Astragaloside IV Attenuates Chronic Prostatitis by Activating Keap1/Nrf2/HO-1 Pathway: Suppressing Ferroptosis and Enhancing Antioxidant Defense

Abstract

Background: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), a chronic inflammatory disorder with complex etiology and limited treatment options, is closely associated with oxidative stress and regulates cell death. Ferroptosis-an iron-dependent cell death driven by lipid peroxidation-amplifies CP/CPPS inflammation by concurrently triggering mitochondrial apoptosis and NLRP3 inflammasome activation, while Keap1/Nrf2/HO-1 axis serves as a central regulator bridging ferroptotic, apoptotic, and inflammatory cell death pathways. Astragaloside IV (AS-IV), a primary bioactive component of Astragalus membranaceus with established clinical use in urological therapies and favorable pharmacokinetics, was prioritized over structural analogs due to its unique dual-phase modulation: enhancing Nrf2 nuclear translocation without suppressing NF-κB-mediated immune surveillance. However, regulatory mechanisms linking AS-IV to ferroptosis inhibition in CP/CPPS remain unknown.

Patients and methods: This study aimed to investigate the therapeutic potential of astragaloside IV (the primary bioactive component of Astragalus membranaceus) for the treatment of CP/CPPS by suppressing ferroptosis via the Keap1/Nrf2/HO-1 pathway. A rat CP/CPPS model was established using complete Freund's adjuvant (CFA), with animals divided into normal control, EAP, and AS-IV high/medium/low-dose groups and treated daily for four weeks. Additionally, a human prostatic epithelial cell (RWPE-1) inflammation model was induced by lipopolysaccharide (LPS), and cells were categorized into control, LPS, AS-IV medium-dose, ferroptosis inhibitor, and Nrf2 inhibitor + AS-IV medium-dose groups.

Results: AS-IV ameliorated prostatic tissue inflammation and fibrosis, reduced lipid peroxidation marker malondialdehyde (MDA) levels, and enhanced antioxidant indicators, including glutathione (GSH) content and glutathione peroxidase 4 (GPX4) activity. Western blotting and immunohistochemical analyses further confirmed that AS-IV activated the antioxidant pathway by suppressing Keap1 expression, promoting Nrf2 nuclear translocation, and upregulating heme oxygenase-1 (HO-1) protein levels. Concurrently, pro-inflammatory cytokine levels, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), were markedly reduced.

Conclusion: This is the first study to demonstrate that AS-IV alleviates type III CP pathological damage by inhibiting ferroptosis via the Keap1/Nrf2/HO-1 axis, thereby providing experimental evidence for the development of multi-target therapeutic strategies based on natural products.

Keywords: Nrf2; cellular redox balance; lipid peroxidation; oxidative stress; pharmacological activation; prostatic inflammation.

Figure 1

Astragaloside IV treatment alleviates EAP-associated prostatitis. (A) Schematic of the experimental protocol for Astragaloside IV administration in EAP rat models. (B) Representative prostate tissue sections from control, EAP model, and Astragaloside IV-treated groups. (C) Hematoxylin and eosin (H&E) staining showed that the infiltrating inflammatory cells in prostate tissues was significantly reduced in EAP rat after AS-VI treatment. (D) Histopathological scoring comparison between EAP model and AS-IV treatment groups. Data presented as mean ± SD. Statistical analysis performed using Kruskal–Wallis non-parametric test. Significance levels: ns (not significant) P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Figure 2

Astragaloside IV treatment enhanced Nrf2 nuclear translocation. (A) Western blot analysis of GPX4, Keap1, Nrf2, and HO-1 protein expression in prostate tissues from EAP model and AS-IV-treated rats. (B–E) Quantitative analysis of GPX4, Keap1, Nrf2, and HO-1 protein expression levels via Western blot. (F–I) Serum biomarkers analysis: Glutathione (GSH), superoxide dismutase (SOD), iron ion levels, and lactate dehydrogenase (LDH) activity. (J and K) Enhanced Nrf2 immunoreactivity demonstrated by immunohistochemical (IHC) and immunofluorescence (IF) staining in AS-IV-treated group. Data represent mean ± SD. Statistical significance determined by one-way ANOVA (B–I). Significance thresholds: ns (not significant) P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Figure 3

LPS induces inflammatory response and ferroptosis in RWPE-1 cells. (A) Western blot analysis of TNF-α, IL-1β and FTH1 expression in LPS-stimulated RWPE-1 cells with/without AS-IV treatment. (B–D) Quantitative analysis of FTH1, TNF-α, and IL-1β protein expression via Western blot. (E and F) Intracellular antioxidant capacity: Glutathione (GSH) levels and superoxide dismutase (SOD) activity. (G) Representative fluorescence images showing intracellular ROS accumulation. (H) Quantitative measurement of ROS fluorescence intensity. Data presented as mean ± SD. Statistical comparisons performed using one-way ANOVA (B-F, H). Significance designations: ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Figure 4

Astragaloside IV attenuates oxidative stress and inhibits ferroptosis in RWPE-1 cells. (A) Western blot analysis of ferritin heavy chain 1 (FTH1) expression in LPS-stimulated RWPE-1 cells with/without AS-IV treatment. (B) Quantitative analysis of FTH1 protein expression via Western blot. (D–G) Biochemical profiling showing lactate dehydrogenase (LDH) release, glutathione (GSH) content, superoxide dismutase (SOD) activity, and iron ion concentrations. (H) Intracellular ROS accumulation visualized through fluorescence microscopy. Data represent mean ± SD. Statistical significance determined by one-way ANOVA (B–G). Significance thresholds: **P<0.01; ***P<0.001; ****P<0.0001.

Figure 5

Continued.

Figure 5

ML385 pretreatment abrogates AS-IV mediated ferroptosis suppression via Nrf2 signaling in RWPE-1 cells. (A) Cell viability assessment under combinatorial drug treatments in LPS-stimulated RWPE-1 cells. (B–D) Biochemical profile analysis: LDH release, iron ion concentration, and GSH content across experimental groups (LPS, AS-IV, ferroptosis inhibitor, ML385+AS-IV). (E) Representative fluorescence imaging of intracellular ROS accumulation. (F) Western blotting assay to measure the protein levels of Nrf2, Keap1, XCT, HO-1, GPX4 and TNF-α in LPS-induced RWPE-1, AS-VI-treated, ferroptosis inhibitor and Nrf2 inhibitor + medium-dose AS-IV. (G–L) Quantitative Western blot analysis of ferroptosis-related proteins and inflammatory factors. (M–P) qRT-PCR analysis of Nrf2 pathway-related gene expression. (Q–T) Subcellular localization analysis via immunofluorescence staining. (U) Co-localization analysis of Keap1-Nrf2 interaction. Data represent mean ± SD. Statistical comparisons performed using one-way ANOVA (A–D and G–P). Significance levels: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Figure 6

Molecular docking analysis of AS-IV with Keap1 and Nrf2. (A) Nrf2 (cyan) PHE-83, GLU-82, THR-80, GLU-79, GLU-78 and ASN-382, ARG-380, ARG-415, SER-555, TYR-525 on Keap1 (blue) could form five hydrogen bond interactions. (B) AS-IV could form five hydrogen bonds with LEU-84, ASP-77, GLU-78 and GLU-79 on Nrf2, and one hydrogen bond with ARG-415 on Keap1.

Figure 7

Therapeutic mechanism of Astragaloside IV in experimental autoimmune prostatitis. AS-IV ameliorates chronic prostatitis/chronic pelvic pain syndrome in EAP rats through three core mechanisms:1. Keap1/Nrf2/HO-1 axis activation; 2. Iron homeostasis regulation; 3. Oxidative damage suppression.