7,8-dihydroxyflavone attenuates NIH IIIA subtype chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) via TrkB/AKT/SIRT3 signaling activation

Introduction

Prostatitis is a common urological disease. According to the classification method established by the National Institutes of Health (NIH) in 1995, it can be divided into four types: Type I, acute bacterial prostatitis; Type II, chronic bacterial prostatitis; Type III, chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS); and Type IV, asymptomatic prostatitis (1,2). Among them, NIH IIIA subtype presents as inflammatory CPPS, while NIH IIIB subtype presents as non-inflammatory CPPS. CP/CPPS has the highest incidence rate, accounting for approximately 90–95% of prostatitis patients, and is the most common urological disease among males under the age of 50 years (2). The incidence rate of prostatitis in males reaches 4.5–9%, and the recurrence rate increases up to 50% with age (3). The main symptoms of CP/CPPS include long-term perineal and lower abdominal pain, urinary abnormalities, and neuropsychiatric symptoms (such as anxiety and tension), leading to sexual dysfunction and decreased fertility (4,5). Clinically, CP/CPPS is characterized by a slow onset, stubborn condition, and recurrent episodes, significantly affecting the quality of life of patients (6). Currently, the pharmacological treatment of CP/CPPS mainly includes α-blockers, nonsteroidal anti-inflammatory drugs (NSAIDs), antidepressant/anxiolytic medications (for pain-induced anxiety), and anti-androgen drugs, among others (3). However, the therapeutic effects are limited, and thus, searching for drugs that can improve CP/CPPS inflammation and thereby alleviate clinical symptoms is of significant practical importance.

Inflammation induced by oxidative stress is a causative factor for many chronic diseases. Multiple studies have indicated that the pathogenesis of CP/CPPS is associated with oxidative stress (7-10). Oxidative stress, resulting from an imbalance between the production and elimination of ROS, may lead to chronic inflammation. When the metabolism of ROS in the prostatic inflammatory foci is disordered, it can produce a large amount of ROS and inhibit the activity of antioxidant enzymes, causing lipid peroxidation damage to cellular proteins, DNA, cell membranes, and organelles. This results in pathological changes such as hypoxia, degeneration, edema, congestion, and fibrosis (11) in the prostatic tissue, and leads to inflammatory cell infiltration (12,13), thereby participating in the development of CP/CPPS. Inhibiting the excessive production of ROS can significantly suppress inflammatory symptoms during the occurrence of CP/CPPS (13,14).

7,8-dihydroxyflavone (7,8-DHF), primarily found in foods such as red wine, citrus fruits, grains, tea, vegetables, fruits, and chocolate, is a specific agonist for the tyrosine kinase B (TrkB) receptor. It activates the TrkB receptor, resulting in significant biological effects, including neuroprotection, cardiovascular function regulation, antioxidation, and anti-inflammation (15). Studies have shown that 7,8-DHF can scavenge excessive ROS and inhibit apoptosis induced by hydrogen peroxide (16). Moreover, 7,8-DHF significantly reduces the activation of nuclear factor kappa-B (NF-κB) caused by lipopolysaccharide (LPS), suppresses the phosphorylation of mitogen-activated protein kinases (MAPKs), and subsequently decreases the secretion of inflammatory mediators nitric oxide (NO), prostaglandin E2 (PGE2), and interleukin-1β (IL-1β) in the LPS-induced RAW264.7 cell line, suggesting a pronounced anti-inflammatory effect of 7,8-DHF (17). Additionally, research has indicated that quercetin can inhibit NF-κB and MAPK signaling to improve rat prostatitis induced by complete Freund’s adjuvant (CFA) (18). Both 7,8-DHF and quercetin belong to the flavonoid class of compounds and can target HSPB1, NRF2, and TrkB to reduce tau aggregation and exert a cytoprotective effect (19). In summary, given that the occurrence of chronic prostatitis is associated with excessive ROS production and that 7,8-DHF possesses antioxidative and anti-inflammatory properties, it raises the question of whether 7,8-DHF could ameliorate CP/CPPS. Currently, this remains unclear. Therefore, this study aims to investigate the effects of 7,8-DHF on CP/CPPS using a rat model of CP/CPPS and to explore its underlying mechanisms. We present this article in accordance with the ARRIVE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-517/rc).

Methods

Materials

7,8-DHF, ANA-12, MK2206, and 3-TYP were procured from GLPBIO. CFA was acquired from Med Chem Express (MCE). Catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) assay kits were purchased from Beyotime Biotechnology Co., Ltd. The SIRT3 antibody was sourced from Abcam. Antibodies against CD3⁺, CD45⁺, P-AKT, and AKT were obtained from Proteintech Group, Inc. (Wuhan, China). Hematoxylin-eosin (HE) stain kit was purchased from Genbiotech (Shanghai, China). Masson staining solution was sourced from Solarbio. Polymerase chain reaction (PCR) primers were obtained from Genepharma. Co., Ltd. (Shanghai, China). All animals were sourced from Charles River. RWPE-1 cells were provided by Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China).

Construction of rat prostatitis model

All male Sprague Dawley rats (200–230 g, Purchased from Charles River) were randomly divided into three groups: Sham, CP/CPPS, and 7,8-DHF. Rats were anesthetized intraperitoneally with sodium pentobarbital (45 mg/kg), followed by shaving the abdominal hair and disinfecting the skin. A longitudinal incision of approximately 1 cm was made in the midline of the lower abdomen. The location of the bladder was identified, and the two lobes of the prostate gland located below the bladder were found and fully exposed on both sides. A 0.1 mL injection of CFA was administered into the rat prostate (18) tissue, and the abdominal incision was closed in layers. All rats had their wounds disinfected and were returned to their cages for normal feeding. All cages were maintained under identical specific pathogen-free (SPF) environmental conditions. If no leakage of CFA injection is observed at all administered doses and the animal survives for 4 weeks post-suturing, it will be included in subsequent experimental procedures. The 7,8-DHF group and the CP/CPPS model group were intraperitoneally injected with 7,8-DHF (5 mg/kg/d) and corresponding volume solvent control, respectively, for four consecutive weeks, while the Sham group had the abdominal incision closed directly after exposure of the prostate lobes (a total of 12 rats per group completed the experimental protocol without any attrition, and all specimens were included in the statistical analysis). Experiments were performed under a project license (No. JUMC2023-152) granted by the Experimental Animal Ethics Committee of the Medical College of Jiaxing University, in compliance with regulations on laboratory animal management at Jiaxing University for the care and use of animals. A protocol was prepared before the study without registration. Only the experimenters who anesthetized the animals knew the groupings of the animals during the sampling process. The other experimenters who collected the samples were blinded to ensure that human factors would not influence the results.

Determination of prostate index

The body weight of the rats was measured. Subsequently, the prostate tissue of the rats was extracted after anesthetizing the rats, excess tissue was removed, and the surface moisture was absorbed. The weight of the prostate was then measured. The prostate index = the weight of the prostate /the body weight of the rat.

Evaluation of chronic pelvic pain

Prior to CP/CPPS modeling (0 week) and at 2 and 4 weeks post-modeling, hyperalgesia was measured. Experimental rats were placed in a quiet room one day before measurement to prevent disturbance from external events. All procedures were conducted within a separate plastic chamber featuring a stainless-steel mesh floor. Referred hyperalgesia were evaluated and quantified using von Frey filaments. A 6 g force was applied to either the pubic region or the scrotal base. This application was repeated 10 times, with each application lasting 1 to 2 seconds and separated by a 2-minute interval. A positive response was defined as immediate licking or scratching of the stimulated site, rapid abdominal contraction, or jumping (20).

Ultrasound examination of rat prostate

Following anesthesia, depilatory cream was applied to the abdominal region of rats for hair removal. Using the VINNO small animal ultrasound imaging system, the bladder was initially identified. Subsequently, the morphological features and ultrasound echogenicity of the peri-bladder prostatic tissues were evaluated, with photographic documentation.

Measurement of water intake and urine output in metabolic cages

Rats were acclimated to metabolic cages for 24 hours. The volume of drinking water was measured before and after 12- and 24-hour periods to calculate water intake. Urine output was simultaneously measured.

Measurement of bladder pressure

After anesthetizing the rats, the bladders were exposed. A three-way cannula was connected to the bladder, an infusion pump, and the pressure measurement channel of the BL-420 Biological Function Experiment System. A microinfusion pump employed a 50-mL syringe as the infusion reservoir. The BL-420 system interface was activated, with the designated channel configured for pressure sensing. Pressure detection was initiated. The infusion pump was started to purge air from the T-tube tubing. Physiological saline at 18 ℃ was infused into the bladder at 1 mL/min via the pump. The bladder pressure curve was recorded using the BL-420 system. The bladder pressure at the occurrence of urine leakage was recorded as the bladder leak point pressure (BLPP). Bladder capacity was simultaneously calculated based on infusion time.

Prostate tissue section staining

Prostate tissues were fixed in 4% paraformaldehyde for 24 hours and embedded in paraffin. Sections (5 µm thick) were stained with HE for morphological examination. Masson’s trichrome staining was performed following the respective kit protocols, including dewaxing, staining, dehydration, and mounting. Finally, sections were observed and photographed under an optical microscope (21).

For immunohistochemical staining, paraffin sections from each group were baked in an oven until dry. Routine dewaxing was performed using xylene, followed by graded alcohol hydration and rehydration. Antigen retrieval was conducted using citrate-based antigen retrieval buffer. Endogenous peroxidase activity was blocked by incubating with 3% H₂O₂ for 10 min at room temperature. Sections were blocked with normal goat serum for 10 min. Primary antibodies (CD3⁺ antibody diluted 1:50, CD45⁺ antibody diluted 1:200) were applied to the slides and incubated overnight at 4 ℃. Subsequently, sections were treated with a polymer enhancer for 20 min and enzyme-labeled anti-mouse/rabbit polymer for 10 min. DAB was used for color development, with reaction time controlled under microscopy. Sections were counterstained with hematoxylin solution, dehydrated, cleared, and mounted with resin. Stained sections were observed and photographed under a microscope. Semi-quantitative analysis was performed using integrated optical density (IOD) analysis.

Detection of inflammatory factor mRNA levels

Total RNA was extracted using a total RNA Kit. Based on the measured RNA concentration, reverse transcription was performed using a reverse transcription PCR (RT-PCR) Kit. Quantitative PCR (qPCR) was conducted using an RT-PCR Kit and the Light Cycler 480 Real-Time PCR System to analyze the expression of pro-inflammatory mediators [monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin 17α (IL-17α), interleukin 1α (IL-1α)]. The CT value correlates with the initial DNA copy number. The CT values for gene amplification were measured using the double CT method, and the relative expression of target genes was calculated.

Western blot

Appropriate amounts of prostate tissue were homogenized. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis, which was terminated once the target bands reached the appropriate position. Gel regions corresponding to the target protein molecular weight were excised and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was immersed from bottom to top in Tris-buffered saline (TBS), then blocked with blocking buffer (5% skim milk in TBST). After washing with TBST to remove residual solution, the membrane was incubated with the primary antibody overnight at 4 ℃. The following day, the membrane was washed three times with TBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse immunoglobulin G (IgG) secondary antibody for 1 hour at room temperature. After three additional TBST washes, protein expression was visualized using enhanced chemiluminescence (ECL). Finally, detection was performed on an Amersham Imager 680 chemiluminescence imaging workstation, and band optical density was analyzed using Image Pro Plus 6.0 software.

Statistical analysis

The outcomes of the data are presented as mean ± standard error of the mean (SEM), and statistical analysis was conducted using Sigma Plot 12.5 software, whereas statistical graphs were created using GraphPad Prism 8 software. For comparisons between two groups, we used an unpaired Student’s t-test for normally distributed data. For comparisons among more than two groups, one-way analysis of variance (ANOVA) was used to test overall differences, followed by either Tukey’s HSD post-hoc test. Randomized block ANOVA was implemented when the control group was 1 without standard error (22). A P value less than 0.05 was considered statistically significant after adjusting for multiple comparisons.

Results

Effects of 7,8-DHF on prostate index and morphology in CP/CPPS rats

A rat model of CP/CPPS was established by injecting CFA into the rat prostate tissue. Following 4 weeks of intervention with 7,8-DHF, its effects were evaluated. Figure 1A demonstrated no difference in body weight among the three groups, which proved the initial safety of 7,8-DHF. However, the prostate index was significantly higher in the CP/CPPS group compared to the Sham group, confirming successful model establishment. Treatment with 7,8-DHF significantly reduced the prostate index in CP/CPPS rats (Figure 1B). Hyperalgesia testing result demonstrated that 7,8-DHF significantly alleviated lower abdominal pain induced by CP/CPPS in rats, an effect confirmed at both week 2 and week 4 (Figure 1C). Ultrasonography revealed large areas of heterogeneous echogenicity in the prostate tissue of the CP/CPPS group compared to the Sham group, suggesting substantial inflammation. This heterogeneous echogenicity was ameliorated by 7,8-DHF intervention (Figure 1D).

Figure 1 Effects of 7,8-DHF intervention on CFA-induced CP/CPPS in rats. (A) Daily changes in rat body weight. (B) Effect of 7,8-DHF intervention on the prostate index in CP/CPPS rats. (C) Effect of 7,8-DHF intervention on the hyperalgesia test in CP/CPPS rats. (D) Effect of 7,8-DHF intervention on prostate ultrasonography in CP/CPPS rats. The red boxed area indicates the prostate, and the white boxed area indicates the bladder. The arrow indicates region of heterogeneous echogenicity. The prostate index = the weight of the prostate / the body weight of the rat. 7,8-DHF, 7,8-dihydroxyflavone; CFA, complete Freund’s adjuvant; CP/CPPS, chronic prostatitis/chronic pelvic pain syndrome.

Effects of 7,8-DHF on prostate function in CP/CPPS rats

Prostatitis patients experiencing urinary frequency often intentionally restrict fluid intake before bedtime to minimize bladder distension during sleep and reduce nocturia episodes (23). Metabolic cage analysis demonstrated that CP/CPPS rats exhibited significantly lower 12- and 24-hour water intake compared to Sham rats. 7,8-DHF treatment increased water intake and consequently increased urine output in CP/CPPS rats (Figure 2A-2D). Furthermore, BLPP and functional bladder capacity were significantly elevated in CP/CPPS rats compared to Sham. 7,8-DHF treatment reduced both BLPP and functional bladder capacity in CP/CPPS group (Figure 2E-2G). Collectively, these results indicate that 7,8-DHF alleviates CFA-induced increases in bladder volume and prostate dysfunction.

Figure 2 Effects of 7,8-DHF intervention on water intake, urine output, and prostate function in CFA-induced CP/CPPS model rats. (A,B) Statistical graphs depicting 12- and 24-h water intake for each group of rats. (C,D) Statistical graphs depicting 12- and 24-h urine output for each group of rats. (E-G) Changes in bladder leak point pressure and functional bladder capacity across groups, along with their corresponding statistical graphs. 7,8-DHF, 7,8-dihydroxyflavone; BLPP, bladder leak point pressure; CFA, complete Freund’s adjuvant; CP/CPPS, chronic prostatitis/chronic pelvic pain syndrome.

Effects of 7,8-DHF on histopathological changes in CP/CPPS rats

To further assess the ameliorative effects of 7,8-DHF on CP/CPPS, rat prostate tissues were subjected to H&E, Masson and toluidine blue staining. H&E staining revealed varying degrees of inflammatory cell infiltration, inflammatory vacuoles, irregular acini, and edema in the prostatic interstitium of CP/CPPS rats, accompanied by significant epithelial cell hyperplasia. 7,8-DHF treatment mitigated inflammatory cell infiltration and epithelial hyperplasia (top row of Figure 3A,3B). Additionally, 7,8-DHF reduced CP/CPPS-induced fibrosis distribution (middle row of Figure 3A,3C). Increased macrophage infiltration in the prostate tissue of CP/CPPS rats was also suppressed by 7,8-DHF (bottom row of Figure 3A,3D).

Figure 3 Effects of 7,8-DHF intervention on immune infiltration in prostate tissues of CFA-induced CP/CPPS rats. (A-D) Representative histological sections stained with HE (40×), Masson (100×), and toluidine blue (200×) from each group, accompanied by corresponding quantitative analyses. The red arrows indicate the mast cells. 7,8-DHF, 7,8-dihydroxyflavone; CFA, complete Freund’s adjuvant; CP/CPPS, chronic prostatitis/chronic pelvic pain syndrome; HE, hematoxylin-eosin.

Effects of 7,8-DHF on prostatic inflammatory levels in CP/CPPS rats

Subsequently, the levels of inflammatory factors in prostate tissue were measured. qPCR analysis showed significantly elevated mRNA levels of MCP-1, TNF-α, IL-6, IL-17α, and IL-1α in the prostate tissue of CP/CPPS rats. The upregulation of these inflammatory cytokines was reduced by 7,8-DHF intervention (Figure 4A-4E). CD3⁺ is a co-receptor on T cells, and CD45⁺ is widely expressed on leukocytes. Immunohistochemistry demonstrated upregulated expression of CD3⁺ and CD45⁺ in the prostate tissue of CP/CPPS rats, which was also ameliorated by 7,8-DHF treatment (Figure 4F-4I).

Figure 4 Effect of 7,8-DHF intervention on inflammatory markers in prostate tissue of CFA-induced CP/CPPS rats. (A-E) Effect of 7,8-DHF intervention on the mRNA levels of inflammatory cytokines (MCP1, TNF-α, IL-6, IL-17α, IL-1α) in rat prostate tissue. (F,G) Effect of 7,8-DHF intervention on CD3⁺ expression in rat prostate tissue by immunohistochemistry (200×) and its quantitative analysis. (H,I) Effect of 7,8-DHF intervention on CD45⁺ expression in rat prostate tissue by immunohistochemistry (200×) and its quantitative analysis. 7,8-DHF, 7,8-dihydroxyflavone; CFA, complete Freund’s adjuvant; CP/CPPS, chronic prostatitis/chronic pelvic pain syndrome; IL-17α, interleukin 17α; IL-1α, interleukin 1α; IL-6, interleukin-6; IOD, integrated optical density; MCP1, monocyte chemoattractant protein-1; TNF-α, tumor necrosis factor-α.

Effects of 7,8-DHF on oxidative stress levels in plasma and prostate tissue of CP/CPPS rats

A feedback regulatory mechanism exists between oxidative stress and inflammation. Oxidative stress can activate inflammation-related transcription factors, promoting the expression of inflammatory cytokines, and is closely linked to prostatitis. We measured the levels of oxidative stress markers—CAT, MDA, and SOD—in plasma and prostate tissue. CP/CPPS rats exhibited significantly decreased CAT and SOD levels alongside increased MDA levels in both plasma and prostate tissue, indicating upregulated oxidative stress. Treatment with 7,8-DHF increased CAT and SOD levels and reduced MDA levels in CP/CPPS rats (Figure 5A-5F). These findings suggest that 7,8-DHF ameliorates CP/CPPS in rats, potentially through its antioxidant effects.

Figure 5 Effect of 7,8-DHF intervention on oxidative stress in plasma and prostate tissue of CFA-induced CP/CPPS rats. (A-C) Effect of 7,8-DHF intervention on plasma levels of CAT, MDA, and SOD in rats from each group. (D-F) Effect of 7,8-DHF intervention on prostate tissue levels of CAT, MDA, and SOD in rats from each group. 7,8-DHF, 7,8-dihydroxyflavone; CAT, catalase; CP/CPPS, chronic prostatitis/chronic pelvic pain syndrome; MDA, malondialdehyde; SOD, superoxide dismutase.

Involvement of the TrkB/AKT/SIRT3 pathway in the protective effects of 7,8-DHF against CP/CPPS

7,8-DHF is an agonist of TrkB receptor, of which its activation stimulates the downstream PI3K/AKT signaling pathway. Western blot analysis confirmed that 7,8-DHF significantly upregulated AKT activation (Figure 6A,6B). SIRT3, a member of the mammalian sirtuin family, exhibits NAD⁺-dependent deacetylase activity and can modulate ROS production and clearance to alleviate mitochondrial oxidative stress. Downregulated SIRT3 expression in the prostate tissue of CP/CPPS rats aligned with the observed increase in oxidative stress. Studies have shown that melatonin protects against focal cerebral ischemia-reperfusion injury in diabetic mice by activating the Akt-SIRT3 pathway to improve mitochondrial dysfunction (24). Other studies also report mutual regulation between SIRT3 and AKT (25-27). In this study, 7,8-DHF activated AKT while concurrently significantly upregulating SIRT3 expression (Figure 6A-6C). Therefore, 7,8-DHF likely ameliorates CP/CPPS in rats through the TrkB/AKT/SIRT3 pathway.

Figure 6 Effect of 7,8-DHF intervention on AKT activity and SIRT3 protein expression in prostate tissue of CFA-induced CP/CPPS rats. (A-C) Effect of 7,8-DHF intervention on the protein expression of P-AKT, AKT, and SIRT3 in rat prostate tissue and its quantitative analysis. 7,8-DHF, 7,8-dihydroxyflavone; CFA, complete Freund’s adjuvant; CP/CPPS, chronic prostatitis/chronic pelvic pain syndrome.

Furthermore, an in vitro prostatitis model was established using lipopolysaccharide (LPS)-stimulated normal human prostatic epithelial RWPE-1 cells. Results showed that LPS induced oxidative stress damage in RWPE-1 cells. 0.5 µM 7,8-DHF attenuated LPS-induced cellular damage. However, the protective effects of 7,8-DHF were abolished by the TrkB inhibitor ANA-12 (10 nM), the AKT inhibitor MK2206 (1 µM), and the SIRT3 activity inhibitor 3-TYP (20 nM) (Figure 7A). ELISA results further demonstrated that LPS significantly promoted the release of the inflammatory cytokine IL-6 from RWPE-1 cells. This effect was reversed by 0.5 µM 7,8-DHF. Co-treatment with ANA-12 (10 nM), MK2206 (1 µM), or 3-TYP (20 nM) counteracted the suppressive effect of 7,8-DHF on IL-6 release (Figure 7B). These findings indicate that 7,8-DHF enhances antioxidant activity and consequently reduces inflammatory cytokine release via the TrkB/AKT/SIRT3 pathway.

Figure 7 Effect of inhibiting TrkB, AKT activity, and SIRT3 activity on the protective role of 7,8-DHF against LPS-induced injury in prostate epithelial cells. (A) Effect of the TrkB antagonist ANA-12, the AKT inhibitor MK2206, and the SIRT3 inhibitor 3-TYP on the protective effect of 7,8-DHF against LPS-induced RWPE-1 cell injury, as determined by CCK-8 assay. (B) Effect of the TrkB antagonist ANA-12, the AKT inhibitor MK2206, and the SIRT3 inhibitor 3-TYP on the reduction of IL-6 levels by 7,8-DHF in LPS-stimulated RWPE-1 cells, as determined by ELISA. 7,8-DHF, 7,8-dihydroxyflavone; CCK-8, Cell Counting Kit-8; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; IL-6, interleukin-6.

Discussion

This study confirms that 7,8-DHF effectively ameliorates prostatic function and tissue damage caused by CP/CPPS by significantly suppressing inflammatory infiltration in prostatic tissues, alleviating pathological hyperplasia of prostatic epithelial and stromal cells, and mitigating fibrotic alterations. Notably, 7,8-DHF markedly inhibits oxidative stress injury, consequently suppressing multiple pro-inflammatory cytokines, providing critical experimental evidence for its therapeutic efficacy in alleviating the pathological manifestations of prostatitis.

Simultaneously, experimental findings demonstrated that the TrkB/AKT/SIRT3 signaling pathway may represent a potential mechanism underlying the protective effects of 7,8-DHF. Our research observed that 7,8-DHF activates the TrkB receptor, promotes phosphorylation of downstream AKT, and concurrently upregulates SIRT3 expression. These alterations in the signaling cascade suggest its potential involvement in the 7,8-DHF-mediated intervention in CP/CPPS.

Research has reported that AKT can regulate the activity and function of SIRT3 through phosphorylation-mediated mechanisms (25-27). Further in vitro experiments, employing specific inhibitors (the TrkB inhibitor ANA-12, the AKT inhibitor MK2206, or the SIRT3 inhibitor 3-TYP), validated this relationship. The results demonstrated that blocking any component within this pathway attenuated the therapeutic effects of 7,8-DHF. These findings support the hypothesis that the TrkB/AKT/SIRT3 signaling pathway potentially plays a significant role in mediating the effects of 7,8-DHF.

Previous studies have demonstrated that 7,8-DHF exerts neuroprotective (28-31), retinoprotective (32,33), and cardioprotective effects (34,35) through activation of the TrkB receptor and its downstream signaling pathways. This study, however, reveals its significant therapeutic efficacy in a CP/CPPS model, thereby expanding its potential clinical applications. This finding not only validates the broad-spectrum anti-inflammatory and antioxidant activities of 7,8-DHF, but more importantly, highlights its novel therapeutic application in genitourinary diseases, particularly for the treatment of CP/CPPS. This research provides a new therapeutic direction for this established compound.

Deeply, the findings of this study closely align with the classical theory in the field of prostatitis that “oxidative stress amplifies inflammation”. Existing literature has explicitly reported that excessive accumulation of ROS in prostate tissue can directly activate inflammatory pathways such as NF-κB, promoting the release of pro-inflammatory factors including TNF-α and IL-6 (9,36,37). This subsequently recruits immune cell infiltration and exacerbates tissue oxidative damage, forming a vicious cycle. This study demonstrates that 7,8-DHF can simultaneously and significantly reduce both ROS levels and the expression of key pro-inflammatory factors in prostate tissue. This dual approach confirms that 7,8-DHF disrupts the “oxidative stress-inflammation” vicious cycle by directly scavenging free radicals, enhancing antioxidant enzyme activity to alleviate oxidative stress, and suppressing the production of downstream inflammatory factors. This provides a solid theoretical foundation for explaining its core protective mechanism in prostatitis.

Activation of TrkB triggers downstream AKT signaling (38), and the activated AKT further modulates the expression of SIRT3 and its downstream targets (39-42), thereby influencing oxidative stress-related markers. This study proposes that targeting the TrkB/AKT/SIRT3 signaling pathway is involved in the treatment of CP/CPPS with 7,8-DHF, particularly chronic or refractory forms, offering a new direction for the clinical management of this disease. Compared to the limitations of conventional therapeutic approaches such as antibiotics or NSAIDs, 7,8-DHF exhibits unique dual anti-inflammatory and antioxidant properties (17,43) by simultaneously activating the TrkB-dependent AKT/SIRT3 pathway. This mechanism not only synergistically suppresses inflammatory responses and oxidative stress damage but may also provide more comprehensive tissue protection by restoring mitochondrial function, highlighting its potential as an optimized therapeutic agent for CP/CPPS.

Nevertheless, the occurrence of CP/CPPS is closely associated with oxidative stress (9,44). This study investigated the involvement of the TrkB/AKT/SIRT3 signaling pathway in alleviating oxidative stress and consequently suppressing CP/CPPS symptoms. However, other oxidative stress-related signaling pathways—such as the ROS-associated Nrf2 (45), FOXO (46), and MAPK (47) pathways—were not thoroughly explored. These will be further examined in our subsequent research.

There are several limitations in this study. CP/CPPS includes two subtypes: inflammatory (Type III A) and non-inflammatory (Type III B). In this study, a CFA-induced prostatitis model was adopted, which indeed better conforms to the pathological characteristics of CP/CPPS of Type III A, with clear histological evidence of inflammation (such as elevated prostatic index and changes in inflammatory factor levels). We acknowledge that this model cannot fully simulate the non-inflammatory pathological process of Type III B patients, which is a limitation of this study. These experiments did not evaluate the safety profile of long-term 7,8-dihydroflavonol administration, and its potential chronic toxicity remains unclear. However, based on previous studies, 7,8-DHF demonstrates a favorable safety profile as a potential clinical therapeutic agent (48), though its effects on the prostate warrant further investigation. Finally, the critical downstream effector molecular network of SIRT3 (e.g., the MnSOD/FOXO3a/PGC-1α pathway) (49-51) has not been fully elucidated, and the specific mechanisms by which it mediates protective effects warrant further in-depth investigation.

This research is the first to reveal the core mechanism by which 7,8-DHF ameliorates inflammatory responses and oxidative damage in CP/CPPS through activation of the TrkB/AKT/SIRT3 signaling pathway. This discovery not only clarifies a key molecular pathway of action but also provides a significant theoretical foundation and experimental basis for developing targeted prostatitis therapeutics based on this pathway.

Conclusions

This study demonstrates at both animal and cellular levels that 7,8-DHF effectively attenuates NIH IIIA subtype CP/CPPS, and reveals the involvement of the TrkB/AKT/SIRT3 signaling pathway in mediating the therapeutic effects of 7,8-DHF on CP/CPPS. By concurrently mitigating oxidative stress and suppressing inflammatory responses, 7,8-DHF disrupts the “oxidative stress-inflammation” vicious cycle in prostate tissue. This investigation provides a novel therapeutic strategy for CP/CPPS.

Acknowledgments

We would like to thank Yulu Qiao for the spiritual support during our work.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-517/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-517/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-517/prf

Funding: This study was supported by Zhejiang Provincial Natural Science Foundation of China (No. LTGD24H050001 to J.G.; No. LMS25H050003 to Y.H.), Jiaxing Public Welfare Science and Technology Program Fund (No. 2023AY40023), the Qin Shen Scholar Program of Jiaxing University (No. CD70623040), Jiaxing Medical Key Subject Funding of Zhejiang Province (No. 2023-ZC-013), Jiaxing Key Laboratory of Precise Diagnosis and Treatment of Urological Tumor (2020-mnzdsys), Jiaxing Municipal Special Project for Technological Industry Development Breakthrough (No. 2025AC014) and Research Training Program (SRT) of Jiaxing University (No. 8517241030).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-517/coif). Y.H. reported receiving funding by Zhejiang Provincial Natural Science Foundation of China (No. LMS25H050003). J.G. reported receiving funding by Zhejiang Provincial Natural Science Foundation of China (No. LTGD24H050001). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. JUMC2023-152) granted by the Experimental Animal Ethics Committee of the Medical College of Jiaxing University, in compliance with regulations on laboratory animal management at Jiaxing University for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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