Effects of a proprietary mixture of extracts from Sabal serrulata fruits and Urtica dioica roots (WS® 1541) on prostate hyperplasia and inflammation in rats and human cells

Abstract

Introduction: Phytotherapeutics, particularly extracts from Sabal serrulata (saw palmetto) fruit or Urtica dioica (stinging nettle) root, are popular for the treatment of male lower urinary symptoms in many countries, but their mechanism of action is poorly understood. We performed in vivo and in vitro studies to obtain deeper insight into the mechanism of action of WS^® 1541, a proprietary combination of a Sabal serrulata fruit and an Urtica dioica root extract (WS^® 1473 and WS^® 1031, respectively) and its components. Methods: We used the sulpiride model of benign prostatic hyperplasia in rats and tested three doses of WS^® 1541 in comparison to finasteride, evaluating weight of prostate and its individual lobes as well as aspects of inflammation, oxidative stress, growth and hyperplasia. In human BPH-1 cells, we studied the effect of WS^® 1473, WS^® 1031, WS^® 1541 and finasteride on apoptosis, cell cycle progression and migrative capacity of the cells. Results: WS^® 1541 did not reduce prostate size in sulpiride treated rats but attenuated the sulpiride-induced changes in expression of most analyzed genes and of oxidized proteins and abrogated the epithelial thickening. In vitro, WS^® 1473 and WS^® 1031 showed distinct profiles of favorable effects in BPH-1 cells including anti-oxidative, anti-proliferative and pro-apoptotic effects, as well as inhibiting epithelial-mesenchymal-transition. Conclusion: This data supports a beneficial effect of the clinically used WS^® 1541 for the treatment of lower urinary tract symptoms associated with mild to moderate benign prostate syndrome and provides a scientific rationale for the combination of its components WS^® 1473 and WS^® 1031.

Keywords: BPH-1 cells; PRO 160/120; Sabal serrulata; Urtica dioica; WS® 1541; benign prostate hyperplasia; epithelial-mesenchymal-transition; inflammation.

FIGURE 1

(A) Experimental outline of the sulpiride-induced prostate hyperplasia model in rats. Sulpiride (40 mg/kg) or vehicle A (acetic acid 1%) was administered intraperitoneally (i.p.) once a day for 30 consecutive days. The proprietary combination of Sabal serrulata fruit and Urtica dioica root extract WS^® 1541 (300, 600 and 900 mg/kg), finasteride (5 mg/kg) or vehicle B (0.2% agar suspension with 1% Tween 80 in distilled water) was given orally daily for 30 consecutive days. Weight normalized to total body weight (BW) of (B) lateral, (C) ventral, and (D) dorsal prostate lobes showing specific induction of hyperplasia in the lateral lobe rather than in the ventral and dorsal lobe (n = 10). (E) Total prostate weight normalized to BW of different treatment groups (n = 10). (F) Tabular presentation of 95% confidence intervals for the data shown in Figures 1B–E. BW was similar in all groups (Online Supplementary Figure S1). No p-values were calculated as these measurements served as model validation only.

FIGURE 2

Heatmap on mRNA expression of indicated genes in lateral lobes of the prostate from treated rats normalized to the reference gene Gapdh (n = 8–10). The expression level of each gene was set to one in the vehicle B treated sulpiride group. Changes in expression vs. the sulpiride-treated vehicle B group are shown in the individual fields.

FIGURE 3

(A) Quantification of immune histological staining (OxyIHC) for oxidized proteins (n = 6/8/10/10/8/9). (B) Representative images showing brownish staining for oxidized proteins and nuclei in blue (scale 100 µm). (C) Histological evaluation of epithelial thickness in H&E stained sections from lateral lobes of treated rats (n = 5/10/10/10/10/9). (D) Representative images of H&E stained cryosections of left lateral lobes (scale 100 µm).

FIGURE 4

(A) Dose-response curve of Sabal (WS^® 1473) and Urtica (WS^® 1031) extracts on reactive oxygen species (ROS) production in treated BPH-1 cells measured based on H2-DCF-DA to fluorescent DCF conversion (n = 4 independent experiments). (B) Quantification of ROS in BPH-1 cells at definite concentrations (25 μg/mL WS^® 1541, 21.9 μg/mL WS^® 1031) according to their presence in WS^® 1541 (25 μg/mL WS^® 1541 + 21.9 μg/mL WS^® 1031) (n = 7 independent experiments). (C) Exemplary gating of BPH-1 cells stained for Annexin V and with propidium iodide (PI). (D) Quantification of the sum of Annexin V+ and Annexin V+/PI + BPH-1 cells after 2 h of treatment with DMSO, Finasteride, Sabal (WS^® 1473), Urtica (WS^® 1031) or its combination (WS^® 1541) (n = 5 individual experiments). (E) Gating strategy on EdU and DNA staining of BPH-1 cells to differentiate between G₁-, S- and G₂-phase of the cell cycle. (F) Percentage of BPH-1 cells in G₁-, S- or G₂-phase during cell cycle progression after treatment with DMSO, finasteride, Sabal (WS^® 1473), Urtica (WS^® 1031) or the combination of Sabal + Urtica (WS^® 1541) (n = 5 individual experiments).

FIGURE 5

(A) Concentration-dependency of Sabal (WS^® 1473) and Urtica (WS^® 1031) extracts on migration of BPH-1 cells (n = 9 independent experiments). (B) Quantification of the migration assay performed with BPH-1 cells in the presence of DMSO, finasteride, 25 μg/mL Sabal (WS^® 1473) or 21.9 μg/mL Urtica (WS^® 1031) or its combination (WS^® 1541; 25 μg/mL WS^® 1473 and 21.9 μg/mL WS^® 1031) showing inhibition of migration in response to Urtica (WS^® 1031) or Sabal + Urtica (WS^® 1541) (n = 7 individual experiments). (C) Representative images of the migration assay.

FIGURE 6

(A) Relative expression levels of TGFb2, TGFb3 and the TGF-β receptors TGFbR1 and TGFbR2 in BPH-1 cells treated for 24 h relative to that of TGFb1 (n = 6 independent experiments). Impact of Sabal (WS^® 1473) and Urtica (WS^® 1031) extracts on the mRNA expression of (B) TGFb1, (C) TGFb2, (D) TGFb3 and the TGF-β receptors (E) TGFbR1 and (F) TGFbR2 in BPH-1 cells treated for 24 h (n = 6 independent experiments).