Mechanism of Kushen Tongguan Pill on benign prostatic hyperplasia: roles of TLR4/NF-κB pathway and macrophage M1 polarization.

objectiveTo investigate the therapeutic effect of Kushen Tongguan Pill (KSTG) on benign prostatic hyperplasia (BPH) induced by castration combined with testosterone propionate in rats, and to explore its underlying mechanism associated with the Toll-like receptor 4/nuclear factor κB (TLR4/NF-κB) signaling pathway.

methodsA rat BPH model was established by castration combined with subcutaneous injection of testosterone propionate. Rats were randomly divided into sham group, BPH group, and low-, medium-, and high-dose KSTG groups (2.52, 5.04, and 10.08 g/kg). After 4 weeks of intervention, prostate wet weight was weighed and prostate index was calculated； histopathological changes were observed by hematoxylin-eosin (HE) staining; collagen deposition and fibrosis were evaluated by Masson's trichrome staining; the expression of α-smooth muscle actin (α-SMA) was detected by immunohistochemistry; serum testosterone (T), estradiol (E2), and dihydrotestosterone (DHT) levels were measured by enzyme-linked immunosorbent assay (ELISA); peripheral white blood cell and neutrophil counts were measured using an automated hematology analyzer; toluidine Blue staining was performed to observe mast cell infiltration in prostate tissue to assess local chronic inflammation. RT-qPCR was used to detect the mRNA expression of key pathway molecules including TLR4, NF-κB p65, IKKα, and IKKβ, as well as inflammatory markers TNF-α, IL-1β, MCP-1, and iNOS in prostate tissue. Western blotting was performed to analyze the protein expression of key pathway molecules including TLR4, MyD88, IκBα, p-IκBα, NF-κB p65, and p-NF-κB p65, as well as the inflammatory marker iNOS in prostate tissue. For in vitro experiments, KSTG-containing serum was used to treat LPS/IFN-γ-induced THP-1-derived macrophages. The mRNA expression of M1 polarization markers (CD86, TNF-α, IL-6) in macrophages was determined by RT-qPCR. Subsequently, the conditioned medium from each macrophage group was collected, and its effect on WPMY-1 cell proliferation was evaluated by CCK-8 assay.

resultsCompared with the BPH group, KSTG dose-dependently reduced prostate volume, wet weight, and prostate index (P < 0.05) and improved the pathological morphology of glandular hyperplasia and interstitial thickening. KSTG dose-dependently reduced serum E2 levels and the E2/T ratio, and significantly decreased the abnormally elevated serum DHT levels (P < 0.05). KSTG also reduced peripheral white blood cell and neutrophil counts, downregulated the mRNA expression of TNF-α and IL-1β, and decreased the protein expression of iNOS in prostate tissue (P < 0.05). Toluidine blue staining further revealed that KSTG treatment reduced mast cell infiltration in the prostate, corroborating the alleviation of local inflammation (P < 0.05). Western blotting showed that KSTG significantly inhibited the protein expression of TLR4 and MyD88 in prostate tissue and suppressed the phosphorylation of IκBα and NF-κB p65 (P < 0.05). In vitro, KSTG-containing serum inhibited the mRNA expression of M1 polarization markers CD86, TNF-α, and IL-6 in THP-1-derived macrophages (P< 0.05). Moreover, the conditioned medium of macrophages pretreated with KSTG significantly attenuated the pro-proliferative effect on WPMY-1 cell proliferation (P < 0.05).

conclusionThis study has demonstrates that the KSTG Pill can significantly regulate M1 macrophage polarization to alleviate the prostatic hyperplasia and related hormonal level in the BPH rats. Its therapeutic mechanism is closely related to suppression of aberrant activation of the TLR4/MyD88/NF-κB signaling pathway in the prostate tissue, thereby reducing the polarization of macrophages to the M1 phenotype and the release of downstream pro-inflammatory factors. These findings provides experimental support for its clinical application, and and valuable insights for the development of novel BPH treatment strategies targeting the immune-inflammatory microenvironment.