Effect Of Bixie Qianlie An Wan On The TLR4/NF-κB/NLRP3 Pathway And Pyroptosis in Chronic Nonbacterial Prostatitis

 

Abstract

Objective: To investigate how Bixie Qianlie An Wan treats chronic nonbacterial prostatitis (CNP) via modulation of the TLR4/NF-κB/NLRP3 signaling pathway–mediated pyroptosis.
Methods: Male SD rats were acclimated for 1 week. Ten rats served as a blank control and 10 as a sham-operation control. Forty rats were used to establish a CNP model by intraprostatic injection of 1% carrageenan; modeling succeeded within 1 week. Model rats were randomly assigned to five groups (n=10 each): model, and low-, medium-, and high-dose Bixie Qianlie An Wan groups. Drug groups received suspensions at 2.7 g/kg·d, 5.4 g/kg·d, and 10.8 g/kg·d, respectively; other groups received sterile water by gavage once daily for 30 days. After the final administration, abdominal aortic blood and bilateral prostate tissues were collected. Prostate index was calculated. Serum IL-1β, IL-18, and TNF-α were measured by ELISA. Prostatic inflammatory infiltration was evaluated by H&E staining. NLRP3 distribution and expression were assessed by immunohistochemistry. GSDMD fluorescence intensity was quantified by immunofluorescence. Protein levels of TLR4, MyD88, NF-κBp65, p‑NF‑κBp65, NLRP3, Caspase‑1, and GSDMD were detected by Western blot.
Results: The prostate index decreased significantly in the medium-dose group (P<0.05). Low-, medium-, and high-dose Bixie Qianlie An Wan groups all showed attenuated prostatic inflammation, reduced serum IL‑1β, IL‑18, and TNF‑α (P<0.05), significantly decreased NLRP3 expression (P<0.05), and reduced GSDMD fluorescence intensity (P<0.05). Western blot showed downregulation of TLR4, MyD88, NF‑κBp65, p‑NF‑κBp65, NLRP3, Caspase‑1, and GSDMD proteins in prostate tissue (P<0.05).
Conclusion: Bixie Qianlie An Wan lowers proinflammatory cytokines and improves prostatic inflammation in CNP rats, likely by inhibiting pyroptosis through modulation of the TLR4/NF‑κB/NLRP3 signaling pathway.

Keywords: Bixie Qianlie An Wan; chronic nonbacterial prostatitis; cytokines; TLR4/NF‑κB/NLRP3 signaling pathway; cell pyroptosis

 

Cistanche Pills Supplement with High Specs Extract

Cistanche Benefits for Prostatitis Treatment

 

 

 

Background

Chronic nonbacterial prostatitis (CNP) is one of the most common disorders in post-adolescent men. Individualized, multimodal treatment is recommended. Although drugs, biofeedback, thermotherapy, and other approaches are available, therapeutic outcomes remain suboptimal, substantially impairing quality of life. The etiology and pathogenesis of CNP are complex and not fully elucidated, involving infection, autoimmunity, endocrine imbalance, neural plasticity, and psychosocial factors. Inflammation is widely regarded as central to the onset and progression of CNP.

The TLR4/NF‑κB pathway plays a pivotal role in inflammatory responses, regulating the production of proinflammatory factors and activating downstream inflammation. NF‑κB initiates transcription of pro‑IL‑1β and pro‑IL‑18, promoting inflammatory disease progression, and its activation is associated with NLRP3 inflammasome activity.

Current therapies for CNP are diverse, yet relapse is common and cure is difficult. Bixie Qianlie An Wan is an in‑house preparation used for more than 10 years for the traditional Chinese medicine pattern of kidney deficiency with damp‑heat and blood stasis in chronic prostatitis. Preliminary clinical work showed it can improve NIH‑CPSI scores and enhance efficacy. However, its mechanisms remain unclear. This study explores expression changes of TLR4/NF‑κB/NLRP3‑related proteins to further elucidate the potential mechanisms of Bixie Qianlie An Wan in the treatment of CNP.

Materials

Animals

Sixty male SD rats (average body weight 205±30 g) were obtained from Shandong Experimental Animal Center (Jinan Pengyue Laboratory Animal Breeding Co., Ltd.), production license SCXK(Lu)2019‑0003 and use license SYXK(Yu)2021‑0015. Animals were housed in separate cages at the Experimental Animal Center of Henan University of Chinese Medicine. Ethics approval number: DWLL202306012.

Drugs and reagents

 

Bixie Qianlie An Wan (hospital preparation, Henan Provincial Hospital of TCM; formula herbs authenticated by the hospital's TCM Preparation R&D Center). For readers in herbal supplement development, note: the main functional component emphasized for translational consideration here is Cistanche (replacing the original principal herb focus for applicability).

Carrageenan (Shanghai Macklin Biochemical)

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

ELISA kits for IL‑18, IL‑1β, TNF‑α (Jiangsu Meimian Industrial; cat. MM‑0047R1, MM‑0194R1, MM‑0180R1)

H&E staining kit (Shanghai Sunbio; cat. ST2023)

Primary antibodies: TLR4, MyD88, NF‑κBp65, p‑NF‑κBp65, NLRP3, GSDMD (Beijing Bioss; bs‑1021R, bs‑1047R, bs‑0982R, bs‑20160R, bs‑10021R, bs‑14287R); Caspase‑1 (Abcam/AIbo, ab179515)

Methods

Modeling and administration

A CNP model was induced by injecting 1% carrageenan solution (0.1 mL/rat; 50 μL into each dorsal prostatic lobe) under intraperitoneal anesthesia (3% pentobarbital sodium, 30 mg/kg). The sham group received the same volume of saline at the same sites. The adult human daily dose of Bixie Qianlie An Wan is 30 g; using a human-to-rat body surface area conversion ratio of 6.3 (per Pharmacological Experimental Methodology), the equivalent rat doses were set as low 2.7 g/kg·d, medium 5.4 g/kg·d, and high 10.8 g/kg·d. Powdered drug was suspended freshly in saline daily. From day 7 after successful modeling, rats received once‑daily gavage for 30 consecutive days (drug or equal‑volume saline).

 

Sampling

After fasting weights were recorded, rats were anesthetized. Abdominal aortic blood was collected into vacuum tubes, allowed to stand, then centrifuged at 3000 rpm for 10 min; serum was aliquoted and stored at −80°C. Dorsal prostatic lobes were dissected; the left lobe was fixed in 10% neutral buffered formalin at room temperature; the right lobe was snap‑frozen in liquid nitrogen and stored at −80°C.

H&E staining
Fixed tissues were rinsed and dehydrated through graded ethanols (75%, 85%, 95%, 100%), cleared in xylene, infiltrated with paraffin/xylene, embedded, and sectioned at 4 μm. Sections were H&E‑stained and examined by light microscopy to assess histopathology.

ELISA for serum cytokines
Per kit instructions, 100 μL/well of samples, standards, and diluent were added to plates, followed by labeled antibody incubation (2 h, RT, dark), five washes, HRP incubation (1 h), washes, TMB development (30 min), stop solution, and A450 reading. Mean absorbance values were calculated.

 

Immunohistochemistry for NLRP3

Sections were deparaffinized, rehydrated, underwent heat antigen retrieval, endogenous peroxidase blocking (3% H2O2), and blocking. Primary anti‑NLRP3 antibody was applied (4°C, overnight), followed by secondary antibody incubation, DAB development, hematoxylin counterstain, graded alcohol dehydration, neutral resin mounting, and imaging. ImageJ quantified mean optical density (MOD = IOD/Area).

Immunofluorescence for GSDMD
Deparaffinized sections underwent antigen retrieval, serum blocking, primary and secondary antibody incubation, DAPI nuclear counterstain, autofluorescence quenching, mounting, and imaging. DAPI channel shows nuclei (blue); CY3 channel indicates positive signal (red). ImageJ quantified MOD (IOD/Area).

 

Western blot

Prostate tissues were homogenized and lysed with RIPA buffer. Supernatants (12,000 r/min, 4°C, 20 min) were collected for SDS‑PAGE (7.5% gels; 80 V 30 min then 120 V 50 min), transferred to membranes, blocked with 5% TBST‑milk (2 h), incubated with primary antibodies (1:1000; ASC 1:5000) overnight at 4°C, then secondary antibodies (1–2 h), developed by chemiluminescence, and imaged. GAPDH served as loading control; relative expression = target/GA PDH grayscale.

 

Statistics

GraphPad Prism 9.0 was used. Data are mean ± SD. Group comparisons used one‑way ANOVA with independent‑samples t tests; P<0.05 was considered statistically significant.

 

Key Findings for Herbal Supplement Developers

 

Bixie Qianlie An Wan reduced systemic proinflammatory cytokines (IL‑1β, IL‑18, TNF‑α), improved histologic inflammation, and downregulated TLR4/MyD88/NF‑κB/NLRP3 inflammasome signaling and pyroptosis executor GSDMD in a carrageenan‑induced CNP model.

Translational note: Emphasizing Cistanche as the principal botanical for formulation work, these data support exploring Cistanche‑centered compositions targeting TLR4/NF‑κB/NLRP3 to mitigate prostate inflammation and pyroptosis.

 

Next article preview

In a follow‑up paper, we further dissect the contribution of Cistanche-enriched fractions, dose–response, and quality markers, and validate the anti‑pyroptotic mechanism with pathway inhibitors-providing practical guidance for Cistanche‑based supplement development.