Exploration Of The Mechanism Of Cistanche Formula Regulating NLRP3 Inflammator-mediated Pyroptosis in The Treatment Of Chronic Prostatitis
Jun 05, 2025
Objective:
This study aimed to investigate the mechanism by which a Cistanche-based herbal formula regulates the activation of the NLRP3 inflammasome and inhibits pyroptosis in the treatment of Type III chronic prostatitis (CP/CPPS).
Methods:
(1) Animal experiment:
Fifty male SD rats were randomly assigned into five groups: control, model, and Cistanche formula-treated groups at low (4.9 g/kg), medium (9.8 g/kg), and high (19.6 g/kg) doses, with 10 rats in each group. Except for the control group, all rats were induced with Type III prostatitis via intraprostatic injection of prostate protein extract combined with CFA. Following successful modeling, rats received daily intragastric administration of either normal saline (control and model groups) or the Cistanche-based formula for 30 days.
Prostate tissue inflammation was assessed by Hematoxylin-Eosin (HE) staining. Serum IL-1β and IL-18 were measured by ELISA, while oxidative stress markers (MDA, SOD, and GSH-Px) were evaluated biochemically. NLRP3 expression in prostate tissue was detected by immunohistochemistry, and NLRP3, Caspase-1, and GSDMD protein levels were analyzed using Western blot.
(2) Cell experiment:
Human prostate epithelial cells (RWPE-1) were divided into control, model, Cistanche formula, and NLRP3 inhibitor (MCC950) groups. Except for the control group, all cells were stimulated with LPS (100 ng/mL, 4 h) and ATP (5 mmol/mL, 30 min) to induce pyroptosis. After modeling, the control and model groups received blank serum, while the Cistanche group received 6.25 μg/mL drug-containing serum. The inhibitor group was treated with MCC950.
PI uptake and Caspase-1 expression were assessed via flow cytometry. LDH release was measured biochemically. Supernatant IL-1β and IL-18 levels were quantified by ELISA. Western blot was used to detect NLRP3, Caspase-1, and GSDMD expression.
Results:
(1) Animal experiment:
Compared to the control group, the model group exhibited significant inflammatory cell infiltration, epithelial degeneration, and interstitial edema in prostate tissue. The Cistanche-treated groups showed dose-dependent reductions in these pathological changes.
Serum IL-1β and IL-18 levels were significantly increased in the model group (P<0.01) and significantly decreased in the Cistanche low, medium, and high-dose groups (P<0.01).
MDA levels were elevated in the model group (P<0.01) but significantly reduced by Cistanche treatment (P<0.01).
SOD and GSH-Px levels were decreased in the model group (P<0.05) and significantly increased in all Cistanche-treated groups (P<0.01, P<0.05).
Immunohistochemistry showed elevated NLRP3 expression in the model group, which was significantly reduced by Cistanche treatment.
Western blot results revealed increased protein levels of NLRP3, Caspase-1, and GSDMD in the model group (P<0.01), all of which were significantly inhibited by Cistanche (P<0.01).
(2) Cell experiment:
The PI uptake rate, Caspase-1 expression, and LDH release were significantly increased in the model group (P<0.01), but significantly reduced in both the Cistanche and MCC950 groups (P<0.01).
IL-1β and IL-18 levels in the supernatant were significantly elevated in the model group (P<0.01) and significantly decreased in the Cistanche and inhibitor groups (P<0.01).
Western blot showed significantly increased NLRP3, Caspase-1, and GSDMD protein expression in the model group (P<0.01), which were significantly downregulated in the Cistanche and inhibitor groups (P<0.01).
Conclusion:
The Cistanche-based herbal formula exerts therapeutic effects on Type III chronic prostatitis by inhibiting NLRP3 inflammasome activation, thereby reducing Caspase-1 activation, preventing GSDMD cleavage and pyroptosis, and suppressing pro-inflammatory cytokine release. These findings suggest that Cistanche may be a promising natural herbal remedy for CP/CPPS, offering anti-inflammatory, antioxidant, and pyroptosis-inhibitory effects via modulation of the NLRP3/Caspase-1/GSDMD signaling pathway.
keywords: chronic prostatitis; Guihuang formula; NLRP3 inflammatome; pyroptosis ; programmed cell death
cistanche benefits from prostate Health
Cistanche-Based Herbal Formula for Chronic Prostatitis: Experimental Study of Mechanism and Efficacy
Chronic prostatitis (CP) is one of the most common conditions in urology and men's health. Epidemiological studies show that approximately 8.4% of adult males in China experience symptoms of prostatitis [1]. According to the National Institutes of Health (NIH) classification, prostatitis is categorized into four types, with Type III prostatitis (chronic pelvic pain syndrome, CP/CPPS) accounting for more than 90% of all chronic cases [2]. Type III prostatitis leads to pelvic pain and urinary dysfunction and is often accompanied by psychological distress such as anxiety and depression, significantly impairing quality of life [3–4].
In Traditional Chinese Medicine (TCM), chronic prostatitis corresponds to syndromes such as "Lin Syndrome" or "Jing Zhuo", and is primarily caused by pathogenic factors like dampness, heat, blood stasis, and stagnation [5–7]. The Guihuang formula, traditionally used to treat CP/CPPS with damp-heat and blood stasis, has been modified in this study to use Cistanche as a core component, forming a novel Cistanche-based herbal formula. This modified formula aims to improve urinary symptoms and pelvic inflammation with both clinical efficacy and safety [8–10].
Cistanche tubulosa in desert
cistanche formula based pills with 75% glycoside
Supportive Service Of Wecistanche-The Contact Us To Get A Wholesale Price
Email:wallence.suen@wecistanche.com
Whatsapp/Tel:+86 15292862950
Background and Mechanism
Our previous studies demonstrated that the Cistanche-based herbal formula significantly alleviated prostatitis-like lesions in rat models by reducing pathological inflammation and suppressing pro-inflammatory cytokines. This effect is associated with the inhibition of PI3K and NF-κB signaling pathways [11].
Further attention has been directed toward the NLRP3 inflammasome, a multiprotein complex that regulates inflammatory responses. Activation of the NLRP3 inflammasome leads to the release of inflammatory cytokines such as IL-1β and IL-18, contributing to the inflammatory cascade in CP/CPPS [12–13]. Investigating whether Cistanche can modulate NLRP3 inflammasome activation is crucial for understanding its anti-inflammatory mechanism in chronic prostatitis treatment.
1. Materials
1.1 Animals and Cells
Male SD rats (6 weeks old, 240–260 g) were obtained from SPF (Beijing) Biotechnology Co., Ltd. [SCXK (Jing) 2019-0010], housed in SPF conditions with free access to food and water. Room temperature was maintained at (23±2)°C and humidity at (60±5)%. All animal procedures were approved by the Animal Ethics Committee of Xiyuan Hospital, China Academy of Chinese Medical Sciences (Ethics No: 2020XLC005-3). normal prostate epithelial cells (RWPE-1, ATCC, Catalog No. IM-H299) were used for in vitro experiments at passage 5.
1.2 Herbal Formula
The Cistanche-based formula consists of the following traditional herbs (in grams):
Cistanche 12 g
Curcuma longa (Turmeric) 10 g
Angelica sinensis 12 g
Magnolia officinalis 10 g
Boswellia carteri (Frankincense) 5 g
Commiphora myrrha (Myrrh) 5 g
Lonicera japonica (Honeysuckle) 15 g
Hedyotis diffusa 15 g
Plantago asiatica (Plantain Herb) 15 g
Citrus reticulata (Tangerine Peel) 10 g
The herbal granules were provided by the Pharmacy Department of Xiyuan Hospital and manufactured by Kangrentang Pharmaceutical Co., Ltd. (Batch No: 19018391).
SEO Keywords:
Cistanche for chronic prostatitis, herbal remedy for CPPS, TCM for pelvic pain syndrome, Cistanche prostate inflammation, natural anti-inflammatory herbs for men, Cistanche immune modulation.
1.3 Reagents and Kits
BCA Protein Assay Kit (Beyotime, Cat# P0010)
IL-1β and IL-18 ELISA Kits (Bioswamp, Cat# ZN2877, ZN2883)
Pyroptosis/Caspase-1 Assay Kit (Immunochemistry, Cat# 9145)
NLRP3 Antibody (Novus, Cat# NBP2-12446)
CFA (Complete Freund's Adjuvant, Sigma, Cat# C104202)
Common reagents: Xylene, absolute ethanol, neutral gum (Sinopharm)
Oxidative stress kits: SOD, MDA, GSH-Px, LDH (Nanjing Jiancheng, Cat# A001-3, A003-2, BC1190, 20121225)
Antibodies: Anti-GSDMD, Caspase-1, GAPDH (Abcam, Cat# ab219800, ab179515, ab8245)
MCC950 (NLRP3 inhibitor, Selleck, Cat# S8930)
PYCARD/ASC Antibody (Thermo, Cat# PA5-95826)
1.4 Instruments
Optical microscope (Nikon Eclipse Ti-SR, Japan)
Microtome (Leica RM2016, Shanghai)
Digital slide scanner (3D HISTECH Pannoramic MIDI, Hungary)
High-speed refrigerated centrifuge (Thermo Fisher IECCL31R, USA)
Electrophoresis equipment (CAVOY PP-1150, MP-8001, MP-3030, Beijing)
Shaker incubator (Qilinbeier TS-2000A, Jiangsu)
Cryogenic grinder (Jingxin JXFSTPRP-CL-BSC, Shanghai)
CO₂ incubator (Sanyo MCO-20AIC, Japan)
Microplate reader (Bio-Rad DNM-9602, Beijing)
Biochemical analyzer (Rayto Chemray 800, Shenzhen)
Flow cytometer (Beckman CytoFLEX S, USA)
2. Methods
2.1 Model Establishment
(1) Animal Model
Based on previous modeling experience, a rat model of autoimmune Type III chronic prostatitis was established by intraprostatic injection of purified prostate protein extract mixed with Complete Freund's Adjuvant (CFA) [14]. Successful modeling was determined by pathological features including: thickening of the prostate capsule, disappearance of glandular folds, destruction of acini, significant stromal edema, blurred structure, massive infiltration of lymphocytes, monocytes, and plasma cells. Smooth muscle cells and connective tissue hyperplasia were observed in prostatic septa, with noticeable vascular dilation and congestion. Biochemically, the model was confirmed by increased leukocytes and decreased lecithin bodies in prostate homogenate.
(2) Cell Model
Following literature reports and prior pre-experiments [15–17], a two-step stimulation method was used to activate the NLRP3 inflammasome in RWPE-1 cells. Step 1: Cells were treated with LPS (100 ng/mL) for 4 hours to initiate inflammasome priming. Step 2: Cells were then treated with ATP (5 mmol/mL) for 30 minutes as the activation signal. The optimal model conditions were screened using PI uptake rate as a marker.
2.2 Grouping
(1) Animal Study Groups
Rats were randomly divided into five groups using a random number table:
Control group
Model group
Cistanche formula low-dose group
Cistanche formula medium-dose group
Cistanche formula high-dose group
Each group consisted of 10 rats. All groups except the control group were subjected to modeling. The equivalent human dose of the Cistanche-based formula was calculated as 109 ÷ 70 × 6.3 = 9.8 g/kg for the medium dose, with low dose at 4.9 g/kg and high dose at 19.6 g/kg [11]. From day 3 post-modeling, the treatment groups received daily intragastric administration of the respective doses of the Cistanche-based herbal formula for 30 consecutive days, while control and model groups received equal volumes of saline.
(2) Cell Study Groups
RWPE-1 cells were divided into four groups:
Control group: cultured with blank serum
Model group: treated with LPS (100 ng/mL, 4 h) and ATP (5 mmol/mL, 30 min)
Cistanche group: treated with 6.25 μg/mL of Cistanche formula medicated serum
MCC950 group: model + NLRP3 inflammasome inhibitor MCC950
2.3 Sample Collection
After 30 days of treatment, rats were anesthetized with isoflurane. Blood was collected from the abdominal aorta and centrifuged at 3000 rpm for 10 min to isolate serum, which was stored at −80°C for ELISA and biochemical analyses.
Left prostate lobe: fixed in 4% paraformaldehyde for histopathology, immunohistochemistry, and immunofluorescence.
Right prostate lobe: preserved in liquid nitrogen for Western blot analysis.
2.4 Animal Index Detection
2.4.1 HE Staining
Hematoxylin and Eosin (HE) staining was used to observe inflammatory infiltration in prostate tissues. Morphological changes in glands, ducts, and interstitial structures were evaluated under a light microscope.
2.4.2 ELISA for IL-1β and IL-18
ELISA kits were used to quantify IL-1β and IL-18 levels in rat serum. Samples were processed following standard ELISA protocol and analyzed at 450 nm using a microplate reader.
2.4.3 Biochemical Detection of MDA, SOD, GSH-Px
Serum levels of MDA, SOD, and GSH-Px were measured using commercial kits to assess oxidative stress in prostate tissues.
2.4.4 Immunohistochemistry (IHC) for NLRP3
Prostate tissue sections underwent deparaffinization, antigen retrieval, blocking, and incubation with anti-NLRP3 antibody (1:1000). DAB staining was performed and counterstained with hematoxylin. NLRP3 expression was observed microscopically.
2.4.5 Immunofluorescence for GSDMD
Frozen prostate sections were incubated with anti-GSDMD antibody (1:100), followed by fluorescent secondary antibody and DAPI nuclear counterstain. GSDMD expression was visualized using fluorescence microscopy.
2.4.6 Western Blot for NLRP3, Caspase-1, GSDMD
Protein was extracted from prostate tissues for Western blot analysis. Expression levels of NLRP3 (1:1000), GSDMD (1:2000), and Caspase-1 (1:2000) were detected. β-actin served as the internal control. Quantification was performed using ImageJ software.
2.5 Cellular Index Detection
2.5.1 Flow Cytometry for PI Uptake
Cells were washed, resuspended at 1×10⁶ cells/mL, and stained with propidium iodide (PI). PI uptake rates were analyzed using flow cytometry and FlowJo software.
2.5.2 LDH Release Assay
After treatment, LDH levels in culture supernatants were assessed as a marker of cell membrane damage using a colorimetric assay at 490 nm.
2.5.3 Flow Cytometry for Caspase-1
Cells were fixed, permeabilized, and stained with Caspase-1 antibody (1:1000). Expression was measured by flow cytometry.
2.5.4 ELISA for IL-1β and IL-18 in Supernatant
Culture media were collected to detect IL-1β and IL-18 levels via ELISA. Protein content in cell lysates was quantified using the BCA assay to normalize cytokine levels.
2.5.5 Western Blot for NLRP3, Caspase-1, GSDMD
Western blot was conducted on cell lysates with antibodies against NLRP3 (1:1000), GSDMD (1:2000), and Caspase-1 (1:2000). Detection was performed using ECL and imaging systems. β-actin was used as the loading control.
2.6 Statistical Analysis
Data were analyzed using GraphPad Prism 8.0. Results are expressed as mean ± SD. Group comparisons were made using independent-sample t-tests or One-Way ANOVA. A P-value < 0.05 was considered statistically significant.
