Serenoa repens and Urtica dioica Fixed Combination: In-Vitro Validation of a Therapy for Benign Prostatic Hyperplasia (BPH)

Abstract

Benign prostatic hyperplasia (BPH) is an age-related chronic disorder, characterized by the hyperproliferation of prostatic epithelial and stromal cells, which drives prostate enlargement. Since BPH aetiology and progression have been associated with the persistence of an inflammatory stimulus, induced both by Nuclear Factor-kappa B (NF-κB) activation and reactive oxygen species (ROS) production, the inhibition of these pathways could result in a good tool for its clinical treatment. This study aimed to evaluate the antioxidant and anti-inflammatory activity of a combined formulation of Serenoa repens and Urtica dioica (SR/UD) in an in vitro human model of BPH. The results confirmed both the antioxidant and the anti-inflammatory effects of SR/UD. In fact, SR/UD simultaneously reduced ROS production, NF-κB translocation inside the nucleus, and, consequently, interleukin 6 (IL-6) and interleukin 8 (IL-8) production. Furthermore, the effect of SR/UD was also tested in a human androgen-independent prostate cell model, PC3. SR/UD did not show any significant antioxidant and anti-inflammatory effect, but was able to reduce NF-κB translocation. Taken together, these results suggested a promising role of SR/UD in BPH and BPH-linked disorder prevention.

Keywords: BPH; Serenoa repens; Urtica dioica; inflammation; oxidative stress.

Figure A1

Effect of NAC 5 mM on ROS generation in BPH-1 and PC3 cells. The cells were treated for 3 h (a,b,e,f) or for 24 h (c,d,g,h). ROS levels were detected by DCF-DA staining at the basal condition (a,c,e,g) and following the exposure to oxidative stimulus H₂O₂ (b,d,f,h). The fluorescence was measured by using a Victor3X multilabel plate counter (Ex 485 nm and Em 535 nm). Results are expressed as the fold change of ROS production. Each bar represents the mean ± SEM (n = 3), and three independent experiments were carried out. * p < 0.05, ** p < 0.01, treatment compared to the control; ° p < 0.05, °°° p < 0.001, oxidative stimulus H₂O₂ vs. basal condition. Differences between various drug concentrations and control were analyzed using one-way ANOVA followed by Bonferroni′s multiple comparison test.

Figure 1

Effect of SR and SR/UD on BPH-1 and PC3 cell viability. BPH-1 cells treated with increasing concentrations of SR and SR/UD (1, 10, 20 µg/mL) for (a) 24 h, (b) 48 h, (c) 72 h. PC-3 cells treated with increasing concentrations of SR and SR/UD (1, 10, 20 µg/mL) for (d) 24 h, (e) 48 h, (f) 72 h. BPH-1 and PC3 cell viability was evaluated with the Crystal Violet Assay. Data represent the mean ± SEM (n = 3). Differences between various drug concentrations and control were analysed for each time point (24, 48, 72 h). The dashed line is the control.

Figure 2

Effect of SR and SR/UD (1, 10, 20 µg/mL) on ROS generation in BPH-1 and PC3 cells. The cells were treated for 3 h (a,b,e,f) or for 24 h (c,d,g,h) with the two compounds. ROS were detected by DCF-DA staining at the basal condition (a,c,e,g) and following the exposure to oxidative stimulus H₂O₂ (b,d,f,h). The fluorescence was measured by using a Victor3X multilabel plate counter (Ex 485 nm and Em 535 nm). Results are expressed as the fold change of ROS production. Each bar represents the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, treatment compared to control. The dashed line is the control.

Figure 3

Effect of 24 h treatments with SR and SR/UD (1, 10 µg/mL) on nuclear translocation of NF-κB in BPH-1 and PC3 cells. (a,b) Quantification of intranuclear NF-KB in BPH-1 cells (a) and PC3 cells (b). Values are expressed as the ratio between treatments and control. Data represent the mean ± SEM (n = 3). *** p < 0.001, treatment compared to the control (indicated as the dashed line). (c,d) Representative images of NF-κB immunofluorescence staining in BPH-1 cells (c) and PC3 cells (d) treated with SR and SR/UD. Images were captured at 60X magnification.

Figure 4

Effect of SR and SR/UD (1, 10, 20 µg/mL) on IL-6 and IL-8 mRNA expression in BPH-1 and PC3 cells. (a) IL-6 expression in BPH-1 cells pre-treated with SR and SR/UD for 48 h and stimulated with LPS (10 µg/mL) for 24 h. (b) IL-8 expression in BPH-1 cells pre-treated with SR and SR/UD for 48 h and stimulated with LPS (10 µg/mL) for 24 h (c) IL-6 expression in PC3 cells treated with increasing concentrations of SR and SR/UD for 72 h. (d) IL-8 expression in PC3 cells treated with increasing concentrations of SR and SR/UD for 72 h. IL-6 and IL-8 were evaluated by qPCR. IL-6 and IL-8 are expressed as the ratio between treatments and LPS stimulus * p < 0.05, treatment compared to LPS, ** p < 0.01, Control compared to LPS. Data represent the mean ± SEM (n = 3).

Figure 5

Human IL-6 and IL-8 production (pg/mL) in BPH-1 and PC3 cells. (a) IL-6 production by BPH-1 cells pre-treated with SR and SR/UD (10, 20 µg/mL) for 24 h and stimulated with LPS (10 µg/mL) for 6 h. (b) IL-8 production by BPH-1 cells pre-treated with SR and SR/UD (10, 20 µg/mL) for 24 h and stimulated with LPS (10 µg/mL) for 6 h. (c) IL-6 expression by PC3 cells treated with SR and SR/UD (10, 20 µg/mL) for 24 h. (d) IL-8 expression by PC3 cells treated with SR and SR/UD (10, 20 µg/mL) for 24 h. IL-6 and IL-8 are expressed as the ratio between treatments and LPS stimulus. * p < 0.05, ** p < 0.01, *** p < 0.001, LPS stimulus compared to the control or treatment compared to the control. Data represent the mean ± SEM (n = 3).