Assortment of Stauntonia hexaphylla and Cornus officinalis protect against testosterone-induced benign prostatic hyperplasia through anti-inflammatory and anti-proliferative activity

Benign prostatic hyperplasia (BPH) is a progressive pathological condition associated with proliferation of prostatic tissues, prostate enlargement, and lower-urinary tract symptoms. However, the mechanism underlying the pathogenesis of BPH is not clear. The aim of this study was to investigate the protective effects of Stauntonia hexaphylla and Cornus officinalis (SC extract) on a testosterone propionate (TP)-induced BPH model. For in vitro experiments, a human prostate adenocarcinoma cell line was used to perform western blotting for androgen receptor (AR), prostate specific antigen (PSA), and 5α-reductase type 2. Male Sprague-Dawley rats were randomly divided into 8 groups as follows for the in vivo experiments: control, BPH, Fina, Saw, SC25, SC50, SC100, and SC200. To induce BPH, all rats, except those in the control group, were daily administered with subcutaneous injections of TP (5 mg/kg), and orally treated with appropriate PBS/drugs for 4 consecutive weeks. Our findings indicated that the SC treatment significantly reduced the prostate size and downregulated the serum testosterone and DHT levels in BPH rats. The histological examination revealed that SC treatment markedly recovered the TP-induced abnormalities and reduced the prostatic hyperplasia. In addition, in vitro and in vivo western blotting indicated that SC treatment significantly downregulated the AR, PSA, and 5α-reductase type 2 expression, while an immunohistochemistry examination revealed that the SC extract significantly reduced the expression of type 2 5α-reductase and proliferating cell nuclear antigen positive cell count. Collectively, our findings demonstrated that SC extract attenuates BPH through anti-proliferative and anti-inflammation activities and might be useful in the clinical treatment of BPH.


Introduction
Many plants have been identified as good sources of natural antioxidants, which protect against 48 BPH and prostate cancer [1,2]. In this study, we investigated the protective effect of a 9:1 49 mixture of Stauntonia hexaphylla and Cornus officinalis, called the SC extract, on BPH. 50 Stauntonia hexaphylla belongs to the family Lardizabalaceae, which is native to Southern 51 Japan and Korea and has been used in medicine owing to its analgesic, sedative, diuretic, and 52 anti-cancer properties [3]. Cornus officinalis, native to Korea, Japan, and China comprises, 53 compounds including terpenoids, flavonoids, sterols, carboxylic acids, polysaccharides, and 54 phenylpropanoids that have been isolated and identified. Owing to its chemical constituents, 55 C. officinalis displays diverse pharmacological activities such as hypoglycemic activity and 56 protective activity toward diabetic target organs, and antioxidant, anti-inflammatory, and 57 anticancer activity [4,5]. 58 Benign prostatic hyperplasia (BPH) is the most frequent, non-cutaneous form of cancer among 59 elderly men and is characterized by progressive glandular and stromal tissue hyperplasia, which 60 leads to an enlarged prostate [6]. The rapid growth of stromal and epithelial elements result in 61 BPH in the prostate, along with lower urinary tract symptoms (LUTS), including obstructive 62 symptoms such as hesitancy, poor intermittent stream, feeling of incomplete bladder emptying, 63 and irritative symptoms such as frequency, urgency, and nocturia [7,8]. Various molecular 64 etiologies of BPH have suggested that hormones, oxidative stress, chronic inflammation, and 65 aging may play a crucial role in BPH development, while researchers consider androgens, 66 especially testosterone-related hormones to be the major contributory factors in the 67 development and progression of BPH [9,10]. The growth and development of prostate gland 68 depends on androgen stimulation, especially through DHT [11,12]; accumulation of DHT with 69 aging results in rapid growth and hyperplasia of prostatic cells [13]. DHT is an active metabolic 4 70 product of the conversion of testosterone by steroid 5α-reductase [14]. Finasteride, a drug that 71 is used as a steroid 5α-reductase Type 2 inhibitor, can be used for the treatment for BPH [15].

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It inhibits the conversion of testosterone to DHT, thereby preventing prostatic hyperplasia. 73 Furthermore, saw palmetto has been widely used as a therapeutic remedy for urinary 74 dysfunction due to BPH and works by ceasing the breakdown of testosterone into its byproduct      Phytochemical compounds in the SC extract such as hederacoside D (Fig 1A) and morroniside 184 (Fig 1C) were identified and quantified by using HPLC. Hederacoside D showed the 185 characteristic peak at 39.52 min (Fig 1B), while morroniside at 6.53 min (Fig 1D) and the 186 concentrations were found as 25 mg/g and 1.5 mg/g respectively.

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Cell viability and in vitro western blotting 188 To study the cytotoxicity of SC extract on LNCaP cells, the MTT assay was performed. As 189 shown in Fig. 2A, there was no significant difference in cell viability between SC treated cells  In this study, we investigated the effect of SC treatment on androgen receptor (AR), prostate 194 specific antigen (PSA), and 5α-reductase type 2 in LNCaP cells using western blot (Fig. 2B). finasteride showed significantly (p < 0.01) decreased PW relative to the BPH group. Similarly, 208 SC50 and SC200 groups also exhibited significantly (p < 0.001, p < 0.01 respectively) 209 decreased PW compared with the BPH group. However, no significant differences in BW were 210 observed among the groups (Fig. 3B). Moreover, relative prostatic weight was significantly (p 211 < 0.001) elevated in the BPH group compared to the control, while Fina group exhibited 212 significantly (p < 0.001) decreased values in comparison to the BPH group (Fig. 3C). The 213 groups SC50 and SC200 also showed these values that were significantly (p < 0.001, p < 214 0.01respectively) decreased than those of the rats in the BPH group. These results proved that 215 SC is a potential treatment for modulating the prostate size of the TP-induced BPH model.

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BPH rats 218 In this experiment, we studied serum testosterone and DHT as they have a fundamental role in 219 progression of BPH. As shown in Fig. 4A, a significant (p < 0.01) increase in serum 220 testosterone level was shown in the BPH group compared with the control group. In contrast, 221 rats in the groups of Fina, SC50 and SC200 showed significantly (p < 0.01) decreased 222 testosterone levels relative to the BPH group. Similarly, the groups Saw, SC25 and SC100 also 223 showed significant (p < 0.05) increase in the testosterone level. Like the serum testosterone, 224 serum DHT level also significantly (p < 0.01) increased in the BPH group (Fig. 4B). By   Immunohistochemical study of rat prostatic tissue 12 252 In this study, we investigated the immunoexpression of 5α-reductase type 2 and PCNA. As 253 shown in Fig. 7A, IHC of prostate tissue indicated that the rats in the control group had no 254 abnormalities; however, those in the BPH and Saw groups showed increased expression of 255 type-2 5α-reductase. Notably, finasteride and SC treatment reversed the effect of TP, and SC 256 treatment reduced the type-2 5α-reductase expression, in a dose-dependent manner. We also and SC treated rats as compared to the BPH group. These data evidenced that SC treatment has 296 an ability to regulate androgens and their byproducts, and thereby attenuate BPH development. it is used as a clinical maker for disease prognosis [35]. In this study, in vitro and in vivo 305 western blotting revealed that the increased AR, PSA and 5α-reductase type 2 in BPH condition 306 can be significantly inhibited with finasteride and SC treatments. These findings proved that 307 SC extract is a potential option to suppress androgen signaling in prostatic cells.

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In the current study, we also performed the IHC to examine the expression of 5α-reductase type  that are associated with prostate proliferation [38]. In this study, significantly increased PCNA 314 expression was detected in the BPH group rats as compared to the control rats. In contrast, both 315 finasteride and SC treated rats exhibited significant reduction in PCNA count relative to the 316 BPH group. Moreover, IHC of type 2 5α-reductase showed elevated expression in BPH group, 317 while both finasteride and SC intervention reversed that effect. Together, these findings proved 318 that SC extract can be a possible treatment for BPH via anti-proliferative activity. In conclusion, the findings of our study revealed that SC treatment significantly reduced the 322 prostate hyperplasia and prostate size. In addition, SC extract had an ability to inhibit type 2 323 5α-reductase expression, thereby preventing the conversion of testosterone into its byproduct The data used to support the finding of this experiment are available from the corresponding 334 author upon request.

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The authors declare that they have no conflicts of interest.