1. Introduction
Benign prostatic hyperplasia (BPH) is one of the most common phenomena related to the aging process, affecting 70–80% of men over the age of 80 [
1]. Nevertheless, according to some epidemiological studies, BPH occurs also in half of the men between 50 and 60 years old [
2,
3]. BPH linkage to age is strictly due to the loss of prostatic function and secretory capacity, which are fundamental in young men for fertility and reproduction but spontaneously decrease during aging progression [
1,
4,
5]. The hallmark of BPH is the hyperproliferation of prostatic epithelial and stromal cells, which drives prostate enlargement and, in most cases, the development of lower urinary tract symptoms (LUTS). Since LUTS often reduce patients’ life quality, it became of interest to find a treatment for BPH, which by itself is normally classified as a non-malignant disease [
6,
7].
One of the most important elements involved in BPH development is chronic inflammation. The critical role of pro-inflammatory cytokines and chemokines during BPH has been confirmed by several studies, suggesting also the use of these molecules as chronic prostate inflammatory biomarkers, potentially suitable for BPH diagnosis [
8,
9]. Particularly, the most relevant cytokines characterizing the prostate hyperplasic state are interleukin 6 (IL-6) and interleukin 8 (IL-8) [
10,
11].
Another important feature involved in BPH aetiology and pathology is oxidative stress. In fact, it has been reported that reactive oxygen species (ROS) play a dramatic role in sustaining the hyperproliferation of epithelial and stromal cells [
12]. ROS production amplifies the recruitment of inflammatory cells, which in turn are responsible for producing a high amount of reactive species via the nicotinamide adenine dinucleotide phosphate (NADPH) pathway. Thus, oxidative stress is also responsible for the complication of the inflammatory state in the hyperplasic prostate [
13].
Medicinal plants, in the form of plant parts or their extracts, are commonly used for the treatment of prostate diseases such as benign hypertrophy, prostatitis, and chronic pelvic pain syndrome. The pharmacological properties that are more interesting for the treatment of prostatic diseases are the anti-androgenic, anti-estrogenic, anti-proliferative, antioxidant, and anti-inflammatory ones [
14].
In this work, two different drugs already approved for the treatment of BPH-associated inflammation, both containing the extract of
Serenoa repens, have been compared with the aim to investigate the molecular mechanisms underlying their effects. The first one is a lipidosterolic extract of
Serenoa repens (SR), while the second one consists of a combined formulation containing a fruit extract of
Serenoa repens and a root extract of
Urtica dioica (SR/UD). Particularly, the fruit extract of
Serenoa repens has been indicated by several studies as a potential treatment for BPH [
15,
16,
17,
18]. The positive effect of this plant extract is due both to the direct inhibition of 5α-reductase and to the prevention of inflammation [
19,
20]. Recent meta-analyses showed that the effectiveness of
Serenoa repens is similar or slightly inferior compared to finasteride and tamsulosin, but clearly higher than the placebo in the treatment of mild and moderate low urinary tract symptoms (LUTS), nocturia, and discomfort [
21]. The combination with
Urtica dioica has been proposed according to its anti-inflammatory and antioxidant activities [
22,
23]. In particular, we investigated whether the combination of
Urtica dioica and
Serenoa repens extracts permits the reduction of inflammation and oxidative stress in an in vitro cell model of prostatic hyperplasia (BPH-1 cell line).
Moreover, the same analyses conducted on BPH-1 cells were performed on the androgen-independent PC3 cell model in order to verify if the antioxidant and anti-inflammatory effects are similar in different prostate tissues or strictly linked to the BPH condition.
3. Discussion
BPH is a common male disease strictly related to aging [
4]. Several studies suggest the positive correlation between prostate aging and the development of a typical inflammatory microenvironment able to enhance cell proliferation and, in the long term, to contribute to the onset of BPH [
9,
33,
34]. According to the critical role of chronic inflammation and ROS production in the pathogenesis and degeneration of BPH, targeting inflammatory mechanisms and oxidative stress could be a promising approach for the treatment of BPH and the consequent prevention of LUTS [
35]. In fact, although the standard treatments for BPH consist of α-Adrenergic blockers and 5α-Reductase inhibitors [
36,
37,
38,
39], the role of plant extracts in BPH symptom counteraction has been recently demonstrated [
37]. In our work, we investigated the antioxidant and anti-inflammatory activity of a drug approved for BPH treatment, SR/UD, in a human BPH in vitro model (BPH-1 cell line). SR/UD contains a patented formulation of
Serenoa repens and
Urtica dioica. Furthermore, we compared it with another approved drug for BPH treatment, consisting of an esanic fruit extract of
Serenoa repens (SR). Previous studies on
Serenoa repens proved its ability to ameliorate inflammation and to prevent ROS production in the BPH scenario [
15,
16,
17,
18].
Urtica dioica was indicated to possess anti-inflammatory activity as well [
40]. Thus, our aim was to investigate for the first time the molecular mechanisms underlying the activity of the
Urtica dioica and
Serenoa repens combination, focusing on the antioxidant and anti-inflammatory properties.
The inflammatory status endurance, which is typical of BPH onset, is mostly triggered by the activation of NF-κB, one of the most important transcription factors involved in the inflammatory response [
28]. The translocation of NF-κB inside the nucleus starts the transcription of different pro-inflammatory agents, such as IL-6 and IL-8 [
8,
41]. IL-6 is a proinflammatory cytokine that acts in the innate immune response [
42], while IL-8 is a chemokine that promotes the migration of neutrophils, basophils, and T lymphocytes [
43]. Thus, both of them are implicated in the recruitment of inflammatory cells and are consequently responsible for promoting BPH when overproduced over time [
44,
45]. Besides, the prostates of patients affected by BPH present higher IL-6 levels compared to the ones with a normal prostate [
46]. The specific level of IL-6 is further recognized to be related to a poor outcome for these prostate diseases [
47]. Several studies confirmed the direct correlation between IL-8 levels and BPH progression [
48]. An important role in the persistence of the inflammatory microenvironment during BPH is also played by oxidative stress. ROS are continuously generated in the prostatic tissue as a result of hypoxia, which occurs after the development of abnormal blood flow patterns [
49]. Our in vitro results show an antioxidant effect of SR/UD on BPH-1 cells. On the other hand, ROS are also responsible for the activation of NF-κB [
50,
51,
52,
53,
54]. Our work highlights the significant downregulation of cytokines resulting from the reduced translocation of NF-κB inside the cells nuclei, suggesting the important anti-inflammatory activity of SR/UD. Thus, the patented combination of
Serenoa repens and
Urtica dioica plays an important role in the inhibition of the most important pro-inflammatory pathways involved in BPH development and progression directly through its antioxidant activity, which results in a decreased NF-κB-triggered inflammation. Accordingly, both the levels of IL-6 and IL-8 mRNA and the release of these cytokines appear to be diminished in the in-vitro model of BPH after the treatment with SR/UD. Particularly, IL-8 is the most affected by SR/UD.
The positive effect of SR/UD in the progression of prostatic diseases is also confirmed by previous clinical trials [
55,
56,
57] and an in-vivo preclinical experiment [
58]. Our in vitro results support the positive observed effect due to a deeper understanding of the molecular mechanism that underpins SR and SR/UD activity. Data show that treatment with SR/UD appears advantageous in the counteraction of oxidative stress and inflammation in BPH-1 cells. In particular, SR/UD has proven to be an effective antioxidant and this activity could be related to the prevention of the onset of inflammation, by reduction of NF-kB activation.
Considering that the aim of our work was to investigate the molecular mechanisms underlying the antioxidant and anti-inflammatory effects of SR/UD, both extracts were also tested in a human androgen-independent prostate cell model (PC3). SR/UD demonstrated the ability to reduce NF-κB translocation inside the nucleus and to slightly decrease the pro-inflammatory markers. Our results highlight the tissue-specificity of SR/UD, confirming its approved use in BPH treatment.
5. Materials and Methods
5.1. Cell Cultures
The benign prostatic hyperplasia cell line (BPH-1) was purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany) and cultured in Roswell Park Memorial Institute medium (RPMI 1640) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20% h.i. FBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 20 ng/mL DHT (Supelco, Merck, Darmstadt, Germany), 5 µg/mL transferrin (Sigma–Aldrich, Merck, Darmstadt, Germany), 5 ng/mL sodium selenite (Sigma–Aldrich, Merck, Darmstadt, Germany) and 5 µg/mL insulin (Sigma–Aldrich, Merck, Darmstadt, Germany). The prostate cancer 3 cell line (PC3) was obtained by ATCC (Manassas, VA, USA) and grown with RPMI 1640, 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Both cell lines were maintained under standard culture conditions at 37 °C and 5% CO2. Cells were collected every 2 days with a minimum amount of 0.05% trypsin–0.02% EDTA.
5.2. SR Composition
Lipido sterolic extract from fruits of S. repens [320 mg S. repens; drug–extract ratio (7–11:1); extraction solvent: hexane].
5.3. SR/UD Composition
WS® 1541, the active portion of SR/UD, is a fixed combination of:
lipophilic extract from fruits of S. repens [160 mg WS® 1473; drug–extract ratio 10.0–14.3:1; extraction solvent: 90% ethanol (m/m)]
aqueous ethanolic extract from roots of U. dioica [120mg WS® 1031, drug–extract ratio 7.6–12.5:1; extraction solvent: 60% ethanol (m/m)].
5.4. Cells Viability Assay
Crystal violet assay was used to assess cell viability after treatment with SR/UD or SR. BPH-1 and PC3 cells were seeded at respective densities of 15,625 × 10
3 cells/cm
2 and 31,125 × 10
3 cells/cm
2 in 96-well plates and treated after 24 h with different concentrations (1, 10, 20 µg/mL) of both SR and SR/UD. The crystal violet assay was performed after 1, 2, and 3 days of treatment. Cells were firstly fixed with 2% formaldehyde (Sigma–Aldrich, Merck, Darmstadt, Germany) for 15 min, washed twice with PBS without bivalent cations (Euroclone, Milan, Italy), and stained with 0.1% Crystal violet solution (Sigma–Aldrich, Merck, Darmstadt, Germany) for 20 min. They were washed three times with PBS and dried overnight. Then, 10% acetic acid (Sigma–Aldrich, Merck, Darmstadt, Germany) was used to lyse the stained cells, and the absorbance was measured at 570 nm using a Victor3X multilabel plate counter (Wallac Instruments, Turku, Finland). Growth curve analysis was carried out according to Ishiyama [
59].
5.5. ROS Assay
BPH-1 and PC-3 cells (5 × 103 cells/well) were seeded in 96-well plates. After 24 h, cells were treated with SR and SR/UD (1, 10, 20 µg/mL) for 3 or 24 h and incubated at 37 °C. Then, cells were incubated with a 100 µM diacetylated 2′,7′-dichlorofluorescein (DCF-DA) probe (Sigma–Aldrich, Merck, Darmstadt, Germany) for 30 min at 37 °C, in the presence or absence of H2O2 0.9 µM (Sigma–Aldrich, Merck, Darmstadt, Germany), and the fluorescence was measured by using a Victor3X multilabel plate counter (Ex 485 nm and Em 535 nm) (Wallac Instruments, Turku, Finland). This assay takes advantage of the fluorescence emitted by the oxidation of the non-fluorescent DCF-DA and evaluates intracellular ROS production.
5.6. Nuclear Factor-kappa B Translocation Assay
To perform the NF-κB translocation assay, BPH-1 and PC3 cells were firstly seeded on glass coverslips in 24-well plates and cultured as previously indicated. When cells reached approximately 30% confluence, they were fixed with 4% formaldehyde (Sigma–Aldrich, Merck, Darmstadt, Germany), permeabilized with 0.1% Triton X-100 (Sigma–Aldrich, Merck, Darmstadt, Germany) in PBS, and stained 1 h with rabbit monoclonal anti-NF-κB p65 (Invitrogen, Life Technologies, Milan, Italy). After PBS washes, they were incubated with anti-rabbit secondary antibody Alexa Fluor 488 (Molecular Probes, Invitrogen, Milan, Italy) for 1 h at room temperature. Cells were then washed with PBS and stained with Hoechst (1:10,000) (Invitrogen, Life Technologies, Milan, Italy). The coverslips were finally mounted on glass slides by using Mowiol 40–88 (Sigma–Aldrich, Merck, Darmstadt, Germany). Images were acquired through a 60x CFI Plan Apochromat Nikon objective with a Nikon C1 confocal microscope and finally analysed using NIS Elements software (Nikon Instruments, Florence, Italy), NIH Image J version 1.52t, and Adobe Photoshop CS4 version 11.0.2 (Adobe, San Jose, CA, USA).
5.7. RNA Expression/Quantitative Real-Time PCR
BPH-1 and PC-3 cells were seeded at respective densities of 15,625 × 10
3 cells/cm
2 and 31,125 × 10
3 cells/cm
2 in 6-well plates. BPH-1 cells were pre-treated with SR or SR/UD (1, 10, 20 µg/mL) and then stimulated with LPS (10 µg/mL) (Sigma–Aldrich, Merck, Darmstadt, Germany) for 24 h. The treatments with SR and SR/UD were maintained during the LPS stimulation. The duration of the SR and SR/UD treatments was a total of 72 h. PC3 cells were treated for 72 h with SR and SR/UD, avoiding the stimulation with LPS. RNA was isolated with the TRIzol method (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), and retro-transcription was performed with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Quantitative PCR (qPCR) reactions were performed using QuantStudio™ 5 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) with Power SYBR™ Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) and the specific primers reported below. Primer sequences were obtained from PrimerBank (
http://pga.mgh.harvard.edu/primerbank/index.html). The GAPDH expression level was used as a reference for the normalization of each value level. The primer sequences used were as follows: IL-6 forward, 5′-TACATCCTCGACGGCATCTC-3′; reverse, 5′-TGCCTCTTTGCTGCTTTCAC-3′. IL-8 forward, 5′-TTGGCAGCCTTCCTGATTTC-3′; reverse, 5′-TTGGGGTGGAAAGGTTTGGAG-3′. GAPDH forward 5′-AATCCCATCACCATCTTCCA-3′; reverse, 5′-TGGACTCCACGACGTACTCA-3′.
5.8. IL-6 and IL-8 ELISA
BPH-1 and PC-3 cells (50 × 103 cells/well) were seeded in 6-well plates. Cells were pre-treated with SR or SR/UD (10, 20 µg/mL) for 24 h and stimulated with LPS (10 µg/mL) for 6 h. The media were then replaced with fresh media without FBS, which were collected after 12 h for the detection of IL-6 and IL-8 levels. Human IL-6 (limit of sensitivity is 3 pg/mL) and IL-8 (limit of sensitivity is 4 pg/mL) levels were determined by an ELISA kit (RayBio, Peachtree Corners, GA, USA and BioLegend, San Diego, CA, USA respectively) according to the protocols. Results are expressed as pg/mL and reported as the means of three independent experiments.
5.9. Statistical Analysis
GraphPad Prism version 3.03 (GraphPad, San Diego, CA, USA) software was used for statistical evaluation of the experimental results. Data were analyzed using one-way analysis of variance (ANOVA), followed by an appropriate post-hoc test (Bonferroni or Tukey-Kramer). A p value of less than 0.05 was considered for determining statistical significance.