Cistanche Aerial-Part Flavonoids Ameliorate Chronic Prostatitis: Rat Validation, Cytokine Modulation, And PI3K–AKT–EGFR–MMP9 Mechanisms
Nov 06, 2025
2.3.1 Animal grouping and model induction
After one week of acclimatization, male SD rats were randomly divided into four groups (n = 6 per group): sham-operated group, model group, positive-drug group (Qianliekang Pule'an tablets), and Cistanche aerial-part alcoholic extract group. Following the "Draft Specification for the Preparation of Animal Models of Chronic Prostatitis" [20], 1% carrageenan in normal saline was injected into the seminal vesicles to induce chronic prostatitis in the model, treatment, and positive-drug groups. The sham group received an equal volume of normal saline. Dosing began after a 3-day recovery.
cistanche extract supplements for chronic prostatitis
2.3.2 Drug administration
The Cistanche aerial-part extract was administered at 63 mg/(kg·d). The positive drug was given at 1.05 g/(kg·d). The vehicle was 0.5% CMC-Na, and the gavage volume was 1 mL per rat. The sham and model groups received an equivalent volume of vehicle. Administration was performed once daily at a fixed time for 28 consecutive days.
2.3.3 Body weight and prostate index
Body weight was recorded weekly. At 24 h after the last dose, rats were anesthetized; prostates were excised and weighed to calculate the prostate index.
Prostate index (%) = prostate mass (g) / body mass (g) × 100%
Results: No significant differences were observed among groups in body weight or prostate mass. The model group showed an increased prostate index versus the sham group (P < 0.05). Both the positive-drug group and the Cistanche extract group exhibited reduced prostate indices compared with the model group (P < 0.05), see Table 3.
Cistanche Introduction for Chronic Prostatitis
2.3.4 Serum inflammatory cytokines by ELISA
At 24 h after the last administration, rats were anesthetized with 20% urethane aqueous solution. Blood was collected from the abdominal aorta, centrifuged, and serum stored at −80°C. Following ELISA kit instructions, serum levels of DHT, IFN-γ, IgG, IL-6, PSA, and TNF-α were determined.
Compared with the sham group, the model group had elevated DHT, IFN-γ, IgG, IL-6, PSA, and TNF-α (P < 0.05, P < 0.01). Versus the model group, the Cistanche aerial-part extract group showed decreased levels of DHT, IFN-γ, IgG, IL-6, PSA, and TNF-α (P < 0.05, P < 0.01), indicating a favorable therapeutic effect against chronic prostatitis; see Figure 4.
2.3.5 HE staining for prostatic histopathology
Prostate tissues were fixed in tissue fixative for more than 24 h, dehydrated, paraffin-embedded, sectioned, and stained with hematoxylin–eosin (HE). Under a light microscope, glandular morphology, stromal inflammatory cell infiltration, and fibrous hyperplasia were recorded.
Macroscopic and histological results after 28 days:
Sham group: prostates were ruddy, soft, and elastic.
Model group: prostates were dark red, firmer, and less elastic.
Cistanche extract and positive-drug groups: notable improvements compared with the model group.
HE staining showed:
Sham: intact glands without atrophy; single-layer epithelial cells intact without hyperplasia; no inflammatory infiltration in interglandular spaces.
Model: marked glandular deformation and atrophy; epithelial exfoliation and hyperplasia; abundant inflammatory cell infiltration in the stroma.
Cistanche extract and positive-drug groups: improved glandular morphology, reduced inflammatory infiltration, and inhibited epithelial hyperplasia; see Figure 5.
2.3.6 Statistical analysis
Data were processed with SPSS 23.0 and expressed as x̄ ± s. Two-group comparisons used the t-test. P < 0.05 was considered statistically significant.
3 Discussion
Chronic prostatitis is a common genitourinary condition with an incidence of up to 8% among men under 50 years worldwide, often presenting with lower urinary tract symptoms and pelvic pain [21]. Its pathogenesis remains incompletely defined; proposed contributors include occult microbial infection, autoimmunity, oxidative stress, and endocrine dysregulation. Current clinical management relies mainly on Western medicine such as α-receptor blockers, NSAIDs, and quinolone antibiotics [22]. Flavonoids from the aerial parts of Cistanche also exhibit therapeutic benefits for chronic prostatitis. In this study, network pharmacology was used to explore mechanisms of anti–chronic prostatitis activity of aerial-part flavonoids from Cistanche, followed by animal validation.
UPLC-Q-Exactive Orbitrap-MS identified 44 flavonoids in the alcoholic extract of Cistanche aerial parts. Database screening yielded 19 active components, 956 disease targets, 138 compound targets, and 88 intersecting targets. Molecular docking was performed between the top 10 PPI-ranked targets and the active components. The five best-scoring pairs were spinacetin–EGFR, liquiritigenin–MMP2, ononin (maackiain glycoside)–MMP9, spinacetin–MMP9, and liquiritigenin–PTGS2. Spinacetin is a major isoflavonoid-related flavone with antioxidant and anti-infective pharmacology, capable of attenuating inflammation and oxidative stress and reducing apoptosis [23]. Liquiritigenin is a dihydroflavonoid that downregulates pro-inflammatory cytokines to exert anti-inflammatory effects [24]. Ononin can reduce downstream pro-inflammatory mediators in diseased tissues and shows antitumor potential [25]. KEGG enrichment implicated PI3K–AKT, EGFR tyrosine kinase inhibitor resistance, and other pathways in the anti–chronic prostatitis action of Cistanche aerial-part flavonoids.
EGFR is a transmembrane receptor critical for regulating cell growth and epithelial integrity [26]. Activated EGFR promotes prostate cell proliferation via downstream pathways including PI3K–AKT; aberrant proliferation may drive stromal and fibrous hyperplasia and abnormal prostatic growth in rats [27]. AKT1, an AKT isoform, is pivotal in proliferation–apoptosis balance, metabolism, and genomic stability [28], with activation largely dependent on the PI3K–AKT pathway. Upon extracellular stimuli binding to membrane receptors, the pathway activates AKT1 to execute its functions [29]. In prostatitis, AKT1 activation promotes production of inflammatory cytokines that further stimulate local cells, causing pain, swelling, tissue damage, and fibrosis. Inhibition of AKT1 expression can mitigate chronic prostatitis [30]. MMP9 is a zinc-dependent endopeptidase that degrades diverse extracellular matrix components [31]. In chronic prostatitis, MMP9 overexpression disrupts tissue architecture, compromises acinar integrity, and facilitates inflammatory cell migration from perivascular regions to lesion sites, expanding inflammation. MMP9 can interact with the PI3K–AKT pathway in multiple ways, and PI3K–AKT activation can upregulate MMP9 expression. We infer that Cistanche aerial-part flavonoids act against chronic prostatitis by regulating EGFR, AKT1, and MMP9 via the PI3K–AKT pathway.
To validate efficacy, carrageenan-induced seminal vesicle injections established the model, followed by treatment. Compared with the model group, the Cistanche group showed decreased serum DHT, IFN-γ, IgG, IL-6, PSA, and TNF-α; improved glandular morphology; and reduced inflammatory cell infiltration.
In summary, flavonoids from the aerial parts of Cistanche act against chronic prostatitis through multi-component, multi-target, and multi-pathway mechanisms, likely regulating EGFR, AKT1, and MMP9 via PI3K–AKT signaling. The present work verified the efficacy of these flavonoids and preliminarily elucidated active constituents and mechanisms, providing a reference for clarifying the pharmacodynamic material basis and mechanism of action of Cistanche aerial parts.
References
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[6]–[19] (Analytical methodology references for UPLC/ESI/Orbitrap/Q-TOF and flavonoid identification; same numbering and journal details as the source, adapted for context consistency.)
[20] Chinese Association of Traditional Chinese Medicine, Professional Committee of Experimental Pharmacology of Chinese Medicine. Draft specification for the preparation of animal models of chronic prostatitis [J]. China Journal of Experimental Traditional Medical Formulae, 2018, 24(19): 10–14.
[21]–[31] Correspond to the cited literature on epidemiology, therapeutics, and molecular targets (EGFR, AKT1, MMP9) as in the source list.
