Targeting benign prostate hyperplasia treatments: AR/TGF-β/NOX4 inhibition by apocynin suppresses inflammation and proliferation

Abstract

Introduction: Apocynin (Apo), an NADPH oxidase (NOX) inhibitor, has been widely used to treat various inflammatory diseases. However, the therapeutic effects of Apo on benign prostatic hyperplasia (BPH), a multifactorial disease associated with chronic inflammation and hormone imbalance, remain unknown.

Objectives: The link between androgen signaling, reactive oxygen species (ROS), and prostate cell proliferation may contribute to the pathogenesis of BPH; therefore, the aim of this study was to identify the specific signaling pathway involved and to demonstrate whether the anti-oxidant Apo plays a role in the prevention and treatment of BPH.

Methods: Ingenuity pathway analysis and si-RNA transfection were conducted to demonstrate the androgen receptor (AR) and NOX4 linkage in BPH. Pathological markers of BPH were measured by H&E staining, immunoblotting, ELISA, qRT-PCR, and immunofluorescence to examine the effect of Apo. Rats stimulated with testosterone and BPH-1 cells were used as BPH models.

Results: AR and NOX4 network-mediated oxidative stress was upregulated in the BPH model. Next, we examined the effects of Apo on oxidative stress and chronic prostatic inflammation in BPH mouse models. In a testosterone-induced BPH rat model, Apo alleviated pathological prostate enlargement and suppressed androgen/AR signaling. Apo suppressed the upregulation of proinflammatory markers and promoted the expression of anti-oxidant factors. Furthermore, Apo regulated the TGF-β/Glut9/activin pathway and macrophage programming. In BPH-1 cells, Apo suppressed AR-mediated proliferation and upregulation of TGFB and NOX4 expression by alleviating oxidative stress. Apo activated anti-oxidant and anti-inflammatory systems and regulated macrophage polarization in BPH-1 cells. AR knockdown partially abolished the beneficial effects of Apo in prostate cells, indicating AR-dependent effects of Apo.

Conclusion: In contrast with existing BPH therapies, Apo may provide a new application for prostatic disease treatment, especially for BPH, by targeting the AR/TGF-β/NOX4 signaling pathway.

Keywords: Apocynin, Androgen receptor; Benign prostatic hyperplasia; Dihydrotestosterone; NOX4; Prostate cancer prevention.

Graphical abstract

Fig. 1

Anti-proliferative, anti-oxidant and anti-inflammatory effects of the NOX inhibitor Apo on BPH-1 cells. Effect of Apo on the viability of BPH-1 cells. BPH-1 cells were treated with different concentrations (7.81–500 μM) of Apo for 24 h. (B) Effect of Apo on oxidative stress in BPH-1 cells. BPH-1 cells were treated without or with 500 µM Apo for 24 h, followed by treatment with or without H₂O₂ for 30 min. (C-F) BPH-1 cells were treated without or with Apo (125, 250, or 500 μM). mRNA levels of HMOX1, SOD1, and GPX1 (C) and NOS2, PTGS2, IL6, and TNFA (D) and MRC1, IL10 (E) and CD68, CD80, CD86 (F) were quantified using quantitative real-time polymerase chain reaction. The expression levels of target genes were normalized to those of GAPDH (housekeeping gene). (G) Protein level of NOX4 was determined. (H) The levels of target protein were normalized to that of ACTB (internal control) and are presented as relative protein levels. *P < 0.05, ^**P < 0.01 and ^***P < 0.001 compared with the vehicle-treated group; analysis of variance (ANOVA), followed by Dunnett’s post-hoc test.

Fig. 2

Inhibitory effect of the Apo on AR-dependent proliferation of BPH-1 cells. (A) Link between AR, TGF-β, and NOX4. Solid lines represent a direct interaction between the two gene products, while the dotted lines indicate an indirect interaction. (B) BPH-1 cells were treated without or with Apo (125, 250, or 500 μM). The cell lysates were subjected to immunoblotting with primary antibodies against AR, KLK3, NCOA1, and PCNA. (C) The levels of target proteins were normalized to those of ACTB (internal control) and expressed as relative protein levels. * P < 0.05, ^** P < 0.01, and ^*** P < 0.001 compared with the vehicle-treated group. (D and E) BPH-1 cells were treated without or with Apo (125, 250, or 500 μM). (D) TGFB level was determined. (E) The level of target protein was normalized to those of ACTB (internal control) and is presented as relative protein level. ^***P < 0.001 compared with the vehicle-treated group; analysis of variance (ANOVA), followed by Dunnett’s post-hoc test.

Fig. 3

Effect of Apo on TGFB/NOX4 signaling in BPH-1 cells. BPH-1 cells were transfected with green fluorescent protein (GFP)-encoding vector or short-interfering RNAs against AR (AR siRNA), and (A) the mRNA levels of AR, TGFB, and NOX4 in BPH-1 cells were determined using qRT-PCR. (B-D) Transfected BPH-1 cells were treated without or with 500 µΜ Apo for 4 h. The cells were harvested, and (B) the mRNA levels of AR, NCOA1, and TGFB and (C) the protein levels of TGFB and NOX were determined. (D) The levels of target proteins were normalized to those of ACTB (internal control) and are presented as relative protein levels. ^**P < 0.01 and ^***P < 0.001 compared with the GFP group. (E) Transfected BPH-1 cells treated with exogenous TGF-β1 or TGF-β2, and then treated without or with 500 µΜ Apo for 4 h. The cells were harvested, and the mRNA levels of NOX4 were determined. ^###P < 0.001 compared with the GFP group; * P < 0.05 compared with the AR siRNA group; ^$$ P < 0.01 compared with the AR siRNA + TGF-β1 group; ^££ P < 0.01 compared with the AR siRNA + TGF-β2 group.

Fig. 4

Inhibitory effects of Apo on prostate gland enlargement in the testosterone-induced BPH rat model. (A-C) Rats were divided into the following two groups: control (Con) and BPH (testosterone-induced BPH). (A) Serum malondialdehyde (MDA) levels were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit. ^###P < 0.001 compared with the Con group. (B) Prostate tissue lysates were subjected to immunoblotting with antibodies against NOX2 and NOX4. ACTB served as an internal control. (C) The density of target protein bands was normalized to that of Actb protein band using ImageJ and is presented as mean ± standard deviation. ^##P < 0.01 and ^###P < 0.001 compared with the Con group. (D-G) The rats were randomly divided into the following four groups: control (Con), BPH (testosterone-induced BPH), Fina (BPH animals treated with finasteride (5 mg/kg bodyweight)), and Apo (BPH animals treated with Apo (5 mg/kg bodyweight)). (D) Representative photographs of prostate tissues from the testosterone-induced BPH rat model. (E) The ratio of prostate weight/bodyweight in the experimental groups. (F) Prostate tissue sections were subjected to hematoxylin and eosin staining. Original magnification: 100×. (G) Based on histological analysis, the thickness of the epithelium from the prostate tissue (TETP) was measured. ^### P < 0.001 compared with the Con group; * P < 0.05, and ^*** P < 0.001 compared with the BPH group; analysis of variance, followed by Dunnett’s post-hoc test.

Fig. 5

Inhibitory effects of Apo on Androgen/AR signaling in the testosterone-induced BPH rat model. (A) The mRNA level of Srd5a2 was quantified using quantitative real-time polymerase chain reaction (qRT-PCR). The expression of Srd5a2 was normalized to that of Gapdh (housekeeping gene). (B) Serum levels of dihydrotestosterone (DHT) were analyzed using an enzyme-linked immunosorbent assay kit. (C) Prostate tissue lysates were subjected to immunoblotting with antibodies against AR and NCOA1. (D) The density of target protein bands was normalized to that of ACTB band using ImageJ and presented as mean ± standard deviation. (E) The Ar and Ncoa1 mRNA levels in the prostate were quantified using qRT-PCR. ^## P < 0.01 and ^### P < 0.001 compared with the Con group; * P < 0.05, * P < 0.01, and ^*** P < 0.001 compared with the BPH group; analysis of variance, followed by Dunnett’s post-hoc test.

Fig. 6

Anti-oxidant and anti-inflammatory effects of Apo in the BPH rat model. (A) Serum malonaldehyde (MDA) concentrations were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit. (B) mRNA levels of Hmox1, Sod1, and Gpx1 in the prostate tissues were determined using quantitative real-time polymerase chain reaction. (C) NOX4 and CD68 levels in the prostate tissue lysates were determined using western blotting with specific antibodies. ACTB served as an internal control. (D) Densitometric analysis of each protein was performed, and the relative protein levels are presented as mean ± standard deviation. mRNA levels of Cd68, Cd80, Il6 (E), Mrc1, Retnla, Il10, and Chi3l3 (F) were quantified using quantitative real-time polymerase chain reaction (qRT-PCR). Il6, Il1b, Tnfa (G), Nos2, and Ptgs2 (H) mRNA levels in the prostate tissues were determined using qRT-PCR. ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 compared with the Con group; * P < 0.05, * P < 0.01, and ^*** P < 0.001 compared with the BPH group; analysis of variance, followed by Dunnett’s post-hoc test.

Fig. 7

Effects of Apo on Tgfb/activin/Glut9 pathway in the BPH rat model. (A) TGFB, TP53, and SLC2A9 levels in the prostate tissue lysates were examined using western blotting with specific antibodies. ACTB served as an internal control. (B) Densitometric analysis of each protein was performed, and the relative protein levels are presented as mean ± standard deviation. (C) Serum Activin A concentrations were determined using an ELISA kit. (D) The intracellular and (E) plasma levels of uric acid were determined using an ELISA kit. ^### P < 0.001 compared with the Con group, ^*** P < 0.001 compared with the BPH group; analysis of variance, followed by Dunnett’s post-hoc test.