The Combination of Curcumae Radix and Syzygium Aromaticum Extracts Mitigates Benign Prostatic Hyperplasia through Anti-Proliferative and Anti-Inflammatory Effects

Abstract

Purpose: Benign prostatic hyperplasia (BPH) is a urinary tract disorder that primarily affects geriatric males. Natural materials have been developed to treat and prevent symptoms of BPH. However, a few natural functional foods has been conclusively proven to cure or prevent symptoms of BPH. The study aimed to investigate the anti-proliferative mechanism of Curcumae Radix (CR) and Syzygium aromaticum (SA) extracts using RWPE-1 cells and testosterone propionate (TP)-induced BPH rats.

Materials and methods: In vitro experiments were performed to assess the synergistic anti-proliferative effects of an equal mixture of CR and SA extracts on TP-treated RWPE-1 cells. In animal experiments, TP-induced BPH rats were administrated with saline, CR and SA extracts at 50, 100, and 200 mg/kg or finasteride at 1 mg/kg daily for 6 weeks. Body weight, prostate weight, dihydrotestosterone (DHT), androgen receptor (AR), and prostate-specific antigen (PSA) levels were measured. Additionally, extracellular signal-regulated kinase and NF-κB levels, oxidative stress, and inflammatory stress responses in the prostate were also analyzed.

Results: In this study, the combination of CR and SA extracts synergistically inhibited cell proliferation compared with the effect of each extract in TP-treated RWPE-1 cells. CR and SA extracts inhibited increasing of prostate weight, thickness of prostate epithelium, the level of PSA and DHT in serum. The expression of protein and gene of PSA and AR in prostate of TP-induced BPH rats were also suppressed by the administration of CR and SA extracts. Furthermore, reactive oxygen species and inflammation axis, NOX4-iNOS-COX2 and its associated representative inflammatory cytokine, interleukin-8 were also reduced in the CR and SA extracts-administered BPH rats.

Conclusions: The results suggest that the combination of CR and SA extracts improves BPH through its anti-inflammatory and anti-oxidative effects, demonstrating great potential as an anti-BPH medicine.

Keywords: Androgen receptors; Curcuma; Prostate-specific antigen; Prostatic hyperplasia; Syzygium aromaticum; Testosterone propionate.

Fig. 1. Effects of CR and SA extracts treatment on cell proliferation in RWPE-1 cells. (A) Cell viability in RWPE-1 cells following treatment with CR, SA, or CR+SA extracts (n=5 per group). (B) Cell proliferation assay in TP-treated RWPE-1 cells under CR, SA or CR+SA extracts (n=5 per group). (C) Immunoblotting analysis of phospho-ERK and ERK (n=3 blots). CR: Curcumae Radix, SA: Syzygium aromaticum, CR+SA: a mixture of CR and SA, TP: testosterone propionate, Fina: finasteride, ERK: extracellular signal-regulated kinases. Values are presented as mean±standard error of the mean. ^ap<0.05 vs. control; ^bp <0.05 vs. TP.

Fig. 2. The histopathological analysis of the prostate tissues in the testosterone propionate (TP)-induced BPH rats treated with CR and SA extracts. (A) Experimental design: seven-week-old male Sprague-Dawley rats underwent castration, and 1 week later, were injected with 3 mg/kg TP and 0.03 mg/kg 17β-estradiol for 4 weeks to establish a BPH model. Concurrently, CR+SA extracts, finasteride or vehicle was administered for 6 weeks. (B) Assessment of prostate size. Measurement of prostate weight and calculation of the prostate index, a ratio between prostate weight and body weight (n=8 per group). (C) Representative images of the prostate in different groups. (D) Representative H&E-stained images illustrating changes in the thickness of prostate epithelial cells at 100× and 200× magnification. (E) Quantification of prostate epithelial cell thickness (µm) and lumen fold using Image J software (n=8 images). CR: Curcumae Radix, SA: Syzygium aromaticum, CR+SA: a mixture of CR and SA, Fina: finasteride, BPH: benign prostate hyperplasia. Values are presented as mean±standard error of the mean. ^ap<0.05 vs. Sham group; ^bp<0.05 vs. BPH group.

Fig. 3. CR and SA extracts downregulate the expression of androgen receptor (AR) and PSA in the testosterone propionate-induced BPH rats. (A) Assessment of PSA and dihydrotestosterone (DHT) levels in serum (n=8 per group). (B) Immunoblotting analysis of PSA and AR in prostate (n=3 blots). (C, D) Evaluation of PSA and AR levels (n=8 per group) and gene expressions in prostate (n=3 blots). CR: Curcumae Radix, SA: Syzygium aromaticum, CR+SA: a mixture of CR and SA, Fina: finasteride, BPH: benign prostate hyperplasia, PSA: prostate-specific antigen. Values are presented as mean±standard error of the mean. ^ap<0.05 vs. Sham group; ^bp<0.05 vs. BPH group.

Fig. 4. Effects of CR and SA extracts on oxidative stress and inflammation in the in the testosterone propionate-induced BPH rats. (A) Evaluation of oxidative stress using DHE staining (n=5 images). (B) Assessment of mitochondrial superoxide using MitoSOX staining (n=5 images). (C) Measurement of NO production in serum (n=8 per group). (D) Immunoblotting analysis of iNOS, COX2, and NOX4 (n=3 blots). CR: Curcumae Radix, SA: Syzygium aromaticum, CR+SA: a mixture of CR and SA, Fina: finasteride, BPH: benign prostate hyperplasia. Values are presented as mean±standard error of the mean. ^ap<0.05 vs. Sham group; ^bp<0.05 vs. BPH group.

Fig. 5. Effects of CR and SA extracts on extracellular signal-regulated kinases (ERK) and NF-kB in the prostate tissues of testosterone propionate-induced BPH rats. (A) Immunoblotting analysis was performed to assess the levels of phospho-ERK and total ERK, along with β-actin in prostate tissues. The expression of phospho-ERK relative to total ERK was quantified (n=3 blots). (B) Immunoblotting analysis was performed to assess the levels of phospho-NF-κB and total NF-κB, along with β-actin in prostate tissues (n=3 blots). (C) Measurement of IL-8 in the prostate through immunohistochemistry (Quantified, right) (n=5 images). IL-8 in prostate was measured by immunohistochemistry (×200). CR: Curcumae Radix, SA: Syzygium aromaticum, CR+SA: a mixture of CR and SA, Fina: finasteride, BPH: benign prostate hyperplasia. Values are presented as mean±standard error of the mean. ^ap<0.05 vs. Sham group; ^bp<0.05 vs. BPH group.