Cordycepin Attenuates Testosterone-Induced Benign Prostatic Hyperplasia in Rats via Modulation of AMPK and AKT Activation

Benign prostatic hyperplasia (BPH) is a disease that commonly affects elderly men. Cordycepin is an adenosine analog with a wide range of pharmacological activities including antiproliferative and prostatic smooth muscle relaxant effects. This study was designed to assess the actions of cordycepin in testosterone-induced BPH in rats. Animals were divided into six treatment groups: control, cordycepin-alone (10 mg/kg), testosterone-alone (3 mg/kg), cordycepin (5 mg/kg) + testosterone, cordycepin (10 mg/kg) + testosterone, and finasteride (0.5 mg/kg) + testosterone. Treatments were continued daily, 5 days a week, for 4 weeks. Cordycepin significantly prevented the increase in prostate weight and prostate index induced by testosterone. This was confirmed by histopathological examinations. Cordycepin antiproliferative activity was further defined by its ability to inhibit cyclin-D1 and proliferating cell nuclear antigen (PCNA) expression. In addition, cordycepin exhibited significant antioxidant properties as proven by the prevention of lipid peroxidation, reduced glutathione diminution, and superoxide dismutase exhaustion. This was paralleled by anti-inflammatory activity as shown by the inhibition of interleukin-6, tumor necrosis factor-α, and nuclear factor-κB expression in prostatic tissues. It also enhanced apoptosis as demonstrated by its ability to enhance and inhibit mRNA expression of Bax and Bcl2, respectively. Western blot analysis indicated that cordycepin augmented phospho-AMP-activated protein kinase (p-AMPK) and inhibited p-AKT expression. Collectively, cordycepin has the ability to prevent testosterone-induced BPH in rats. This is mediated, at least partially, by its antiproliferative, antioxidant, anti-inflammatory, and pro-apoptotic actions in addition to its modulation of AMPK and AKT activation.


Introduction
Benign prostatic hyperplasia (BPH) is characterized by enhanced multiplication of prostatic stromal and epithelial cells. PBH is a common disease within the elderly male population [1]. According to several reports, the incidence of BPH rises with age [2]. BPH symptoms are thought to affect about half of all men over the age of 60 [3]. Symptoms of BPH can significantly and negatively impact the quality of life of affected men [4]. These symptoms of BPH include decreased urinary flow and urinary incontinence, nocturia, dribbling, and difficulty initiating flow [5]. The pathogenesis of BPH has been linked

Animals
Experimental work involving animals was conducted according to the approved procedures by the Faculty of Pharmacy's Research Ethics Committee, King Abdulaziz University (Ref # PH-1443-23). Male Wistar rats (200-230 g) of 10-week of age were obtained from the animal facility of Faculty of Pharmacy, King Abdulaziz University. The rats were maintained under an air-conditioned environment (20-24 • C), alternating 12-h light/12-h dark cycles, and ad libitum feeding with a standard diet and water. The animals were acclimatized for 1 week to our experimental facility before the start of experiments.

Toxicity Study
To assess the acute toxicity of cordycepin based on the OECD Test Guideline 423, animals were administered an oral cordycepin dose of 2000 mg/kg. After that, the rats were observed closely during the first hour and regularly for the following 24 h of administration. As all animals survived, the experimental protocol was carried out again utilizing three additional rats.

Experimental Design
Thirty-six male rats were separated into 6 groups randomly (6 animals per group). Group 1 (control) was subcutaneously (s.c.) administered normal saline (10 mL/kg, p.o.) and corn oil (1 mL/kg). Group 2 (cordycepin) was administered cordycepin dissolved in normal saline (10 mg/kg p.o.) and corn oil (s.c.). Group 3 (testosterone) was given Pharmaceutics 2022, 14, 1652 3 of 14 normal saline (10 mL/kg, p.o.) and testosterone (3 mg/kg dissolved in corn oil, s.c.). Group 4 (testosterone + cordycepin 5 mg/kg) was administered cordycepin (5 mg/kg, p.o.) and testosterone (5 mg/kg s.c.). Group 5 (testosterone + cordycepin 10 mg/kg) was administered cordycepin (10 mg/kg, p.o.) and testosterone (5 mg/kg s.c.). Group 6 (positive control) was given finasteride (0.5 mg/kg p.o.) and testosterone (5 mg/kg s.c.). Administration of oral doses was carried out before s.c. injections by 1 h. Treatment of animals was administered once daily every 5 days per week for 4 consecutive weeks. All specified doses and treatment schedules were according to a pilot study. Prostate tissues from each animal group were harvested immediately after the sacrifice and 72 h after the last treatment. Parts of the prostatic ventral lobes were kept in a 10% neutral buffered formalin for histopathology and immunohistochemistry studies. The remaining tissues were flash-frozen in liquid nitrogen and stored at −80 • C for assessment of mRNA expression and biochemical markers.

Prostate Index and Weight
Prostate weights were registered after dissection, and hence the prostate index was calculated relative to the total body weight of the animal by dividing the prostate weight by the total body weight before multiplying by 1000.

Histopathological Examination
Paraffin sections of 4 µm were obtained from the fixed prostate tissues. Following de-paraffinization and rehydration, the paraffin sections were stained with hematoxylin and eosin (H&E). The height of the prostate glandular epithelia was determined using Image J software (Image J, 1.46a, NIH, Bethesda, MD, USA).

Statistical Analysis
Data are reported as mean ± SD. For statistical analysis with multiple comparisons using GraphPad Prism version 8.1. (GraphPad, La Jolla, CA, USA), one-way ANOVA with Tukey's post-hoc test was utilized and a p-value of less than 0.05 is considered statistically significant.

Acute Toxicity Assessment
After 24 h of oral administration of cordycepin at 2000 mg/kg, there were no deaths detected among the three male rats utilised in this study. Hence, a following study was performed on three additional rats utilising the same experimental protocol, which also led to no deaths observed in the tested animals.

Prostate Weights and Indices
Principally, cordycepin at both doses tested was safe and well-tolerated as it resulted in no mortality or significant changes in prostate weights. In contrast, testosterone enanthate significantly increased prostate weight and index by 178.2% and 153.0%, respectively, relative to the control value (Table 1). However, coadministration with cordycepin at both 5 and 10 mg/kg led to a substantial decrease in prostate weight by 32.2% and 45.2% and prostate index by 29.5% and 41.5%, respectively, compared to rats that received testosterone alone. It was also noticeable that cotreatment of rats with finasteride at 0.5 mg/kg significantly decreased the prostate weight and index by 52.5% and 48.4% associated with testosterone administration. 1.50 c,d ± 0.23 Data (n = 6) are expressed as mean ± SD. COR = cordycepin, T = testosterone, FIN = finasteride. a, b, c, or d: statistically different (p < 0.05) from control, cordycepin, testosterone or T + COR (5 mg/kg), respectively.

Histopathological Examination
Microscopic examination of the prostate gland from the control and cordycepin-alone groups revealed normal histology of acini that were lined by cuboidal to low columnar epithelium with occasional papillary infoldings ( Figure 1A,B). However, testosterone injection resulted in hyperplasia of prostatic acini that was characterized by frequent inward folding of the epithelial lining forming intraluminal projections. The hyperplastic cells appeared cuboidal to tall columnar with granular eosinophilic cytoplasm. Some examined sections showed expansion of the interstitial tissue with edema and inflammatory cell infiltration ( Figure 1C). Regarding cordycepin (5 mg/kg)-treated animals, moderate improvement was detected in the examined sections as several acini showed hyperplasia of the epithelial lining accompanied by fewer areas of interstitial inflammatory regions ( Figure 1D). The highest protection was observed in cordycepin (10 mg/kg)-treated animals as acinar epithelia appeared almost normal. Only fewer sections showed the epithelial lining hyperplasia with intraluminal projections ( Figure 1E). The positive control group has obvious improvements against testosterone hyperplasia with almost normal prostatic architecture. Few acini showed desquamated epithelial cells into the acinar lumen admixed with eosinophilic tissue debris and inflammatory cells infiltration ( Figure 1F).

Proliferation Markers
Immunohistochemical analysis of the key regulators of cell proliferation cyclin-D1 and PCNA revealed an average number of positive cells for either of them in the control group (Figure 2A). Similarly, cordycepin resulted in no significant changes in the number of stained cells compared to the control group ( Figure 2B). However, testosterone significantly induced a noticeable increase in the number of stained cells, indicating an elevated proliferation rate ( Figure 2C). As shown in Figure 2D,E, the coadministration of cordycepin was capable of significantly ameliorating the substantial increment of cyclin D1 and PCNA-positive cells associated with administering testosterone alone. Interestingly, finasteride resulted in comparable findings to cordycepin at 10 mg/kg ( Figure 2F). Densitometry data in Figure 2 revealed that cordycepin (10 mg/kg) significantly reduced the expression of cyclin D1 and PCNA by 48.8% and 31.7%, respectively.

Proliferation Markers
Immunohistochemical analysis of the key regulators of cell proliferation cyclin-D1 and PCNA revealed an average number of positive cells for either of them in the control group (Figure 2A). Similarly, cordycepin resulted in no significant changes in the number of stained cells compared to the control group ( Figure 2B). However, testosterone significantly induced a noticeable increase in the number of stained cells, indicating an elevated proliferation rate ( Figure 2C). As shown in Figure 2D,E, the coadministration of cordycepin was capable of significantly ameliorating the substantial increment of cyclin D1 and PCNApositive cells associated with administering testosterone alone. Interestingly, finasteride resulted in comparable findings to cordycepin at 10 mg/kg ( Figure 2F). Densitometry data in Figure 2 revealed that cordycepin (10 mg/kg) significantly reduced the expression of cyclin D1 and PCNA by 48.8% and 31.7%, respectively.

Oxidative Stress Markers
The protective activity of cordycepin against oxidative stress induced by testosterone in prostatic tissues was also evaluated. Table 2 shows that testosterone exposure significantly elevated the levels of MDA, a lipid peroxidation marker, by around 3.5 folds while markedly depleting the content of GSH and reducing the activity of SOD by 64.0% and 71.4%, respectively, relative to the control values. Interestingly, treatment with cordycepin at 5 mg/kg and 10 mg/kg significantly attenuated the testosterone-induced increase in

Oxidative Stress Markers
The protective activity of cordycepin against oxidative stress induced by testosterone in prostatic tissues was also evaluated. Table 2 shows that testosterone exposure significantly elevated the levels of MDA, a lipid peroxidation marker, by around 3.5 folds while markedly depleting the content of GSH and reducing the activity of SOD by 64.0% and 71.4%, respectively, relative to the control values. Interestingly, treatment with cordycepin at 5 mg/kg and 10 mg/kg significantly attenuated the testosterone-induced increase in MDA levels by 33.0% and 42.1%. Moreover, cordycepin significantly ameliorated GSH depletion and SOD exhaustion by both doses tested. In a similar vein, finasteride was effective in ameliorating testosterone-induced oxidative stress as indicated by the MDA and GSH content and SOD activity levels. 8.6 c,d,e ± 0.96 Data (n = 6) are expressed as mean ± SD. COR = cordycepin, T = testosterone, FIN = finasteride. a, b, c, d or e: statistically different (p < 0.05) from control, cordycepin, testosterone, T + COR (5 mg/kg) or T + COR (10 mg/kg), respectively.

Inflammatory Markers
Further immunohistochemical analysis was carried out to examine the inflammatory state of prostatic tissues in cordycepin-treated rats following testosterone administration ( Figure 3). Testosterone significantly stimulated the expression of IL-6, while cordycepin treatment significantly ameliorated this increased expression of IL-6 by 13.2% and 54.2% at 5 mg/kg and 10 mg/kg, respectively. Furthermore, the expression of TNF-α and NF-κB were also induced with testosterone and these expression levels were markedly attenuated by cordycepin treatment at 5 mg/kg by 15.7% and 17.5%, respectively. Cordycepin at 10 mg/kg caused even further reduction in TNF-α expression by 49.7% and NF-κB expression by 40.5% compared to testosterone alone ( Figure 3).

Expression of Bax and Bcl2
Cordycepin's antiapoptotic activity was examined according to Bax and Bcl2 mRNA and protein expression in prostatic tissues of testosterone-treated rats. As shown in Figure 4A,D, testosterone markedly decreased the mRNA and protein expression of the proapoptotic protein Bax, respectively. Yet, cordycepin significantly attenuated this decrease in the expression of Bax at all doses tested in this study. With regards to the antiapoptotic protein Bcl2, testosterone significantly increased its mRNA and protein expression, however, cordycepin significantly attenuated these changes at 5 mg/kg and 10 mg/kg ( Figure 4B,E). Finasteride also showed significant anti-apoptotic activity, as indicated by the induced expression of Bax and the reduced expression of Bcl2 relative to the testosterone-alone group (Figure 4). at 5 mg/kg and 10 mg/kg, respectively. Furthermore, the expression of TNF-α and NF-κB were also induced with testosterone and these expression levels were markedly attenuated by cordycepin treatment at 5 mg/kg by 15.7% and 17.5%, respectively. Cordycepin at 10 mg/kg caused even further reduction in TNF-α expression by 49.7% and NF-κB expression by 40.5% compared to testosterone alone ( Figure 3).

Expression of Bax and Bcl2
Cordycepin's antiapoptotic activity was examined according to Bax and Bcl2 mRNA and protein expression in prostatic tissues of testosterone-treated rats. As shown in Figure  4A,D, testosterone markedly decreased the mRNA and protein expression of the proapoptotic protein Bax, respectively. Yet, cordycepin significantly attenuated this decrease in the expression of Bax at all doses tested in this study. With regards to the antiapoptotic protein Bcl2, testosterone significantly increased its mRNA and protein expression, however, cordycepin significantly attenuated these changes at 5 mg/kg and 10 mg/kg ( Figure  4B,E). Finasteride also showed significant anti-apoptotic activity, as indicated by the induced expression of Bax and the reduced expression of Bcl2 relative to the testosteronealone group (Figure 4).

Expression of p-AKT and p-AMPK
Finally, the phosphorylated and total protein forms of AMPK and AKT were analyzed to study the cellular targets driving the beneficial effects of cordycepin that were observed throughout this study. As can be seen in Figure 5A, testosterone treatment led to a noticeable decrease in p-AMPK concomitant with an increase in p-AKT expression. However, cordycepin treatment at doses of 5 mg/kg and 10 mg/kg administered to testosterone-challenged animals resulted in significant amelioration of p-AMPK inhibition as well as p-AKT enhanced expression. The 5-α reductase inhibitor finasteride also enhanced and Bcl2 (B) mRNAs and proteins (C) in testosterone-treated rats. Lanes I, II, III, IV, V, and VI represent control, cordycepin, testosterone, testosterone + cordycepin 5 mg/kg, testosterone + cordycepin 10 mg/kg, and testosterone + finasteride, respectively. Densitometric data of Bax and Bcl2 protein expression are demonstrated in bar charts (D,E), respectively. Data presented in the charts are mean ± SD. For mRNA and protein expression studies, n = 6 and 3, respectively. COR = cordycepin, T = testosterone, FIN = finasteride. a, b, c, d or e: significantly different (p < 0.05) from control, cordycepin, testosterone, T + COR (5 mg/kg) or T + COR (10 mg/kg), respectively.

Expression of p-AKT and p-AMPK
Finally, the phosphorylated and total protein forms of AMPK and AKT were analyzed to study the cellular targets driving the beneficial effects of cordycepin that were observed throughout this study. As can be seen in Figure 5A, testosterone treatment led to a noticeable decrease in p-AMPK concomitant with an increase in p-AKT expression. However, cordycepin treatment at doses of 5 mg/kg and 10 mg/kg administered to testosteronechallenged animals resulted in significant amelioration of p-AMPK inhibition as well as p-AKT enhanced expression. The 5-α reductase inhibitor finasteride also enhanced p-AMPK expression and inhibited p-AKT protein content associated with testosterone administration (Figure 5B,C).

Expression of p-AKT and p-AMPK
Finally, the phosphorylated and total protein forms of AMPK and AKT were analyzed to study the cellular targets driving the beneficial effects of cordycepin that were observed throughout this study. As can be seen in Figure 5A, testosterone treatment led to a noticeable decrease in p-AMPK concomitant with an increase in p-AKT expression. However, cordycepin treatment at doses of 5 mg/kg and 10 mg/kg administered to testosterone-challenged animals resulted in significant amelioration of p-AMPK inhibition as well as p-AKT enhanced expression. The 5-α reductase inhibitor finasteride also enhanced p-AMPK expression and inhibited p-AKT protein content associated with testosterone administration (Figure 5B,C). Figure 5. Effect of cordycepin on prostatic protein expression of p-AKT and p-AMPK (A) in testosterone-treated rats. Lanes I, II, III, IV, V, and VI represent control, cordycepin, testosterone, testosterone + cordycepin 5 mg/kg, testosterone + cordycepin 10 mg/kg, and testosterone + finasteride, respectively. Densitometric data of p-AKT and p-AMPK expression presented in bar charts (B,C) are mean ± SD (n = 6). COR = cordycepin, T = testosterone, FIN = finasteride. a, b, c, d or e: significantly different (p < 0.05) from control, cordycepin, testosterone, T + COR (5 mg/kg) or T + COR (10 mg/kg), respectively. Figure 5. Effect of cordycepin on prostatic protein expression of p-AKT and p-AMPK (A) in testosterone-treated rats. Lanes I, II, III, IV, V, and VI represent control, cordycepin, testosterone, testosterone + cordycepin 5 mg/kg, testosterone + cordycepin 10 mg/kg, and testosterone + finasteride, respectively. Densitometric data of p-AKT and p-AMPK expression presented in bar charts (B,C) are mean ± SD (n = 6). COR = cordycepin, T = testosterone, FIN = finasteride. a, b, c, d or e: significantly different (p < 0.05) from control, cordycepin, testosterone, T + COR (5 mg/kg) or T + COR (10 mg/kg), respectively.

Discussion
BPH is one of the most prevalent benign tumors in men, with increasing prevalence after 40 years of age [23]. Uncontrolled cell proliferation, oxidative stress, disturbed cell apoptosis, and inflammation participate in the development of BPH [24][25][26]. Symptoms of BPH such as lower urinary tract symptoms can negatively influence the quality of life of affected men. The main available approaches for pharmacological management of this disease involve using adrenergic α-1 antagonists and 5-α reductase inhibitors [27]. However, it is known that these pharmacological management options may cause intolerable side effects including postural hypotension, impotence, and gynecomastia, which can affect patient compliance [28,29]. Cordycepin is a nucleoside analog that is commonly isolated from the fermentative fluid and fruiting bodies of the entomopathogenic fungus Cordyceps militaris [30]. Cordycepin has several biological and pharmacological properties reported in the literature including the regulation of steroidogenesis, cell proliferation, inflammation, and apoptosis through various signaling pathways [31][32][33][34]. Hence, our study was conducted to investigate the potential of cordycepin to mitigate BPH induced by testosterone in rats.
In this study, it was observed that treatment with cordycepin for 4 weeks significantly attenuated increased prostate weights and indices associated with testosterone administration. Furthermore, histopathological examination confirmed these results as cordycepin cotreatment was associated with almost normal acinar epithelia. In accordance with these findings, it was reported that Cordyceps militaris fruiting body extract tended to inhibit the increase in prostate weight induced by the administration of testosterone over 30 days [35]. Our findings are also in line with the established antiproliferative activity of cordycepin against various cells, including those of the prostate [36,37]. This can be explained by the ability of cordycepin to activate the A3 adenosine receptor, and its activation is associated with the inhibition of prostate cancer cell proliferation [16][17][18].
Several studies also highlight a major role of oxidative stress in the pathogenesis of testosterone-induced BPH [38]. Interestingly, our findings demonstrated that cordycepin possesses significant antioxidant activity as evidenced by the reduced lipid peroxidation and antioxidant enzyme exhaustion. This is in harmony with previous findings reporting the significant antioxidant activity of cordycepin [39][40][41]. In addition, these results are parallel with several reports correlating the anti-oxidation of many compounds with protection against PBH [42][43][44].
Accumulating evidence indicates a significant role of inflammation in the development of testosterone-induced BPH [45]. In this study, cordycepin demonstrated significant anti-inflammatory activity supported by the decreased expression of IL-6, TNF-α, and NF-κB in the prostatic tissues of testosterone-challenged rats. These findings gain support from previous reports highlighting a significant anti-inflammatory activity of cordycepin mediated through suppression of NF-κB signaling pathways [41,[46][47][48]. Oxidative stress can trigger certain inflammatory pathways via activating transcription factors including NF-κB, which in turn control the gene expression of proinflammatory mediators such as TNF-α [49][50][51]. Hence, the decreased expression of inflammatory mediators associated with cordycepin could be partly due to its considerable antioxidant activity.
Prostatic inflammation can lead to stromal and glandular hyperplasia by disturbing the balance between cell proliferation and cell death in the prostate [52]. In the same vein, testosterone is known to induce pro-proliferative and anti-apoptotic changes in cycline-D1, PCNA, Bax, and Bcl2 mRNA content in the prostate gland [8]. In this study, cordycepin at both tested doses significantly ameliorated these pro-proliferative and anti-apoptotic changes induced by testosterone administration. These findings are in harmony with those in the literature describing the antiproliferative activity of cordycepin against prostate cells and other types of cells that involve regulating the expression of cyclin-D1, PCNA, Bax, and Bcl2 [37,[53][54][55][56].
Interestingly, cordycepin was noted to inhibit cell proliferation by modulating AKT signaling in several types of cancer, including prostate cancer [57][58][59][60][61]. It is known that AKT signaling plays a significant role in BPH development as its overexpression is associated with an increased prostate size [62,63]. AKT signaling activation is also associated with the dysregulation of cell death by reducing the expression of Bax and inducing the expression of Bcl-2 [64]. Hence, the potential of cordycepin to inhibit AKT activity in BPH was explored in this study. It was found that testosterone alone significantly induced the expression levels of p-AKT in rat prostate. This is in line with reported findings that PI3K/AKT activation is essential for cell proliferation in testosterone-induced BPH [65]. Remarkably, cordycepin in the current study significantly attenuated this rise in p-AKT expression in the prostatic tissues of testosterone-treated rats. Moreover, cordycepin caused significant amelioration of testosterone-induced decrease in the prostatic expression of p-AMPK. Similar findings were reported in several studies where cordycepin has been shown to activate AMPK and inhibit AKT activation [58][59][60][61]. In this regard, it has been demonstrated that BPH involves lower levels of p-AMPK, and its activation has been proposed to counter-modulate the signaling of pro-inflammatory mediators [66][67][68][69]. This is also in harmony with previous findings demonstrating the beneficial effects of targeting the PI3K/AKT pathway in BPH [63,[70][71][72]. The suggested mechanism of actions of cordycepin are illustrated in Figure 6. sion of p-AMPK. Similar findings were reported in several studies where cordycepin has been shown to activate AMPK and inhibit AKT activation [58][59][60][61]. In this regard, it has been demonstrated that BPH involves lower levels of p-AMPK, and its activation has been proposed to counter-modulate the signaling of pro-inflammatory mediators [66][67][68][69]. This is also in harmony with previous findings demonstrating the beneficial effects of targeting the PI3K/AKT pathway in BPH [63,[70][71][72]. The suggested mechanism of actions of cordycepin are illustrated in Figure 6.

Conclusions
Overall, cordycepin offers protection against testosterone-induced BPH in rats. This protective activity can be at least partially explained by its antiproliferative, antioxidant, anti-inflammatory, and pro-apoptotic actions as well as its modulation of AMPK and AKT activation.

Conclusions
Overall, cordycepin offers protection against testosterone-induced BPH in rats. This protective activity can be at least partially explained by its antiproliferative, antioxidant, anti-inflammatory, and pro-apoptotic actions as well as its modulation of AMPK and AKT activation.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Faculty of Pharmacy's Research Ethics Committee, King Abdulaziz University (Reference # PH-1443-23).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.