N-acetylcysteine alleviates experimental autoimmune prostatitis in rats by activating PI3K/Akt/CREB and Keap1/Nrf2 pathways

Introduction

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a prevalent male genitourinary disorder affecting approximately 8.2% of the global male population and constituting 90% of all prostatitis cases (1,2). This condition demonstrates age-dependent prevalence, with peak occurrence observed in young to middle-aged men (2,3). Clinically, CP/CPPS manifests as urogenital pain involving perineal, testicular, penile, suprapubic, inguinal, and lumbosacral regions, frequently accompanied by lower urinary tract symptoms, sexual dysfunction, and psychosocial comorbidities (3). However, although a significant number of patients suffer from CP/CPPS, the precise etiology, diagnostic criteria, and therapeutic strategies for CP/CPPS remain incompletely defined. Current evidence suggests that the occurrence of CP/CPPS is involved in multiple mechanisms including infection, chronic inflammation, autoimmunity, pelvic floor dysfunction, oxidative stress, and neurological dysregulation (4). Therefore, the UPOINT phenotypic classification system [encompassing urinary symptoms (U), psychosocial dysfunction (P), organ-specific symptoms (O), infection-related symptoms (I), neurological/systemic conditions (N), tenderness of skeletal muscles (T)] has been established as a clinical framework for diagnosis and management of CP/CPPS (5). However, conventional therapeutic modalities including antibiotics, non-steroidal anti-inflammatory drugs, α-adrenergic receptor antagonists, pelvic floor rehabilitation, extracorporeal shock wave treatment and Chinese herb remedy demonstrate limited efficacy (6-9). This therapeutic gap underscores the urgent need for enhanced mechanistic understanding and development of novel treatment strategy for CP/CPPS.

N-acetylcysteine (NAC) is a well-established antioxidant that modulates oxidative balance through sulfhydryl group donation and glutathione (GSH) synthesis (10). Its therapeutic efficacy has been validated across multiple pathological conditions including cardiomyocyte dysfunction (11), erectile dysfunction (12), and acute lung injury (13), demonstrating robust antioxidant, anti-fibrotic, and anti-inflammatory properties. Mechanistically, NAC enhances antioxidant defenses by promoting GSH peroxidase and superoxide dismutase (SOD) activity (4), while simultaneously reducing inflammatory mediators such as TNF-α, IL-6 and cyclooxygenase 2 to mitigate inflammatory pain (13,14). Furthermore, it attenuates TGF-β signaling activation triggered by inflammation and/or oxidative stress, thereby preventing fibrosis in pulmonary and penile tissues (12,15). Given the multifactorial nature of CP/CPPS and NAC’s pleiotropic effects, this study hypothesizes that NAC may represent a promising therapeutic strategy for CP/CPPS management.

Several studies have identified oxidative stress and NLRP3 inflammasome activation as key pathological drivers in autoimmune prostatitis. Recent pharmacological studies demonstrate that compounds such as isoliquiritigenin, sodium butyrate, 4-octyl itaconate, and resolvin D1 effectively alleviate experimental autoimmune prostatitis (EAP) through activating nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway, which simultaneously suppresses oxidative stress and NLRP3 inflammasome activity (2,16-18). Notably, NAC has been extensively validated as a potent modulator of the Nrf2/HO-1 axis, demonstrating therapeutic efficacy in diverse pathological conditions such as diabetes-related skeletal muscle atrophy (19), carbon tetrachloride-induced hepatic injure (20), renal ischemia-reperfusion damage (21), and busulfan-associated male reproductive dysfunction (22). This collective evidence strongly supports the hypothesis that NAC may represent a viable therapeutic strategy for CP/CPPS, particularly in its autoimmune EAP manifestation.

To validate the precise therapeutic effects of NAC on CP/CPPS, we established an EAP rat model following established protocols (6). Considering that pain represents a hallmark symptom of CP/CPPS, we specifically monitored mechanical allodynia in NAC-treated EAP rats through intragastric administration. Our findings demonstrated that NAC significantly attenuated pain responses in EAP rats through suppression of inflammatory cytokines and oxidative makers. Further study suggests that NAC may ameliorate the prostatic microenvironment in EAP rats via modulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/cAMP-response element binding protein (CREB) and Nrf2/HO-1 signaling pathways. These results collectively indicate NAC’s potential as a therapeutic agent for CP/CPPS through its multifaceted regulatory effects on pain, inflammation, and oxidative stress. We present this article in accordance with the ARRIVE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0022/rc).

Methods

Animals and experimental design

In this study, all animal experiments were performed under a project license (No. 20211293A) granted by the Animal Care and Animal Ethics Committee of West China Hospital, Sichuan University, in compliance with institutional guidelines for the care and use of animals. A total of thirty-four 10-week-old male Sprague-Dawley rats (weighing 280–330 g) were purchased from Chengdu Dossy Experimental Animals, Co., Ltd. (Chengdu, China) and housed in the Animal Center of West China Hospital, Sichuan University under pathogen-free conditions with free access to water and food, and a 12-hour light/dark cycle. After three days of adaptation, the experiment commenced.

According to previous studies (17,18,23), ten rats were used to prepare autologous prostate tissue homogenate supernatant (PTHS), and the remaining 24 rats were randomly divided into three groups (n=8 rat per group) using a random number table: control (CON), EAP and EAP + NAC. Rats in the EAP and EAP + NAC groups were induced as described previously (6,23). Briefly, under aseptic conditions and anesthesia with 1.0–1.5% isoflurane, each rat received an intraprostatic injection of 1.0 mL of an isovolumetric mixture of PTHS (20 mg/mL) and Freund’s complete adjuvant (Beyotime, P2036, Shanghai, China). In contrast, rats in the CON group were injected with the same volume of saline. To simulate a therapeutic rather than a prophylactic intervention, seven days after model induction, rats in the EAP + NAC group were administered 500 mg/kg NAC (Hainan Zambon Pharmaceutical Co., Ltd., Haikou, China) daily by gavage, while the CON group received an equal volume of saline instead.

Von Frey filament in mechanical allodynia

The Von Frey test is a widely used method for assessing mechanical allodynia in rodents. In this study, Aesthesio von Frey filaments (IITC Life Science Inc., Woodland Hills, CA, USA) with varying forces (0.4, 1.0, 2.0, 4.0, 6.0 g) were used to evaluate mechanical allodynia and hyperalgesia before and after NAC treatment. Before formal testing, all rats underwent a three-day habituation period. Briefly, according to previously established protocols (24), each rat was placed in a customized cage with a mesh bottom. Once the rat was acclimated and remained calm, the Von Frey monofilament was applied perpendicularly to plantar surface of the hind paw and urogenital regions until it bent. Each force was applied 5 times per rat, with a 5-second interval between stimulations. A positive response was recorded as a brisk paw withdrawal, licking, or shaking of the paw. The average percentage of positive response for each force and each group was subsequently analyzed. Data are presented as the mean response frequency percentage ± standard error of the mean (SEM). Notably, to ensure the accuracy and objectivity of the behavioral data, all Von Frey tests were conducted by a single, blinded researcher, who received standardized operational training, with all assessments performed at a fixed time period each day (between 9:00 a.m. and 12:00 p.m.) to control for potential circadian influences. In addition, environmental conditions (including temperature, humidity, and lighting) were also kept as consistent as possible across each experimental session.

Histological studies

Following behavioral testing, rats were euthanized via isoflurane overdose for the collection of prostate tissues. The retrieved prostate tissues (each group contained eight individuals) were rinsed using phosphate buffered solution and stabilized in 10% neutral paraformaldehyde overnight. Subsequently, they were delivered to the pathological laboratory of West China Hospital (Sichuan University) for standard processing, such as dehydration, embedding, and sectioning. Finally, 5-µm prostate sections were obtained for further histological studies.

Hematoxylin and eosin (H&E)

After dewaxing and stepwise dehydration, serial prostate sections were stained with H&E. Then, the pathophysiological characteristics and morphology of prostate tissue in each group were observed under the Zeiss Ax10 Imager (ZEISS, Oberkochen, Germany). In accordance with previously described methods (17,18), the severity of inflammation was graded on a four-point scale (0–3). A score of 0 indicated no inflammation; 1, mild, but definite perivascular cuffing with mononuclear cells; 2, moderate perivascular cuffing with mononuclear cells; and 3, marked perivascular cuffing accompanied by hemorrhage and numerous mononuclear cells within the parenchyma. The average score from multiple fields per slide was used for analysis.

Immunohistochemistry and immunofluorescence assays

Immunohistochemistry and immunofluorescence were performed as follows. After dewaxing and hydration, paraffin sections were subjected to antigen retrieval by heating in ethylene diamine tetraacetic acid (EDTA) buffer (pH 8.0) at 98 ℃ for 20 minutes. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide, and nonspecific sites were blocked with 10% normal horse serum. The sections were then incubated with primary antibodies overnight at 4 ℃, and the relevant antibody information is shown in Table 1. For negative controls, the primary antibody was replaced with normal serum from the host species. Following this, the sections were incubated with secondary antibodies at 37 ℃ for 1 h. For immunohistochemical staining, sections were reacted with 3,3'-diaminobenzidine (DAB; ZLI-9018, Beijing ZSbio) to show the brown positive signal, and counterstained with hematoxylin to visualize cell nuclei. For immunofluorescence staining, nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Beyotime, China) for 5 min in the dark. Finally, all the sections were captured with the Zeiss Ax10 Imager at ×200, ×400 magnifications. The percentage of positive area was quantified using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA) on three sections per rat (n=8/per group) by applying a uniform threshold for hue, saturation, and intensity across all images. All histological and immunohistochemical evaluations were independently assessed by two experienced experimenters who were completely blinded to the group information.

Western blot analysis

Prostate tissues were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer (Meilunbio, MA0151, Dalian, China) supplemented with protease and phosphatase inhibitor cocktail I (MedChemExpress, Shanghai, China). Protein concentrations were measured using Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific Inc., 23227, Waltham, MA, USA). After calculating and adjusting the concentration, equal amounts of protein (40 µg per lane) were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; BAIHE Science & Technology, Chengdu, China) and transferred onto polyvinylidene fluoride membranes (Millipore Corporation, Belford, MA, USA). The membranes were blocked with 5% skimmed milk to prevent nonspecific binding, and then incubated at 4 ℃ overnight with primary antibodies (detailed in Table 1). The next day, the membranes were reacted with secondary antibodies. Protein signals were visualized using immobilon Western Chemilumine Substrate (WBKLS0500, Millipore), and images were captured with the ChemiDoc MP imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and relative quantification of each molecular to β-actin was performed using Image J 1.46r (National Institutes of Health, Wayne Rasband, MD, USA).

Detection of oxidative stress related indicators

Reactive oxygen species (ROS) levels in prostate tissues were measured utilizing the Reactive Oxygen Species Assay kit (S0033S, Beyotime Biotechnology, Shanghai, China). Fresh 10-µm frozen prostate sections were rinsed with phosphate buffer solution and incubated with the diluted 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) solution for 1 hour at 37 ℃ in the dark. Subsequently, nuclei were labelled with 4,6-diamino-2-phenyl indole (DAPI). Fluorescent images were captured using a Zeiss Ax10 fluorescent microscope, and the mean fluorescence intensity [the total fluorescence intensity of this Area (IntDen)/the area of this area (Area)] was analyzed with the Image J software 1.46r (National Institutes of Health). In parallel, reduced GSH content and total SOD activity were determined using commercial kits (A006-2-1 and A001-1, respectively; Nanjing Jiancheng Bioengineering institute, Nanjing, China) according to the manufacturer’s instructions.

Enzyme-linked immunosorbent assay (ELISA)

In order to investigate whether inflammation plays a role in autoimmune prostatitis. Concentrations of NF-κB, IL-1β in rat prostate and serum were determined utilizing enzyme-linked immunosorbent assay kits (Sunredbio Technology Co., Ltd, Shanghai, China) and the absorbance microplate reader (SpectraMax 190, Molecular Device, San Jose, CA, USA). All the procedures were performed in strict accordance with the instructions.

Statistical analysis

In this study, all statistical analyses were performed by a researcher blinded to the group allocations. Prior to analysis, the normality of the data was assessed using the Shapiro-Wilk test, and homogeneity of variances was examined using Bartlett’s test. For data satisfying both parametric assumptions, parametric tests were employed: one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test were used for single-factor designs; two-way ANOVA was used to evaluate the effects of different treatment groups and treatment phase, with a focus on assessing the interaction between these two factors. When a significant interaction was detected, the Bonferroni test was subsequently used for multiple comparisons. For data not satisfying these two points, the nonparametric Kruskal-Wallis test followed by Dunn’s multiple comparison test was conducted using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA). All data are presented as mean ± SEM. A P value <0.05 was considered statistically significant. In the figures, significant levels are indicated as follows: *, P<0.05; **, P<0.01; ***, P<0.001.

Results

NAC could alleviate the level of inflammation in the prostate of EAP rats

Consistent with prior research demonstrating robust inflammatory responses in EAP rat prostate tissue (2,16,17), our H&E staining observed extensive inflammatory cell infiltration in EAP model prostates (Figure 1), accompanied by a significant elevation in prostate index (prostate weight/body weight ratio) (Figure 1F). Immunofluorescence analyses further identified massive macrophage recruitment (F4/80⁺ cells) to the prostate (Figure 1B,1G), with concurrent upregulation of pro-inflammatory mediators including NF-κB, IL-1β, and TNF-α in both prostatic tissue and serum (Figure 1C,1D,1H-1L). Given NAC’s well-documented anti-inflammatory properties, we demonstrated that 4-week NAC administration substantially mitigated these inflammatory alterations across all measured parameters (Figure 1), suggesting its potential to counteract EAP-associated inflammation through multiple mechanisms.

Figure 1 NAC could alleviate the level of inflammation in the prostate of EAP rats (n=8/group). (A) Hematoxylin-eosin staining (×100 and ×200 magnification) of rat prostatetissues, and (E) histopathological scores of rats in each group. (B) Representative images (×200 and ×400 magnification) of immunofluorescence of F4/80+ macrophages in rat prostate, and (G) quantification of F4/80+ fluorescence staining. (C) Exhibiting the immunohistochemistry images of NF-κB, and (D) IL-1β (×100 and ×200 magnification) of prostate tissues, (H) NF-κB and (I) IL-1β statistical data. (F) The prostate index (prostate weight/body weight) of each group. The amount of NF-κB (J), IL-1β (K) and TNF-α (L) in the prostate and serum were determined using ELISA kit. The data were expressed as mean ± standard error of mean. *, P<0.05; **, P<0.01; ***, P<0.001. EAP, experimental autoimmune prostatitis; ELISA, enzyme linked immunosorbent assay; IL-1β, interleukin-1β; NAC, N-acetylcysteine; NF-κB, nuclear transcription factor-κB.

NAC could effectively relieve the pain response of EAP rats

Pain represents a predominant non-specific symptom in CP/CPPS patients, manifesting across multiple urogenital regions including the perineum, testes, penis, suprapubic area, groin, lower back, and rectum (3). Given its clinical significance, pain management constitutes a cornerstone of CP/CPPS treatment. In this study, we employed the Von Frey test to systematically evaluate mechanical allodynia in both hindfoot (Figure 2A-2C) and urogenital regions (Figure 2D-2F) at different experimental phases. Our data revealed that on day 7 post-immunization, compared to the CON group, EAP rats exhibited a trend toward increased pain sensitivity in the paw (Figure 2B; 95% CI of diff. −3.152 to 47.59), alongside a markedly enhanced pain sensitivity in the genitourinary region (Figure 2E; 95% CI of diff. 3.486 to 42.17). Whereas compared to the EAP group, sustained 30-day NAC administration significantly attenuated these pain response (Figure 2C; 95% CI of diff. −46.41 to −5.645; Figure 2F; 95% CI of diff. −53.00 to −13.66), demonstrating the potential of NAC to mitigate inflammatory pain in this model.

Figure 2 NAC could effectively relieve the pain response of EAP rats (n=8/group). (A) Representing the frequencies of pain response to mechanical stimulation on left plantar surface using von Frey filaments prior to immunization, and (B) the frequencies of pain response on the 7th day of immunization, and (C) after NAC treatment for 30 days. (D) Showing the frequencies of pain response to mechanical stimulation in the lower abdominal area near the prostate prior to immunization, and on the 7th day of immunization (E), and after NAC treatment for 30 days (F). The data were expressed as mean ± standard error of mean. *, P<0.05; **, P<0.01; ***, P<0.001. EAP, experimental autoimmune prostatitis; NAC, N-acetylcysteine.

NAC could regulate the expression of pro-oxidation and anti-oxidation related molecules in EAP rats

Oxidative stress has been considered as a pivotal pathological mechanism in CP/CPPS or EAP (2,4). As a potent antioxidant, NAC demonstrated significant efficacy in mitigating oxidative damage in this study. First, our DCFH-DA fluorescence analysis revealed substantial elevation of ROS in EAP rat prostates (Figure 3A,3B), which was effectively counteracted by 30-day NAC administration through enhanced reduced GSH production and total SOD activity (Figure 3C,3D). The oxidative imbalance in EAP rats was further evidenced by upregulated pro-oxidant enzymes (iNOS, NOX2, NOX4) (Figure 4A-4F) and downregulated antioxidant markers (GPX1, SOD1, SOD2) (Figure 4G-4L), all of which were corrected following NAC treatment. These findings collectively substantiated that NAC’s therapeutic effects in EAP are mediated through its robust capacity to restore redox homeostasis by simultaneously suppressing oxidative pathways and activating endogenous antioxidant defenses.

Figure 3 NAC could significantly decrease the level of oxidative stress in the prostate tissue of EAP rats (n=8/group). (A) Displaying the ROS in prostate tissues stained by DCFH-DA reagent, and (B) quantification of DCFH-DA fluorescence intensity of each group. (C) Representing the concentration of reduced GSH in prostate of each group, and (D) the total SOD activity in the prostate of each group. The data were expressed as mean ± standard error of mean. *, P<0.05; ***, P<0.001. DCFH-DA, dichlorodihydrofluorescein diacetate; EAP, experimental autoimmune prostatitis; GSH, glutathione; NAC, N-acetylcysteine; SOD, superoxide dismutase.

Figure 4 NAC could regulate the expression of pro-oxidation and anti-oxidation related molecules in EAP rats (n=8/group). (A) Displaying the immunohistochemistry pictures of iNOS, (B) NOX2, (C) NOX4, and (D) quantification of iNOS, (E) NOX2, (F) NOX4 immunohistochemistry staining intensity of each group. (G) Displaying the immunohistochemistry pictures of GPX1, (H) SOD1, (I) SOD2, and (J) quantification of GPX1, (K) SOD1, (L) SOD2 immunohistochemistry staining intensity of each group. The data were expressed as mean ± standard error of mean. *, P<0.05; **, P<0.01; ***, P<0.001. EAP, experimental autoimmune prostatitis; GPX1, glutathione peroxidase 1; iNOS, inducible nitric oxide synthase; NAC, N-acetylcysteine; NOX, nicotinamide adenine dinucleaotide phosphate hydrogen (NADPH) oxidases; SOD, superoxide dismutase.

NAC could activate the PI3K/Akt/CREB signaling pathway in EAP rats

Emerging evidence underscores the pivotal role of PI3K-Akt signaling dysregulation in diverse pathologies spanning cancer, diabetes, cardiovascular disorders, and neurological conditions. Notably, IGF-1-mediated activation of PI3K-Akt-CREB signaling has demonstrated neuroprotection effects by mitigating hippocampal inflammation and oxidative stress while ameliorating cognitive dysfunction and anxiety in high-fat diet models (25). Our study revealed a novel regulatory axis in EAP pathogenesis, where the PI3K/Akt/CREB pathway exhibits significant downregulation in prostatic tissues (Figure 5A-5H). Crucially, NAC administration effectively reactivated this way (Figure 5A-5H), demonstrating its dual capacity to suppress oxidative stress and autoimmune prostatitis through PI3K/Akt/CREB-mediated mechanisms, thereby establishing a previously unrecognized CP/CPPS therapeutic nexus between antioxidant pathways and urogenital inflammation.

Figure 5 NAC could activate the PI3K/Akt/CREB signaling pathway in EAP rats (n=8/group). (A) Displaying the immunohistochemistry pictures of PI3K, (B) pAkt (Ser473), (C) pCREB (Ser133), and (D) quantification of PI3K, (E) pAkt (Ser473), (F) pCREB (Ser133) immunohistochemistry staining intensity of each group. (G) Exhibiting the WB protein bands of PI3K, pAKT (Ser473), Akt, pCREB(Ser133), CREB and β-Actin in the prostates, and (H) the statistical data of WB. The data were expressed as mean ± standard error of mean. *, P<0.05; **, P<0.01; ***, P<0.001. Akt, protein kinase B; CREB, cyclic AMP responsive element-binding protein; EAP, experimental autoimmune prostatitis; NAC, N-acetylcysteine; PI3K, phosphatidylinositol 3-kinase; WB, western blot.

NAC could activate keap1/Nrf2 signaling pathway in EAP rats

A substantial body of research has unequivocally demonstrated the potential antioxidant and anti-inflammatory properties of the Nrf2/HO-1 axis across diverse pathological conditions (26). Notably, Nrf2 exhibits dual regulatory mechanisms by not only neutralizing ROS but also directly suppressing macrophage-mediated inflammation through inhibition of proinflammatory cytokine transcription-a process independent of ROS concentration (27). Given NAC’s established capacity to modulate the Nrf2 pathway, we systematically evaluated the Nrf2/KEAP1/HO-1 signaling axis in our model. Immunohistochemistry and quantitative analysis revealed a dysregulated expression pattern in EAP rats characterized by diminished Nrf2 and HO-1 levels concomitant with KEAP1 upregulation-the latter serving as a key negative regulator of Nrf2 (Figure 6A-6H). Crucially, NAC administration effectively restored the physiological balance of this redox-sensitive pathway, as evidenced by the corrected protein expression (Figure 6A-6H), thereby establishing a mechanistic link between NAC’s therapeutic effects and Nrf2 pathway activation in EAP pathogenesis.

Figure 6 NAC could activate Keap1/Nrf2 signaling pathway in EAP rats (n=8/group). (A) Displaying the immunohistochemistry pictures of Nrf2, (B) KEAP1, (C) HO-1, and (D) quantification of Nrf2, (E) KEAP1, (F) HO-1 immunohistochemistry staining intensity. (G) Exhibiting the WB protein bands of Nrf2, KEAP1, HO-1 and β-Actin in prostate of each group, and (H) the statistical data of WB. The data were expressed as mean ± standard error of mean. *, P<0.05; **, P<0.01; ***, P<0.001. EAP, experimental autoimmune prostatitis; HO-1, heme oxygenase-1; KEAP1, kelch-like ECH-associated protein 1; NAC, N-acetylcysteine; Nrf2, Nuclear factor erythroid 2-related factor 2; WB, western blot.

Discussion

CP/CPPS has emerged as a focal area of research in recent years (7-9,16-19,24), with EAP rat models gaining particular prominence due to their superior translational validity. In the present study, EAP induction was achieved through intraprostatic injection of prostate tissue homogenate supernatant emulsified with Freund’s complete adjuvant, a protocol that successfully recapitulated key clinical features as evidenced by pronounced mechanical allodynia compared to control animals. Comprehensive histopathological analysis revealed profound architectural disruption in EAP prostates characterized by dense inflammatory infiltrates, paralleled by significant elevation of proinflammatory mediators including NF-κB and IL-1β. The oxidative component of EAP pathogenesis was further substantiated by marked upregulation of ROS-generating enzymes (iNOS, NOX2, NOX4), thereby establishing a multifactorial disease model that integrates neuropathic pain, inflammatory signaling, and oxidative stress dysregulation-all hallmarks of human CP/CPPS pathophysiology.

The multifaceted antioxidant mechanisms of NAC have been extensively documented, with its therapeutic efficacy in various pathological conditions being well-established, including its prominent role in mitigating oxidative stress in this study (12). NAC exerts its antioxidant effects through dual pathways: (I) direct radical scavenging via its reactive thiol moiety, which effectively neutralizes hydroxyl radicals (·OH), nitrogen dioxide (·NO₂), and carbon trioxide ion (CO₃·⁻)-key ROS generated by leukocytes; (II) indirect antioxidant activity as a precursor for GSH synthesis, the body’s master antioxidant that serves critical functions in detoxification, mitochondrial DNA stabilization, and cellular defense against oxidative damage. Notably, GSH depletion has been implicated in the pathogenesis of numerous diseases spanning cardiovascular disorders, metabolic syndromes, and chronic inflammatory conditions (28). Beyond its role as a GSH precursor, NAC has been shown to augment antioxidant enzyme systems, including potentiation of GSH peroxidase activity and upregulation of SOD.

Previous studies have shown that in the treatment of respiratory diseases, NAC has a wide dosage range, with daily doses up to 3,000 mg not significantly increasing the incidence of adverse reactions (29). Our previous research confirmed that in a rat model of bilateral cavernous nerve injury, NAC at a dose of 500 mg/kg effectively replenished GSH levels in penile cavernosal cells, exerting significant antioxidant and antifibrotic effects (12). In the present study, we administered NAC to rats by gavage at a dose of 500 mg/kg, which corresponds to a human equivalent dose of approximately 81 mg/kg for a 60 kg adult. This dose aligns with the high-dose range of 600–3,000 mg/day reported in clinical reviews. We found that, at this dose, NAC: (I) attenuated ROS production, (II) downregulated the expression of pro-oxidative enzymes (iNOS, NOX2, NOX4), (III) elevated the expression of antioxidant defense enzymes (SOD1, SOD2, GPX1), and (IV) enhanced GSH levels and GPX1 activity, thereby exerting a key role in the EAP model.

Another therapeutic mechanism of NAC comes from its anti-inflammatory activity. Numerous studies have discovered that NAC can suppress the activation and translocation of NF-κB. While NF-κB, as a transcription factor, plays a significant role in regulating inflammation and immune response (28,30). Previous studies have reported that NAC can prevent NF-κB activation by inhibiting ubiquitination-dependent degradation of the NF-κB inhibitor (I-κB), and direct scavenging free radicals that trigger NF-κB translocation (28). This inhibitory effect culminates in significant downregulation of proinflammatory cytokines including TNF-α, IL-1β and IL-6. Consistent with these studies, our results revealed marked elevation of NF-κB and its downstream mediators (IL-1β, TNF-α) in both prostate and serum of EAP rats, all of which were effectively decreased following NAC treatment. The observed anti-inflammatory effects may synergize with NAC’s ROS-scavenging capacity, as oxidative stress is a known activator of NF-κB signaling. Furthermore, emerging evidence suggests that NAC extends its anti-inflammatory repertoire by modulating transcription factors such as c-Fos/c-Jun, STAT proteins, and cyclin inhibitors (30,31), thereby establishing a comprehensive anti-inflammatory profile. Collectively, these mechanistic insights provide compelling evidence for NAC’s dual role as both antioxidant and anti-inflammatory agent, with its capacity to suppress NF-κB-mediated inflammation providing particularly instrumental in ameliorating EAP.

The intricate interplay between inflammation and oxidative stress, while not strictly causal, collectively drives disease pathogenesis, underscoring the therapeutic rationale for simultaneous modulation of both pathways. NAC emerges as simple, ingenious, safe, cheap and yet effective treatments to improve outcomes for many diseases (32). Nevertheless, there is no document on NAC in CP/CPPS until now. In the context of EAP, our study found that NAC could downregulate KEAP1 and release Nrf2. Furthermore, the nuclear localization of Nrf2 not only suppressed pro-oxidant enzymes (iNOS, NOX2, NOX4) but also enhance antioxidant defenses (HO-1, GPX1, SOD2), which collectively contributed to mitigating oxidative stress in EAP rats.

Beyond oxidative stress regulation, our study elucidates a novel synergistic mechanism involving the Akt/CREB signaling axis. CREB, as a transcription factor, is usually activated through phosphorylation of Ser133 by other kinases including Akt. Once it’s activated, CREB will be transferred into nucleus together with other transcriptional coactivators, and further bind the CREB promotor to regulate gene expression (33). Currently, growing evidence suggests that the activation of PKA/CREB involves in anti-oxidative, anti-inflammation and neuroprotective, etc. (34-37). For example, CREB binds to CREB-binding protein, a member of the family of histone acetyltransferases, which furtherly catalyzes histone acetylation and promotes its target gene transcription, including anti-inflammatory cytokines IL-10 and dual specificity phosphatase 1. Furtherly, IL-10 can transform M1-type macrophages to M2-type, namely, converting the macrophages with inflammation phenotype to anti-inflammation phenotype (37). This molecular convergence is manifested in our EAP model where NAC treatment simultaneously: (I) enhanced Akt/CREB phosphorylation, (II) attenuated NF-κB-driven cytokine production (IL-1β, TNF-α), and (III) reduced ROS burden—effects that collectively implicate CREB as a critical mediator linking Nrf2/HO-1 antioxidant signaling with inflammatory resolution. Taken together, our data suggest that NAC treatment is associated with the regulation of PI3K/Akt/CREB and Keap1/Nrf2 signaling pathways. As depicted in Figure 7, NAC administration was associated with increased Akt and CREB phosphorylation, enhanced Nrf2 nuclear translocation, and expression of its downstream targets. These associative activations suggest that NAC may act as a multifunctional therapeutic agent capable of disrupting the vicious cycle of oxidative stress and chronic inflammation characteristic of EAP pathogenesis. Finally, we are responsible to clarify several limitations of this study. On the one hand, the dose of NAC administrated was not optimized. According to previous literature (12,29), we used a single dose of 500 mg·kg⁻¹ as an exploratory approach, without conducting a dose-response study to determine the optimal effective dose; On the other hand, in this study, we did not employ pathway-specific inhibitors or gene silencing approaches to verify whether changes in the PI3K/Akt/CREB and Keap1/Nrf2 pathways are directly responsible for the ameliorative effects of NAC on EAP. In the future, we will systematically explore this issue and assess its relevance to clinical exposure levels.

Figure 7 A proposed mechanism of NAC treatment on CP/CPPS. On the one hand, NAC could directly activate GPxs to produce GSH. On the other hand, NAC could upregulate the PI3K/Akt signaling to activate downstream Nrf2 and CREB proteins. With the nuclear translocation of Nrf2 and pCREB, which would increase the expression of an-oxidative stress molecules HO-1 and SODs, and decrease the synthesis of pro-inflammatory and pro-oxidative stress proteins NF-κB and NOXs. Finally, the reduced production of ROS and raised consumption of ROS would contribute to the improvement of CP/CPPS. Akt, protein kinase B; CP/CPPS, chronic prostatitis/chronic pelvic pain syndrome; CREB, cyclic AMP responsive element-binding protein; GPxs, glutathioneperoxidases; GSH, glutathione; HO-1, heme oxygenase-1; NAC, N-acetylcysteine; NF-κB, nuclear transcription factor-κB; NOXs, NADPH oxidases; Nrf2, Nuclear factor erythroid 2-related factor 2; PI3K, phosphatidylinositol 3-kinase; SODs, superoxide dismutases.

Conclusions

In this study, the mechanical allodynia (Von Frey test) served as the primary observational index to systematically assess pain-related behaviors in the EAP model. Combined with histological scoring of prostatic inflammation and the detection of related molecular markers, we further explored the underlying mechanisms. The results demonstrates that oral NAC effectively ameliorated symptoms in EAP model rats, providing dual therapeutic benefits by alleviating both prostatic inflammation and pelvic pain. Mechanistically, NAC exerts its protective effects not only to mediate its free thiol side-chain or Nrf2-HO-1 pathway to inhibite oxidative stress, but also to directly regulate NF-κB or PI3K-Akt-CREB to attenuate inflammation. These findings collectively underscore NAC’s remarkable therapeutic potential for CP/CPPS treatment, offering a robust theoretical basis for its clinical application.

Acknowledgments

The authors express thanks to the pathology Platform of the Public Laboratory Technology Center of West China Hospital, Sichuan University for the service of paraffin sectioning.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0022/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0022/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0022/prf

Funding: This work was supported by the Sichuan Science and Technology Program (Nos. 2025YFHZ0212 and 2026NSFSC1832).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0022/coif). The 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. All animal experiments were performed under a project license (No. 20211293A) granted by the Animal Care and Animal Ethics Committee of West China Hospital, Sichuan University, in compliance with institutional guidelines 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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