Nrf2-related signaling in prostatitis-associated inflammation, fibrosis, and ferroptosis [46, 47]. Therefore, we selected an Nrf2 inhibitor to determine whether Nrf2 activation is required for the antioxidant and anti-inflammatory effects of MYR. Nevertheless, MAPK, STAT3, and NF-κB may also contribute to the effects of MYR, and future studies using pathway-specific inhibitors or genetic approaches are needed to define their relative contributions. MYR inhibited MAPK signaling in LPS- induced RWPE-1 cells, suggesting its involvement in the protective effect of MYR. However, this pathway was not further validated in prostate tissues from the EAP model. Therefore, the involvement of MAPK signaling in vivo remains inferential and should be interpreted with caution. Future studies should examine MAPK activation in prostate tissues and use pathway-specific inhibitors or genetic approaches to confirm its contribution to the therapeutic effects of MYR.

To date, the phytochemical characteristics and pharmacological activities of MYR have been extensively studied; however, its clinical translation still faces certain challenges. First, existing studies indicate that the absolute oral bioavailability of MYR is relatively low, which may result in insufficient systemic exposure and thereby limit its therapeutic efficacy. Therefore, further human pharmacokinetic studies are warranted to gain a deeper understanding of its in vivo disposition characteristics and pharmacological activity. Second, the presence of the blood–prostate barrier, combined with the abundance of fibrous connective tissue in the prostate, the local slightly alkaline microenvironment, and P-glycoprotein (P-gp)-mediated active efflux, may collectively restrict the distribution and accumulation of MYR within the prostate tissue. Consequently, the 10 µM MYR concentration used in vitro was selected based on cell viability assays and its protective effects against LPS-induced inflammatory and oxidative injury. However, whether this concentration is achievable in prostate tissue after oral administration remains uncertain, as plasma and prostate tissue MYR levels were not directly measured in the present study. This limits the translational interpretation of the in vitro findings. Future pharmacokinetic and tissue distribution studies are needed to determine the relationship between the in vivo dose, systemic exposure, prostate tissue concentration, and effective cellular concentration of MYR.

Several limitations should be acknowledged. First, although LPS-induced RWPE-1 cells are useful for studying epithelial inflammatory and oxidative stress responses, this model only reflects a limited TLR4-mediated epithelial response and does not fully reproduce the autoimmune-like microenvironment of CP/CPPS, which involves interactions among epithelial cells, stromal fibroblasts, macrophages, T cells, and Th17-associated cytokines. Therefore, the in vitro signaling results should be interpreted as partial mechanistic evidence. Second, although MYR inhibited MAPK, STAT3, and NF-κB signaling in vitro, these pathways require further validation in prostate tissues in vivo. Third, plasma and prostate tissue concentrations of MYR were not measured, and thus the pharmacological relevance of the in vivo dose and in vitro concentration remains to be clarified. Future studies using more physiologically relevant models, pathway-specific interventions, and pharmacokinetic analyses are needed to further confirm the therapeutic potential of MYR in CP/CPPS.

Conclusion

In conclusion, MYR attenuated inflammatory, oxidative, and pain-related changes in experimental models relevant to CP/CPPS. These protective effects were associated with reduced activation of MAPK/STAT3/NF-κB signaling and enhanced Nrf2-dependent antioxidant responses. Our findings provide preclinical evidence supporting further investigation of MYR in CP/CPPS.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Hang Zhang: Conceptualization, Methodology, Software; Dahong Zhang: Data curation, Writing – original draft; Yanping Zhang: Visualization, Investigation; Zhenwei Han: Supervision; Zhihai Teng: Software, Validation; Yaxuan Wang: Writing – review & editing. All authors have read and approved the final manuscript.

Funding

This study was supported by the Medical Scientific Research Project of Hebei Province (grant NO. 20250377).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The research was reviewed and approved by the Ethics Committee of the Second Hospital of Hebei Medical University (2024-AE244).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.