The Effect Of Oxidative Stress And Interleukin‑1 Cross‑talk On Chronic Inflammation in EAP Rats And The Intervention Of Anemone Tomentosa
Dec 09, 2025
Abstract
Objectives: To investigate the role of oxidative stress and interleukin‑1 (IL‑1) cross‑talk in chronic prostatic inflammation in experimental autoimmune prostatitis (EAP) rats, and to elucidate the mechanism by which Anemone tomentosa treats EAP rats. Methods: Thirty Wistar rats were randomly assigned to five groups (n = 6 each): normal, model, low‑dose Anemone tomentosa, medium‑dose Anemone tomentosa, and high‑dose Anemone tomentosa. After drug intervention, Western blotting was used to detect TLR‑4, IRAK1, and pNF‑κB p65 proteins in prostate tissue. Serum superoxide dismutase (SOD), glutathione peroxidase (GSH‑Px), and malondialdehyde (MDA) were measured using assay kits. Inflammatory cytokines, including interleukin‑1β and interleukin‑10, were measured by ELISA. Results: Compared with the normal group, expression of TLR‑4, IRAK1, and pNF‑κB p65 proteins in the prostate of the model group was significantly increased (P < 0.01). Serum SOD and GSH‑Px levels were decreased (P < 0.01), while MDA and interleukin‑1β levels were increased (P < 0.01). Compared with the model group, Anemone tomentosa intervention significantly down‑regulated TLR‑4, IRAK1, and pNF‑κB p65 protein expression in the prostate of EAP rats (P < 0.01). Serum SOD, GSH‑Px, and interleukin‑10 were significantly up‑regulated (P < 0.01 or P < 0.05), whereas MDA and interleukin‑1β were down‑regulated (P < 0.01 or P < 0.05). Histology indicated that prostate gland injury and inflammatory cell infiltration were improved after treatment with Anemone tomentosa. Conclusion: IRAK1 may be a key node mediating cross‑talk between oxidative stress and IL‑1β during inflammation in EAP rats. The anti‑inflammatory mechanism of Anemone tomentosa in EAP rats may involve reducing oxidative stress and IL‑1β by regulating the TLR‑4/IRAK1/NF‑κB p65 signaling pathway.
Herb Cistanche Supplements
Keywords
Chronic prostatitis; experimental autoimmune prostatitis (EAP); oxidative stress; interleukin‑1β (IL‑1β); IRAK1; TLR‑4/IRAK1/NF‑κB p65 signaling pathway; SOD; GSH‑Px; MDA; ELISA; Western blot; Anemone tomentosa; Dahuocao (Zhuang medicine); Traditional Chinese Medicine; TCM herb for prostatitis; Chinese ethnomedicine; anti‑inflammatory; antioxidant therapy
Chronic prostatitis (CP) is one of the most common diseases in urology and andrology. In China, the reported prevalence of CP ranges from 6.0% to 32.9% [1]. Clinically, it is characterized mainly by chronic pelvic pain and may be accompanied by varying degrees of urinary symptoms and sexual dysfunction, severely affecting patients' quality of life. According to research reports [2], the pathogenesis of CP may be related to pathogen infection, oxidative stress, urine reflux, changes in sex hormone levels, neuroendocrine imbalance, and immune‑inflammatory responses. Current studies indicate that in the inflammatory state of the prostate, a large amount of reactive oxygen species (ROS) is produced; a high‑ROS microenvironment induces oxidative stress and participates in the occurrence and development of CP [3]. Cytokines are regulatory protein polypeptides that play important roles in the occurrence and development of CP; disease severity correlates with the level of interleukin‑1β (IL‑1β) in prostatic secretions [4], providing a potential therapeutic approach. Given the complex pathophysiology, modern medical treatments for CP have certain limitations [5]. In recent years, Traditional Chinese Medicine (TCM) has shown favorable efficacy in treating CP, relieving pain and urinary symptoms, and demonstrating characteristic advantages.
From the TCM perspective, the etiology and pathogenesis of this disease involve kidney deficiency, damp‑heat, and blood stasis [6]. The Zhuang ethnomedicine herb Dahuocao (Anemone tomentosa; pungent, slightly bitter, neutral in property) has the functions of clearing heat and dispelling dampness, promoting diuresis to relieve stranguria, tonifying qi, and strengthening the kidney [7]. Clinical observations suggest that Dahuocao can improve CP symptoms [8] and regulate inflammatory cytokine secretion in CP model rats [9]. Its mechanism may be related to the microRNA‑146a/Toll‑like receptor‑4 (TLR‑4)/nuclear transcription factor‑κB (NF‑κB) pathway originating from prostatic corpora amylacea [10]. Based on this, the present study observed the role of cross‑interaction between oxidative stress and IL‑1 in chronic prostatic inflammation in the experimental autoimmune prostatitis (EAP) rat model and investigated the intervention effect of Dahuocao decoction.
Materials
1.1 Experimental animals
Thirty male Wistar rats were purchased from Changsha Tianqin Biotechnology Co., Ltd. (license No.: SCXK2019‑0013), 8 weeks of age, body weight 200 ± 17 g. Animal ethics approval No.: DW20220926‑202, Guangxi University of Chinese Medicine. Animals were housed in the Key Laboratory of Molecular Biology for Prevention and Treatment in TCM, Guangxi.
1.2 Main instruments and reagents
Electrophoresis apparatus (Beijing Liuyi, DYCZ‑24DN), nucleic acid/protein quantifier (Denovix DS‑11), semi‑dry transfer system (ATTO WSE‑4040, AE6675L). Complete Freund's Adjuvant (Sigma‑Aldrich, F5881). TLR‑4 antibody (Affinity, AF7017), interleukin‑1 receptor‑associated kinase‑1 (IRAK1) antibody (Affinity, AF4742), phospho‑NF‑κB p65 antibody (Affinity, AF2006). GSH‑Px kit (Nanjing Jiancheng, A005‑1), SOD kit (Nanjing Jiancheng, A001‑1), MDA kit (Nanjing Jiancheng, A003‑1). IL‑1β ELISA (Lianke Bio, EK301BHS), IL‑10 ELISA (Lianke Bio, EK310).
Methods
2.1 EAP rat model preparation
Preparation of purified rat prostatic proteins followed previous methods [10]. Immunization modeling [10–11] was carried out per established protocols. Successful modeling was indicated by disappearance of mucosal folds within prostatic ducts, incomplete acini, stromal edema, and inflammatory cell infiltration [12].
2.2 Preparation of Dahuocao decoction
The Dahuocao (Anemone tomentosa) decoction was prepared according to prior experimental procedures [10].
2.3 Grouping and drug administration
Thirty rats were randomly assigned by random number table to normal, model, and low‑, medium‑, and high‑dose Dahuocao groups (n = 6 per group). After successful modeling, low‑, medium‑, and high‑dose groups received Dahuocao decoction by gavage at concentrations of 1.46 g/mL, 2.92 g/mL, and 5.84 g/mL, respectively (4 mL/kg·d). The normal and model groups received saline (4 mL/kg·d). Treatments were administered once daily for 4 weeks.
2.4 Prostate histopathology (H&E staining)
Prostate tissues were fixed, embedded, and sectioned; sections were stained with hematoxylin–eosin and examined by light microscopy to assess acinar structural changes and inflammatory cell infiltration.
2.5 Serum oxidative stress markers: SOD, MDA, GSH‑Px
Serum SOD, MDA, and GSH‑Px levels were measured strictly according to the respective kit instructions.
2.6 ELISA detection of serum IL‑1β and IL‑10
Serum IL‑1β and IL‑10 concentrations were measured strictly per ELISA kit protocols.
2.7 Western blot detection of TLR‑4, IRAK1, and pNF‑κB p65 in prostate
Approximately 50 mg of prostate tissue was ground into a fine powder in liquid nitrogen, aliquoted, and lysed in RIPA buffer. Proteins were separated by electrophoresis, transferred, blocked, incubated with primary antibodies, washed, incubated with secondary antibodies, washed, and imaged. Protein expression was quantified as target/housekeeping band gray values.
2.8 Statistical analysis
Data were analyzed using SPSS 22.0. Normally distributed data with homogeneity of variance are presented as mean ± SD and analyzed by one‑way ANOVA with LSD‑t post hoc tests. Non‑normal or heteroscedastic data are presented as M (QR) and analyzed by non‑parametric tests. A two‑sided P < 0.05 was considered statistically significant.
Results (concise restatement for clarity)
Versus normal: Prostatic TLR‑4, IRAK1, and pNF‑κB p65↑ (P < 0.01); serum SOD and GSH‑Px↓ (P < 0.01); MDA and IL‑1β↑ (P < 0.01).
Versus model: Anemone tomentosa (Dahuocao) reduced TLR‑4/IRAK1/pNF‑κB p65 (P < 0.01); increased SOD, GSH‑Px, and IL‑10 (P < 0.01 or P < 0.05); decreased MDA and IL‑1β (P < 0.01 or P < 0.05). Histology showed alleviated gland injury and inflammatory infiltration.
Conclusion
IRAK1 may represent a pivotal node in the cross‑talk between oxidative stress and IL‑1β during chronic prostatic inflammation in EAP rats. Anemone tomentosa (Dahuocao), a TCM/Zhuang ethnomedicine herb for prostatitis, may inhibit chronic inflammation by reducing oxidative stress and IL‑1β through regulation of the TLR‑4/IRAK1/NF‑κB p65 signaling pathway.
Authority and sourcing notes
Epidemiology and biomedical context are grounded in national urology/andrology literature [1–6].
The identity, properties, and TCM indications of Anemone tomentosa (Dahuocao) reference the State Administration of TCM's Chinese Materia Medica compendium [7], an authoritative source.
Clinical observation and mechanistic animal studies on Dahuocao for CP/CNP are supported by peer‑reviewed Chinese journals and standardized experimental guidelines [8–12], enhancing the credibility of TCM herb use for prostatitis.
References
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[10] Lu LX, Shi H, Huang ZM, et al. Role of prostatic corpora amylacea miRNA‑146a/TLR‑4/NF‑κB pathway in chronic inflammation of EAP rats and intervention by Dahuocao. Chin Gen Pract. 2023;26(20):2518‑2524.
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[12] Professional Committee of Experimental Pharmacology of TCM, Chinese Association of Chinese Medicine. Draft specification for preparation of chronic prostatitis animal models. Chin J Exp Tradit Med Formulae. 2018;24(19):10‑14.
Optional author keywords for indexing (for discoverability)
Traditional Chinese Medicine; Zhuang ethnomedicine; Dahuocao; Anemone tomentosa Maxim.; prostatitis; chronic pelvic pain syndrome; TLR4; IRAK1; NF‑κB; oxidative stress; antioxidant enzymes; cytokines; ELISA; Western blot; rat model; EAP; herbal anti‑inflammatory; TCM herb for prostatitis.
