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Paljung-San, a traditional herbal medicine, attenuates benign prostatic hyperplasia in vitro and in vivo
Author’s Accepted Manuscript Paljung-San, a Traditional Herbal Medicine, Attenuates Benign Prostatic Hyperplasia In Vitro and In Vivo Eunsook Park, Mee-Young Lee, Woo-Young Jeon, Chang-Seob Seo, Sooseong You, Hyeun-Kyoo Shin www.elsevier.com/locate/jep
PII: DOI: Reference:
S0378-8741(18)30030-8 https://doi.org/10.1016/j.jep.2018.02.037 JEP11248
To appear in: Journal of Ethnopharmacology Received date: 4 January 2018 Revised date: 23 February 2018 Accepted date: 23 February 2018 Cite this article as: Eunsook Park, Mee-Young Lee, Woo-Young Jeon, ChangSeob Seo, Sooseong You and Hyeun-Kyoo Shin, Paljung-San, a Traditional Herbal Medicine, Attenuates Benign Prostatic Hyperplasia In Vitro and In Vivo , Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2018.02.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Paljung-San, a Traditional Herbal Medicine, Attenuates Benign Prostatic Hyperplasia In Vitro and In Vivo
Eunsook Park1, Mee-Young Lee1, Woo-Young Jeon1, Chang-Seob Seo1, Sooseong You2, and Hyeun-Kyoo Shin1*
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K-herb Research Center, Korea Institute of Oriental Medicine, 1672 Yuseong-daero,
Yuseong-gu, Daejeon 34054, Republic of Korea 2
KM Fundamental Research Division, Korea Institute of Oriental Medicine, 1672
Yuseong-daero, Yuseong-gu, Daejeon 34054, Republic of Korea Eunsook Park: [email protected] Mee-Young Lee: [email protected] Woo-Young Jeon: [email protected] Chang-Seob Seo: [email protected] Sooseong You: [email protected] Hyeun-Kyoo Shin: [email protected]
*Corresponding author: Dr. Hyeun-Kyoo Shin, K-herb Research Center, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon, 34054, Republic of Korea. Phone: +82-42-868-9464; Fax: +82-42-868-2120
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Abstract Ethnopharmacological relevance: Paljung-san is a traditional herbal medicine used widely for the treatment of urogenital diseases in East Asia. However, scientific evidence of the efficacy of Paljung-san and its mechanisms of action against benign prostatic hyperplasia (BPH) is not clearly established. Aim of the study: We investigated the inhibitory effect of Paljung-san water extract (PSWE) and its mechanisms against BPH in vitro and in vivo. Materials and methods: Active compounds of PSWE were analyzed quantitatively by High-performance liquid chromatography (HPLC). For in vitro study, PSWE treated BPH-1 cells were used to perform western blot analysis, cell cycle analysis and enzymelinked immunosorbent assay. For in vivo BPH model, male rats were subcutaneously injected with 10 mg/kg of testosterone propionate (TP) every day for four weeks. 200 and 500 mg/kg of PSWE was administrated daily by oral gavage with s.c. injection of TP, respectively. Results: HPLC revealed that PSWE contains 1.21, 1.18, 2.27, 3.56, 4.23, 3.00, 6.78, and 0.004 mg/g of gallic acid, 5-caffeoylquinic acid, chlorogenic acid, geniposide, liquiritin apioside, liquiritin, glycyrrhizin, and chrysophanol components, respectively. In human BPH-1 cells, PSWE treatment reduced cell proliferation through arresting the cell cycle in the DNA synthesis phase. Moreover, PSWE suppressed prostaglandin E2 production with reduced cyclooxygenase-2 expression. In TP -induced BPH rat model, PSWE administration showed reduced prostate weights and dihydrotestosterone levels and led to a restoration of normal prostate morphology. PSWE also decreased TPinduced Ki-67 and cyclin D1 protein levels in the prostatic tissues. Decreased 2
glutathione reductase activity and increased malondialdehyde levels in the BPH groups were reversed by PSWE administration. Conclusion: PSWE attenuates the progression of BPH through anti-proliferative, antiinflammatory and anti-oxidant activities in vitro and in vivo. Therefore, these data provide the scientific evidence of pharmacological efficacy of PSWE against BPH.
Keywords: Paljung-san water extract; traditional herbal medicine; high-performance liquid chromatography; benign prostatic hyperplasia; BPH-1 cells; rat model
1. Introduction Benign prostatic hyperplasia (BPH), a noncancerous enlargement of the prostate gland, is a common disease found in men aged over 50 years (Berry et al., 1984). BPH involves overgrowth of both epithelial and stromal cells in the transitional zone of the prostate gland around the urethra, and leads to lower urinary tract symptoms (LUTS) including problems with urinary storage (hesitancy, weak streaming and retention) or voiding (frequency, urgency and nocturia) (O'Leary, 2003). Although the cause of BPH is not fully understood, age-related imbalances in androgen levels are associated with an increased risk of BPH (Roehrborn, 2008). Dihydrotestosterone (DHT), a metabolite of testosterone converted by 5-reductase (5R), acts as an active androgen for prostatic growth (McConnell, 1995). Therefore, excessive DHT levels in the prostate gland induce overproliferation of both epithelial and stromal cells and can lead to the 3
development and progression of BPH (Steers, 2001). As mentioned above, many clinical studies have found increases in serum DHT levels and DHT/testosterone ratios in elderly patients with BPH (Horton et al., 1975). Whereas men aged 40–49 years make up around 20–40% of all patients with BPH, men aged over 60 years account for over 60% in this cohort, so the prevalence of histological BPH increases with aging (Berry et al., 1984). In addition to the effects of androgens, oxidative stress and inflammation in the prostate gland also influence the development and progression of BPH (De Nunzio et al., 2016). Among the pharmacological therapies used for the treatment of BPH, 5R inhibitors such as finasteride and dutasteride, which block the conversion of testosterone to DHT, are effective synthetic agents with anti-proliferative effects on the enlarged prostate. Despite this therapeutic efficacy, taking these medications for prolonged times leads to adverse side effects associated with sexual function such as erectile dysfunction, lowered sexual drive (libido) and reduced semen volume during ejaculation (Gormley et al., 1992). Therefore, many men who desire fewer side effects and effective treatments for BPH have been interested in phytotherapeutic agents as complementary and alternative medicines (Ma et al., 2013). In fact, several herbal medicines derived from natural products have been reported as having therapeutic efficacy for the treatment of BPH (Lowe and Fagelman, 2002). Extract of Pygeum africanum, the African prune tree, is well known as a phytotherapeutic agent used for treating men with BPH, and for alleviating LUTS (Wilt et al., 2002). Paljung-san, also known as Bazheng-san in Chinese, is a traditional herbal medicine based on the classic Korean book Donguibogam in which the principles and 4
practice of Eastern traditional medicine are described. Paljung-san is commonly used for the treatment of urogenital diseases such as acute nephritis, cystitis, kidney stones and urethral syndrome (Kim et al., 2007; Lee et al., 2000). Moreover, it has been reported as being therapeutic against diseases of the male reproductive system. Modified Paljung-san in a rodent model showed inhibition against chronic prostatitis via its anti-inflammatory and anti-oxidant activities (Xiong et al., 2017). A clinical study showed that Paljung-san treatment in patients with BPH ameliorated LUTS such as urinary frequency and retention (Song et al., 2010). However, scientific evidence of the efficacy of Paljung-san for the treatment of BPH is not fully defined. Therefore, we investigated the therapeutic efficacy of Paljung-san water extract (PSWE) and its mechanisms of action in human BPH-1 cells in vitro and in a rat model of BPH in vivo.
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2. Materials and methods 2.1. Chemicals and reagents Reference standard marker compounds, gallic acid (purity 99.0%) and 5caffeoylquinic acid (purity 99.2%) were purchased from Merck KGaA (Darmastadt, Germany). Gepinoside (purity 98.0%), liquiritin (purity 99.6%), and glycyrrhizin (purity 99.0%) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Chlorogenic acid (purity 99.6%), liquiritin apioside (purity 98.0%), and chrysophanol (purity 99.0%) were purchased from Acros Organics (Pittsburgh, PA, USA), Shanghai Sunny Biotech (Shanghai, P. R. China), and Biopurify Phytochemicals (Chengdu, P. R. China), respectively. Solvents, methanol, acetonitrile and distilled water, used for HPLC analysis were HPLC grade and obtained from J. T. Baker (Phillipsburg, NJ, USA). Formic acid, the American Chemical Society (ACS) reagent grade, was obtained from Merck KGaA. 2.2. Plant materials The component medicinal components of Paljung-san (Rhei Radix et Rhizoma, Akebiae Caulis, Dianthi Herba, Polygoni Avicularis Herba, Talcum, Gardeniae Fructus, Plantaginis Semen, Glycyrrhizae Radix et Rhizoma, and Junci Medulla) were purchased from Kwangmyungdag Medicinal Herbs (Ulsan, S. Korea) in May 2015. A voucher specimen (2015–KE56–1 to KE56–9) has been deposited at the K-herb Research Center, Korea Institute of Oriental Medicine. 2.3. Preparation of PSWE Paljung-san was mixed from the nine traditional medicinal herbs listed above and in Table 1 combined in equal amounts, and extracted in a 10-fold volume of water 6
at 100 °C for 2 h using an electric extractor (COSMOS-660; Kyungseo Machine Co., Incheon, S. Korea). The extracted solution was filtered using a standard sieve (No. 270, 53 m; Chung Gye Sang Gong Sa, Seoul, S. Korea) and then the filtered solution was processed to a powder using a PVT100 freeze–dryer (IlShin Bio Base, Yangju, S. Korea). The amount of lyophilized water extract was 467 g (yield: 9.34%). 2.4.High-performance liquid chromatography (HPLC) analysis of PSWE HPLC analysis was performed using a Shimadzu Prominence LC-20A series (Kyoto, Japan) as described previously (Park et al., 2016b). The separation of the major marker components was achieved on a Waters SunFire C18 column (250 4.6 mm, 5 m, Milford, MA, USA) with a column oven temperature of 40 °C. The mobile phases consisted of distilled water (A) and acetonitrile (B), both with 0.1% (v/v) formic acid. The gradient system of mobile phase was as follows: 5–60% B for 0–30 min; 60–100% B for 30–40 min; 100% B for 40–45 min; 100–5% B for 45–50 min; and 5% B for 50– 60 min. The flow rate was 1.0 mL/min and the injection volume was 10 L. For HPLC analysis of PSWE, 200 mg of lyophilized PSWE was dissolved in 20 mL of 50% methanol and then extracted using a sonicator for 30 min. The extracted solution was filtered through a 0.2 m pore membrane filter (PALL Life Sciences, Ann Arbor, MI, USA) before HPLC injection. 2.5. Cell culture BPH-1 cells (Park et al., 2017) were maintained in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 20% FBS (Invitrogen, Carlsbad, CA) at 37 °C under an atmosphere of 5% CO2 in air. 7
2.6. Cell proliferation assay Cell viability was analyzed using a nonradioactive Cell Counting Kit (CCK)-8 system (Dojindo, Tokyo, Japan) according to the manufacturer’s instructions (Park et al., 2017). To determine the effect of PSWE on DNA synthesis, 5-Ethynyl-2′deoxyuridine (EdU) incorporation assay were performed using Click-iT Plus EdU Imaging kits (Invitrogen, Carlsbad, CA, USA) as described previously (Park et al., 2017). 2.7. Western blot analysis Protein levels were determined by western blot analysis as described (Park et al., 2016a). Extracts of total proteins from BPH-1 cells or rat prostatic tissues were prepared using RIPA buffer (Sigma-Aldrich). The following antibodies were used: anticyclin D1 (ab134175; Abcam, Cambridge, UK), anti-PCNA (ab29; Abcam), anti-COX2 (160112; Cayman Chemical Co., Ann Arbor, MI, USA) and anti--actin (#4967; Cell Signaling, Danvers, MA, USA). 2.8. Flow cytometry analysis Cells were stained with propidium iodide using flow cytometry kits (Abcam) according to the manufacturer’s instructions. FACS analysis of cell cycles was performed on BD FACSCalibur system (BD Biosciences, Heidelberg, Germany) and Modifit LT version 4.1 software (Verity Software House, Topsham, ME, USA). 2.9. Measurement of PGE2 levels Levels of PGE2 in BPH-1 cells were measured using human PGE2 enzymelinked immunosorbent assay (ELISA) kits (Cayman Chemical, Ann Arbor, MI, USA) 8
according to the manufacturer’s instructions. Briefly, BPH-1 cells (1 104 cells/well) were seeded onto 48-well plates in triplicates in complete medium. Next day, the medium was refreshed and DMSO vehicle, finasteride or PSWE was added to the medium. After 24 h, the supernatants were harvested for measuring PGE2 levels. 2.10. Animals Seven-week-old male Sprague Dawley rats weighing 200–220 g were purchased from Orient Bio Inc. (Seoul, S. Korea). Rats were maintained under a 12/12h light/dark cycle at 18–23 °C and a relative humidity of 40–60% in our animal handling facility. Standard laboratory chow (Harlan Teklad, Madison, WI, USA) and water were available ad libitum. All experimental procedures were approved by the Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee and performed in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals. Animals were cared for in accordance with the dictates of the National Animal Welfare Law of S. Korea. 2.11. Experimental design Experimental procedures for studying the efficacy of PSWE in the rat model of TP-induced BPH were designed based on a previous study (Park et al., 2016a). Briefly, rats were divided randomly into five groups (n = 5 per group) and treated daily for 4 weeks as follows: a negative control (NC) group that received phosphate-buffered saline (PBS) orally with a subcutaneous (s.c.) injection of corn oil; a BPH group that received PBS orally with a s.c. injection of 10 mg/kg of TP (Tokyo Chemical Industry Co., Tokyo, Japan) dissolved in corn oil; a finasteride group as a positive control that 9
received 10 mg/kg of finasteride (Sigma-Aldrich) orally with s.c. injection of 10 mg/kg of TP; PSWE-200 and PSWE-500 groups that received 200 and 500 mg/kg of PSWE orally together with an s.c. injection of 10 mg/kg of TP, respectively. A dose of PSWE in rat was calculated based on guideline (Food and Administration, 2005), which is described the conversion of animal doses to human equivalent doses based on body surface area. Given that rat dose to human equivalent dose is 347.448 mg/kg, we chosen 200 and 500 mg/kg, a lower and higher dosage than that, respectively. 2.12. Histology and immunohistochemistry For histology, deparaffinized and rehydrated sections from the fixed rat prostate tissues were stained with Mayer’s hematoxylin (MHS-16, Sigma-Aldrich) and eosin (HT110-1-32, Sigma-Aldrich) (H&E) solution. Immunohistochemistry for Ki-67 was performed using Vectastain Elite ABC kits (Vector Laboratories Inc., Burlingame, CA, USA) as described (Park et al., 2016a). 2.13. Measurement of DHT and MDA levels, and GR activity Measurement of DHT and MDA levels, and GR activity was performed as described previously (Park et al., 2016a). Briefly, GR activity in prostate lysates was analyzed using Glutathione Reductase Assay kits (Cayman) according to the manufacturer’s instructions. 2.14. Statistical analysis Data are presented as the mean ± standard error of the mean (SEM). Statistical significance was calculated using one-way analysis of variance with Dunnett’s test (Dunnett, 1964). Differences were considered significant at P<0.05. 10
3. Results 3.1. HPLC analysis of eight marker components in PSWE Using optimized HPLC, eight marker components were eluted within 45 min, with a resolution of 1.12. The retention times of gallic acid, 5-caffeoylquinic acid, chlorogenic acid, geniposide, liquiritin apioside, liquiritin, glycyrrhizin, and chrysophanol were 6.31, 10.98, 12.58, 13.60, 16.22, 16.62, 30.12, and 40.53 min, respectively (Fig. 1). The concentrations of these markers were 1.21, 1.18, 2.27, 3.56, 4.23, 3.00, 6.78, and 0.004 mg/g, respectively. 3.2. PSWE reduces cell viability in vitro The epithelial cell line BPH-1 was used to address the effects of PSWE in vitro. PSWE treatment for 48 h significantly inhibited cell viability (Fig. 2). Among the tested concentrations of PSWE, those over 125 g/mL repressed cell viability in a concentration-dependent manner. Unlike PSWE, the finasteride treatment used as a positive control had no effect on cell viability. 3.3. PSWE inhibits cell proliferation in vitro The pathogenesis of BPH is associated with the overproliferation of epithelial cells in prostatic ducts. Therefore, we examined the effect of PSWE on proliferationrelated protein levels. PSWE treatment in BPH-1 cells decreased both cyclin D1 and proliferating cell nuclear antigen (PCNA) protein levels in concentration-dependent manners (Fig. 3A). Control finasteride treatment also decreased the levels of these proteins. To evaluate DNA synthesis in the cell cycle, 5-ethynyl-2-deoxyuridine (EdU) assays were performed. The number of EdU-positive cells was markedly decreased by 11
treatment with PSWE as well as finasteride compared with vehicle alone (Fig. 3B), suggesting that PSWE inhibits DNA synthesis in BPH-1 cells. In addition, fluorescenceactivated cell sorting (FACS) analysis revealed that PSWE treatment resulted in a reduced proportion of cells in the DNA synthesis (S) phase compared with vehicle treatment alone (Fig. 3C). Finasteride treatment showed a similar pattern of cell cycle alterations. Taken together, these results indicate that PSWE inhibits the proliferation of BPH-1 cells through arrest at the S phase of the cell cycle. 3.4. PSWE suppresses inflammation in vitro To elucidate whether PSWE would regulate BPH-related inflammatory responses, we examined the effect of PSWE on the levels of prostaglandin E2 (PGE2), which is produced during inflammation. PSWE treatment for 24 h as well as finasteride (which had no toxicity for BPH-1 cells; data not shown), significantly suppressed PGE2 production in concentration-dependent manners (Fig. 4A). Moreover, both finasteride and PSWE treatments reduced the protein levels of cyclooxygenase-2 (COX-2), an inducible enzyme responsible for PGE2 production (Fig. 4B), suggesting that PSWE represses the inflammatory response in BPH-1 cells. 3.5. PSWE represses pathogenesis in a testosterone propionate (TP)-induced rat model of BPH To further confirm the inhibitory effect of PSWE against BPH in vitro, the therapeutic efficacy of PSWE in vivo was determined using a rat model of TP-induced BPH. As shown in Figure 5A, the relative prostate weight of the TP-treated group (BPH group) was significantly higher than of the negative control (NC) group. However, the relative prostate weight of the group administered TP plus finasteride as a positive 12
control (Fin group), was decreased compared with the BPH group. The relative prostate weight of rats administered PSWE at 200 mg/kg and 500 mg/kg with TP (PSWE-200 and PSWE-500 groups, respectively) were also markedly reduced compared with the BPH group. In addition, increased DHT levels in the prostates of the BPH group compared with the NC group were repressed by 200 and 500 mg/kg PSWE as well as by finasteride (Fig. 5B). Histologically, the BPH group exhibited prostatic hyperplasia with multiple layers of epithelial cells compared with the NC group while the Fin, PSWE-200 and PSWE-500 groups showed amelioration of the disrupted prostatic morphology compared with the BPH group (Fig. 5C). Taken together, these results suggest that PSWE reverses the pathogenesis of BPH in vivo. 3.6. PSWE exerts anti-proliferative effects in a rat model of TP-induced BPH We examined the expression of prostatic proliferation markers to evaluate the mechanisms underlying the inhibition of PSWE on BPH pathogenesis. Figure 6A shows an increased expression of Ki-67, a cell proliferation marker, in both stromal and epithelial prostatic cells in the BPH group but not in the NC group. On the other hand, prostatic tissues in the Fin and both PSWE groups showed lower expression of Ki-67 than in the BPH group. Consistent with these changes in Ki-67 expression, the protein levels of cyclin D1, another marker of proliferation, were also regulated by PSWE administration. In prostatic lysates, the cyclin D1 protein level was significantly elevated in the BPH group compared with the NC group but was reduced in Fin and both PSWE groups compared with the BPH group (Fig. 6B). These results indicate that PSWE has anti-proliferative effects against BPH in vivo. 3.7. PSWE has anti-oxidative effects in a rat model of TP-induced BPH 13
Oxidative stress is associated with the pathogenesis of BPH. To test whether PSWE would affect oxidative stress in BPH, glutathione reductase (GR) activity and malondialdehyde (MDA) levels, both markers of oxidative stress, were determined in prostates of the BPH group. As shown in Figure 7A, the activity of GR, an antioxidant enzyme, was markedly reduced in the BPH group compared with the NC group but was restored by PSWE as well as finasteride treatments. Lipid peroxidation involves the degradation of lipids induced by oxidative stress and the release of reactive oxygen species (ROS) (Nishida et al., 2016). The BPH group showed higher levels of MDA, an indicator of lipid peroxidation, than did the NC group, whereas the Fin and both PSWE groups exhibited lower levels of MDA than in the BPH group (Fig.7B). Taken together, these results suggest that PSWE has anti-oxidant activity against BPH in vivo.
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4. Discussion BPH is the most common urogenital disease in aged men and requires clinical intervention in about one-third of those aged over 60 years (Berry et al., 1984). Surgical and pharmacological approaches against BPH are the main medical treatments by removing hypertrophic prostatic tissues and improvement of ULTS, respectively. Nevertheless, adverse effects reported with these approaches and concern for the quality of life have led to increased interest in the use of natural products with fewer side effects against BPH (Lowe and Fagelman, 2002). A Korean group reported that four men with BPH and LUTS who were treated with Paljung-san for 10 days showed improvements in LUTS, suggesting the possibility of using this herbal medicine against BPH (Song et al., 2010). However, no scientific study of the effect of Paljung-san treatment on BPH has been carried out. Therefore, this study is the first to establish scientific evidence of the efficacy of PSWE and its mechanisms of action against BPH. We found that PSWE treatment markedly inhibited cell proliferation and PGE2 production in the BPH-1 cell line through reduced DNA synthesis and COX-2 protein levels, respectively. Furthermore, the repressive effect of PSWE in vitro was verified using a rat model of BPH in which PSWE administration attenuated pathogenesis through its anti-proliferative and anti-oxidant activities. Overproliferation in epithelial and stromal cells is a major pathogenic feature of BPH and its progression (Steers, 2001). Cell proliferation is governed by the cell cycle, which is comprised of four phases including G1, S (DNA synthesis), G2, and M (mitosis) (Golias et al., 2004). The G1 to S, or G2 to M transitions are regulated by the expression or activation of various cell cycle-regulatory proteins (Matson and Cook, 15
2016). Among them, cyclin D1, a protein that acts during the G1 phase and is required for the initiation of the S phase, PCNA, a nuclear protein that is elevated during the G1/S phase transition, and Ki-67, an antigen expressed in all phases except for resting cells, are widely used as markers of proliferating cells (Scholzen and Gerdes, 2000; Stacey, 2003). PSWE treatment in BPH-1 cells and in our rat model of BPH reduced the expressions of these markers. Furthermore, the proportion of cells in the S phase of the cell cycle as well as the number of EdU-positive cells, reflecting DNA synthesis, were significantly decreased by PSWE. Therefore, these findings indicate that PSWE inhibits cell proliferation by arresting the S phase of the cell cycle. Several studies have demonstrated that pathways involving inflammation and oxidative stress contribute to cell proliferation in BPH. The presence of heterogeneous bacterial and viral strains in BPH biopsy specimens suggests a possible contribution of prostatic inflammation to its pathogenesis (De Nunzio et al., 2016). Histopathology of prostatic tissues from patients with BPH revealed that the levels of inflammatory markers are increased along with prostate growth (Di Silverio et al., 2003). In addition, COX-2, an enzyme associated with inflammation, is highly expressed in epithelial cells from BPH samples, and its repression inhibits their overgrowth (Ammar et al., 2015). Given that Paljung-san is effective against inflammatory diseases such as acute nephritis, cystitis and prostatitis (Xiong et al., 2016), PSWE might inhibit cell proliferation through its anti-inflammatory activity in BPH tissues. Here, PSWE treatment reduced the levels of PGE2, an inflammatory modulator regulated by COX-2 activity, as well as COX-2 protein levels in BPH-1 cells. These findings suggest that the effect of PSWE on inhibiting cell proliferation in BPH is associated with its anti16
inflammatory activity. Furthermore, there are well-known interactions between inflammation and oxidative stress in various other diseases (Li et al., 2016). Oxidative stress associated with ROS production leads to lipid peroxidation, increases the levels of proinflammatory cytokines such as tumor necrosis factor alpha and interleukin-6, and this then leads to inflammation (Nishida et al., 2016). Therefore, many herbal medicines target oxidative stress and inflammation simultaneously. Thus, Ghrelin activity and Geraniol treatments attenuate liver injury caused by oxidative stress, inflammation and apoptosis (Li et al., 2013). Extracts of pomegranate fruit and of the fern Abacopteris penangiana protect against BPH through their anti-inflammatory and anti-oxidant properties (Ammar et al., 2015; Yang et al., 2014). Consistent with the above findings, treatment with PSWE in our rat model of BPH also restored the activity of anti-oxidant enzymes and repressed lipid peroxidation, inhibiting inflammation and oxidative stress simultaneously. Here, the concentrations of distinct chemical components in PSWE were measured by HPLC. It has been reported that glycyrrhizin, which is at a higher level in PSWE than other components, has a range of bioactivities in various diseases, including anti-proliferative, anti-inflammatory and anti-tumorigenic effects (Ma et al., 2016; Rackova et al., 2007). In addition, liquiritin apioside and liquiritin, which are also found in PSWE, are naturally occurring polyphenols that exhibit anti-obesity and anti-oxidant properties (Guan et al., 2012; Kamisoyama et al., 2008). These findings are consistent with the therapeutic efficacy of PSWE against BPH through its anti-proliferative, antiinflammatory and anti-oxidant activities.
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5. Conclusions We found that PSWE inhibited the progression of BPH in vitro and in vivo. This involved inhibition of cell proliferation through cell cycle arrest, and anti-inflammatory and anti-oxidant activities. Accordingly, our findings provide the first scientific evidence of the efficacy of Paljung-san treatment against BPH.
Conflict of interests The authors declare that they have no competing interest.
Acknowledgements This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number; H115C0025).
Authors’ contributions HKS participated in the design of the study and manuscript editing. EP conducted in vitro and in vivo experiments, data interpretation and manuscript preparation. MYL carried out in vivo experiments, data interpretation and edited the manuscript. WYJ conducted in vivo experiments and data interpretation. CSS carried out the preparation of PSWE, HPLC analysis and manuscript preparation. SY helped in the experiment. All authors red and approved the final manuscript.
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6. References Ammar, A.E., Esmat, A., Hassona, M.D., et al. (2015) The effect of pomegranate fruit extract on testosterone-induced BPH in rats. Prostate, 75: 679-692. Berry, S.J., Coffey, D.S., Walsh, P.C., et al. (1984) The development of human benign prostatic hyperplasia with age. J Urol, 132: 474-479. De Nunzio, C., Presicce, F., Tubaro, A. (2016) Inflammatory mediators in the development and progression of benign prostatic hyperplasia. Nat Rev Urol, 13: 613626. Di Silverio, F., Gentile, V., De Matteis, A., et al. (2003) Distribution of inflammation, pre-malignant lesions, incidental carcinoma in histologically confirmed benign prostatic hyperplasia: a retrospective analysis. Eur Urol, 43: 164-175. Dunnett, C.W. (1964) New tables for multiple comparisons with a control. Biometrics, 20: 482-491. Food, Administration, D. (2005) Guidance for industry: estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers. Center for Drug Evaluation and Research (CDER), 7. Golias, C.H., Charalabopoulos, A., Charalabopoulos, K. (2004) Cell proliferation and cell cycle control: a mini review. Int J Clin Pract, 58: 1134-1141. Gormley, G.J., Stoner, E., Bruskewitz, R.C., et al. (1992) The effect of finasteride in men with benign prostatic hyperplasia. The Finasteride Study Group. N Engl J Med, 327: 1185-1191. Guan, Y., Li, F.F., Hong, L., et al. (2012) Protective effects of liquiritin apioside on cigarette smoke-induced lung epithelial cell injury. Fundam Clin Pharmacol, 26: 47319
483. Horton, R., Hsieh, P., Barberia, J., et al. (1975) Altered blood androgens in elderly men with prostate hyperplasia. The Journal of Clinical Endocrinology & Metabolism, 41: 793-796. Kamisoyama, H., Honda, K., Tominaga, Y., et al. (2008) Investigation of the antiobesity action of licorice flavonoid oil in diet-induced obese rats. Biosci Biotechnol Biochem, 72: 3225-3231. Li, S., Hong, M., Tan, H.Y., et al. (2016) Insights into the Role and Interdependence of Oxidative Stress and Inflammation in Liver Diseases. Oxid Med Cell Longev, 2016: 4234061. Li, Y., Hai, J., Li, L., et al. (2013) Administration of ghrelin improves inflammation, oxidative stress, and apoptosis during and after non-alcoholic fatty liver disease development. Endocrine, 43: 376-386. Lowe, F.C., Fagelman, E. (2002) Phytotherapy in the treatment of benign prostatic hyperplasia. Curr Opin Urol, 12: 15-18. Ma, C.H., Lin, W.L., Lui, S.L., et al. (2013) Efficacy and safety of Chinese herbal medicine for benign prostatic hyperplasia: systematic review of randomized controlled trials. Asian J Androl, 15: 471-482. Ma, Y.-F., Guo, N.-N., Chu, J., et al. (2016) Glycyrrhizin treatment inhibits proliferation and invasive potential of lung cancer cells. INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE, 9: 10592-10596. Matson, J.P., Cook, J.G. (2016) Cell cycle proliferation decisions: the impact of single cell analyses. FEBS J, McConnell, J.D. (1995) Prostatic growth: new insights into hormonal regulation. Br J 20
Urol, 76 Suppl 1: 5-10. Nishida, N., Yada, N., Hagiwara, S., et al. (2016) Unique features associated with hepatic oxidative DNA damage and DNA methylation in non-alcoholic fatty liver disease. J Gastroenterol Hepatol, 31: 1646-1653. O'Leary, M.P. (2003) Lower urinary tract symptoms/benign prostatic hyperplasia: maintaining symptom control and reducing complications. Urology, 62: 15-23. Park, E., Lee, M.Y., Jeon, W.Y., et al. (2016a) Inhibitory Effect of Yongdamsagan-Tang Water Extract, a Traditional Herbal Formula, on Testosterone-Induced Benign Prostatic Hyperplasia in Rats. Evid Based Complement Alternat Med, 2016: 1428923. Park, E., Lee, M.Y., Seo, C.S., et al. (2017) Yongdamsagan-tang, a traditional herbal formula, inhibits cell growth through the suppression of proliferation and inflammation in benign prostatic hyperplasia epithelial-1 cells. J Ethnopharmacol, 209: 230-235. Park, E., Lee, M.Y., Seo, C.S., et al. (2016b) Acute and subacute toxicity of an ethanolic extract of Melandrii Herba in Crl:CD sprague dawley rats and cytotoxicity of the extract in vitro. BMC Complement Altern Med, 16: 370. Rackova, L., Jancinova, V., Petrikova, M., et al. (2007) Mechanism of antiinflammatory action of liquorice extract and glycyrrhizin. Nat Prod Res, 21: 1234-1241. Roehrborn, C.G. (2008) Pathology of benign prostatic hyperplasia. Int J Impot Res, 20 Suppl 3: S11-18. Scholzen, T., Gerdes, J. (2000) The Ki-67 protein: from the known and the unknown. J Cell Physiol, 182: 311-322. Song, M., Park, S., Kang, J., et al. (2010) Report of four cases of Paljung-san on lower urinary tract symptoms in patients with benign prostatic hyperplasia. J Korean Oriental Med, 31: 153-161. 21
Stacey, D.W. (2003) Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells. Curr Opin Cell Biol, 15: 158-163. Steers, W.D. (2001) 5alpha-reductase activity in the prostate. Urology, 58: 17-24; discussion 24. Wilt, T., Ishani, A., Mac Donald, R., et al. (2002) Pygeum africanum for benign prostatic hyperplasia. Cochrane Database Syst Rev, 1: Xiong, Y., Qiu, X., Shi, W., et al. (2016) Anti-inflammatory and antioxidant effect of modified Bazhengsan in a rat model of chronic bacterial prostatitis. J Ethnopharmacol, Xiong, Y., Qiu, X., Shi, W., et al. (2017) Anti-inflammatory and antioxidant effect of modified Bazhengsan in a rat model of chronic bacterial prostatitis. J Ethnopharmacol, 198: 73-80. Yang, X., Yuan, L., Xiong, C., et al. (2014) Abacopteris penangiana exerts testosteroneinduced benign prostatic hyperplasia protective effect through regulating inflammatory responses, reducing oxidative stress and anti-proliferative. J Ethnopharmacol, 157: 105113.
Figure legends Figure 1. Three-dimensional chromatogram of Paljung-san decoction using HPLC with photodiode array (PDA) detection. Figure 2. Effect of PSWE on cell viability in BPH-1 cells. BPH-1 cells were treated with DMSO vehicle, finasteride or a series of PSWE concentrations for 48 h. Cell viability was measured using CCK-8 assays. Data are representative of three independent experiments and values represent the mean ± SEM of triplicate samples 22
from one experiment. *P<0.05, **P<0.01 and ***P<0.001, compared with vehicle alone. Figure 3. Effect of PSWE on cell proliferation in BPH-1 cells. BPH-1 cells were treated with vehicle, finasteride or PSWE for 48 h. (A) Cell lysates (10 g) were subjected to western blot analysis. (B) EdU-incorporated cells (green) and nuclei (blue) in BPH-1 cells were detected by immunofluorescence (left). Scale bar = 100 m. The percentage of EdU-positive cells undergoing proliferation was determined by counting five areas per slide (right). (C) BPH-1 cells were fixed and stained with propidium iodide, and DNA contents were analyzed by flow cytometry. Data are representative of three independent experiments and values represent the mean ± SEM of these. *P<0.05 and **P<0.01, compared with vehicle control. Figure 4. Effect of PSWE on inflammation in BPH-1 cells. (A) PGE2 levels in BPH-1 cells were measured in the supernatants of cultured cells treated with vehicle, finasteride or PSWE for 24 h. (B) BPH-1 cells were treated with vehicle, finasteride or PSWE for 48 h. The expression of COX-2 protein was determined by western blot analysis. Data are representative of three independent experiments and values represent the mean ± SEM of triplicate samples from one experiment. ***P<0.001, compared with vehicle control. Figure 5. Effect of PSWE on BPH pathogenesis in TP-treated rats. (A) The relative mean prostate weight (g) was calculated as a ratio of prostate weight (g)/body weight (g). (B) DHT concentrations were measured in prostate lysates using ELISA kits. (C) H&E stained prostate sections from BPH rats (magnification, 200). Values are presented as the mean ± SEM (n = 5). ##P<0.01, compared with the NC group; *P<0.05 23
and **P<0.01, compared with the BPH group. Key: NC, negative control; BPH, TPinduced BPH; Fin, administration of finasteride and s.c. injection of TP; PSWE-200, administration of 200 mg/kg of PSWE and s.c. injection of TP; PSWE-500, administration of 500 mg/kg of PSWE and s.c. injection of TP. Figure 6. Effect of PSWE on cell proliferation in the prostates of rats with BPH. (A) Ki-67 expression in the prostate from rats with BPH was determined by immunohistochemistry (magnification, 200). (B) Total prostatic protein (30 g) was subjected to western blot analysis for the detection of cyclin D1 protein levels. Data are representative and values represent the mean ± SEM (n = 5). ##P<0.01, compared with the NC group; *P<0.05, compared with the BPH group. Key: NC, negative control; BPH, TP-induced BPH; Fin, administration of finasteride and s.c. injection of TP; PSWE-200, administration of 200 mg/kg of PSWE and s.c. injection of TP; PSWE-500, administration of 500 mg/kg of PSWE and s.c. injection of TP. Figure 7. Effect of PSWE on oxidative stress in the prostates of rats with BPH. Prostate lysates were subjected to measurements of GR activity (A) and MDA levels (B). Values are presented as the mean ± SEM (n = 5). ##P<0.01, compared with the NC group; *P<0.05 and **P<0.01, compared with the BPH group. Key: NC, negative control; BPH, TP-induced BPH; Fin, administration of finasteride and s.c. injection of TP; PSWE-200, administration of 200 mg/kg of PSWE and s.c. injection of TP; PSWE500, administration of 500 mg/kg of PSWE and s.c. injection of TP.
24
Table 1. Composition of Paljung-san
Herbal medicine
Scientific name of plant source
Rhei Radix et Rhizoma
Rheum tanguticum Maximowicz ex Balf.
Akebiae Caulis
Family
Origin
Ratio (%)
Polygonaceae
P. R. China 11.1
Akebia quinata Decaisne
Lardizabalaceae
Yeongcheon, 11.1 S. Korea
Dianthi Herba
Dianthus superbus L.
Caryophyllaceae
Yeongcheon, 11.1 S. Korea
Polygoni Avicularis Herba
Polygonum aviculare L.
Polygonaceae
Yeongcheon, 11.1 S. Korea
Talcum
Hydrated magnesium silicate
–
P. R. China 11.1
Gardeniae Fructus
Gardenia jasminoides Ellis
Rubiaceae
P. R. China 11.1
Plantaginis Semen
Plantago asiatica L.
Glycyrrhizae Radix et Rhizoma
Glycyrrhiza uralensis Fischer
Junci Medulla
Juncus effusus L.
25
Plantaginaceae P. R. China 11.1 Leguminosae
P. R. China 11.1
Juncaceae
P. R. China 11.1
Graphical Abstract Paljung-San HPLC
BPH-1 cell line
TP-induced BPH rat model TP
Cyclin D1 expression ↓
COX2 expression ↓
DNA synthesis ↓
PGE2 synthesis ↓
Proliferation
Inflammation
Cyclin D1 & Ki-67 expression ↓
GR activity & MDA levels ↓
Proliferation
Oxidative stress
DHT levels ↓ Prostate weight ↓
Figure 1.
Figure 2.
Cell viability (% of control)
120 100
* *** ***
80 60 40 20 0
0
1
10
50
Finasteride (µM)
0
31.25 62.5 125 250 500
PSWE (µg/mL)
Figure 3. B
A
Vehicle Finasteride (µM)
40
0
125 250 500
% of EdU-positive cells
50
EdU
1
500 µg/mL PSWE
PSWE (µg/mL)
WB: anti-cyclin D1 WB: anti-PCNA WB: anti-β actin
Merge
0
50µM Fin
*
30 20 10 0 Vehicle
C Vehicle G1: 71.41 % S: 20.27 % G2/M: 8.32 %
**
50 µM Fin G1: 72.59 % S: 19.42 % G2/M: 7.98 %
500 µg/mL PSWE G1: 72.32 % S: 15.94 % G2/M: 11.74 %
Fin
PSWE
Figure 4. A 12
B
PGE2 (ng/mL)
10
***
***
8
4
***
0
10
50
Finasteride (µM)
0
31.2
0 125 500
62.5
125
PSWE (µg/mL)
50
WB: anti-β actin
***
2
1
1
WB: anti-COX-2
***
***
0
PSWE (µg/mL)
0
***
6
Finasteride (µM)
250
500
Figure 5. C
B 3 2.5 2 1.5 1 0.5 0
##
1000
* **
**
DHT level in prostate (% of control)
Relative prostate weight ratio
A
NC
BPH
PSWE-200
PSWE-500
##
800 600
**
**
**
400 200
0
Fin
Figure 6.
A
B
PSWE 200 500
NC
BPH
Fin WB: Anti-Cyclin D1
PSWE-200
PSWE-500
Relative Cyclin D1 protein levels
WB: Anti-β-actin 1.4 1.2
##
1 0.8 0.6
0.4 0.2 0
*
GR activity (U/mg protein) 15
**
10
0 1 2 3 4
**
##
5
5
MDA concentraion (nM/mg protein)
Figure 7.
A B
20 80
0
6 60
*
40
20
0
0 1 2 3
* **
4 5 6
PII: DOI: Reference:
S0378-8741(18)30030-8 https://doi.org/10.1016/j.jep.2018.02.037 JEP11248
To appear in: Journal of Ethnopharmacology Received date: 4 January 2018 Revised date: 23 February 2018 Accepted date: 23 February 2018 Cite this article as: Eunsook Park, Mee-Young Lee, Woo-Young Jeon, ChangSeob Seo, Sooseong You and Hyeun-Kyoo Shin, Paljung-San, a Traditional Herbal Medicine, Attenuates Benign Prostatic Hyperplasia In Vitro and In Vivo , Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2018.02.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Paljung-San, a Traditional Herbal Medicine, Attenuates Benign Prostatic Hyperplasia In Vitro and In Vivo
Eunsook Park1, Mee-Young Lee1, Woo-Young Jeon1, Chang-Seob Seo1, Sooseong You2, and Hyeun-Kyoo Shin1*
1
K-herb Research Center, Korea Institute of Oriental Medicine, 1672 Yuseong-daero,
Yuseong-gu, Daejeon 34054, Republic of Korea 2
KM Fundamental Research Division, Korea Institute of Oriental Medicine, 1672
Yuseong-daero, Yuseong-gu, Daejeon 34054, Republic of Korea Eunsook Park: [email protected] Mee-Young Lee: [email protected] Woo-Young Jeon: [email protected] Chang-Seob Seo: [email protected] Sooseong You: [email protected] Hyeun-Kyoo Shin: [email protected]
*Corresponding author: Dr. Hyeun-Kyoo Shin, K-herb Research Center, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon, 34054, Republic of Korea. Phone: +82-42-868-9464; Fax: +82-42-868-2120
1
Abstract Ethnopharmacological relevance: Paljung-san is a traditional herbal medicine used widely for the treatment of urogenital diseases in East Asia. However, scientific evidence of the efficacy of Paljung-san and its mechanisms of action against benign prostatic hyperplasia (BPH) is not clearly established. Aim of the study: We investigated the inhibitory effect of Paljung-san water extract (PSWE) and its mechanisms against BPH in vitro and in vivo. Materials and methods: Active compounds of PSWE were analyzed quantitatively by High-performance liquid chromatography (HPLC). For in vitro study, PSWE treated BPH-1 cells were used to perform western blot analysis, cell cycle analysis and enzymelinked immunosorbent assay. For in vivo BPH model, male rats were subcutaneously injected with 10 mg/kg of testosterone propionate (TP) every day for four weeks. 200 and 500 mg/kg of PSWE was administrated daily by oral gavage with s.c. injection of TP, respectively. Results: HPLC revealed that PSWE contains 1.21, 1.18, 2.27, 3.56, 4.23, 3.00, 6.78, and 0.004 mg/g of gallic acid, 5-caffeoylquinic acid, chlorogenic acid, geniposide, liquiritin apioside, liquiritin, glycyrrhizin, and chrysophanol components, respectively. In human BPH-1 cells, PSWE treatment reduced cell proliferation through arresting the cell cycle in the DNA synthesis phase. Moreover, PSWE suppressed prostaglandin E2 production with reduced cyclooxygenase-2 expression. In TP -induced BPH rat model, PSWE administration showed reduced prostate weights and dihydrotestosterone levels and led to a restoration of normal prostate morphology. PSWE also decreased TPinduced Ki-67 and cyclin D1 protein levels in the prostatic tissues. Decreased 2
glutathione reductase activity and increased malondialdehyde levels in the BPH groups were reversed by PSWE administration. Conclusion: PSWE attenuates the progression of BPH through anti-proliferative, antiinflammatory and anti-oxidant activities in vitro and in vivo. Therefore, these data provide the scientific evidence of pharmacological efficacy of PSWE against BPH.
Keywords: Paljung-san water extract; traditional herbal medicine; high-performance liquid chromatography; benign prostatic hyperplasia; BPH-1 cells; rat model
1. Introduction Benign prostatic hyperplasia (BPH), a noncancerous enlargement of the prostate gland, is a common disease found in men aged over 50 years (Berry et al., 1984). BPH involves overgrowth of both epithelial and stromal cells in the transitional zone of the prostate gland around the urethra, and leads to lower urinary tract symptoms (LUTS) including problems with urinary storage (hesitancy, weak streaming and retention) or voiding (frequency, urgency and nocturia) (O'Leary, 2003). Although the cause of BPH is not fully understood, age-related imbalances in androgen levels are associated with an increased risk of BPH (Roehrborn, 2008). Dihydrotestosterone (DHT), a metabolite of testosterone converted by 5-reductase (5R), acts as an active androgen for prostatic growth (McConnell, 1995). Therefore, excessive DHT levels in the prostate gland induce overproliferation of both epithelial and stromal cells and can lead to the 3
development and progression of BPH (Steers, 2001). As mentioned above, many clinical studies have found increases in serum DHT levels and DHT/testosterone ratios in elderly patients with BPH (Horton et al., 1975). Whereas men aged 40–49 years make up around 20–40% of all patients with BPH, men aged over 60 years account for over 60% in this cohort, so the prevalence of histological BPH increases with aging (Berry et al., 1984). In addition to the effects of androgens, oxidative stress and inflammation in the prostate gland also influence the development and progression of BPH (De Nunzio et al., 2016). Among the pharmacological therapies used for the treatment of BPH, 5R inhibitors such as finasteride and dutasteride, which block the conversion of testosterone to DHT, are effective synthetic agents with anti-proliferative effects on the enlarged prostate. Despite this therapeutic efficacy, taking these medications for prolonged times leads to adverse side effects associated with sexual function such as erectile dysfunction, lowered sexual drive (libido) and reduced semen volume during ejaculation (Gormley et al., 1992). Therefore, many men who desire fewer side effects and effective treatments for BPH have been interested in phytotherapeutic agents as complementary and alternative medicines (Ma et al., 2013). In fact, several herbal medicines derived from natural products have been reported as having therapeutic efficacy for the treatment of BPH (Lowe and Fagelman, 2002). Extract of Pygeum africanum, the African prune tree, is well known as a phytotherapeutic agent used for treating men with BPH, and for alleviating LUTS (Wilt et al., 2002). Paljung-san, also known as Bazheng-san in Chinese, is a traditional herbal medicine based on the classic Korean book Donguibogam in which the principles and 4
practice of Eastern traditional medicine are described. Paljung-san is commonly used for the treatment of urogenital diseases such as acute nephritis, cystitis, kidney stones and urethral syndrome (Kim et al., 2007; Lee et al., 2000). Moreover, it has been reported as being therapeutic against diseases of the male reproductive system. Modified Paljung-san in a rodent model showed inhibition against chronic prostatitis via its anti-inflammatory and anti-oxidant activities (Xiong et al., 2017). A clinical study showed that Paljung-san treatment in patients with BPH ameliorated LUTS such as urinary frequency and retention (Song et al., 2010). However, scientific evidence of the efficacy of Paljung-san for the treatment of BPH is not fully defined. Therefore, we investigated the therapeutic efficacy of Paljung-san water extract (PSWE) and its mechanisms of action in human BPH-1 cells in vitro and in a rat model of BPH in vivo.
5
2. Materials and methods 2.1. Chemicals and reagents Reference standard marker compounds, gallic acid (purity 99.0%) and 5caffeoylquinic acid (purity 99.2%) were purchased from Merck KGaA (Darmastadt, Germany). Gepinoside (purity 98.0%), liquiritin (purity 99.6%), and glycyrrhizin (purity 99.0%) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Chlorogenic acid (purity 99.6%), liquiritin apioside (purity 98.0%), and chrysophanol (purity 99.0%) were purchased from Acros Organics (Pittsburgh, PA, USA), Shanghai Sunny Biotech (Shanghai, P. R. China), and Biopurify Phytochemicals (Chengdu, P. R. China), respectively. Solvents, methanol, acetonitrile and distilled water, used for HPLC analysis were HPLC grade and obtained from J. T. Baker (Phillipsburg, NJ, USA). Formic acid, the American Chemical Society (ACS) reagent grade, was obtained from Merck KGaA. 2.2. Plant materials The component medicinal components of Paljung-san (Rhei Radix et Rhizoma, Akebiae Caulis, Dianthi Herba, Polygoni Avicularis Herba, Talcum, Gardeniae Fructus, Plantaginis Semen, Glycyrrhizae Radix et Rhizoma, and Junci Medulla) were purchased from Kwangmyungdag Medicinal Herbs (Ulsan, S. Korea) in May 2015. A voucher specimen (2015–KE56–1 to KE56–9) has been deposited at the K-herb Research Center, Korea Institute of Oriental Medicine. 2.3. Preparation of PSWE Paljung-san was mixed from the nine traditional medicinal herbs listed above and in Table 1 combined in equal amounts, and extracted in a 10-fold volume of water 6
at 100 °C for 2 h using an electric extractor (COSMOS-660; Kyungseo Machine Co., Incheon, S. Korea). The extracted solution was filtered using a standard sieve (No. 270, 53 m; Chung Gye Sang Gong Sa, Seoul, S. Korea) and then the filtered solution was processed to a powder using a PVT100 freeze–dryer (IlShin Bio Base, Yangju, S. Korea). The amount of lyophilized water extract was 467 g (yield: 9.34%). 2.4.High-performance liquid chromatography (HPLC) analysis of PSWE HPLC analysis was performed using a Shimadzu Prominence LC-20A series (Kyoto, Japan) as described previously (Park et al., 2016b). The separation of the major marker components was achieved on a Waters SunFire C18 column (250 4.6 mm, 5 m, Milford, MA, USA) with a column oven temperature of 40 °C. The mobile phases consisted of distilled water (A) and acetonitrile (B), both with 0.1% (v/v) formic acid. The gradient system of mobile phase was as follows: 5–60% B for 0–30 min; 60–100% B for 30–40 min; 100% B for 40–45 min; 100–5% B for 45–50 min; and 5% B for 50– 60 min. The flow rate was 1.0 mL/min and the injection volume was 10 L. For HPLC analysis of PSWE, 200 mg of lyophilized PSWE was dissolved in 20 mL of 50% methanol and then extracted using a sonicator for 30 min. The extracted solution was filtered through a 0.2 m pore membrane filter (PALL Life Sciences, Ann Arbor, MI, USA) before HPLC injection. 2.5. Cell culture BPH-1 cells (Park et al., 2017) were maintained in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 20% FBS (Invitrogen, Carlsbad, CA) at 37 °C under an atmosphere of 5% CO2 in air. 7
2.6. Cell proliferation assay Cell viability was analyzed using a nonradioactive Cell Counting Kit (CCK)-8 system (Dojindo, Tokyo, Japan) according to the manufacturer’s instructions (Park et al., 2017). To determine the effect of PSWE on DNA synthesis, 5-Ethynyl-2′deoxyuridine (EdU) incorporation assay were performed using Click-iT Plus EdU Imaging kits (Invitrogen, Carlsbad, CA, USA) as described previously (Park et al., 2017). 2.7. Western blot analysis Protein levels were determined by western blot analysis as described (Park et al., 2016a). Extracts of total proteins from BPH-1 cells or rat prostatic tissues were prepared using RIPA buffer (Sigma-Aldrich). The following antibodies were used: anticyclin D1 (ab134175; Abcam, Cambridge, UK), anti-PCNA (ab29; Abcam), anti-COX2 (160112; Cayman Chemical Co., Ann Arbor, MI, USA) and anti--actin (#4967; Cell Signaling, Danvers, MA, USA). 2.8. Flow cytometry analysis Cells were stained with propidium iodide using flow cytometry kits (Abcam) according to the manufacturer’s instructions. FACS analysis of cell cycles was performed on BD FACSCalibur system (BD Biosciences, Heidelberg, Germany) and Modifit LT version 4.1 software (Verity Software House, Topsham, ME, USA). 2.9. Measurement of PGE2 levels Levels of PGE2 in BPH-1 cells were measured using human PGE2 enzymelinked immunosorbent assay (ELISA) kits (Cayman Chemical, Ann Arbor, MI, USA) 8
according to the manufacturer’s instructions. Briefly, BPH-1 cells (1 104 cells/well) were seeded onto 48-well plates in triplicates in complete medium. Next day, the medium was refreshed and DMSO vehicle, finasteride or PSWE was added to the medium. After 24 h, the supernatants were harvested for measuring PGE2 levels. 2.10. Animals Seven-week-old male Sprague Dawley rats weighing 200–220 g were purchased from Orient Bio Inc. (Seoul, S. Korea). Rats were maintained under a 12/12h light/dark cycle at 18–23 °C and a relative humidity of 40–60% in our animal handling facility. Standard laboratory chow (Harlan Teklad, Madison, WI, USA) and water were available ad libitum. All experimental procedures were approved by the Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee and performed in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals. Animals were cared for in accordance with the dictates of the National Animal Welfare Law of S. Korea. 2.11. Experimental design Experimental procedures for studying the efficacy of PSWE in the rat model of TP-induced BPH were designed based on a previous study (Park et al., 2016a). Briefly, rats were divided randomly into five groups (n = 5 per group) and treated daily for 4 weeks as follows: a negative control (NC) group that received phosphate-buffered saline (PBS) orally with a subcutaneous (s.c.) injection of corn oil; a BPH group that received PBS orally with a s.c. injection of 10 mg/kg of TP (Tokyo Chemical Industry Co., Tokyo, Japan) dissolved in corn oil; a finasteride group as a positive control that 9
received 10 mg/kg of finasteride (Sigma-Aldrich) orally with s.c. injection of 10 mg/kg of TP; PSWE-200 and PSWE-500 groups that received 200 and 500 mg/kg of PSWE orally together with an s.c. injection of 10 mg/kg of TP, respectively. A dose of PSWE in rat was calculated based on guideline (Food and Administration, 2005), which is described the conversion of animal doses to human equivalent doses based on body surface area. Given that rat dose to human equivalent dose is 347.448 mg/kg, we chosen 200 and 500 mg/kg, a lower and higher dosage than that, respectively. 2.12. Histology and immunohistochemistry For histology, deparaffinized and rehydrated sections from the fixed rat prostate tissues were stained with Mayer’s hematoxylin (MHS-16, Sigma-Aldrich) and eosin (HT110-1-32, Sigma-Aldrich) (H&E) solution. Immunohistochemistry for Ki-67 was performed using Vectastain Elite ABC kits (Vector Laboratories Inc., Burlingame, CA, USA) as described (Park et al., 2016a). 2.13. Measurement of DHT and MDA levels, and GR activity Measurement of DHT and MDA levels, and GR activity was performed as described previously (Park et al., 2016a). Briefly, GR activity in prostate lysates was analyzed using Glutathione Reductase Assay kits (Cayman) according to the manufacturer’s instructions. 2.14. Statistical analysis Data are presented as the mean ± standard error of the mean (SEM). Statistical significance was calculated using one-way analysis of variance with Dunnett’s test (Dunnett, 1964). Differences were considered significant at P<0.05. 10
3. Results 3.1. HPLC analysis of eight marker components in PSWE Using optimized HPLC, eight marker components were eluted within 45 min, with a resolution of 1.12. The retention times of gallic acid, 5-caffeoylquinic acid, chlorogenic acid, geniposide, liquiritin apioside, liquiritin, glycyrrhizin, and chrysophanol were 6.31, 10.98, 12.58, 13.60, 16.22, 16.62, 30.12, and 40.53 min, respectively (Fig. 1). The concentrations of these markers were 1.21, 1.18, 2.27, 3.56, 4.23, 3.00, 6.78, and 0.004 mg/g, respectively. 3.2. PSWE reduces cell viability in vitro The epithelial cell line BPH-1 was used to address the effects of PSWE in vitro. PSWE treatment for 48 h significantly inhibited cell viability (Fig. 2). Among the tested concentrations of PSWE, those over 125 g/mL repressed cell viability in a concentration-dependent manner. Unlike PSWE, the finasteride treatment used as a positive control had no effect on cell viability. 3.3. PSWE inhibits cell proliferation in vitro The pathogenesis of BPH is associated with the overproliferation of epithelial cells in prostatic ducts. Therefore, we examined the effect of PSWE on proliferationrelated protein levels. PSWE treatment in BPH-1 cells decreased both cyclin D1 and proliferating cell nuclear antigen (PCNA) protein levels in concentration-dependent manners (Fig. 3A). Control finasteride treatment also decreased the levels of these proteins. To evaluate DNA synthesis in the cell cycle, 5-ethynyl-2-deoxyuridine (EdU) assays were performed. The number of EdU-positive cells was markedly decreased by 11
treatment with PSWE as well as finasteride compared with vehicle alone (Fig. 3B), suggesting that PSWE inhibits DNA synthesis in BPH-1 cells. In addition, fluorescenceactivated cell sorting (FACS) analysis revealed that PSWE treatment resulted in a reduced proportion of cells in the DNA synthesis (S) phase compared with vehicle treatment alone (Fig. 3C). Finasteride treatment showed a similar pattern of cell cycle alterations. Taken together, these results indicate that PSWE inhibits the proliferation of BPH-1 cells through arrest at the S phase of the cell cycle. 3.4. PSWE suppresses inflammation in vitro To elucidate whether PSWE would regulate BPH-related inflammatory responses, we examined the effect of PSWE on the levels of prostaglandin E2 (PGE2), which is produced during inflammation. PSWE treatment for 24 h as well as finasteride (which had no toxicity for BPH-1 cells; data not shown), significantly suppressed PGE2 production in concentration-dependent manners (Fig. 4A). Moreover, both finasteride and PSWE treatments reduced the protein levels of cyclooxygenase-2 (COX-2), an inducible enzyme responsible for PGE2 production (Fig. 4B), suggesting that PSWE represses the inflammatory response in BPH-1 cells. 3.5. PSWE represses pathogenesis in a testosterone propionate (TP)-induced rat model of BPH To further confirm the inhibitory effect of PSWE against BPH in vitro, the therapeutic efficacy of PSWE in vivo was determined using a rat model of TP-induced BPH. As shown in Figure 5A, the relative prostate weight of the TP-treated group (BPH group) was significantly higher than of the negative control (NC) group. However, the relative prostate weight of the group administered TP plus finasteride as a positive 12
control (Fin group), was decreased compared with the BPH group. The relative prostate weight of rats administered PSWE at 200 mg/kg and 500 mg/kg with TP (PSWE-200 and PSWE-500 groups, respectively) were also markedly reduced compared with the BPH group. In addition, increased DHT levels in the prostates of the BPH group compared with the NC group were repressed by 200 and 500 mg/kg PSWE as well as by finasteride (Fig. 5B). Histologically, the BPH group exhibited prostatic hyperplasia with multiple layers of epithelial cells compared with the NC group while the Fin, PSWE-200 and PSWE-500 groups showed amelioration of the disrupted prostatic morphology compared with the BPH group (Fig. 5C). Taken together, these results suggest that PSWE reverses the pathogenesis of BPH in vivo. 3.6. PSWE exerts anti-proliferative effects in a rat model of TP-induced BPH We examined the expression of prostatic proliferation markers to evaluate the mechanisms underlying the inhibition of PSWE on BPH pathogenesis. Figure 6A shows an increased expression of Ki-67, a cell proliferation marker, in both stromal and epithelial prostatic cells in the BPH group but not in the NC group. On the other hand, prostatic tissues in the Fin and both PSWE groups showed lower expression of Ki-67 than in the BPH group. Consistent with these changes in Ki-67 expression, the protein levels of cyclin D1, another marker of proliferation, were also regulated by PSWE administration. In prostatic lysates, the cyclin D1 protein level was significantly elevated in the BPH group compared with the NC group but was reduced in Fin and both PSWE groups compared with the BPH group (Fig. 6B). These results indicate that PSWE has anti-proliferative effects against BPH in vivo. 3.7. PSWE has anti-oxidative effects in a rat model of TP-induced BPH 13
Oxidative stress is associated with the pathogenesis of BPH. To test whether PSWE would affect oxidative stress in BPH, glutathione reductase (GR) activity and malondialdehyde (MDA) levels, both markers of oxidative stress, were determined in prostates of the BPH group. As shown in Figure 7A, the activity of GR, an antioxidant enzyme, was markedly reduced in the BPH group compared with the NC group but was restored by PSWE as well as finasteride treatments. Lipid peroxidation involves the degradation of lipids induced by oxidative stress and the release of reactive oxygen species (ROS) (Nishida et al., 2016). The BPH group showed higher levels of MDA, an indicator of lipid peroxidation, than did the NC group, whereas the Fin and both PSWE groups exhibited lower levels of MDA than in the BPH group (Fig.7B). Taken together, these results suggest that PSWE has anti-oxidant activity against BPH in vivo.
14
4. Discussion BPH is the most common urogenital disease in aged men and requires clinical intervention in about one-third of those aged over 60 years (Berry et al., 1984). Surgical and pharmacological approaches against BPH are the main medical treatments by removing hypertrophic prostatic tissues and improvement of ULTS, respectively. Nevertheless, adverse effects reported with these approaches and concern for the quality of life have led to increased interest in the use of natural products with fewer side effects against BPH (Lowe and Fagelman, 2002). A Korean group reported that four men with BPH and LUTS who were treated with Paljung-san for 10 days showed improvements in LUTS, suggesting the possibility of using this herbal medicine against BPH (Song et al., 2010). However, no scientific study of the effect of Paljung-san treatment on BPH has been carried out. Therefore, this study is the first to establish scientific evidence of the efficacy of PSWE and its mechanisms of action against BPH. We found that PSWE treatment markedly inhibited cell proliferation and PGE2 production in the BPH-1 cell line through reduced DNA synthesis and COX-2 protein levels, respectively. Furthermore, the repressive effect of PSWE in vitro was verified using a rat model of BPH in which PSWE administration attenuated pathogenesis through its anti-proliferative and anti-oxidant activities. Overproliferation in epithelial and stromal cells is a major pathogenic feature of BPH and its progression (Steers, 2001). Cell proliferation is governed by the cell cycle, which is comprised of four phases including G1, S (DNA synthesis), G2, and M (mitosis) (Golias et al., 2004). The G1 to S, or G2 to M transitions are regulated by the expression or activation of various cell cycle-regulatory proteins (Matson and Cook, 15
2016). Among them, cyclin D1, a protein that acts during the G1 phase and is required for the initiation of the S phase, PCNA, a nuclear protein that is elevated during the G1/S phase transition, and Ki-67, an antigen expressed in all phases except for resting cells, are widely used as markers of proliferating cells (Scholzen and Gerdes, 2000; Stacey, 2003). PSWE treatment in BPH-1 cells and in our rat model of BPH reduced the expressions of these markers. Furthermore, the proportion of cells in the S phase of the cell cycle as well as the number of EdU-positive cells, reflecting DNA synthesis, were significantly decreased by PSWE. Therefore, these findings indicate that PSWE inhibits cell proliferation by arresting the S phase of the cell cycle. Several studies have demonstrated that pathways involving inflammation and oxidative stress contribute to cell proliferation in BPH. The presence of heterogeneous bacterial and viral strains in BPH biopsy specimens suggests a possible contribution of prostatic inflammation to its pathogenesis (De Nunzio et al., 2016). Histopathology of prostatic tissues from patients with BPH revealed that the levels of inflammatory markers are increased along with prostate growth (Di Silverio et al., 2003). In addition, COX-2, an enzyme associated with inflammation, is highly expressed in epithelial cells from BPH samples, and its repression inhibits their overgrowth (Ammar et al., 2015). Given that Paljung-san is effective against inflammatory diseases such as acute nephritis, cystitis and prostatitis (Xiong et al., 2016), PSWE might inhibit cell proliferation through its anti-inflammatory activity in BPH tissues. Here, PSWE treatment reduced the levels of PGE2, an inflammatory modulator regulated by COX-2 activity, as well as COX-2 protein levels in BPH-1 cells. These findings suggest that the effect of PSWE on inhibiting cell proliferation in BPH is associated with its anti16
inflammatory activity. Furthermore, there are well-known interactions between inflammation and oxidative stress in various other diseases (Li et al., 2016). Oxidative stress associated with ROS production leads to lipid peroxidation, increases the levels of proinflammatory cytokines such as tumor necrosis factor alpha and interleukin-6, and this then leads to inflammation (Nishida et al., 2016). Therefore, many herbal medicines target oxidative stress and inflammation simultaneously. Thus, Ghrelin activity and Geraniol treatments attenuate liver injury caused by oxidative stress, inflammation and apoptosis (Li et al., 2013). Extracts of pomegranate fruit and of the fern Abacopteris penangiana protect against BPH through their anti-inflammatory and anti-oxidant properties (Ammar et al., 2015; Yang et al., 2014). Consistent with the above findings, treatment with PSWE in our rat model of BPH also restored the activity of anti-oxidant enzymes and repressed lipid peroxidation, inhibiting inflammation and oxidative stress simultaneously. Here, the concentrations of distinct chemical components in PSWE were measured by HPLC. It has been reported that glycyrrhizin, which is at a higher level in PSWE than other components, has a range of bioactivities in various diseases, including anti-proliferative, anti-inflammatory and anti-tumorigenic effects (Ma et al., 2016; Rackova et al., 2007). In addition, liquiritin apioside and liquiritin, which are also found in PSWE, are naturally occurring polyphenols that exhibit anti-obesity and anti-oxidant properties (Guan et al., 2012; Kamisoyama et al., 2008). These findings are consistent with the therapeutic efficacy of PSWE against BPH through its anti-proliferative, antiinflammatory and anti-oxidant activities.
17
5. Conclusions We found that PSWE inhibited the progression of BPH in vitro and in vivo. This involved inhibition of cell proliferation through cell cycle arrest, and anti-inflammatory and anti-oxidant activities. Accordingly, our findings provide the first scientific evidence of the efficacy of Paljung-san treatment against BPH.
Conflict of interests The authors declare that they have no competing interest.
Acknowledgements This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number; H115C0025).
Authors’ contributions HKS participated in the design of the study and manuscript editing. EP conducted in vitro and in vivo experiments, data interpretation and manuscript preparation. MYL carried out in vivo experiments, data interpretation and edited the manuscript. WYJ conducted in vivo experiments and data interpretation. CSS carried out the preparation of PSWE, HPLC analysis and manuscript preparation. SY helped in the experiment. All authors red and approved the final manuscript.
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6. References Ammar, A.E., Esmat, A., Hassona, M.D., et al. (2015) The effect of pomegranate fruit extract on testosterone-induced BPH in rats. Prostate, 75: 679-692. Berry, S.J., Coffey, D.S., Walsh, P.C., et al. (1984) The development of human benign prostatic hyperplasia with age. J Urol, 132: 474-479. De Nunzio, C., Presicce, F., Tubaro, A. (2016) Inflammatory mediators in the development and progression of benign prostatic hyperplasia. Nat Rev Urol, 13: 613626. Di Silverio, F., Gentile, V., De Matteis, A., et al. (2003) Distribution of inflammation, pre-malignant lesions, incidental carcinoma in histologically confirmed benign prostatic hyperplasia: a retrospective analysis. Eur Urol, 43: 164-175. Dunnett, C.W. (1964) New tables for multiple comparisons with a control. Biometrics, 20: 482-491. Food, Administration, D. (2005) Guidance for industry: estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers. Center for Drug Evaluation and Research (CDER), 7. Golias, C.H., Charalabopoulos, A., Charalabopoulos, K. (2004) Cell proliferation and cell cycle control: a mini review. Int J Clin Pract, 58: 1134-1141. Gormley, G.J., Stoner, E., Bruskewitz, R.C., et al. (1992) The effect of finasteride in men with benign prostatic hyperplasia. The Finasteride Study Group. N Engl J Med, 327: 1185-1191. Guan, Y., Li, F.F., Hong, L., et al. (2012) Protective effects of liquiritin apioside on cigarette smoke-induced lung epithelial cell injury. Fundam Clin Pharmacol, 26: 47319
483. Horton, R., Hsieh, P., Barberia, J., et al. (1975) Altered blood androgens in elderly men with prostate hyperplasia. The Journal of Clinical Endocrinology & Metabolism, 41: 793-796. Kamisoyama, H., Honda, K., Tominaga, Y., et al. (2008) Investigation of the antiobesity action of licorice flavonoid oil in diet-induced obese rats. Biosci Biotechnol Biochem, 72: 3225-3231. Li, S., Hong, M., Tan, H.Y., et al. (2016) Insights into the Role and Interdependence of Oxidative Stress and Inflammation in Liver Diseases. Oxid Med Cell Longev, 2016: 4234061. Li, Y., Hai, J., Li, L., et al. (2013) Administration of ghrelin improves inflammation, oxidative stress, and apoptosis during and after non-alcoholic fatty liver disease development. Endocrine, 43: 376-386. Lowe, F.C., Fagelman, E. (2002) Phytotherapy in the treatment of benign prostatic hyperplasia. Curr Opin Urol, 12: 15-18. Ma, C.H., Lin, W.L., Lui, S.L., et al. (2013) Efficacy and safety of Chinese herbal medicine for benign prostatic hyperplasia: systematic review of randomized controlled trials. Asian J Androl, 15: 471-482. Ma, Y.-F., Guo, N.-N., Chu, J., et al. (2016) Glycyrrhizin treatment inhibits proliferation and invasive potential of lung cancer cells. INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE, 9: 10592-10596. Matson, J.P., Cook, J.G. (2016) Cell cycle proliferation decisions: the impact of single cell analyses. FEBS J, McConnell, J.D. (1995) Prostatic growth: new insights into hormonal regulation. Br J 20
Urol, 76 Suppl 1: 5-10. Nishida, N., Yada, N., Hagiwara, S., et al. (2016) Unique features associated with hepatic oxidative DNA damage and DNA methylation in non-alcoholic fatty liver disease. J Gastroenterol Hepatol, 31: 1646-1653. O'Leary, M.P. (2003) Lower urinary tract symptoms/benign prostatic hyperplasia: maintaining symptom control and reducing complications. Urology, 62: 15-23. Park, E., Lee, M.Y., Jeon, W.Y., et al. (2016a) Inhibitory Effect of Yongdamsagan-Tang Water Extract, a Traditional Herbal Formula, on Testosterone-Induced Benign Prostatic Hyperplasia in Rats. Evid Based Complement Alternat Med, 2016: 1428923. Park, E., Lee, M.Y., Seo, C.S., et al. (2017) Yongdamsagan-tang, a traditional herbal formula, inhibits cell growth through the suppression of proliferation and inflammation in benign prostatic hyperplasia epithelial-1 cells. J Ethnopharmacol, 209: 230-235. Park, E., Lee, M.Y., Seo, C.S., et al. (2016b) Acute and subacute toxicity of an ethanolic extract of Melandrii Herba in Crl:CD sprague dawley rats and cytotoxicity of the extract in vitro. BMC Complement Altern Med, 16: 370. Rackova, L., Jancinova, V., Petrikova, M., et al. (2007) Mechanism of antiinflammatory action of liquorice extract and glycyrrhizin. Nat Prod Res, 21: 1234-1241. Roehrborn, C.G. (2008) Pathology of benign prostatic hyperplasia. Int J Impot Res, 20 Suppl 3: S11-18. Scholzen, T., Gerdes, J. (2000) The Ki-67 protein: from the known and the unknown. J Cell Physiol, 182: 311-322. Song, M., Park, S., Kang, J., et al. (2010) Report of four cases of Paljung-san on lower urinary tract symptoms in patients with benign prostatic hyperplasia. J Korean Oriental Med, 31: 153-161. 21
Stacey, D.W. (2003) Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells. Curr Opin Cell Biol, 15: 158-163. Steers, W.D. (2001) 5alpha-reductase activity in the prostate. Urology, 58: 17-24; discussion 24. Wilt, T., Ishani, A., Mac Donald, R., et al. (2002) Pygeum africanum for benign prostatic hyperplasia. Cochrane Database Syst Rev, 1: Xiong, Y., Qiu, X., Shi, W., et al. (2016) Anti-inflammatory and antioxidant effect of modified Bazhengsan in a rat model of chronic bacterial prostatitis. J Ethnopharmacol, Xiong, Y., Qiu, X., Shi, W., et al. (2017) Anti-inflammatory and antioxidant effect of modified Bazhengsan in a rat model of chronic bacterial prostatitis. J Ethnopharmacol, 198: 73-80. Yang, X., Yuan, L., Xiong, C., et al. (2014) Abacopteris penangiana exerts testosteroneinduced benign prostatic hyperplasia protective effect through regulating inflammatory responses, reducing oxidative stress and anti-proliferative. J Ethnopharmacol, 157: 105113.
Figure legends Figure 1. Three-dimensional chromatogram of Paljung-san decoction using HPLC with photodiode array (PDA) detection. Figure 2. Effect of PSWE on cell viability in BPH-1 cells. BPH-1 cells were treated with DMSO vehicle, finasteride or a series of PSWE concentrations for 48 h. Cell viability was measured using CCK-8 assays. Data are representative of three independent experiments and values represent the mean ± SEM of triplicate samples 22
from one experiment. *P<0.05, **P<0.01 and ***P<0.001, compared with vehicle alone. Figure 3. Effect of PSWE on cell proliferation in BPH-1 cells. BPH-1 cells were treated with vehicle, finasteride or PSWE for 48 h. (A) Cell lysates (10 g) were subjected to western blot analysis. (B) EdU-incorporated cells (green) and nuclei (blue) in BPH-1 cells were detected by immunofluorescence (left). Scale bar = 100 m. The percentage of EdU-positive cells undergoing proliferation was determined by counting five areas per slide (right). (C) BPH-1 cells were fixed and stained with propidium iodide, and DNA contents were analyzed by flow cytometry. Data are representative of three independent experiments and values represent the mean ± SEM of these. *P<0.05 and **P<0.01, compared with vehicle control. Figure 4. Effect of PSWE on inflammation in BPH-1 cells. (A) PGE2 levels in BPH-1 cells were measured in the supernatants of cultured cells treated with vehicle, finasteride or PSWE for 24 h. (B) BPH-1 cells were treated with vehicle, finasteride or PSWE for 48 h. The expression of COX-2 protein was determined by western blot analysis. Data are representative of three independent experiments and values represent the mean ± SEM of triplicate samples from one experiment. ***P<0.001, compared with vehicle control. Figure 5. Effect of PSWE on BPH pathogenesis in TP-treated rats. (A) The relative mean prostate weight (g) was calculated as a ratio of prostate weight (g)/body weight (g). (B) DHT concentrations were measured in prostate lysates using ELISA kits. (C) H&E stained prostate sections from BPH rats (magnification, 200). Values are presented as the mean ± SEM (n = 5). ##P<0.01, compared with the NC group; *P<0.05 23
and **P<0.01, compared with the BPH group. Key: NC, negative control; BPH, TPinduced BPH; Fin, administration of finasteride and s.c. injection of TP; PSWE-200, administration of 200 mg/kg of PSWE and s.c. injection of TP; PSWE-500, administration of 500 mg/kg of PSWE and s.c. injection of TP. Figure 6. Effect of PSWE on cell proliferation in the prostates of rats with BPH. (A) Ki-67 expression in the prostate from rats with BPH was determined by immunohistochemistry (magnification, 200). (B) Total prostatic protein (30 g) was subjected to western blot analysis for the detection of cyclin D1 protein levels. Data are representative and values represent the mean ± SEM (n = 5). ##P<0.01, compared with the NC group; *P<0.05, compared with the BPH group. Key: NC, negative control; BPH, TP-induced BPH; Fin, administration of finasteride and s.c. injection of TP; PSWE-200, administration of 200 mg/kg of PSWE and s.c. injection of TP; PSWE-500, administration of 500 mg/kg of PSWE and s.c. injection of TP. Figure 7. Effect of PSWE on oxidative stress in the prostates of rats with BPH. Prostate lysates were subjected to measurements of GR activity (A) and MDA levels (B). Values are presented as the mean ± SEM (n = 5). ##P<0.01, compared with the NC group; *P<0.05 and **P<0.01, compared with the BPH group. Key: NC, negative control; BPH, TP-induced BPH; Fin, administration of finasteride and s.c. injection of TP; PSWE-200, administration of 200 mg/kg of PSWE and s.c. injection of TP; PSWE500, administration of 500 mg/kg of PSWE and s.c. injection of TP.
24
Table 1. Composition of Paljung-san
Herbal medicine
Scientific name of plant source
Rhei Radix et Rhizoma
Rheum tanguticum Maximowicz ex Balf.
Akebiae Caulis
Family
Origin
Ratio (%)
Polygonaceae
P. R. China 11.1
Akebia quinata Decaisne
Lardizabalaceae
Yeongcheon, 11.1 S. Korea
Dianthi Herba
Dianthus superbus L.
Caryophyllaceae
Yeongcheon, 11.1 S. Korea
Polygoni Avicularis Herba
Polygonum aviculare L.
Polygonaceae
Yeongcheon, 11.1 S. Korea
Talcum
Hydrated magnesium silicate
–
P. R. China 11.1
Gardeniae Fructus
Gardenia jasminoides Ellis
Rubiaceae
P. R. China 11.1
Plantaginis Semen
Plantago asiatica L.
Glycyrrhizae Radix et Rhizoma
Glycyrrhiza uralensis Fischer
Junci Medulla
Juncus effusus L.
25
Plantaginaceae P. R. China 11.1 Leguminosae
P. R. China 11.1
Juncaceae
P. R. China 11.1
Graphical Abstract Paljung-San HPLC
BPH-1 cell line
TP-induced BPH rat model TP
Cyclin D1 expression ↓
COX2 expression ↓
DNA synthesis ↓
PGE2 synthesis ↓
Proliferation
Inflammation
Cyclin D1 & Ki-67 expression ↓
GR activity & MDA levels ↓
Proliferation
Oxidative stress
DHT levels ↓ Prostate weight ↓
Figure 1.
Figure 2.
Cell viability (% of control)
120 100
* *** ***
80 60 40 20 0
0
1
10
50
Finasteride (µM)
0
31.25 62.5 125 250 500
PSWE (µg/mL)
Figure 3. B
A
Vehicle Finasteride (µM)
40
0
125 250 500
% of EdU-positive cells
50
EdU
1
500 µg/mL PSWE
PSWE (µg/mL)
WB: anti-cyclin D1 WB: anti-PCNA WB: anti-β actin
Merge
0
50µM Fin
*
30 20 10 0 Vehicle
C Vehicle G1: 71.41 % S: 20.27 % G2/M: 8.32 %
**
50 µM Fin G1: 72.59 % S: 19.42 % G2/M: 7.98 %
500 µg/mL PSWE G1: 72.32 % S: 15.94 % G2/M: 11.74 %
Fin
PSWE
Figure 4. A 12
B
PGE2 (ng/mL)
10
***
***
8
4
***
0
10
50
Finasteride (µM)
0
31.2
0 125 500
62.5
125
PSWE (µg/mL)
50
WB: anti-β actin
***
2
1
1
WB: anti-COX-2
***
***
0
PSWE (µg/mL)
0
***
6
Finasteride (µM)
250
500
Figure 5. C
B 3 2.5 2 1.5 1 0.5 0
##
1000
* **
**
DHT level in prostate (% of control)
Relative prostate weight ratio
A
NC
BPH
PSWE-200
PSWE-500
##
800 600
**
**
**
400 200
0
Fin
Figure 6.
A
B
PSWE 200 500
NC
BPH
Fin WB: Anti-Cyclin D1
PSWE-200
PSWE-500
Relative Cyclin D1 protein levels
WB: Anti-β-actin 1.4 1.2
##
1 0.8 0.6
0.4 0.2 0
*
GR activity (U/mg protein) 15
**
10
0 1 2 3 4
**
##
5
5
MDA concentraion (nM/mg protein)
Figure 7.
A B
20 80
0
6 60
*
40
20
0
0 1 2 3
* **
4 5 6