Regulatory Effects Of Bixie Qianlie’an Pill On The TLR4/NF-κB/NLRP3 Signaling Pathway And Pyroptosis in Chronic Nonbacterial Prostatitis

 

Abstract

Objective: To investigate the mechanism by which Bixie Qianlie'an Pill (BQAP) treats chronic nonbacterial prostatitis (CNP), focusing on pyroptosis mediated via the TLR4/NF-κB/NLRP3 signaling pathway.

Methods: Male Sprague–Dawley (SD) rats were acclimatized for 1 week. Ten rats each were randomly assigned to a blank control group and a sham-operated group. CNP models were induced in the remaining 40 rats by intraprostatic injection of 1% κ-carrageenan; modeling success was assessed at 1 week. Successfully modeled rats were randomly divided into a model group and three BQAP treatment groups (low, medium, and high dose; n=10/group). The treatment groups received BQAP suspensions by oral gavage at 2.7, 5.4, and 10.8 g/kg·d, respectively; other groups received sterile water. Dosing was once daily for 30 days. After the last administration, blood was collected from the abdominal aorta and bilateral prostate tissues were harvested. Prostate index was calculated. Serum IL-1β, IL-18, and TNF-α were measured by ELISA. Inflammatory infiltration in prostate tissue was evaluated by HE staining. NLRP3 localization and expression were assessed by immunohistochemistry; GSDMD fluorescence intensity was detected by immunofluorescence. Protein expression of TLR4, MyD88, NF-κB p65, p-NF-κB p65, NLRP3, Caspase-1, and GSDMD in prostate tissue was determined by Western blot.

Results: The prostate index decreased significantly in the BQAP medium-dose group (p<0.05). Low-, medium-, and high-dose BQAP reduced inflammatory cell infiltration in prostate tissue and decreased serum IL-1β, IL-18, and TNF-α levels (all p<0.05). Quantitative immunohistochemistry showed a significant reduction in NLRP3 expression (p<0.05), and quantitative immunofluorescence demonstrated a significant decrease in GSDMD fluorescence intensity (p<0.05). Western blot analysis revealed reduced expression of TLR4, MyD88, NF-κB p65, p-NF-κB p65, NLRP3, Caspase-1, and GSDMD in prostate tissue (all p<0.05).

Conclusion: Bixie Qianlie'an Pill lowers proinflammatory cytokine levels and ameliorates inflammation in the prostate of CNP rats. Its mechanism may involve modulation of the TLR4/NF-κB/NLRP3 pathway to inhibit pyroptosis.

Keywords: Bixie Qianlie'an Pill; chronic nonbacterial prostatitis; cytokines; TLR4/NF-κB/NLRP3 signaling pathway; pyroptosis

 

Cistanche herb supplements for chronic nonbacterial prostatitis

 

 

Buy Now

 

 

Introduction

Chronic nonbacterial prostatitis (CNP) is one of the most common conditions in post-pubertal males. Although individualized multimodal therapy-pharmacotherapy, biofeedback, thermotherapy, etc.-is recommended, outcomes remain suboptimal and patients' quality of life is substantially impaired [1]. The etiology and pathogenesis are complex and not fully elucidated, involving infection, autoimmunity, endocrine imbalance, neural plasticity, and psychosocial factors [2]. Inflammation is thought to be central to the onset and progression of CNP [3].

The TLR4 (toll-like receptor 4)/NF-κB (nuclear transcription factor kappa B) pathway plays a pivotal role in inflammatory responses by regulating proinflammatory cytokine production [4]. NF-κB activation can initiate transcription of pro–IL-1β and pro–IL-18, driving inflammatory disease progression; this activation is associated with the activity of NLRP3 (nucleotide-binding oligomerization domain-like receptor protein 3) [5].

Current CNP therapies are varied, yet relapse is frequent and cure is difficult [3]. Bixie Qianlie'an Pill (a hospital-prepared formula) has been used clinically for over 10 years to treat CNP of the "kidney deficiency with damp-heat and blood stasis" pattern in traditional Chinese medicine. Previous clinical observations showed BQAP improves NIH-CPSI scores and overall efficacy [6]. However, its mechanism remains unclear. This study explores the expression of TLR4/NF-κB/NLRP3 pathway–related proteins to further clarify the potential mechanism of BQAP in CNP.

Materials

 

Animals

Sixty male SD rats were purchased from Shandong Provincial Laboratory Animal Center (Jinan Pengyue Experimental Animal Breeding Co., Ltd.). Production and use licenses: SCXK (Lu) 2019-0003 and SYXK (Yu) 2021-0015. Mean body weight 205±30 g. Rats were housed separately at the Experimental Animal Center of Henan University of Chinese Medicine. The protocol was approved by the Institutional Animal Ethics Committee (DWLL202306012).

Cistanche herb supplements

Drugs and Reagents

 

Bixie Qianlie'an Pill (Henan Provincial Hospital of TCM, hospital preparation, approval No. Yu Yao Zhi Zi 204010164). The 12 herbal constituents-Dioscoreae Hypoglaucae Rhizoma (Bixie), Plantaginis Semen, Abutili Semen, Rehmanniae Radix Preparata, Cuscutae Semen, Astragali Radix, Hirudo, Phellodendri Cortex, Succinum, Epimedii Herba, Cnidii Fructus, Achyranthis Bidentatae Radix-were authenticated by the Hospital's Center for R&D of TCM Preparations (Chief Pharmacist Hongyan Yu).

κ-Carrageenan (Macklin, Shanghai).

10% neutral buffered formalin (Shanghai Enzyme-linked Biotechnology).

ELISA kits for IL-18, IL-1β, TNF-α (Jiangsu Enzyme Immunoassay Industry Co., Ltd.; Cat. Nos. MM-0047R1, MM-0194R1, MM-0180R1).

HE staining kit (Shanghai Shangbao Biotechnology, Cat. No. ST2023).

Primary antibodies: TLR4, MyD88, NF-κB p65, phospho–NF-κB p65, NLRP3, GSDMD (Bioss, Beijing; Cat. Nos. bs-1021R, bs-1047R, bs-0982R, bs-20160R, bs-10021R, bs-14287R); Caspase-1 (Abcam via Ai Bo Antibody, Shanghai; ab179515).

 

Methods

 

Modeling and Drug Administration

CNP was induced by intraprostatic injection of 1% κ-carrageenan in normal saline (0.1 mL/rat) into the left and right dorsolateral lobes of the prostate under intraperitoneal anesthesia (3% sodium pentobarbital, 30 mg/kg), as described in [7–8]. The seminal vesicles were exposed and elevated; 50 μL carrageenan solution was injected into each prostatic lobe, and the muscle and skin were sutured. The sham group received equal volumes of saline at the same sites.

Adult human daily dose of BQAP is 30 g. Using a 70-kg adult as reference, the human-to-rat equivalent dose was converted by body surface area (conversion factor 6.3 per Pharmacological Experimental Methodology). The equivalent rat dose was defined as the low dose: 2.7 g/kg·d; the medium and high doses were 5.4 and 10.8 g/kg·d, respectively. BQAP was ground into fine powder and suspended in saline freshly each day. From day 7 after modeling confirmation, rats in treatment groups received the respective dose by oral gavage once daily for 30 days; other groups received equal volumes of saline.

Tissue Collection

After overnight fasting and anesthesia, the abdominal skin and subcutaneous tissue were opened along the ventral midline. Abdominal aorta blood was collected into vacuum tubes and allowed to clot at room temperature, then centrifuged at 3000 rpm for 10 min; serum was aliquoted and stored at −80°C. The seminal vesicles were lifted to expose the dorsolateral prostate; tissues were rinsed in saline. The left lobe was fixed in 10% neutral buffered formalin; the right lobe was snap-frozen in liquid nitrogen and stored at −80°C.

 

HE Staining for Histopathology

Formalin-fixed tissues were washed with saline and dehydrated through graded ethanol (75%, 85%, 95%, 100%), cleared with xylene, infiltrated with paraffin/xylene (1:1), then paraffin. Blocks were cooled at −20°C overnight and sectioned at 4 μm. Sections were stained with HE and examined under light microscopy to evaluate inflammatory changes.

 

ELISA for Serum Proinflammatory Cytokines

Per kit instructions, 100 μL of sample or standard was added per well, followed by labeled antibody. Plates were covered and incubated 2 h at room temperature in the dark, washed 5 times, then 100 μL HRP conjugate was added and incubated 1 h. After five washes, 100 μL TMB substrate was added and incubated 30 min in the dark. Reaction was stopped with 50 μL stop solution and absorbance at 450 nm was read. Mean OD was calculated for duplicates.

 

Immunohistochemistry for NLRP3

Paraffin sections were deparaffinized and rehydrated at room temperature, followed by heat-induced antigen retrieval. Endogenous peroxidase was quenched with 3% H2O2. After blocking, primary anti-NLRP3 antibody was applied (4°C overnight). Sections were washed and incubated with secondary antibody, developed with DAB, counterstained with hematoxylin, dehydrated, mounted, and imaged. Quantification was performed in ImageJ. MOD (mean optical density) = IOD (integrated optical density) / area.

 

Immunofluorescence for GSDMD

Paraffin sections were deparaffinized, underwent antigen retrieval, blocked, and incubated with primary and fluorescent secondary antibodies. Nuclei were counterstained with DAPI. Autofluorescence quenching was performed, and sections were mounted with antifade medium. Nuclei appeared blue (DAPI channel); positive signals were red (Cy3 channel). Quantification was performed with ImageJ: MOD = IOD / area.

 

Western Blot

 

Lysis: Prostate tissues were homogenized and lysed with RIPA buffer. Lysates were centrifuged at 12,000 r/min for 20 min at 4°C, and supernatants collected.

SDS-PAGE: 7.5% gels; samples were resolved at 80 V for 30 min, then 120 V for 50 min.

Transfer and Blocking: Proteins were transferred to membranes and blocked in 5% skim milk in TBST for 2 h; membranes were washed 3× (10 min each).

Primary antibodies: Applied at 1:1000 (ASC 1:5000 where applicable) and incubated overnight at 4°C.

Secondary antibodies: Membranes were incubated 1–2 h, then washed 3–5× with TBST.

Detection: Chemiluminescent substrate was added and bands were imaged.

Quantification: GAPDH served as the loading control. Relative expression = target band gray value / GAPDH.

 

Statistical Analysis

Data were analyzed and graphed with GraphPad Prism 9.0. Results are expressed as mean ± SD. One-way ANOVA and independent-samples t tests were used for group comparisons. p<0.05 was considered statistically significant.

 

Results

 

Prostate index: Significantly reduced in the BQAP medium-dose group versus model (p<0.05).

Histopathology: Low-, medium-, and high-dose BQAP groups showed alleviated inflammatory cell infiltration compared with the model group.

Serum cytokines: IL-1β, IL-18, and TNF-α levels decreased in all BQAP groups (p<0.05).

IHC for NLRP3: Quantitative analysis showed significantly lower NLRP3 expression after BQAP (p<0.05).

IF for GSDMD: GSDMD fluorescence intensity was significantly reduced (p<0.05).

Western blot: Expressions of TLR4, MyD88, NF-κB p65, p–NF-κB p65, NLRP3, Caspase-1, and GSDMD in prostate tissue were all decreased in BQAP groups (p<0.05).

 

Discussion for Herbal-therapy Developers (concise implications)

 

Pathway target: Data support that BQAP modulates the TLR4→MyD88→NF-κB axis and downstream NLRP3 inflammasome, attenuating Caspase-1–dependent GSDMD activation and pyroptosis. This validates TLR4/NF-κB/NLRP3 as a rational mechanistic target in CNP herbal development.

Bioactivity hypothesis by constituents: Several BQAP herbs have literature-reported anti-inflammatory or inflammasome-modulating effects (e.g., Astragalus: NF-κB inhibition; Phellodendron: berberine-related NLRP3 modulation; Epimedium flavonoids: anti-inflammatory signaling). Future fractionation could correlate these with observed pathway suppression.

Dosing translation: Rat effective ranges (2.7–10.8 g/kg·d crude herb equivalent) align with human clinical dosage after BSA conversion. For product development, standardize by key marker compounds and set human-equivalent dose margins based on the medium dose, which showed optimal prostate index improvement.

Biomarkers/endpoints: Cytokines (IL-1β, IL-18, TNF-α), prostate index, and histology are sensitive. Include NLRP3, p-p65, and GSDMD as pharmacodynamic markers in preclinical screening of refined extracts.

Next steps:

Validate in additional CNP models (e.g., autoimmune or hormone-driven) to generalize efficacy.

Perform time-course and dose–response for pyroptosis markers.

Conduct component synergy studies and quality control fingerprints.

Safety pharmacology and reproductive toxicity profiling given long-term urogenital use.

 

Conclusions

BQAP mitigates inflammatory injury in CNP rats and suppresses pyroptosis by downregulating the TLR4/NF-κB/NLRP3 pathway and its downstream effectors Caspase-1 and GSDMD. These findings provide mechanistic support for the development and modernization of herbal therapeutics targeting inflammasome-mediated pathology in chronic prostatitis.

 

References

References (English translation)

[1] Liang Chaochao, Xia Shujie, Deng Chunhua, et al. Chinese Expert Consensus on Prostatitis-Pelvic Syndrome (2024 edition). Journal of Modern Urology. 2024;29(09):756-761.

[2] Cyril AC, Jan RK, Radhakrishnan R. Pain in chronic prostatitis and the role of ion channels: a brief overview. British Journal of Pain. 2022;16:50-59.

[3] Andrology Branch of the Chinese Association of Integrative Medicine. Integrated Traditional Chinese and Western Medicine Guidelines for the Diagnosis and Treatment of Chronic Prostatitis. Chinese Journal of Andrology. 2023;37(1):3-17.

[4] Huang Jianhua, Liu Rui, Zhou Yifei, et al. Mechanistic study of Mai Men Dong Decoction on rat pulmonary fibrosis via regulation of the TLR4–NF-κB pathway. Liaoning Journal of Traditional Chinese Medicine. 2024;51(11):188-193, 227-228.

[5] Bae WJ, Shin D, Piao JJ, et al. Extracorporeal shockwave therapy alleviates inflammatory pain by down-regulating the NLRP3 inflammasome in experimental chronic prostatitis and chronic pelvic pain syndrome. World Journal of Men's Health. 2024;42(1):157-167.

[6] Zhai Zhaoyong, Sun Zixue, Zhang Yizheng, et al. Clinical efficacy of Bixie Qianlie'an Pill combined with conventional therapy in patients with type IIIB prostatitis of the "kidney deficiency with damp-heat obstruction of collaterals" pattern. Medical Forum Magazine. 2024;45(14):1544-1548, 1553.

[7] Professional Committee of Experimental Pharmacology of Traditional Chinese Medicine, China Association of Chinese Medicine. Draft specification for the preparation of animal models of chronic prostatitis. Chinese Journal of Experimental Traditional Medical Formulae. 2018;24(19):10-14.

[8] Zhu Peixuan, Fan Qiongyin, Zhang Jingxuan, et al. Factors influencing the κ-carrageenan–induced rat model of chronic prostatitis. World Journal of Traditional Chinese Medicine. 2021;16(14):2094-2100.