Ameliorative effect of Abeliophyllum distichum Nakai on benign prostatic hyperplasia in vitro and in vivo

Abstract

Background/objectives: Benign prostatic hyperplasia (BPH) is the most common prostate disease and one of the most common chronic diseases caused by aging in men. On the other hand, there has been no research on BPH using Abeliophyllum distichum Nakai (A. distichum). Therefore, this study investigated the effects of A. distichum on BPH.

Materials/methods: A. distichum leaves were extracted with distilled water, 70% ethanol, and 95% hexane as solvents. Subsequently, the inhibitory effects of each A. distichum extract on androgen receptor (AR) signaling were evaluated in vitro. The testosterone-induced BPH model was then used to confirm the efficacy of A. distichum leaves in 70% ethanol extract (ADLE).

Results: ADLE had the strongest inhibitory effect on AR signaling. A comparison of the activity of ADLE by harvest time showed that the leaves of A. distichum harvested in autumn had a superior inhibitory effect on AR signaling to those harvested at other times. In the BPH rat model, the administration of ADLE reduced the prostate size and prostate epithelial cell thickness significantly and inhibited AR signaling. Subsequently, the administration of ADLE also reduced the expression of growth factors, thereby inactivating the PI3K/AKT pathway.

Conclusions: An analysis of the efficacy of ADLE to relieve BPH showed that the ethanol extract grown in autumn exhibited the highest inhibitory ability of the androgen-signaling related factors in vitro. ADLE also inhibited the expression of growth factors by inhibiting the expression of the androgen-signaling related factors in vivo. Overall, ADLE is proposed as a functional food that is effective in preventing BPH.

Keywords: Androgen receptor; cell proliferation; dihydrotestosterone; prostatic hyperplasia.

Fig. 1. Comparison of the AR signal inhibitory effect by extraction methods of A. distichum. (A) Representative western blot showing the bands of AR, 5AR2, and PSA. LNCaP cells were incubated for 24 h in a culture medium containing TP (100 nmol), ADL (A. distichum leaves, 100 μg/mL; DW, 70% EtOH or 95% hexane), or Fi (1 μg/mL). (B) IF detection of AR in LNCaP cells. LNCaP cells were plated on glass slides and processed for IF. The cells were stained with the anti-AR antibody and then with AlexaFluor 588 secondary antibody. Nuclear staining was done by DAPI staining. Image merging of AR and DAPI was analyzed with a Zeiss fluorescence microscope program.

AR, androgen receptor; TP, testosterone propionate; 5AR2, 5α-reductase type2; PSA, prostate specific antigen; ADL, A. distichum leaf; DW, distilled water; EtOH, ethanol; Fi, finasteride; IF, immunofluorescence; ADLD, A. distichum leaf distilled water extract; ADLE, A. distichum leaf ethanol extract; ADLH, A. distichum leaf hexane extract; DAPI, 4′,6-diamidino-2-phenylindole. ^##P < 0.01 compared to untreated LNCaP cell; ^**P < 0.01, ^***P < 0.001 and ^****P < 0.0001 vs. TP-treated LNCaP cell.

Fig. 2. Effect of ADLE on inhibiting androgen receptor signaling according to harvest season. LNCaP cells were incubated for 24 h in culture medium containing TP (100 nmol), spring_ADLE (50, 100, or 200 μg/mL), autumn_ADLE (50, 100, or 200 μg/mL) or Fi (1 μg/mL). Thereafter, cell lysates (30 μg) were analyzed for the expression of 5AR2 and PSA. The relative protein expression levels were normalized to those of β-actin.

ADLE, A. distichum leaf ethanol extract; TP, testosterone propionate; Fi, finasteride; 5AR2, 5α-reductase type2; PSA, prostate specific antigen. ^#P < 0.05 and ^##P < 0.01 vs. untreated cells; ^*P < 0.05 and ^**P < 0.01 vs. TP-treated cells.

Fig. 3. Effect of ADLE on prostate tissue in TP-induced BPH rats. (A) Photographs of the prostate tissues (VP, DLP, AP). (B) Total prostate tissue weight and (C) prostate indices of the rats. (D) H&E-stained prostate tissues (magnification 100×). (E) Epithelial thickness of the prostate tissues.

ADLE, A. distichum leaf ethanol extract; TP, testosterone propionate; BPH, benign prostatic hyperplasia; VP, ventral prostate; DLP, dorsolateral prostate; AP, anterior prostate; Fi, finasteride; H&E, hematoxylin and eosin; DW, distilled water; Con, corn oil subcutaneous injection and DW oral intake; BPH, TP subcutaneous injection (3 mg/kg) and oral intake; BPH + ADLE, TP subcutaneous injection (3 mg/kg) and A. distichum leaves 70% ethanol extract (100 mg/kg) oral intake; BPH + Saw, TP subcutaneous injection (3 mg/kg) and saw palmetto extract (100 mg/kg) oral intake; BPH + Fi, subcutaneous injection and finasteride (1 mg/kg) oral intake. ^##P < 0.01 vs. the Con group; ^**P < 0.01 vs. the BPH group (n = 8 per group).

Fig. 4. Effect of ADLE on the AR-signaling-related factors in prostate tissues of BPH rats. (A) Representative western blot showing the bands of 5AR2, SRC1, AR, ER, and PSA. (B) densitometer of the protein expression level. (C) Concentration of DHT in the serum. (D) Immunochemical staining of AR in prostate tissue.

ADLE, A. distichum leaf ethanol extract; TP, testosterone propionate; AR, androgen receptor; BPH, benign prostatic hyperplasia; 5AR2, 5α-reductase type2; SRC1, steroid receptor co-activator 1; ER, estrogen receptor; PSA, prostate specific antigen; DHT, dihydrotestosterone; DW, distilled water; Con, corn oil subcutaneous injection and DW oral intake; BPH, TP subcutaneous injection (3 mg/kg) and oral intake; BPH + ADLE, TP subcutaneous injection (3 mg/kg) and A. distichum leaves 70% ethanol extract (100 mg/kg) oral intake; BPH + Saw, TP subcutaneous injection (3 mg/kg) and saw palmetto extract (100 mg/kg) oral intake; BPH + Fi, subcutaneous injection and finasteride (1 mg/kg) oral intake. ^##P < 0.01 vs. the Con group; ^**P < 0.01 vs. the BPH group (n = 8 per group).

Fig. 5. Expression of growth factors and PI3K/AKT in prostate tissue after the administration of ADLE in BPH. (A) Representative western blot showing the bands of EGF, IGF-1, TGF-β1, and VEGF. (B) Representative western blot showing the bands of PI3K, p-AKT and t-AKT.

TP, testosterone propionate; ADLE, A. distichum leaf ethanol extract; BPH, benign prostatic hyperplasia; EGF, epidermal growth factor; IGF-1, insulin-like growth factor-1; VEGF, vascular endothelial growth factor; TGF-β1, transforming growth factor-beta 1; DW, distilled water; Con, corn oil subcutaneous injection and DW oral intake; BPH, TP subcutaneous injection (3 mg/kg) and oral intake; BPH + ADLE, TP subcutaneous injection (3 mg/kg) and A. distichum leaves 70% ethanol extract (100 mg/kg) oral intake; BPH + Saw, TP subcutaneous injection (3 mg/kg) and saw palmetto extract (100 mg/kg) oral intake; BPH + Fi, subcutaneous injection and finasteride (1 mg/kg) oral intake. ^#P < 0.05 and ^##P < 0.01 vs. the Con group; ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs. the BPH group (n = 8 per group).

Fig. 6. Expression of proliferation and apoptosis-related factor in the prostate tissue after the administration of ADLE in BPH. (A) Representative western blot showing the bands of PCNA and cyclin D1. (B) Immunochemical staining of AR in prostate tissue. (C) Representative western blot showing the bands of Bcl-2 and Bax.

ADLE, A. distichum leaf ethanol extract; BPH, benign prostatic hyperplasia; PCNA, proliferative cell nuclear antigen; AR, androgen receptor; DW, distilled water; Con, corn oil subcutaneous injection and DW oral intake; BPH, TP subcutaneous injection (3 mg/kg) and oral intake; BPH + ADLE, TP subcutaneous injection (3 mg/kg) and A. distichum leaves 70% ethanol extract (100 mg/kg) oral intake; BPH + Saw, TP subcutaneous injection (3 mg/kg) and saw palmetto extract (100 mg/kg) oral intake; BPH + Fi, subcutaneous injection and finasteride (1 mg/kg) oral intake. ^#P < 0.05 and ^##P < 0.01 vs. the Con group; *P< 0.05 and ^**P < 0.01 vs. the BPH group (n = 8 per group).