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. 2026 Feb 4;49(1):79. doi: 10.1007/s10753-026-02468-9

Leonurine Mitigates Experimental Autoimmune Prostatitis by Modulating Macrophage M1 Polarization Through the TLR4/NF-κB Signaling Pathway

Rui-Jie Hu 1,2,3, Xiao-Long Ying 1,2,3, Cheng Zhang 1,2,3, Xu Wang 1,2,3, Chang-Sheng Zhan 1,2,3,✉
PMCID: PMC12916543  PMID: 41639329

Abstract

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) constitutes a clinically complex urological condition defined by the persistence of pelvic pain and chronic inflammation. Emerging evidence underscores the critical involvement of macrophage-mediated immune dysregulation, particularly the dominance of pro-inflammatory M1 macrophages, in driving CP/CPPS pathogenesis. Leonurine, a bioactive alkaloid derived from leonuri, exhibits various pharmacological properties and has been shown to regulate macrophage polarization in rheumatoid arthritis. This study aimed to evaluate leonurine’s therapeutic efficacy in a murine experimental autoimmune prostatitis (EAP) model, established by subcutaneous injection of complete Freund’s adjuvant-emulsified prostate antigens. Leonurine administration in EAP mice markedly reduced prostatic inflammatory responses, mitigated chronic pain, and inhibited the expression of pro-inflammatory cytokines. Likewise, leonurine decreased inducible nitric oxide synthase (iNOS) expression levels, an established marker for M1 macrophage polarization. Leonurine has been found to suppress M1 polarization and decrease the secretion of M1-related cytokines (IL-1β and TNF-α) in immortalized bone marrow-derived macrophages (iBMDMs) under in vitro conditions. Mechanistic investigations demonstrated that leonurine mediates its therapeutic effects by modulating the TLR4/NF-κB signaling pathway in both macrophages and EAP models. Molecular docking and dynamics simulations demonstrated stable binding interactions between leonurine and key proteins involved in the TLR4/NF-κB signaling cascade. As a whole, these findings verify that leonurine relieves experimental autoimmune prostatitis (EAP) by regulating M1 macrophage polarization through the TLR4/NF-κB signaling cascade.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-026-02468-9.

Keywords: Chronic prostatitis/Chronic pelvic pain syndrome, Leonurine, Macrophage polarization, Toll-like receptor 4

Introduction

A common urological issue among adult men, prostatitis includes NIH Category III prostatitis——otherwise termed chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS)——which constitutes over 90% of chronic cases and is the most frequent clinical type [1]. As shown by epidemiological studies, the mean prevalence of prostatitis-like symptoms in the general population is about 8.2% [2], and nearly half of men will experience prostatitis symptoms of varying degrees at a certain stage in life [3]. Typically, the main clinical manifestations involve lower urinary tract symptoms (e.g., frequency, urgency, dysuria, nocturia) with no urinary tract infection detected, as well as chronic pelvic and perineal pain [4].Currently, the etiology and pathogenesis of CP/CPPS remain poorly understood, resulting in limited efficacy of conventional pharmacological treatments [3] and a high recurrence rate. Accordingly, there is an urgent requirement to clarify the pathological mechanisms underlying CP/CPPS and develop novel, effective therapeutic agents with targeted actions. In recent years, accumulating evidence has suggested that immune dysregulation——particularly autoimmune responses directed against prostate-specific antigens——serves a critical role in the pathogenesis of CP/CPPS [5–7].

Macrophages, as integral components of the innate immune system, are widely distributed across various tissues. These cells perform a critical function in immune responses, tissue repair, and sustaining homeostasis [8]. When exposed to various microenvironmental stimuli, macrophages can polarize into two distinct phenotypes: the pro-inflammatory M1 and anti-inflammatory M2 subtypes. During inflammatory processes, these two subsets are dynamically activated, with their balanced ratio being critical for sustaining physiological homeostasis. While M1 macrophages promote inflammation, M2 macrophages function to suppress inflammatory responses. Dysregulation of this balance can significantly impact disease progression [9]. As key effector cells in innate immunity, macrophages make substantial contributions to various inflammatory disorders. Emerging data suggest that an imbalance in macrophage polarization may also participate in CP/CPPS pathogenesis [10–12].

Leonurine, a natural alkaloid from Leonurus japonicus, has diverse immunomodulatory properties—including anti-inflammatory, antioxidant, and anti-angiogenic actions [13]. Notably, it has demonstrated remarkable efficacy in combating inflammatory diseases. Previous studies have shown that leonurine alleviates asthma by modulating the p38 MAPK/NF-κB signaling pathway [14] and relieves rheumatoid arthritis through regulating macrophage polarization and scavenging reactive oxygen species [15]. Nonetheless, no research has examined the therapeutic role of leonurine in CP/CPPS. The present research intends to investigate the protective effects of leonurine in CP/CPPS and elucidate the molecular mechanisms that underlie them.

Methods

Reagents and Pharmaceuticals

Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) supplied Leonurine (purity ≥ 98%). Complete Freund’s Adjuvant (CFA) was sourced from Sigma-Aldrich (Germany), and mouse ELISA kits for TNF-α, IL-1β, and IL-6 were obtained from Bioswamp (Wuhan, China). Primary antibodies included anti-mouse F4/80 (BioLegend, #566787), anti-CD11b (BD Biosciences, #553312), anti-CD86 (BD Biosciences, #553692), anti-iNOS (Affinity, #AF0199), anti-Arg1 (ABclonal, #A25808), anti- Toll-like receptor 4 (TLR4) (Affinity, #AF7017), anti-MYD88 (Affinity, #AF5195), anti–β-Actin (Affinity, #AF7018), anti–NF-κB P65 (ABclonal, #A19653), and anti–phospho-NF-κB P65 (ABclonal, #AP1294). Corresponding HRP-conjugated secondary antibodies were also utilized, including anti-rabbit (#S0001) and anti-mouse (#S0002) secondary antibodies obtained from Affinity. Additionally, LPS and the TLR4 agonist RS09 were procured from MedChemExpress (China).

Animal Models and Administration Treatment

Six-week-old NOD immunodeficient mice [16] were acquired from the Nanjing Institute of Biomedical Sciences, Nanjing University (Nanjing, China). Before initiating the experiment, the animals underwent a one-week acclimatization period to enhance their adaptation to the novel environment. Standard chow and autoclaved water were provided to all animals, and the housing environment was maintained at 18–22 °C with a 12-hour light/dark cycle. The Animal Care and Use Committee of Anhui Medical University’s Animal Center (Approval No.: LLSC20200813) approved all animal experiments. The experimental autoimmune prostatitis (EAP) model was generated based on previously reported methods [17, 18]. Prostate antigens (PAgs) were extracted from Sprague-Dawley rats and subjected to homogenization treatment. Briefly, prostate tissue was aseptically excised from male Sprague-Dawley rats. After rinsing with ice-cold phosphate-buffered saline (PBS), the tissue was minced and homogenized in PBS supplemented with 0.5% Triton X-100. The total protein concentration was quantified using a bicinchoninic acid (BCA) assay and standardized to 40 mg/mL. The homogenate was centrifuged at 8,500 × g for 30 min at 4 °C. The resulting supernatant was collected, aliquoted, and stored at −80 °C overnight. For primary immunization, the antigen preparation was emulsified with an equal volume of complete Freund’s adjuvant (CFA) by repeated passage through a three-way stopcock to form a stable water-in-oil emulsion, as confirmed by its stability in a water droplet test. The final immunogen was stored at −80 °C until it was used. Mice were randomly assigned to the following groups (n = 7 per group): Control, EAP, EAP + LEO (Low, 15 mg/kg/d), EAP + LEO (High, 30 mg/kg/d). Following the experimental protocol, except for the Control group, each treatment group received an immunization dose of PAgs (300 µg per mouse) on days 0 and 28. Leonurine (purity ≥ 98%, Shanghai Aladdin) was initially solubilized in dimethyl sulfoxide to yield a concentrated stock (100 mg/mL). Prior to each administration, this stock was appropriately diluted in sterile physiological saline to achieve the designated doses of 15 or 30 mg/kg body weight. The compound was delivered once daily by oral gavage (10 mL/kg) from day 28 post‑immunization until the experimental endpoint (day 42), at which time all animals were humanely euthanized. Control and EAP groups received an equal volume of vehicle (saline containing 0.1% DMSO) on the identical schedule. The selected doses were based on previously reported anti‑inflammatory efficacy and safety of Leonurine in other murine inflammatory models [13, 19, 20]. In subsequent recovery experiments, RS09—a TLR4 agonist—was introduced [21]. Mice were randomly assigned to four groups: control, EAP, high-concentration LEO (30 mg/kg/d), and RS09 (n = 7 per group). The RS09-treated mice received intraperitoneal injections of RS09 (2 mg/kg/d) for two weeks, while members of both control and EAP groups continued being given normal saline by gavage for comparative analysis.

Pain Abnormality Test

Forty days post-immunization, abdominal mechanical allodynia was evaluated in all experimental groups with von Frey filaments. Six calibrated filaments, each with a distinct force threshold (0.008–2.0 g), were applied perpendicularly to the lower abdomen. To prevent desensitization, stimulation sites were alternated systematically, thereby avoiding repeated testing on identical areas. Ten trials per filament were performed on each mouse, with a positive nociceptive response defined by any of the following observable behaviors: (1) sudden abdominal contraction; (2) vigorous licking or biting of the stimulated site; (3) jumping withdrawal.

Histopathological Evaluation

10% neutral-buffered formalin was used for immediate fixation of prostate tissue samples, after which the samples were embedded in paraffin and cut into 4–5 μm-thick sections. Sections were subjected to H&E staining first, and then imaged via a digital whole-slide scanner to enable all-round histopathological evaluation. Inflammation severity was quantitatively graded (0–3) based on standard criteria: 0, no inflammatory infiltration; 1, mild perivascular mononuclear cell cuffing; 2, moderate perivascular mononuclear infiltration; 3, prominent perivascular cuffing with hemorrhage and dense mononuclear cell infiltration.

Tissue Immunochemistry

Xylene was used for the deparaffinization of prostate tissue sections, after which the sections were rehydrated using graded ethanol solutions and subjected to antigen retrieval with citrate buffer (pH 6.0). The SABC-HRP Kit (Beyotime Biotechnology, #P0615) was used for immunohistochemical staining, following the manufacturer’s protocol. Stained sections were digitized using a Pannoramic MIDI slide scanner (3DHistech, Hungary), and quantitative analysis of immunoreactivity was performed utilizing ImageJ software.

Immunofluorescence

Subsequent to deparaffinization and rehydration, prostate tissue sections underwent antigen retrieval. Endogenous peroxidase activity was quenched using 3% H₂O₂ in methanol for a duration of 10 min. Incubation of sections with primary antibodies at 4 °C overnight preceded 2-hour room temperature incubation with the corresponding secondary antibodies. Nuclei underwent counterstaining with DAPI in the dark. A laser scanning confocal microscope (Olympus Fluoview FV3000) was used to acquire immunofluorescence images.

Elisa

All protocols should be conducted in strict compliance with the instructions detailed in the reagent kit.

Real-Time Polymerase Chain Reaction

Using the Fastagen RNA Extraction Kit (Pioneer Biotechnology, #220011), total RNA was isolated from prostate tissues and cultured cells. Using NanoDrop 2000 spectrophotometry, we quantified RNA concentration and purity, ensuring 260/280 absorbance ratios remained within 1.8–2.0. Reverse transcription and qPCR were performed following the manufacturer’s instructions using the PrimeScript™ RT Reagent Kit and TB Green® Premix Ex Taq™ II (Takara Bio). The ABI 7500 Real-Time PCR System (Thermo, MA, USA) was used for amplification, and relative gene expression was calculated via the 2 − ΔΔCT method. Supplementary Table S1 provides detailed primer sequences.

Flow Cytometry

The spleens of mice were placed in a folded sieve for homogenization, resulting in the generation of a single-cell suspension. Sieve aspiration was followed by the addition of red blood cell lysis buffer to achieve full red blood cell lysis. M1 macrophages were stained fluorescently with APC-labeled anti-CD11b, FITC-labeled anti-F4/80, and PE-labeled anti-CD86 antibodies, with staining completed via 1-hour incubation at 4 °C. A CytoFLEX flow cytometer (Beckman Coulter, Brea, CA) analyzed the stained cells, and data were processed using CytExpert software.

Western Blotting

Individual tissue samples were homogenized using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. After SDS-PAGE separation of proteins from each sample, the resolved proteins were transferred to nitrocellulose membranes. Primary antibodies were added for overnight incubation after 1 h of blocking with 5% skim milk powder. Post-incubation, the membrane was rinsed with TBST prior to treatment with the secondary antibody. Again, prior to detecting protein bands with an enhanced chemiluminescence (ECL) system (ChemiScope 5600; Hengmei Technology, China), the strips were thoroughly cleaned. Finally, protein band quantification was performed using ImageJ software.

Cell Culture

For this research, immortalized bone marrow-derived macrophages (iBMDMs) from Academician Shao Feng’s team (Chinese Academy of Sciences) were used. iBMDMs were incubated at a consistent 37 °C with 5% CO2, following culture in high-glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. iBMDMs were pretreated for 2 h with serum-free medium containing leonurine (10 µM, 20 µM) before the experiment. After such pretreatment, the cells underwent 24-hour stimulation with 100 ng/ml LPS, while the control group was left untreated. In the recovery experiment, the RS09 group received treatment involving the addition of TLR4 agonist RS09 at a concentration of 5 µM on a basis of leonurine 20 µM.

Molecular Docking

Molecular docking experiments examined leonurine’s binding affinity to TLR4/NF-κB signaling pathway proteins. Leonurine’s 2D structure (from PubChem, http://pubchem.ncbi.nlm.nih.gov/) was converted to 3D via Chemoffice and saved in mol2 format. Target proteins’ high-resolution crystal structures (from RCSB PDB, http://www.rcsb.org/) were refined with PyMOL (removing water and phosphate groups) and saved as PDB files. AutoDock Vina 1.5.6 served for molecular docking. Protein and ligand structures underwent preprocessing via AutoDockTools: polar hydrogens were added to proteins, while water molecules were eliminated; for ligands (leonurine), hydrogen atoms and rotatable bonds were assigned appropriately. A docking grid box was delineated around the binding site of interest. After comprehensive sampling of conformational space, the optimal binding pose was determined via the lowest binding affinity score (kcal/mol). PyMOL (v2.5.4) and Discovery Studio 2019 were employed to visualize protein-ligand interactions.

Molecular Dynamics Simulation

GROMACS 2022 was employed to perform molecular dynamics (MD) simulations. Using GROMACS’ pdb2gmx tool and the AutoFF web server, force field parameters were generated. The CHARMM36 force field was applied to the receptor protein, with the ligand using the CGenFF force field. A cubic TIP3P water box was used to solvate the system, extending 1 nm from the solute. Long-range electrostatic interactions were managed using the Particle Mesh Ewald (PME) method with a 1.0 nm cutoff. The SHAKE algorithm was used to constrain all bonds, while simulations operated with the Verlet leapfrog integrator at a 1 fs timestep. Three stages made up energy minimization: 3,000 steepest descent steps, with 2,000 conjugate gradient steps subsequent. Production MD simulations were run under NPT conditions (310 K, 1 bar) for 100 ns. Trajectory analyses included RMSD (root-mean-square deviation; gmx rms), RMSF (root-mean-square fluctuation; gmx rmsf), SASA (solvent-accessible surface area; gmx sasa), hydrogen bond analysis (gmx hbond), Rg (radius of gyration; gmx gyrate), RMSF (root-mean-square fluctuation; gmx rmsf), and SASA (solvent-accessible surface area; gmx sasa).

Statistics

Statistical analyses utilized GraphPad Prism 9.0. All experiments had ≥ 3 independent repeats, data expressed as mean ± SD. Two-tailed Student’s t-tests for two groups and one-way ANOVA with Bonferroni’s post hoc test for multiple groups were used, with significance at p < 0.05.

Results

Leonurine Alleviates Inflammation and Diminishes Macrophage Infiltration in Mice with EAP

The structural composition of leonurine is illustrated in Fig. 1A. A series of animal experiments were conducted following the protocol shown in Fig. 1B. Mice were subcutaneously injected with PAgs and CFA mixture on days 0 and 28 to induce the EAP model. Following secondary immunization, the treatment group received oral leonurine at doses of 30 mg/kg/d (high) or 15 mg/kg/d (low). Model validation and therapeutic efficacy were assessed using von Frey filaments testing alongside H&E histopathology. H&E staining confirmed the successful induction of EAP, with EAP mice showing marked inflammatory infiltration relative to control animals. Notably, treatment with leonurine led to a dose-dependent reduction in inflammatory cell infiltration within prostatic tissues (Fig. 1C). Mechanical allodynia assessments revealed exacerbated nociceptive responses in EAP-affected mice; however, administration of leonurine consistently mitigated pelvic pain responses across increasing filament forces (Fig. 1E). Immunohistochemical analysis revealed markedly decreased levels of inflammatory cell markers denoted by CD45⁺ cells in mice treated with leonurine when compared to EAP controls (Fig. 1F). Collectively, our findings establish that leonurine represents a promising therapeutic agent for alleviating pelvic pain and inflammation induced by EAP.

Fig. 1.

Fig. 1

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Leonurine reduces prostatitis infiltration in EAP mice and alleviates pelvic pain. (A) Chemical formula of leonurine. (B) Experimental procedure. (C) H&E staining showed that after oral administration of Leonurine, the infiltrative inflammatory cells in the prostate tissue of EAP mice were significantly reduced. (D) Histopathological scores of the mice in the Control, EAP, EAP + Leo(Low), and EAP + Leo(High) groups. (E) Pain response frequency for the Control, EAP, EAP + Leo(low), and EAP + Leo(High) groups to mechanical stimulation. (F-H) Immunohistochemical staining of CD45 and quantification in different groups

Leonurine Decreases the Proportion of pro-inflammatory M1 Macrophages and Downregulates Key Inflammatory Mediators

Macrophages play a key role in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) pathogenesis, so we investigated how leonurine affects splenic macrophages in EAP mice. Immunofluorescence analysis revealed a significantly elevated F4/80⁺ immunoreactivity in EAP mice compared to controls (*p* < 0.001), indicating robust infiltration of macrophages. Leonurine treatment markedly reduced F4/80⁺ signal intensity (Fig. 2A), demonstrating an attenuation in macrophage recruitment. Further flow cytometric analysis demonstrated that EAP mice had a higher proportion of M1-type macrophages in the spleen than controls. Notably, regardless of whether treated with low or high doses, both groups receiving leonurine demonstrated varying degrees of reduction in splenic macrophage numbers (Fig. 2B). Moreover, this phenomenon was corroborated by RT-qPCR and Western blot analyses which revealed decreased levels of iNOS—an established marker for M1-type macrophages—following leonurine treatment (Fig. 2D). Furthermore, inflammatory cytokines such as TNF-α, IL-6, and IL-1β secreted by M1-type macrophages exhibited significant elevation within the model group; however, leonurine effectively inhibited these alterations (Fig. 2F). We also assessed serum levels of these inflammatory factors (Fig. 2I), revealing corresponding trends consistent with our earlier findings. Collectively, this series of results indicates that leonurine possesses commendable anti-inflammatory properties.

Fig. 2.

Fig. 2

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Leonurine reduces the proportion of M1 in the prostate of EAP mice and lowers pro-inflammatory factors. (A) Immunofluorescence showing the levels of F4/80+ macrophages in the Control, EAP, EAP + Leo (Low), and EAP + Leo (High) groups. (B, C) Flow cytometry analysis and quantitative assessment of M1 macrophage proportions in splenic lymphocytes from the Control, EAP, EAP + Leo (Low), and EAP + Leo (High) groups. (D) Real-time PCR analysis of iNOS mRNA levels in the prostate tissues of mice from the Control, EAP, EAP + Leo (Low), and EAP + Leo (High) groups. (E) Western blot analysis and quantification of iNOS levels in the prostate tissues of mice from each group. (F-H) Real-time PCR analysis of TNF-α, IL-6, and IL-1β mRNA levels in the prostate tissues of mice from the Control, EAP, EAP + Leo (Low), and EAP + Leo (High) groups. (I-K) Measurement of serum levels of the proinflammatory cytokines TNF-α, IL-6, and IL-1β in mice from the Control, EAP, EAP + Leo (Low), and EAP + Leo (High) groups by enzyme-linked immunosorbent assay (ELISA)

Leonurine Exhibits Stable Binding Affinities To Key Proteins Within the TLR4/NF-κB Signaling Pathway

TLR4, functioning as a transcription factor, initiates downstream signaling pathways to enhance NF-κB activation—essential for M1 macrophage polarization. Building upon this established mechanistic link, we sought to investigate whether the anti-inflammatory effect of leonurine in EAP operates through this pivotal axis. Molecular docking analysis revealed stable binding interactions of leonurine with TLR4 (− 5.6 kcal/mol), MYD88 (− 5.3 kcal/mol), and NF-κB p65 (− 5.8 kcal/mol), with all binding energies being less than − 5 kcal/mol (Fig. 3A). Subsequent 100-ns molecular dynamics simulations demonstrated system equilibration after 20 ns; the leonurine-protein complexes maintained a stable root-mean-square deviation (RMSD) of approximately 2.1 Å (Fig. 3D). The consistent structural integrity was evidenced by minimal fluctuations in both the Rg (Fig. 3E) and SASA (Fig. 3F), indicating no significant expansion or contraction within the complexes occurred. Persistent hydrogen bonding was observed, ranging from 0 to 4 bonds with an average of 2 bonds (Fig. 3G), while low residue-specific flexibility was recorded as indicated by root-mean-square fluctuation values between 0.8 and 2.1 Å (Fig. 3H). Taken together, these computational analyses reveal that leonurine interacts with key proteins in the TLR4/NF-κB pathway with high affinity and stability, supporting its mechanistic role in the regulation of inflammatory signaling.

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Fig. 3 Leonurine demonstrates favorable binding with proteins associated with the TLR4/NF-κB axis. (A-C) Predicted three-dimensional binding mode of leonurine to TLR4, MYD88, and RELA. (D-F) Root mean square deviation (RMSD), radius of gyration (Rg), and solvent accessible surface area (SASA) of the ligand-protein complex. (G) Hydrogen bond interactions of the leonurine-target protein complex. (H) Root mean square fluctuation (RMSF) of the leonurine-target protein complex

Leonurine Inhibits the TLR4/NF-κB Pathway

Relative to control groups, EAP mouse prostate tissues displayed marked upregulation of TLR4, MYD88, and phospho-NF-κB p65 expression levels; Western blot analysis revealed that these increases were effectively reversed by leonurine treatment (Fig. 4A). IHC analysis of TLR4 and MYD88 corroborated the Western blot findings, demonstrating consistent reductions in expression (Fig. 4E). Additionally, the EAP group exhibited elevated NF-κB p65 levels as revealed by immunofluorescence analysis, while leonurine treatment mitigated these increases (Fig. 4G). In parallel, iBMDM-based in vitro validation revealed LPS stimulation led to heightened expression of TLR4, MYD88, and p65, while leonurine treatment resulted in a notable dose-dependent reduction of these proteins (Fig. 4H).

Fig. 4.

Fig. 4

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Leonurine inhibits the TLR4/NF-κB axis. (A-D) Representative Western blots and quantitative analysis of TLR4, MYD88, p-p65, and p65 expression across the indicated groups. (E, F) Immunohistochemical analysis of TLR4 and MYD88 levels in the Control, EAP, EAP + Leo (Low), and EAP + Leo (High) groups. (G) Immunofluorescence analysis of p65 in the Control, EAP, EAP + Leo (Low), and EAP + Leo (High) groups. (H-K) Western blot analysis of TLR4, MYD88, p-p65, and p65 levels in the Control, LPS, LPS + Leo (10 µM), and LPS + Leo (20 µM) groups

The TLR4 Agonist Undermined the Therapeutic Effect of Leonurine in EAP Mice

In EAP mice, RS09 (a TLR4 agonist commonly used in related studies [22]) was co-administered with leonurine via oral gavage for two weeks (Fig. 5 A), reversing leonurine’s therapeutic effect on EAP. Specifically, the RS09 co-treatment group exhibited greater inflammatory cell infiltration in prostatic tissues (Fig. 5B-C), elevated pathological scores (Fig. 5D), and more severe pelvic pain (Fig. 5E) than the group treated with leonurine alone. TLR4 activation led to marked upregulation of pro-inflammatory cytokine mRNA levels TNF-α, IL-1β, and IL-6 among them in prostate tissues (Fig. 5F-H), with parallel serum level elevations (Fig. 5I-K).

Fig. 5.

Fig. 5

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The TLR4 agonist RS09 reverses the protective effects of leonurine on prostatitis in the EAP mouse model. (A) Flowchart. (B) H&E staining shows inflammatory cell infiltration in the prostate tissues of the Control, EAP, EAP + Leo (High), and EAP + Leo (High) + RS09 groups. (C) Immunohistochemical staining of CD45 in different groups. (D) Histopathological scores of the mice in the Control, EAP, EAP + Leo (High), and EAP + Leo (High) + RS09 groups. (D) Pain response frequency for the Control, EAP, EAP + Leo (High), and EAP + Leo (High) + RS09 groups to mechanical stimulation. (F-H) Real-time PCR analysis of IL-6, IL-1β, and TNF-α mRNA levels in the prostate tissues of mice from the Control, EAP, EAP + Leo (High), and EAP + Leo (High) + RS09 groups. (I-K) Measurement of serum levels of the proinflammatory cytokines IL-6, IL-1β, and TNF-α in mice from the Control, EAP, EAP + Leo (High), and EAP + Leo (High) + RS09 groups by enzyme-linked immunosorbent assay (ELISA)

RS09 Antagonizes leonurine-mediated Suppression of M1 Macrophage Polarization in EAP Mice

Subsequent immunofluorescence analysis, conducted to explore whether leonurine targets the TLR4/NF-κB pathway. to mitigate M1 macrophage polarization in EAP mice, revealed marked increases in macrophage infiltration in treated groups after TLR4 agonism (Fig. 6A). Concurrently, both protein and transcript analyses revealed that leonurine decreased iNOS expression in EAP prostates, and RS09 co-treatment reversed this effect. (Fig. 6B). Flow cytometry confirmed a higher proportion of CD11b⁺F4/80⁺CD86⁺(M1) cells within the RS09 group compared to those treated with leonurine (Fig. 6D). These results show that TLR4 agonism counteracts leonurine’s suppression of M1 polarization. Immunohistochemical and immunofluorescence analyses revealed that the inhibitory effect of leonurine on the TLR4/NF-κB signaling pathway was suppressed by RS09 (Fig. 7A), with protein expression levels exhibiting a consistent trend (Fig. 7D).

Fig. 6.

Fig. 6

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Reversal of the leonurine-mediated reduction in M1 macrophage proportion by the TLR4 agonist RS09 in EAP mice. (A) Immunofluorescence showing F4/80⁺ macrophage levels in the Control, EAP, EAP + Leo (High), and EAP + Leo (High) + RS09 groups. (B) Real-time PCR analysis of iNOS mRNA levels in the prostate tissues of mice from the Control, EAP, EAP + Leo (High), and EAP + Leo (High) + RS09 groups. (C) Western blot analysis and quantification of iNOS levels in the prostate tissues of mice from each group. (D, E) Flow cytometry analysis and quantitative assessment of M1 macrophage proportions in splenic lymphocytes from the Control, EAP, EAP + Leo (High), and EAP + Leo (High) + RS09 groups

Fig. 7.

Fig. 7

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The TLR4 agonist RS09 reverses the inhibitory effect of leonurine on the TLR4/NF-κB signaling pathway. (A, B) Immunohistochemical analysis of TLR4 and MYD88 levels in the Control, EAP, EAP + Leo (High), and EAP + Leo (High) + RS09 groups. (C) Immunofluorescence analysis of p65 in the Control, EAP, EAP + Leo (High), and EAP + Leo (High) + RS09 groups. (D, G) Representative Western blots and quantitative analysis of TLR4, MYD88, p-p65, and p65 expression across the indicated groups

In vitro, TLR4 Agonists Disrupted the Effect of Leonurine on M1 Polarization in Macrophages

A cellular inflammatory model was established by treating immortalized iBMDMs with 100 ng/ml LPS. RT-qPCR/Western blot analyses revealed marked iNOS upregulation in inflamed cells (Fig. 8A) and concurrent enhancement of M1 macrophage polarization (Fig. 8C). Treatment with leonurine reduced iNOS levels and decreased the proportion of M1 cells, whereas co-administration of the TLR4 agonist RS09 markedly reversed these anti-inflammatory effects (Fig. 8A). Notably, M1-linked cytokine mRNA levels—specifically IL-6, IL-1β, and TNF-α—showed comparable regulatory patterns (Fig. 8E). Subsequent Western blot assessment revealed that leonurine treatment significantly lowered protein levels of TLR4, MYD88, and phosphorylated p65. While RS09 co-treatment effectively reversed this suppression by restoring pathway protein expression (Fig. 8H). Collectively, these findings indicate that leonurine mitigates prostatic inflammation in EAP through inhibition of TLR4/NF-κB pathway activation, thereby limiting M1 macrophage polarization progression (Fig. 9).

Fig. 8.

Fig. 8

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The TLR4 agonist RS09 reactivates the TLR4/NF-κB pathway and counteracts the anti-inflammatory effects of leonurine in vitro. (A) The levels of iNOS mRNA in the Control, LPS, LPS + Leo (20 µM), and LPS + Leo (20 µM) + RS09 groups were detected by Real-time PCR. (B) Western blot analysis and quantification of iNOS levels in the prostate tissues of mice from each group. (C, D) Flow cytometry analysis and quantitative assessment of M1 macrophage proportions in iBMDMs from the Control, LPS, LPS + Leo (20 µM), and LPS + Leo (20 µM) + RS09 groups. (E, G) Real-time PCR analysis of TNF-α, IL-1β, and IL-6 mRNA levels in the iBMDMs of mice from the Control, LPS, LPS + Leo (20 µM), and LPS + Leo (20 µM) + RS09 groups. (H-K) Western blot analysis of TLR4, MYD88, p-p65, and p65 levels in the Control, LPS, LPS + Leo (20 µM), and LPS + Leo (20 µM) + RS09 groups

Fig. 9.

Fig. 9

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Schematic of the proposed mechanism for Leonurine-mediated alleviation EAP. Leonurine suppresses M1 macrophage polarization by inhibiting activation of the TLR4/MyD88/NF‑κB signaling pathway through interaction with its key components. This inhibition reduces the production of pro‑inflammatory cytokines including TNF‑α, IL‑1β, and IL‑6, thereby attenuating tissue inflammation and ameliorating pelvic pain in the EAP model. (By Figdraw)

Discussion

CP/CPPS, a common urological disorder, is categorized as NIH Category III prostatitis. It mainly affects young to middle-aged men and is marked by persistent pelvic or perineal pain, lower urinary tract symptoms, and psychological burden. Symptoms frequently recur or persist for three months or longer, substantially compromising the physical and psychological health of affected individuals [4]. Owing to poorly characterized pathological mechanisms in CP/CPPS, current management approaches emphasize symptomatic relief rather than targeted therapies; given its escalating prevalence and considerable burden, clarifying its complex pathophysiology and developing novel therapeutics are of critical urgency.

Leonurine, a bioactive component isolated from the traditional Chinese herb Leonurus japonicus, possesses broad-spectrum anti-inflammatory properties and is employed in managing diverse inflammatory disorders, though its therapeutic potential for CP/CPPS remains uninvestigated [19, 20, 23]. To bridge this research gap, we developed an EAP mouse model and administered oral leonurine at low (15 mg/kg/day) and high (30 mg/kg/day) doses; leonurine treatment notably attenuated prostatic inflammation and alleviated pelvic pain in these mice. Recent studies have indicated that leonurine modulates macrophage polarization as a therapeutic approach for rheumatoid arthritis [15] and inflammation induced by monosodium urate (MSU) crystals [24]. Given the established role of macrophages in CP/CPPS pathogenesis and progression [18, 25], with M1 macrophages exacerbating pathology via heightened inflammation [26, 27], we explored leonurine’s impact on M1 polarization. Leonurine was observed to dose-dependently decrease splenic M1 macrophage (CD11b⁺F4/80⁺CD86⁺) proportions and suppress the release of M1-associated inflammatory cytokines in EAP mice, as our findings indicated.

The TLR4/NF-κB signaling axis exerts central regulatory functions in macrophage polarization and various inflammatory disorders [28–31]. Activation of TLR4 initiates pro-inflammatory signaling via the MyD88 (myeloid differentiation factor 88)-dependent pathway. As a critical adaptor in TLR4 downstream transduction, MyD88 mediates NF-κB nuclear translocation and the consequent amplification of inflammatory responses [32, 33]. We thus hypothesize that leonurine controls M1 macrophage polarization in EAP mice by inhibiting the TLR4/MyD88/NF-κB signaling pathway. To verify this hypothesis, molecular docking and kinetic simulation experiments were performed, and results showed leonurine could stably bind to components of the TLR4/MyD88/NF-κB pathway. Furthermore, evaluation of proteins associated with this signaling cascade demonstrated that leonurine potently suppressed TLR4/MyD88/NF-κB pathway activation in EAP mice. Consistent outcomes were noted in cell models, with the TLR4 agonist RS09. ultimately utilized in both animal and cell systems to further validate these results. Studies indicate that leonurine’s anti-inflammatory effect on alleviating EAP is reversed by administration of TLR4 agonists, thereby counteracting its inhibitory influence on M1 polarization.

The strategic targeting of the TLR4/NF‑κB signaling axis, a central hub in innate immune activation, presents a dual‑edged therapeutic profile. On one hand, its pleiotropic role in inflammatory cascades suggests that Leonurine may elicit a broad‑spectrum anti‑inflammatory response, which could be particularly beneficial in managing complex, multifactorial conditions such as CP/CPPS where multiple inflammatory pathways are concurrently dysregulated [34]. On the other hand, the ubiquitous nature of this pathway necessitates careful evaluation of potential systemic immune modulation or unintended immunosuppression, especially during prolonged administration [35]. Unlike highly selective synthetic TLR4 antagonists that aim for precise pathway inhibition [36], Leonurine—as a phytochemical—may operate through a mild, multi‑target “network pharmacology” mode, subtly recalibrating immune homeostasis rather than imposing stark suppression. This characteristic, often observed in natural product‑based interventions, might offer a favorable balance between efficacy and safety by dampening excessive inflammation while preserving physiological immune surveillance [37]. Further preclinical toxicology and detailed immune‑profiling studies will be essential to delineate this balance and to guide the rational translation of Leonurine into clinical settings for CP/CPPS.

Collectively, our work is the first to establish that leonurine mitigates prostatitis in EAP mice via TLR4/NF-κB-mediated modulation of M1 macrophage polarization. Although our results emphasize leonurine’s potential in treating CP/CPPS, additional systematic clinical research is needed to assess its safety and efficacy for clinical applications—with the final goal of providing new therapeutic choices for patients with the condition.

Conclusion

This study reveals that Leonurine alleviates EAP pathology through inhibition of M1 macrophage polarization, mediated by TLR4/NF-κB pathway regulation. This finding suggests leonurine may act as a potential therapeutic agent for treating CP/CPPS.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (17.2KB, docx)
Supplementary Material 2 (1.3MB, jpg)
Supplementary Material 3 (1.6MB, jpg)
Supplementary Material 4 (833.5KB, docx)

Author Contributions

CSZ and RJH oversaw study design and all analyses, while RJH, XLY, and CZ drafted the manuscript, WX revised it, and all authors contributed to and approved the final version.

Funding

Funded by the Chinese National Natural Science Foundation (82000720).

Data Availability

Unique, study-specific results are present in the article/supplementary material; the corresponding author should be contacted for further inquiries.

Declarations

Competing Interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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Supplementary Materials

Supplementary Material 1 (17.2KB, docx)
Supplementary Material 2 (1.3MB, jpg)
Supplementary Material 3 (1.6MB, jpg)
Supplementary Material 4 (833.5KB, docx)

Data Availability Statement

Unique, study-specific results are present in the article/supplementary material; the corresponding author should be contacted for further inquiries.

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