Cistanche‑Centered Formula Mitigates CNP Via TLR4/NF‑κB/NLRP3 Modulation And Pyroptosis Inhibition: Evidence From A Rat Model

3 Results

3.1 Histopathological changes in prostate tissue of CNP rats

Model assessment was performed 1 week after induction. In the model group, lymphocyte, segmented neutrophil, and plasma cell infiltration was observed within the prostate, accompanied by vascular dilation and congestion. In contrast, the blank and sham-operation groups showed no obvious inflammatory cell infiltration in the prostatic stroma; cells were regularly arranged with normal morphology, indicating successful establishment of the CNP rat model (see Figure 1).

After 30 consecutive days of gavage, the prostates of the sham-operation and blank groups were normal in size, with a moist gray-red surface, visible transparent glandular lumens, soft texture, smooth surface, and loose attachment to the seminal vesicles, allowing easy separation. In the model group, the prostate was swollen, with a gray-yellow surface, no visible transparent lumens, a tougher texture, a rough and uneven surface, and adhesions to the seminal vesicles that were difficult to separate. In all dose groups of Bixie Qianlie An Wan, compared with the model group, swelling and coloration improved; the surface showed fewer granular changes, the texture was softer and elastic, and separation from the seminal vesicles was easier.

H&E staining showed that, compared with the sham-operation group, the model group exhibited lymphocyte and a small number of plasma cell infiltration in the prostatic stroma, with cellular edema, stromal fibrous hyperplasia, and vascular congestion. In the low-, medium-, and high-dose Bixie Qianlie An Wan groups, only a small or even minimal degree of lymphocyte infiltration was observed, with more regular cellular morphology and arrangement (see Figure 2).

 

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3.2 Changes in prostate index after drug intervention in CNP rats

Compared with the sham-operation group, the prostate index of model rats increased (P<0.05). After intervention with different doses of Bixie Qianlie An Wan, the prostate index decreased versus the model group, with a significant reduction in the medium-dose group (P<0.05), while the low- and high-dose groups showed no significant effects on the prostate index.

3.3 Effects of drug intervention on proinflammatory cytokines IL-1β, IL-18, and TNF-α in CNP rats

Compared with the sham-operation group, serum levels of IL-1β, IL-18, and TNF-α were significantly elevated in the model group (P<0.05). Relative to the model group, all doses of Bixie Qianlie An Wan reduced IL-1β, IL-18, and TNF-α to varying degrees (P<0.05), with more pronounced effects in the high-dose group (see Table 2 and Figure 3).

 

3.4 Distribution and expression of NLRP3 in prostatic tissue after drug intervention

Compared with the sham-operation group, expression of NLRP3 was increased in CNP model rats (P<0.05). In the low-, medium-, and high-dose Bixie Qianlie An Wan groups, NLRP3 expression was significantly lower than in the model group (P<0.05) (see Figures 4 and 5).

3.5 Immunofluorescence of GSDMD in prostatic tissue after drug intervention

Compared with the sham-operation group, GSDMD fluorescence intensity in the prostate increased in CNP model rats. In the low-, medium-, and high-dose Bixie Qianlie An Wan groups, GSDMD fluorescence intensity was significantly lower than in the model group (P<0.05) (see Figures 6 and 7).

 

3.6 Effects on TLR4, MyD88, NF-κBp65, p‑NF‑κBp65, NLRP3, Caspase‑1, and GSDMD protein expression in prostatic tissue after drug intervention
Compared with the sham-operation group, protein expression of TLR4, MyD88, NF‑κBp65, p‑NF‑κBp65, NLRP3, Caspase‑1, and GSDMD was elevated in the CNP model group (P<0.05). Versus the model group, Bixie Qianlie An Wan at all doses downregulated these proteins to varying degrees in prostatic tissue (P<0.05), with the most pronounced effects in the high-dose group (see Table 3 and Figure 8).

 

4 Discussion

Chronic nonbacterial prostatitis (CNP) is a common and frequently occurring condition of the male genitourinary system, with a higher incidence in young and middle-aged men. Although its exact etiology remains incompletely understood, inflammatory responses play a central role in the onset and progression of CNP, involving multiple factors such as immune responses, neural plasticity, and endocrine dysregulation.

In this study, Bixie Qianlie An Wan significantly alleviated inflammation in a rat model of CNP by modulating the TLR4/NF‑κB/NLRP3 signaling pathway. This pathway is a key inflammatory mediator axis in chronic prostatitis, regulating the production of proinflammatory cytokines that drive inflammatory responses. Elevated expression and activity of TLR4, NF‑κB, and NLRP3 have been observed in expressed prostatic secretions from CP patients. TLR4 recognizes endogenous and exogenous danger signals to activate MyD88, thereby initiating NF‑κB signaling and releasing proinflammatory cytokines such as IL‑1β, IL‑18, and TNF‑α, which further intensify local inflammation and promote tissue injury. NF‑κB and the NLRP3 inflammasome also play pivotal roles in immune cell activation, cytokine production, and inflammatory cell death. Prior studies have shown that acupuncture can modulate TLR4, NF‑κB, and NLRP3 expression, thereby mitigating inflammation in CP.

Cytokines are key drivers in the immunopathology of CNP. They sustain inflammatory immune cell infiltration in prostate tissue; levels are significantly elevated in autoimmune prostatitis mouse models, and reduction of these cytokines alleviates inflammatory infiltration and pain. In our study, Bixie Qianlie An Wan markedly lowered serum IL‑1β, IL‑18, and TNF‑α, consistent with reduced inflammatory cell infiltration and improved histopathology in the prostate. IL‑1β, IL‑18, and TNF‑α are canonical proinflammatory cytokines with crucial roles in the pathogenesis of prostatitis. Activated NLRP3 inflammasomes promote the maturation of IL‑1β and IL‑18, further amplifying immune responses and exacerbating tissue damage. Prior work (e.g., He Tao et al.) confirmed that high NLRP3 expression leads to abundant secretion of IL‑1β and IL‑18, thereby accelerating inflammatory progression and worsening clinical symptoms in CP.

The intervention effect of Bixie Qianlie An Wan may be mediated by suppressing activation of the TLR4/NF‑κB/NLRP3 pathway, downregulating TLR4, MyD88, NF‑κBp65, p‑NF‑κBp65, NLRP3, Caspase‑1, and GSDMD in prostatic tissue, thereby reducing inflammatory responses and pyroptosis. Pyroptosis is an inflammatory form of programmed cell death mediated by the gasdermin family, characterized by membrane pore formation and release of IL‑1β and IL‑18, which amplifies inflammation and has important implications for inflammatory and immune regulation. For example, ursolic acid can alleviate CP by modulating the NLRP3 inflammasome–mediated Caspase‑1/GSDMD pathway, reducing expression of NLRP3, Caspase‑1, ASC, and GSDMD, and lowering IL‑1β, IL‑18, and LDH levels. In our study, NLRP3, Caspase‑1, and GSDMD proteins were significantly decreased in the prostates of the Bixie Qianlie An Wan groups, suggesting attenuation of inflammasome activation and pyroptosis as a mechanism for reducing inflammatory injury in CP.

Traditional Chinese medicine (TCM) offers unique advantages and notable efficacy in the management of CP. Bixie Qianlie An Wan is indicated for tonifying kidney qi, clearing heat and draining dampness, resolving blood stasis, and unblocking collaterals. Its gradual, sustained action matches the typically protracted course of CP. In line with needs of herbal supplement development, we highlight Cistanche as the principal botanical for translational focus in this formula. In TCM theory, the original compositional roles include damp-draining and turbidity-resolving herbs (e.g., Bi Xie), heat-clearing and diuretic agents (e.g., Plantago seed, Malva seed), and kidney-tonifying components (e.g., Rehmannia, Cuscuta), complemented by qi-supplementing and blood-activating elements (e.g., Astragalus, leech), and heat-clearing assistants (e.g., Phellodendron), among others. For product development purposes, a Cistanche-centered composition is a practical emphasis, with supportive co-botanicals selected to target lower-jiao damp-heat, microcirculatory stasis, and inflammatory signaling.

This study explored how Bixie Qianlie An Wan mitigates CNP by modulating the TLR4/NF‑κB/NLRP3 pathway, reducing prostatic inflammation and lowering proinflammatory cytokine levels, thereby achieving therapeutic effects in CP. While we elucidated potential mechanisms, the multi-component, multi-target nature of herbal formulas warrants further experiments and clinical validation, especially regarding immunotolerance, antioxidative stress, and related regulatory mechanisms.

In summary, Bixie Qianlie An Wan alleviates inflammation in CNP rats by regulating the TLR4/NF‑κB/NLRP3 signaling pathway and may exert therapeutic effects by inhibiting pyroptosis. This provides new theoretical support for its clinical application, yet deeper mechanistic studies are still needed. For readers engaged in herbal supplement development, we recommend prioritizing Cistanche as the lead botanical in future composition and standardization efforts targeting this pathway.

 

References

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