Combined Sabal and Urtica Extracts (WS® 1541) Exert Anti-proliferative and Anti-inflammatory Effects in a Mouse Model of Benign Prostate Hyperplasia

Abstract

WS^® 1541 is a phytopharmaceutical drug combination containing a lipophilic extract from fruits of Sabal serrulata (WS^® 1473) and an aqueous ethanolic extract from roots of Urtica dioica (WS^® 1031). It is approved in several countries worldwide for the treatment of lower urinary tract syndrome (LUTS) linked to benign prostate hyperplasia (BPH). Clinical studies have demonstrated the efficacy of this unique combination in the treatment of BPH-related LUTS. However, its mechanisms of action in vivo remain partly uncharacterized. The aim of this study was to take advantage of a validated mouse model of BPH to better characterize its growth-inhibitory and anti-inflammatory properties. We used the probasin-prolactin (Pb-PRL) transgenic mouse model in which prostate-specific overexpression of PRL results in several features of the human disease including tissue hypertrophy, epithelial hyperplasia, increased stromal cellularity, inflammation, and LUTS. Six-month-old heterozygous Pb-PRL male mice were randomly distributed to five groups (11-12 animals/group) orally treated for 28 consecutive days with WS^® 1541 (300, 600, or 900 mg/kg/day), the 5α-reductase inhibitor finasteride used as reference (5 mg/kg/day) or vehicle (olive oil 5 ml/kg/day). Administration of WS^® 1541 was well tolerated and caused a dose-dependent reduction of prostate weight (vs. vehicle) that was statistically significant at the two highest doses. This effect was accompanied by a reduction in prostate cell proliferation as assessed by lower Ki-67 expression (qPCR and immunohistochemistry). In contrast, finasteride had no or only a mild effect on these parameters. The growth-inhibitory activity of WS^® 1541 was accompanied by a strong anti-inflammatory effect as evidenced by the reduced infiltration of cells expressing the leukocyte common antigen CD45. In sharp contrast, finasteride significantly increased the prostate inflammatory status according to this readout. Molecular profiling (qPCR) of 23 selected pro-inflammatory genes confirmed the strong anti-inflammatory potency of WS^® 1541 compared to finasteride. Since treatment of WS^® 1541 did not interfere with transgene expression and activity in the prostate of Pb-PRL mice, the effects observed in this study are entirely attributable to the intrinsic pharmacological action of the drug combination.

Keywords: BPH; COX-2; WS® 1541; chemokines; cytokines; iNOS; inflammation; lower urinary tract syndrome (LUTS).

FIGURE 6

Prostate fibrosis. (A) Representative images of picrosirius red staining showing stromal fibrosis in the ventral prostate. Arrows point to dense, isolated fibrotic stained foci in the vehicle- and finasteride-treated groups whereas staining is evenly distributed in the WS^® 1541-treated groups, especially at the highest dose shown here. (B) Illustration of fibrosis score calculation by image analysis using Calopix^® software (see the section “Materials and Methods” for explanations). (C) Fibrosis score in the various treatment groups. Symbol: ^∗ vs. vehicle group, ANOVA, followed by Tukey’s, n = 11–12 per group. (D) Expression level of profibrotic genes COL1A1 and COL3A1 was assessed by RT-qPCR in ventral lobe. Results are expressed as fold expression vs. vehicle group.

FIGURE 1

Treatment tolerance and validation. (A) The weight of each animal (g) was measured at 0, 7, 14, 21, and 28 days of treatment. Each curve represents one single animal, n = 11–12 animals per group. (B) Averaged animal weight change in each treatment group between day 0 (dotted line) and sacrifice at day 28 of treatment (black dots). No significant (NS) difference was observed for any treatment group compared to vehicle (Mann–Whitney test). (C) Evaluation of the PRL/STAT5 pathway in all groups. Left: the mRNA expression of the rPRL transgene was determined by qPCR in the three lobes. Results of dorsal lobe are shown as fold expression vs. vehicle group. Right: the level of STAT5 activation in the various lobes and various treatment groups was analyzed by immunohistochemistry using anti-phosphorylated STAT5 antibodies. One typical image of dorsal lobe is shown (vehicle group), in which some nuclei positive for pSTAT5 are identified by black arrows. The ratio of STAT5-positive versus total nuclei in the epithelium was quantified as described in the section “Materials and Methods,” and the results of dorsal lobe are expressed as fold expression vs. vehicle group. ANOVA was used as statistical test followed by Tukey’s (n = 11–12 animals per group).

FIGURE 2

Prostate weights. (A) Ratio between tissue weight of the half prostate (sum of the three lobes, mg) and the corresponding animal weight (g). Data are normalized to the control group (vehicle). (B) Lobe-specific effect of the various treatments as in panel A. Data are normalized to the vehicle group. Symbol: ^∗ vs. vehicle group, ANOVA followed by Tukey’s.

FIGURE 3

Prostate cell proliferation. (A) The epithelial cell proliferation index was determined by Ki-67 immunohistochemistry (one typical image is shown with some positive nuclei identified by arrows). The data obtained for half prostates (top) or each individual lobe (bottom) are shown for each treatment relative to the vehicle group. (B) Ki-67 and Bcl-2 expression as determined by qPCR is represented for each individual prostate lobe and is normalized to the vehicle group. Symbol: ^∗ vs. vehicle group (n = 11–12 per group), ANOVA, followed by Tukey’s.

FIGURE 4

Prostate histopathology. Representative images of dorsal and ventral prostate histology in the various treatment groups (magnification 20×). For WS^® 1541 only the highest dose is shown as an example. Intraluminal mineralizations (identified by arrows) were frequently observed in the finasteride group. Symbols: S, stroma; L, lumen.

FIGURE 5

Prostate inflammatory status. (A) Representative illustrations of CD45 immunostaining of half prostate sections from the various groups (only the highest dose is shown for WS^® 1541). CD45-positive cell foci are circled in black, and one typical foci is shown at high magnification. The number of CD45-positive cell clusters was quantified for half prostate (B) and for each individual lobe (C) by image analysis as described in the section “Materials and Methods.” The results are normalized to the vehicle condition (see Table 1C for averaged crude values). Symbol: ^∗ vs. vehicle group, ANOVA, followed by Tukey’s. D, dorsal prostate; L, lateral prostate; V, ventral prostate; U, urethra.

FIGURE 7

WS^® 1541 treatment reduces the pro-inflammatory molecular profile of Pb–PRL prostates. Expression of 23 selected pro-inflammatory factors in the dorsal, lateral, and ventral prostate lobes from the various treatment groups was assessed by RT-qPCR (n = 11–12). Results are presented as a heatmap with respect to the vehicle group [the highest induction (red) corresponds to 32-fold, the lowest (blue) to 0.003-fold]. Fina, finasteride.

FIGURE 8

Differential regulation of pro-inflammatory cytokines/chemokines by WS^® 1541. Typical examples of gene regulation by WS^® 1541: genes that are (i) significantly downregulated in all lobes and at all doses (IL-15), (ii) significantly downregulated in all lobes but not at all doses (CCL2), (iii) significantly downregulated in some but not all lobes (CCL8), (iv) unaltered (CXCL10), and (v) regulated in opposite ways in different lobes (CD40lg). See text for the section “Discussion.” Statistical analysis was performed by ANOVA, followed by Tukey’s.