Anti-proliferation Effects Of Cistanches Salsa On The Progression Of Benign Prostatic Hyperplasia Ⅰ
Oct 18, 2022
Abstract: Cistanche Tubulosa has been used in traditional medicine for the treatment of kidney deficiency, neurasthenia, sexual dysfunction diseases, and benign prostatic hyperplasia(BPH). The aim of this study was to investigate the mechanism by which C. tubulosa extract(CSE) elicits an anti-proliferative effect on the prostate tissue of BPH-induced rats. The effects of CSE on BPH were evaluated in terms of prostate weight, production of seruln dihydrotestosterone (DHT), and the mRNA expression of 5o-reductase type 1 and type 2 in the prostate tissue of BPH-induced rats. In addition, hematoxylin and eosin (H&E) staining was performed for histological examination of prostate gland morphology, and protein expression levels in prostate tissue were investigated by western blot analysis. CSE treatment decreaseed prostate weight, serum DHT concentration, and the mRNA expression of 5o-reductase type 1 and type 2 in prostate tissue of BPH-induced rats. In addition, CSE treatment suppressed cell proliferation by regulating the expression levels of inflamnnatory-related proteins (inducible nitric oxide synthase and cyclooxygenase 2) and apoptosis-associated proteins (caspase-3 and Bcl-2 family proteins). CSE may be a potential therapeutic candidate for BPH owing to its ability to regulate the expression of inflammatory and apoptosis-related proteins.
Keywords: benign prostatic hyperplasia, Cistanche Tubulosa, dihydrotestosterone, inflammation, apoptosis, Bcl-2.
Introduction
Cistanche Tubulosa is a parasitic plant native to the northwest of China. Its stem is an important traditional oriental medicine that is used for the treatment of kidney deficiency and neurasthenia (Deng et al.2004).C.tubulosa has also been reported to be efficacious in the treatment of sexual dysfunction diseases, such as impotence and benign prostatic hyperplasia (BPH) in Korean medicine book called the DonguiBogam. Phenylethanoid glycosides (PhGs)are the major active constituents of this herb (Jimenez and Riguera 1994).Many PhGs have been shown to have a wide range of bio-logical properties, including antioxidant and antitumor effects. Acteoside, a PhG from C. tubulosa, inhibits apoptosis induced by the 1-methyl-4-phenylpyridinium ion (MMP*) in cerebellar granule neurons (Pu et al. 2003). Although studies on the physiological functions of C. tubulosa have been performed, the molecular mecha-nism(s)underlying the effects of C. tubulosa on BPH have not been investigated.
BPH is characterized by an enlargement of the prostate, owing to non-cancerous growth within the gland (Andriole et al. 2004). Increases in BPH prevalence and incidence are occurring within the context of an aging population. By 2030,20% of the US population will be 65 years of age or older, a figure that will include more than 20 million men (Parsons 2010).Although BPH is a com-mon disorder in elderly men, the etiology and pathology of BPH are not completely defined, and there is currently no perfect treat-ment for BPH (Djavan et al.2010).
Treatment options for BPH include pharmacological and surgical strategies.
In recent years, the number of surgical interventions in patients with BPH patients decreased in favor of pharmacological treatments (Emberton et al. 2011). Owing to the nature of BPH, pharmacological treatment requires long and continuous use of medication. Therefore, long and continuous tolerability of the medication is a very important issue. Various kinds of pharmaco-logical agents such as 5a-reductase inhibitors (finasteride and du-tasteride) and a-adrenoceptor antagonists (alfuzosin, terazosin, doxazosin, tamsulosin) are used for BPH treatment (Lepor 2011;
Untergasser et al.2005).The main pharmacological agents for BPH treatment are 5a-reductase inhibitors, which are known to regu-late 5o-dihydrotestosterone levels, and o-adrenergic blockers (Tiwari et al. 2005). Although these pharmacological agents are considered to significantly inhibit the progress of BPH, numerous studies have reported their undesirable side effects (Patel and Chapple 2006).For example,finasteride has been reported to cause various side effects, including dizziness, impaired muscle growth, and severe myopathy, owing to its structural similarity to steroidal hormones (McConnell et al.2003; Uygur et al.1998).Because these pharmacological agents exhibit undesirable side effects, finding herbal remedies that effectively treat BPH has considerable medicinal value.
To evaluate the effect of C. tubulosa on BPH, we demonstrated that C. tubulosa extract (CSE) could modulate the imbalance between prostatic cell growth and apoptosis in testosterone-induced BPH rat models.
Materials and methods Materials and reagents
C.tubulosa was purchased from Omniherb Co.Ltd. (Daegu, Republic Korea) and finasteride was obtained from Merck and Co.Inc. (New Jersey, USA). Testosterone, phenylmethylsulfonylfluoride (PMSF), Triton X-100, propidium iodide (PI), Nonidet P-40(NP-40), and protein inhibitor cocktail (PIC) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Missouri, USA). GAPDH oligonucleotide primers and 5o-reductase type 1 and type 2 were purchased from Bioneer (Deajeon, Korea), and SYBR Primix Ex Taq was purchased from Takara(Shuzo, Shiga,Japan).Antibodies for anti-iNOS(M-19), COX-2(C-20),poly(ADP-ribose)polymerase-1(PARP-1,(D-1),caspase-3 (H-277),Bcl-2(C-2),Bcl-xL(H-5),Bax(B-9),and anti-β-actin(ACTBD11B7)were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). The antibody for anti-PCNA was purchased from BD Biosciences,Pharmingen (San Diego,California,USA). Preparation of C. tubulosa extract (CSE)
The herbs of C. tubulosa were purchased from Omniherb Co.Ltd. (Yeongcheon, Kyungpook, Republic of Korea). They had a moisture content of <13% by weight and were air-dried. Afterward, herbs underwent extraction with 50%(v/v) ethanol-water at 60 ℃for 8 h. The extract obtained was filtered through 20 μm cartridge paper, and ethanol was removed by vacuum rotary evaporation (Eyela, Japan). The concentrate was subsequently freeze-dried, and the yield was found to be 12% w/w. The experiment was performed by dissolving the dry extract in distilled water, and the rest of extract was stored at -20 ℃. The identities of the active components of the extract,as determined by HPLC, were reported previously(Jiang and Tu 2009).
Animals
Ten-week-old male Sprague-Dawley rats (200±20 g) were purchased from Daehan Biolink (Daejeon, Republic of Korea). The animals were housed under conditions that were in accordance with the guidelines for the care and use of laboratory animals, adopted and promulgated by the Institutional Animal Care Committee of Sangji University (Reg. No.2014-09). The rats were acclimatized to the laboratory conditions for 2 weeks before starting the experiment. They were provided free access to food and water for 4 weeks and were maintained under a 12 h light/12 h dark cycle at a constant temperature of 22±2℃ and relative humidity of 55±9% throughout the experiment. The rats were randomly assigned to one of 4 groups (n=6): the control group (Con, normal prostate with vehicle solution: phosphate buffered saline (PBS)200μL， p.o.）； BPH-induced group （BPH）， BPH-induced group treated with finasteride (5 mg/kg/day, p.o.,(Fina)); BPH-induced group treated with CSE(200 mg/kg/day; p.o.). The rats in the control group were cut open and then sewn up without removing the testicles after anaesthetization with zoletil@ 50 (20 mg/kg, i.p.);rats in the other groups were castrated. After 3 days of recovery, the BPH-induced groups were injected with testosterone propionate (10mg/kg/day,i.p., dissolved in corn oil) alone or along with CSE or finasteride daily for 4 weeks. Twenty-four hours after the last administration, all rats were sacrificed after anaesthetization with zoletil@ 50 (20 mg/kg,i.p.).Blood samples were drawn from the caudal vena cava, and serum was separated by centrifugation and stored at-80 ℃. The entire prostate gland was removed and weighed (Guo et al.2004).
Prostate weight to body weight ratio
Prostatic tissues were excised, rinsed, and weighed immediately. Prostate weight to body weight ratio(PW/BW ratio) was calculated as described below.
PW/BW ratio
= (each prostate weight of animal from experimental group/each body weight of animal from experimental group) x 100 Growth inhibition of prostate weight
On study completion, prostates were removed and weighed. The percentage of growth inhibition was calculated as follows:100-[(treated group-control group)(BPH group-control group)×100]. Serum concentrations of dihydrotestosterone (DHT)analysis
Serum concentrations of DHT were determined with commer-cial ELISA (ALPCO Diagnostics, Salem, New Hampshire, USA). As-says were performed according to the manufacturer's instructions.
Histological analysis
The prostate tissue in each group was fixed in 4% formalin solution, embedded in paraffin, and then cut into 4 pm sections. The sections were stained with hematoxylin and eosin (H&E) for histological examination. Images were acquired using an SZX10 microscope (Olympus, Tokyo, Japan).Histological analysis and the thickness of epithelium tissue from prostate (TETP) was measured by a professional histologist from Seoul Medical Science Institute (Seoul, Korea) using Leica Application Suite (LAS, ver. 3.3.0) soft-ware for objective assessment in histological analysis. Quantitative real-time polymerase chain reaction (PCR)analysis
The prostatic tissue from each animal was homogenized, and total RNA was isolated using Easy-Blue@ Reagent (Intron Biotech-nology Inc., Gyeongi-do, Republic of Korea) according to the man-ufacturer's instructions. Total RNA was quantified using an Epoch@micro-volume spectrophotometer system (BioTek Instruments, Inc. Winooski, Vermont, USA). Total RNA from the liver and visceral adipose tissues was converted in to cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, California, USA).PCR amplification was performed using the in-corporation of SYBR green (Applied Biosystems). The oligonucle-otide primers for 5o-reductase type 1, designed from rat, were GGAAGG TTT CAT GGCCTT CG(forward) and TCG AGG GCA GCT GTA TG(reverse), the 5o-reductase type 2 primers, designed from rat, were ATG GGG ACC CTG ATC CTG TG (forward) and CGA CAC CACAAA GGA AGG CA (reverse), and the GAPDH primers, used as a house-keeping gene and designed from rat, were TGA TTC TAC CCA CGG CAA GT (forward) and AGC ATC ACC CCA TTT GAT GT (reverse).Reverse transcription was carried out using a thermocy-cler (Gene Amp⑧ PCR system 9700, Applied Biosystems), and the results were expressed as the ratio of optimal density to GAPDH. Western blot analysis
Prostate tissue from each animal was homogenized in the commercial lysis buffer PRO-PREP⑧(Intron Biotechnology Inc.) and incubated for 25 min on ice to induce cell lysis. Tissue extracts were centrifuged at 16 000g(4°C)for20min, and the supernatant was transferred to a clean tube. The protein concentration was deter showed typical histologic changes, including a thickened glandular epithelium, a vacuolated cytoplasm pointing toward the glandular lumen, and a reduced glandular luminal area (Fig. 3A). Administration of CSE for 4 weeks suppressed these typical hyperplastic patterns, which represent a histological change from normal prostatic tissue to prostatic hyperplasia. In brief, histological examination of finasteride-and CSE-treated groups showed a clear trend for ameliorating prostatic hyperplasia. In addition, as shown in Fig. 3B, TETP analysis showed that the most thickened epithelial tissue was observed in BPH-induced rats compared to that in any other study groups, and finasteride- and CSE-treated groups showed a significant reduction in prostate thickness.
To evaluate the proliferation of prostate epithelial cells, we examined the protein expression of PCNA in the prostate tissue of BPH-induced rats. As shown in Fig. 3C, western blot analysis indi-cated an increase in PCNA protein expression in the BPH-induced group compared to PCNA levels in the control group. Compared to the BPH-induced group, however, the finasteride- and CSE-treated groups displayed reduced PCNA protein expression, indicating their anti-proliferation effects in BPH.
Fig.2.Effect of Cistanche Tubulosa extract (CSE) administration on serum dilhydrotestosterone (DHT) production and 5a-reductase type 1 and type 2 mRNA levels in prostate tissues of benign prostatic hyperplasia(BPH)-induced rat models. (A) Serum concentrations of DHT were determined using ELISA assay. (B) 5a-Reductase type 1 and type 2 mRNA expressionin prostate tissue were analyzed by quantitative real-time PCR. The data shown represent mean±SEM of 6 rats per group."p<0.05,***p<0.001 vs.Con group;""p<0.001 vs.BPH group.
