Regulatory Effects Of Bixie Qianlie’an Pill On The TLR4/NF-κB/NLRP3 Pathway And Pyroptosis in A Rat Model Of Chronic Nonbacterial Prostatitis
Nov 27, 2025
Results
3.1 Histopathological changes in prostate tissue of CNP rats
Model evaluation was performed 1 week after modeling. In the model group, lymphocyte, polymorphonuclear neutrophil, and plasma cell infiltration was observed within the prostate, along with vascular dilation and congestion. In the blank and sham-operated groups, no obvious inflammatory cell infiltration was found 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-operated and blank groups were of normal gland size, with a moist gray-red surface, transparent glandular lumina, soft texture, smooth surface, and loose attachment to the seminal vesicles, allowing easy separation. In contrast, the model group exhibited prostate swelling, a gray-yellow surface without visible transparent lumina, a firmer texture, a rough, uneven surface, and adhesions to the seminal vesicles that were difficult to separate. Compared with the model group, all Bixie Qianlie'an Pill (BQAP) dose groups showed improvements in swelling and color; the surface had fewer granular changes, the texture was softer and elastic, and separation from the seminal vesicles was easier.
HE staining showed that, compared with the sham-operated group, the model group had lymphocyte and a small number of plasma cell infiltration within the prostatic stroma, cellular edema, proliferative stromal connective tissue, and vascular congestion. In the BQAP low-, medium-, and high-dose groups, only a small to minimal degree of lymphocyte infiltration was observed, and cellular morphology and arrangement were more regular (see Figure 2).
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3.2 Changes in prostate index after drug intervention in CNP rats
Compared with the sham-operated group, the prostate index of the model group was increased (p<0.05). After intervention with different doses of BQAP, the prostate index was reduced versus the model group; the reduction was significant in the medium-dose BQAP group (p<0.05), while the effects in the low- and high-dose groups were not significant.
3.3 Effects of drug intervention on serum proinflammatory factors IL-1β, IL-18, and TNF-α in CNP rats
Compared with the sham-operated group, serum IL-1β, IL-18, and TNF-α levels were significantly elevated in the model group (p<0.05). Relative to the model group, BQAP at all doses reduced IL-1β, IL-18, and TNF-α to varying degrees (p<0.05), with the high-dose group showing a more marked effect (see Table 2 and Figure 3).
3.4 Distribution and expression of NLRP3 in prostate tissue of CNP rats after drug intervention
Compared with the sham-operated group, NLRP3 expression was increased in the model group (p<0.05). NLRP3 expression in the BQAP low-, medium-, and high-dose groups was significantly lower than in the model group (p<0.05) (see Figures 4 and 5).
3.5 Immunofluorescence results for GSDMD in prostate tissue of CNP rats after drug intervention
Compared with the sham-operated group, GSDMD fluorescence intensity in the prostate tissue of model rats was increased. The BQAP low-, medium-, and high-dose groups showed significantly lower GSDMD fluorescence intensity than the model group (p<0.05) (see Figures 6 and 7).
3.6 Effects of drug intervention on TLR4, MyD88, NF-κB p65, p–NF-κB p65, NLRP3, Caspase-1, and GSDMD protein expression in prostate tissue of CNP rats
Compared with the sham-operated group, the model group showed increased expression of TLR4, MyD88, NF-κB p65, p–NF-κB p65, NLRP3, Caspase-1, and GSDMD proteins (p<0.05). Relative to the model group, BQAP at different doses downregulated these proteins in prostate tissue to varying degrees (p<0.05), with a more pronounced effect in the high-dose group (see Table 3 and Figure 8).
Discussion
CNP is a common and frequently occurring disease of the male genitourinary system, particularly prevalent among young and middle-aged men [9]. Although its exact etiology remains incompletely understood, inflammatory responses play a central role in its onset and progression, involving factors such as immune responses, neural plasticity, and endocrine dysregulation [10].
In this study, BQAP significantly attenuated inflammatory responses in a rat model of chronic nonbacterial prostatitis by modulating the TLR4/NF-κB/NLRP3 signaling pathway. This pathway is an important inflammation-mediating axis in chronic prostatitis, participating in the regulation of proinflammatory cytokine production [11]. Studies have shown increased expression and activity of TLR4, NF-κB, and NLRP3 in prostatic fluid from CP patients [12]. The TLR4 receptor recognizes endogenous and exogenous danger signals to activate MyD88, thereby initiating NF-κB signaling and promoting the release of proinflammatory factors such as IL-1β, IL-18, and TNF-α, which further exacerbate local inflammation and tissue injury [13]. Moreover, NF-κB and the NLRP3 inflammasome play key roles in immune cell activation, cytokine production, and cell death [14–15]. Acupuncture has been shown to modulate TLR4, NF-κB, and NLRP3 protein expression, thereby alleviating inflammatory responses in CP [16].
Cytokines are pivotal in the immunopathological process of CNP [17]. They are a major cause of sustained inflammatory immune cell infiltration in prostatic tissue [18]; their concentrations are markedly elevated in autoimmune prostatitis mouse models, and lowering their levels can alleviate inflammatory infiltration and pain symptoms [19]. Our findings show that BQAP significantly reduced serum IL-1β, IL-18, and TNF-α, consistent with reduced inflammatory cell infiltration and improved pathology in the prostate. IL-1β, IL-18, and TNF-α are prototypical proinflammatory cytokines with crucial roles in the onset and development of prostatitis [20]. Activated NLRP3 inflammasomes promote the maturation of IL-1β and IL-18, further amplifying immune responses and worsening tissue damage [21]. He Tao et al. [22] confirmed that high NLRP3 expression leads to massive secretion of IL-1β and IL-18, thereby promoting inflammatory progression and aggravating clinical symptoms in CP.
The therapeutic effects of BQAP may be mediated by suppressing activation of the TLR4/NF-κB/NLRP3 pathway, downregulating TLR4, MyD88, NF-κB p65, p–NF-κB p65, NLRP3, Caspase-1, and GSDMD in prostatic tissue, and thereby mitigating inflammation and pyroptosis. Pyroptosis is an inflammatory form of programmed cell death mediated by the gasdermin family, characterized by pore formation in the cell membrane and the release of IL-1β and IL-18, which intensify inflammation [23–24], and is of great importance in inflammatory and immune regulation [25]. Studies have shown that ursolic acid can attenuate CP by regulating the NLRP3 inflammasome–mediated Caspase-1/GSDMD pathway, suppressing pyroptosis-related proteins NLRP3, Caspase-1, ASC, and GSDMD, and reducing levels of IL-1β, IL-18, and lactate dehydrogenase [26]. In our study, NLRP3, Caspase-1, and GSDMD protein expression in prostate tissue was significantly decreased in the BQAP groups, suggesting that BQAP may alleviate inflammatory injury in CP by inhibiting inflammasome activation and reducing pyroptosis.
Traditional Chinese medicine (TCM) has unique advantages and notable efficacy in treating CP [27]. BQAP is formulated to tonify kidney qi, clear heat and drain dampness, resolve blood stasis, and unblock collaterals. Its pill form exerts effects more gradually, aligning with the chronic course of the disease. In this formula, Dioscoreae Hypoglaucae Rhizoma (Bixie) promotes diuresis and separates the turbid from the clear; Plantaginis Semen and Abutili Semen clear heat and drain dampness and promote urination-together serving as sovereign drugs to clear damp-heat in the lower jiao and address the branch (symptoms). Rehmanniae Radix Preparata nourishes kidney yin and replenishes essence; Cuscutae Semen tonifies liver and kidney and secures essence and reduces urination-combined with heat-clearing, damp-draining herbs to expel pathogens and tonify the kidney, ensuring that once the pathogen is removed, the kidney is stabilized. Astragali Radix benefits qi, raises yang, and promotes diuresis; Hirudo activates blood and unblocks channels. The combination of Astragalus and Hirudo supplements qi without retaining pathogens and disperses blood stasis without harming the healthy qi, serving as ministerial drugs. Phellodendri Cortex is bitter and cold, enters the kidney channel, clears heat and dries dampness, and reduces ministerial fire of the lower jiao, assisting the sovereign herbs. Succinum resolves stasis, unblocks collaterals, clears heat, and promotes diuresis. Epimedii Herba and Cnidii Fructus warm and tonify kidney yang, dispel wind and dampness, and prevent excessive cold bitterness from damaging healthy qi, serving as adjuvant drugs. Achyranthis Bidentatae Radix not only invigorates blood, promotes diuresis, and relieves strangury, but also guides the formula downward, acting both as adjuvant and envoy, thereby achieving significant clinical efficacy. This study explored BQAP's mechanism in alleviating CNP via modulation of the TLR4/NF-κB/NLRP3 pathway, providing theoretical support for its clinical application. By reducing inflammatory responses in prostatic tissue and lowering proinflammatory cytokines, BQAP achieves therapeutic effects in CP.
Although this study reveals the potential mechanism of BQAP in treating CNP, due to the complexity of multi-component TCM formulas and their multi-target, multi-pathway actions, particularly in modulating immune tolerance and antioxidant stress–related mechanisms, further experimental studies and clinical validation are needed for deeper understanding.
In summary, BQAP alleviates inflammatory responses in CNP rats by modulating the TLR4/NF-κB/NLRP3 signaling pathway and may exert therapeutic effects by inhibiting pyroptosis. This finding provides new theoretical evidence for its clinical application, though its mechanisms warrant further in-depth investigation.
References
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