Sildenafil Citrate Induces Prostatic Hyperplasia in BPH Model Rats and Aged Rats

doi:10.21203/rs.3.rs-4131702/v1

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Sildenafil Citrate Induces Prostatic Hyperplasia in BPH Model Rats and Aged Rats

https://doi.org/10.21203/rs.3.rs-4131702/v1

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Erectile dysfunction (ED), a prevalent disease among middle-aged and elderly males, significantly impacts both patient and partner quality of life. Phosphodiesterase type 5 inhibitor (PDE5i) represents an effective therapeutic method for ED. Given their widespread global utilization, concerns arise regarding potential reproduction-related problems arising from clinical use. During the extensive development of PDE5i, we speculated that the potential of these inhibitors to variably induce prostatic hyperplasia, but this field remains unexplored. In order to verify the male reproductive toxicity of PDE5i, sildenafil citrate at doses of 5, 10 and 20 mg/kg was administered in BPH model rats and aged rats. Anatomical and pathological analyses indicate a compelling association between sildenafil citrate administration and the promotion of prostatic hyperplasia in both BPH model rats and aged rats. Serum analyses revealed a notable increase in serum prostate binding protein (PBP) in BPH model rats following sildenafil citrate administration. Furthermore, significant increase in serum levels of E2 and T, as well as T in dorsal lobe prostate tissue of aged rats, were observed compared to the model control group. The epithelial-mesenchymal transition (EMT) microarray demonstrated that sildenafil citrate upregulated Fgfb1 and Tmeff1 within the EMT signaling pathway of the dorsal lobe prostate in BPH model rats, concurrently down-regulating Itga5, Versican and Vimentin. These results confirm the hypothesis that sildenafil citrate has reproductive toxicity in males and suggest that the EMT signaling pathway has a potential role in the proliferation of the dorsal lobe prostate in BPH model rats.

Health sciences/Diseases

Health sciences/Urology

Erectile dysfunction (ED), defined as the inability to achieve or sustain a penile erection sufficient for satisfactory sexual intercourse, is one of the common diseases within the domains of in andrology and urology^1,2. Previous studies have projected that ED affects 322 million men globally by 2025, denoting a remarkable surge of 111% from the figures recorded in 1995³. The etiological factors contributing to ED are broadly classified into organic and psychogenic categories, with the former including vascular, nervous, endocrine, and penile determinants⁴. Vascular ED is the most common cause of organic ED, especially in elderly men and patients with risk factors such as diabetes mellitus, hypertension, dyslipidemia, atherosclerosis, obesity, smoking and other cardiovascular disorder^5–7. At present, the therapeutic methods of ED mainly includes oral medications, physical therapy, injection of active drugs in the cavernosal body, intraurethral alprostadil injection, and surgical treatment⁵. Among them, oral phosphodiesterase inhibitor type 5 (PDE5i) is the most commonly used first-line treatment of ED in clinic practice, and it is still the most popular treatment option owing to its notable efficacy, safety, and non-invasive nature⁸.

Specifically, the underlying mechanism of PDE5i involves intricate processes occurring during sexual stimulation. Nitric oxide synthase (NOS) of the corpus cavernosum's non-adrenergic non-cholinergic neurons and vascular endothelial cells facilitates the catalysis of L-arginine, leading to the synthesis of nitric oxide (NO). Subsequently, NO diffuses into smooth muscle tissue, while stimulating the conversion of guanosine triphosphate (GTP) to the second messenger cyclic guanosine monophosphate (cGMP)^9–11. The resultant cGMP binds to and activates protein kinase (PKG), thereby accelerating protein phosphorylation, reducing calcium concentration, inducing cavernous smooth muscle diastole, facilitating blood influx into the cavernous sinus of the penis, culminating in the process of cavernous tissue congestion and erection, and contributing to the maintenance of subsequent erectile states^12–14. Meanwhile, cGMP undergoes hydrolysis by phosphodiesterase¹⁵. Therefore, the inhibition of PDE serves to increase the level of cGMP, effectively realizing the therapeutic purpose of treating ED.

The utilization of specific PDE5i can increase the activity and duration of cGMP. Specifically, in penile tissue, the regulatory role of cGMP is terminated by PDE5, which destroys its phosphodiester bond, a process not shared by PDEs in other cellular contexts⁹. The inhibitory impact of PDE5 on cGMP can be effectively countered by PDE5i, wherein these inhibitors occupy the catalytic site, impeding direct interaction with cGMP^16,17. Owing to additional molecular interactions with the catalytic site, the synthetic PDE5i exhibits an affinity approximately 1000–5000 times higher than that of cGMP^18–20. Consequently, throughout the continuous process of cGMP synthesis, specific PDE5i facilitates the accumulation of cGMP in the corpus cavernosum, thereby helping to improve erectile function²⁰.

Currently, four PDE5i drugs have been approved by the Food and Drug Administration (FDA), namely sildenafil, tadalafil, vardenafil, and avanafil¹⁷. Sildenafil citrate, recognized as the first approved, safe and efficacious oral drug for ED⁸, obtained FDA approval in April 1998, marking its global introduction to the market²¹. On February 18, 2012, with the expiration of the patent protection of sildenafil citrate, a wave of rapid imitation and development of PDE5is based on sidenafil was launched at home and abroad. At present, approximately 20 structurally analogous compounds are undergoing various phases of preclinical and clinical development within the realm of PDE5i in China.

While early clinical studies of sildenafil citrate have shown that a singular oral dose of 100 mg sildenafil citrate does not exert adverse effect on sperm function or ejaculation quality in healthy volunteers²², it has been observed to enhance human sperm motility, maintain membrane integrity, and augment sperm penetration in infertile patients^23,24. However, conflicting perspectives exist within scholarly research, positing that sildenafil citrate usage may adversely impact sperm motility. A large number of studies have shown the association between prolonged sildenafil citrate utilization and the induction of oxidative stress, inflammatory responses, and structural changes within the testicular milieu. This long-term exposure has been linked to diminished sperm count and motility, elevated serum levels of testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH), as well as an escalated risk of penile fibrosis, culminating in irreversible damage to the corpus cavernosum tissue^25–30.

Owing to the localized presence of PDE4 and PDE5 in prostatic tissue, and excessive NO has been implicated in sperm damage. Therefore, it is reasonable to speculate that the potential male reproductive toxicity associated with prolonged usage of PDE5i. However, a comprehensive examination of the association between PDE5i and benign prostatic hyperplasia (BPH) has not been systematically studied. In light of this, our investigation used sildenafil citrate as a representative drug PDE5i. Using BPH model rats and aged rats, our findings indicate that PDE5i may exhibit an inducing effect on prostatic hyperplasia within clinically relevant dosages. Furthermore, our study shows the involvement of the epithelial-mesenchymal transition (EMT) signaling pathway, indicating its potential regulatory role in this process. Our study complements the current understanding of the reproductive toxicity associated with sildenafil citrate, particularly concerning its mechanisms of action. Such insights contribute to a detailed comprehension of the intricate relationship between PDE5i and BPH, laying a foundation for informed and rational utilization of sildenafil citrate in clinical practice.

Study 1: Toxic Effects of Sildenafil Citrate on BPH Model Rats

Anatomical Analysis of Prostate in BPH Model Rats Treated with Sildenafil Citrate

Anatomical data showed that 4 weeks after administration, sildenafil citrate, administered at a dosage equivalent to the clinical standard, could increase the prostate volume, prostate weight and organ coefficient of BPH model rats (Table 1), reflecting the weight gain of prostate induced by sildenafil citrate. Notably, the group administered with sildenafil citrate (20 mg/kg) exhibited statistically significant differences across all three indexes, indicating the pronounced prostate toxicity associated with high dose of sildenafil citrate.

Sildenafil citrate has the potential to increase the weight and visceral body coefficient of ventral prostate (VP) and dorsal lobe of the prostate (DLP) (Table 2). Notably, with the increase dose of sildenafil citrate, the organ coefficient of VP and DLP showed an increasing trend, suggesting that sildenafil citrate has a dose-dependent effect on prostatic physiology. In the castration group, due to testicular removal and no testosterone propionate, it is difficult to divide the prostate into VP and DLP, so the anatomical data for the VP and DLP levels of this group are omitted from presentation.

Table 1

Effects of sildenafil citrate on prostate volume, prostate weight and organ coefficient in BPH model rats. Results were performed as means ± SD (n = 12), analyzed using ANOVA followed by LSD post hoc test. Organ coefficient = 100 × organ weight / terminal body weight. ^a Significantly different from model control (p < 0.05). ^b Significantly different from model control (p < 0.01).
Treatment	Prostate volume (ml)	Prostate weight (g)	Organ coefficient
Negative control	0.758 ± 0.149	0.704 ± 0.105	0.187 ± 0.030
Castration group	0.038 ± 0.009^b	0.083 ± 0.021^b	0.024 ± 0.007^b
Model control	0.931 ± 0.137	0.783 ± 0.126	0.215 ± 0.035
Sildenafil (5 mg/kg)	1.076 ± 0.164	0.962 ± 0.156	0.284 ± 0.052^a
Sildenafil (10 mg/kg)	1.106 ± 0.214	0.954 ± 0.203	0.291 ± 0.029^a
Sildenafil (20 mg/kg)	1.165 ± 0.167^a	1.047 ± 0.156^b	0.305 ± 0.051^b

Table 2

Effects of sildenafil citrate on prostate lobes of BPH model rats. Results were performed as means ± SD (n = 12), analyzed using ANOVA followed by LSD post hoc test. Organ coefficient = 100 × organ weight / terminal body weight. ^a Significantly different from model control (p < 0.05). ^b Significantly different from model control (p < 0.01).
	VP		DLP
Treatment	Weight (g)	Organ coefficient	Weight (g)	Organ coefficient
Negative control	0.443 ± 0.079	0.118 ± 0.021	0.261 ± 0.048	0.069 ± 0.014
Model control	0.476 ± 0.092	0.130 ± 0.024	0.307 ± 0.044	0.085 ± 0.014
Sildenafil (5 mg/kg)	0.559 ± 0.120	0.161 ± 0.033^b	0.403 ± 0.103	0.117 ± 0.028^b
Sildenafil (10 mg/kg)	0.567 ± 0.144	0.170 ± 0.044^a	0.387 ± 0.077	0.116 ± 0.024^b
Sildenafil (20 mg/kg)	0.634 ± 0.090^b	0.188 ± 0.023^b	0.413 ± 0.083^a	0.122 ± 0.021^b

Histopathological Analysis of Prostates in BPH Model Rats Induced by Sildenafil Citrate

The results of pathological observation revealed that compared with the model control group, administration of 5,10 and 20 mg/kg sildenafil citrate exhibited a significant augmentation in both thickness and size of the prostatic epithelium and glandular lumen, as well as a notable increase of the number and dense distribution of glands, acinar deformation and other morphological changes in BPH model rats (Fig. 1). After dosing, sildenafil citrate may lead to abnormal changes in prostatic lumen and epithelial height (Table 3). The epithelial height of VP decreased, and the epithelial height of DLP increased. In the castration group, due to testicular removal and no testosterone propionate, it is difficult to divide the prostate into VP and DLP, so the pathological data for the VP and DLP levels of this group are omitted from presentation.

Table 3

Effect of sildenafil citrate on the prostatic cavity area and height of epithelium epithelial height of prostate lobes in BPH model rats. Results were performed as means ± SD (n = 12), analyzed using ANOVA followed by LSD post hoc test. ^a Significantly different from model control (p < 0.05). ^b Significantly different from model control (p < 0.01).
	VP		DLP
Treatment	Prostatic cavity area (µm²)	Height of epithelium (µm)	Prostatic cavity area (µm²)	Height of epithelium (µm)
Negative control	11165 ± 4815^a	10.75 ± 1.83^b	10525 ± 5561^b	10.33 ± 0.18
Model control	16435 ± 4639	13.66 ± 2.86	21850 ± 10540	10.90 ± 2.68
Sildenafil (5 mg/kg)	16507 ± 14497	11.41 ± 2.66^b	13589 ± 9829^b	10.70 ± 2.35
Sildenafil (10 mg/kg)	11487 ± 9134	12.08 ± 2.27^b	17813 ± 10624^a	11.87 ± 2.79^a
Sildenafil (20 mg/kg)	21101 ± 13964^a	12.33 ± 2.71^b	15193 ± 9881^b	11.36 ± 2.67

Analysis of Serum Hormone levels in BPH Model Rats Induced by Sildenafil Citrate

The serum hormone level analysis indicates that administration of sildenafil citrate to BPH model rats resulted in a discernible increase in serum prostate binding protein (PBP) levels, exhibiting a dose-dependent relationship with sildenafil citrate dosage when compared to the model control group. Notably, a significant increase in serum estradiol (E2) hormone levels was observed in BPH model rats exposed to high-dose sildenafil, while little effects were discerned in testosterone (T) and prolactin (PRL) levels (Table 4).

Table 4

Effect of sildenafil citrate on serum hormone levels in BPH model rats. Results were performed as means ± SD (n = 12), analyzed using ANOVA followed by LSD post hoc test. ^a Significantly different from model control (p < 0.05). ^b Significantly different from model control (p < 0.01).
Treatment	E2 (pg/ml)	T (ng/ml)	PRL (ng/ml)	PBP (pg/ml)
Negative control	2.918 ± 0.019	0.773 ± 0.03	103.3 ± 3.5	46.5 ± 13.4
Castration group	2.939 ± 0.099	0.605 ± 0.033^b	113.6 ± 11.0^a	44.5 ± 16.7
Model control	2.939 ± 0.019	0.793 ± 0.052	107.1 ± 7.5	50.5 ± 9.4
Sildenafil (5 mg/kg)	2.971 ± 0.031	0.779 ± 0.065	108.4 ± 7.1	53.6 ± 15.2
Sildenafil (10 mg/kg)	2.974 ± 0.029	0.774 ± 0.042	108.9 ± 6.5	58.1 ± 16.0
Sildenafil (20 mg/kg)	2.988 ± 0.035^a	0.799 ± 0.044	108.8 ± 6.5	59.9 ± 15.6

Evaluation of EMT Gene Expression by a Microarray Analysis in the Dorsal Lobe of BPH Model Rats Induced by Sildenafil Citrate

Investigation of gene expression microarray associated with EMT was conducted to ascertain a potential causal relationship between sildenafil citrate and the EMT signaling pathway. The results from quantitative polymerase chain reaction demonstrated that the sample purity adhered to the stipulated experimental criteria. A comprehensive analysis of 89 EMT-associated genes revealed noteworthy outcomes, of which two genes exhibited significant up-regulation, exceeding a cutoff value of 2, while three genes displayed significant down-regulation, also exceeding a cutoff value of 2 (Table 5). Statistical analysis showed that sildenafil citrate exerted an upregulatory effect on Fgfb1 and Tmeff1 and a down-regulatory effect on Itga5, Versican and Vimentin within the EMT signaling pathway of the DLP in BPH model rats.

Table 5

Effect of sildenafil on the expression of EMT pathway genes in the dorsal lobe prostate of BPH rats (n = 4).
Gene symbol	Fold change	P-value	Gene description
Fgfbp1	2.33	0.0234	Fibroblast growth factor binding protein 1
Tmeff1	2.25	0.0024	Transmembrane protein with EGF-like and two follistatin-like domains 1
Itga5	-1.91	0.0228	Integrin subunit alpha 5
Vcan	-2.57	0.0229	Versican
Vim	-2.16	0.0405	Vimentin

Study 2: Toxic Effects of Sildenafil Citrate on Aged Rats

Anatomical Analysis of Prostate in Aged Rats Treated with Sildenafil Citrate

Ten weeks after administration of sildenafil citrate, the body weight of aged rats in sildenafil citrate (10 mg/kg) and sildenafil citrate (20 mg/kg) exhibited a slight elevation compared to the model control group, though without statistical significance (Table 6). Notably, the prostate weight (Table 6), prostate volume (Table 6), organ coefficient (Table 6), the prostate weight in VP (Fig. 2A) and the corresponding organ coefficient (Fig. 2B), the prostate weight in DLP (Fig. 2A) and the corresponding organ coefficient (Fig. 2B) were significantly increased in sildenafil citrate (10 mg/kg) and sildenafil citrate (20 mg/kg). and there was a dose-effect relationship, and the prostate weight in the dorsal lobe was more than that in the ventral lobe. More significantly, the prostatic weight in the dorsal lobe exhibiting a more obvious effect than that in the ventral lobe.

Table 6

Effect of sildenafil citrate on prostate volume and prostate weight in aged rats. Results were performed as means ± SD (n = 8), analyzed using ANOVA followed by LSD post hoc test. ^a Significantly different from model control (p < 0.05).
Treatment	Body weight (g)	Prostate weight (g)	Organ coefficient	Prostate volume (ml)
Control	706.7 ± 42.8	1.08 ± 0.13	1.53 ± 0.19	1.10 ± 0.13
Sildenafil (5 mg/kg)	705.0 ± 55.7	1.12 ± 0.23	1.60 ± 0.39	1.21 ± 0.20^a
Sildenafil (10 mg/kg)	724.8 ± 34.5	1.18 ± 0.17^a	1.62 ± 0.20^a	1.25 ± 0.16^a
Sildenafil (20 mg/kg)	735.0 ± 66.2	1.21 ± 0.15^a	1.65 ± 0.25^a	1.17 ± 0.13^a

Histopathological Analysis of Prostates in Aged Rats Induced by sildenafil Citrate

Microscopic image analysis revealed noteworthy findings in the examination of prostatic morphology. Specifically, within the dorsal lobe, the sildenafil citrate treatment group exhibited a significantly augmented prostatic cavity area compared to the model control group, whereas in the ventral lobe, the sildenafil citrate group manifested a diminished prostatic cavity area relative to the control group (Fig. 2C). Additionally, the height of epithelium in both VP and DLP of the sildenafil citrate group exceeded that of the control group (Fig. 2D). Furthermore, in comparison to the model control group, sildenafil citrate administration leaded to abnormal thickening of the ventral and dorsal prostate epithelium, a heightened glandular count, acinar deformation, and irregular glandular cavities in aged rats (Fig. 2E).

Analysis of Serum Hormone and Prostate Hormone Levels in Dorsal Lobe of Aged Rats Induced by Sildenafil Citrate

The analysis of hormone levels in serum and DLP revealed a significant increase in E2 and T levels in serum, as well as an increase in T levels in the DLP of aged rats, as compared to the model control group, following sildenafil citrate administration (Table 7&Table 8). Notably, the influence of sildenafil citrate on testosterone exhibited a discernible dose-dependent relationship. Furthermore, the examination of insulin concentrations in both serum and the DLP indicated no significant difference between the sildenafil citrate treatment group and the model control group. This suggests that sildenafil citrate is unlikely to exert an impact on insulin levels, leading to the inference that its influence on insulin remains minimal or negligible.

Table 7

Effect of sildenafil citrate on serum hormone levels in aged rats. Results were performed as means ± SD (n = 8), analyzed using ANOVA followed by LSD post hoc test. a Significantly different from model control (p < 0.05). b Significantly different from model control (p < 0.01).
Treatment	E2 (pg/ml)	T (ng/ml)	Insulin (µIU/ml)
Control	18.85 ± 6.28	0.23 ± 0.12	4.84 ± 0.33
Sildenafil (5 mg/kg)	30.51 ± 4.41^b	0.40 ± 0.26	4.60 ± 0.44
Sildenafil (10 mg/kg)	20.94 ± 6.10	0.61 ± 0.48^a	4.46 ± 0.17
Sildenafil (20 mg/kg)	22.79 ± 8.83^a	0.63 ± 0.18^b	4.67 ± 1.00

Table 8

Effect of sildenafil citrate on prostate hormone levels in the dorsal lobe of aged rats. Results were performed as means ± SD (n = 8), analyzed using ANOVA followed by LSD post hoc test. ^a Significantly different from model control (p < 0.05).
Treatment	E2 (pg/ml)	T (ng/ml)	Insulin (µIU/ml)
Control	6.29 ± 2.31	0.33 ± 0.17	311.6 ± 43.3
Sildenafil (5 mg/kg)	6.27 ± 1.79	0.35 ± 0.34	277.0 ± 30.3
Sildenafil (10 mg/kg)	5.25 ± 2.11	0.59 ± 0.37	337.1 ± 63.4
Sildenafil (20 mg/kg)	8.58 ± 2.79	0.76 ± 0.38^a	301.4 ± 38.7

Penile erection is a neurovascular phenomenon intricately regulated by psychological factors and coordinated through the collaboration of endocrine, vascular, and nervous systems³¹. This process involves penile vasodilation, relaxation of penile smooth muscle, increased blood flow within the penile cavernous body, and the maintenance of normal venous occlusion function³². When its dysfunction occurs, it not only significantly affects the quality of life for men and their families but can also serve as an early indicator of severe coronary or peripheral vascular diseases³³. Approximately 80% of case related to ED can be attributed to penile vascular diseases deriving from endothelial dysfunction linked to the NO-cGMP system³². Sildenafil citrate, a widely utilized therapeutic agent, is capable of increasing cGMP levels and inducing smooth muscle relaxation, thereby constituting a prominent intervention in the management of ED³⁴. However, although sildenafil citrate is effective in treating ED, 20–50% of patients who initially respond to sildenafil citrate will discontinue its use³⁵. In recent times, scholarly attention has shifted towards investigating the potential male reproductive toxicity associated with sildenafil citrate. Nevertheless, its specific role in the context of prostatic hyperplasia remains an area necessitating further exploration.

In this study, we found that sildenafil citrate administration elicited a promotive effect on prostatic hyperplasia in BPH model rats. Comparative analysis against the model control group revealed a statistically significant increase in the prostate organ coefficient across sildenafil citrate treatment groups at low, moderate, and high doses, directly indicated the effect of sildenafil citrate on prostatic hyperplasia in BPH model rats. The pathogenesis of prostatic hyperplasia entails increments in glandular count and epithelial height. Notably, glandular proliferation may not necessarily correlate with an enlargement in glandular luminal area. Histomorphological analysis of the prostate from our BPH model rats showed heightened epithelial thickness in the DLP, increased glandular proliferation in the VP, accompanied by glandular luminal compression and deformation. Consequently, our findings suggest that the adverse impact of sildenafil citrate on the prostate of BPH model rats primarily manifests as glandular hyperplasia within the VP and epithelial hyperplasia within the DLP.

ED is a condition that can manifest in men across all age groups, yet its prevalence is notably after the age of 60, with a risk three times higher in individuals aged 60 and above in comparison to those aged 40^36,37. Therefore, it is of great significance to investigate the effect of sildenafil citrate on prostatic hyperplasia in aged rats. Our findings reveal that sildenafil citrate administration elicited a notable increase in prostate weight, organ coefficient, and height of epithelium in aged rats, suggesting a great potential for sildenafil citrate to induce prostatic proliferation in aged rats. These observations collectively emphasize the anatomical and morphological alterations induced by sildenafil citrate in the prostate issue, providing valuable insights into its impact on prostatic hyperplasia in the context of aging.

Prostatic fluid contains at least three specific proteins, namely prostate acid phosphatase (PAP), prostate specific antigen (PSA) and prostate binding protein (PBP)³⁸. PBP, also recognized as prostaglandin and a protein, is the primary secretory product unique to the ventral lobe of the prostate³⁹. As an androgen-dependent protein, PBP serves as a valuable tissue-specific marker for assessing androgen response and facilitating the functional differentiation of ventral prostate⁴⁰. In our study, we found that after administration of sildenafil citrate to BPH model rats, the serum PBP tended to increase compared with the model control group, suggesting the possibility of prostate toxicity caused by sildenafil citrate in BPH model rats.

Endocrine homeostasis serves as the basis for the physiological function of the human body. Disturbance in hormone homeostasis can precipitate aberrations in organ parenchyma, benign neoplasms, and potentially malignant tumors⁴¹. It is well known that the progression of BPH involves a collaborative combination of androgens, including testosterone, dihydrotestosterone (DHT), androstenedione (A4), dehydroepiandrosterone (DHEA), and androsterone (A) and estrogens, primarily including E2 and E1, which collectively regulate the growth and development of the normal pros-tate, thereby driving the pathogenesis of BPH⁴². Therefore, comprehending the precise metabolic dynamics and associated fluctuations of androgens and estrogens in both serum and prostate tissue is essential for the accurate diagnosis and preventive strategies against BPH. Testosterone, an imperative factor for normal prostate development, causes regeneration and cellular proliferation in castrated animals, accompanied by an increase in prostate volume⁴³. Estradiol, on the other hand, stimulates the proliferation of both stromal and epithelial cells in the prostate and induces the phenotypic differentiation of stromal cells into smooth muscle cells^44,45. In our study, it was discerned that sildenafil citrate could significantly increase the levels of E2 and T in serum and T in DLP of aged rats, and the effect of sildenafil citrate on T had a dose-dependent relationship. This is consistent with the confirmed finding that E2 and T synergistically promote prostatic hyperplasia in BPH model rats^42,46.

In the pathogenesis and progression of BPH, the pivotal role of EMT signaling pathway is noteworthy. The secretory protein FGFBP1 assumes significance by selectively binding to immobilized fibroblast growth factor (FGF) in the extracellular matrix, thereby facilitating its release⁴⁷. FGFBP1 is associated with diverse cellular processes, including apoptosis, proliferation, invasion, migration, angiogenesis and metastasis^48,49. During embryonic development, FGFBP1 proves beneficial to proliferation, differentiation, and wound healing⁵⁰. In contrast to its low expression in normal adult tissues, FGFBP1 exhibits a significant upregulation in various tumor types. Notably, the silencing of FGFBP1 demonstrates inhibitory effects on cell proliferation and migration^51,52. Transmembrane protein 1 (TMEFF1), characterized by epidermal growth factor-like and two follicle-like domains, is a member of the tumor-testicular antigen family, actively participating in biological processes such as the physiological function and embryonic development of the central nervous system⁵³. TMEFF1 was initially found to be differentially expressed in brain tissues and tumors, and its anticancer effect was verified in brain tumors. Subsequently, it was found that the expression of TMEFF1 was significantly up-regulated in breast cancer, colon cancer and ovarian cancer cells with high metastasis and drug resistance⁵⁴. ITGA5, functioning as a heterodimeric fibronectin receptor binding to integrin β1, exerts obvious influence on both extracellular matrix dynamics and intracellular signal transduction, exhibiting a close association with the occurrence and progression of numerous neoplastic conditions⁵⁵. Many studies have confirmed that ITGA5 as a proto-oncogene, pivotal in modulating the processes of proliferation, apoptosis, invasion, and metastasis across various malignancies⁵⁶. The multifaceted proteoglycan, Versican, emerges not only as a large chondroitin sulfate proteoglycan (CS) in the extracellular matrix but also as a constituent of the transparent protein family in this matrix⁵⁷. Remarkably, multifunctional proteoglycans stand out as key contributors to immune and inflammatory responses in diverse diseases, such as cardiovascular and pulmonary diseases, autoimmune diseases, and multiple forms of malignancies⁵⁸. Vimentin is a major component of the intermediate filament protein family, widely expressed in normal mesenchymal cells, contributing indispensably to cellular integrity⁵⁹. A large number of studies have confirmed that vimentin can regulate EMT signaling pathway, thereby affecting a variety of physiological and pathological processes, such as cellular growth, wound healing and the occurrence and progression of tumor⁶⁰. In the context of our EMT gene microarray analysis, we observe that sildenafil citrate induces an upregulation of Fgfb1 and Tmeff1 in the EMT signaling pathway of the DLP of BPH model rats, and a downregulation is noted in the expression levels of Itga5, Versican, and Vimentin. Notably, this observation diverges from our anticipated outcomes. We postulate that sildenafil citrate may not induce or aggravate prostatic toxicity through the EMT signaling pathway, or the discernible transformation between epithelium and mesen-chyme might be less pronounced, resulting in a weak role of the EMT signaling pathway in this intricate process. The specific channels implicated in these responses need further validation.

To sum up, our findings show that has the propensity to enhance prostatic hyperplasia in both BPH model rats and aged rats within clinically relevant dosage, suggesting that prolonged administration of sildenafil citrate to individuals with BPH may aggravate the progression of the disease. Notably, sildenafil citrate administration resulted in increased levels of E2 and T in serum, along with increased T levels in DLP of aged rats. In addition, the EMT microarray showed that sildenafil citrate up-regulated Fgfb1 and Tmeff1 and down-regulated Itga5, Versican and Vimentin in EMT signaling pathway in DLP of BPH model rats. However, further illustration of the specific pathways implicated in EMT requires additional confirmation.

Study 1: Toxic Effects of Sildenafil Citrate on BPH Model Rats

Animal Treatment

Seventy-two Specific Pathogen-Free (SPF) male Sprague-Dawley (SD) rats, weighing 110-120g, aged 4–5 weeks, were purchased from Shanghai Bikaiyi Biotechnology Co., Ltd. (Shanghai, China). Maintained in a controlled environment with a 12h:12h light/dark cycle, all animals, with free feeding (Shanghai Shilin Science & Tech Co., Ltd., Shanghai China) and drinking, were housed under conditions of 20–26°C and 40–70% humidity. The reporting of all animal experiments in the manuscript follows the recommendations in the ARRIVE guidelines. This study and included experimental protocols were approved by the institutional animal care and use committee of Shanghai lnstitute for Biomedical and Pharmaceutical Technologies.

Following a 5-day acclimatization period, all the rats exhibited optimal health. The animals were randomly assigned to 6 groups (n = 12) based on body weight. These groups included the negative control, castration, model control, sildenafil citrate (5 mg/kg), sildenafil citrate (10 mg/kg) and sildenafil citrate (20 mg/kg). Except for the negative control group, orchiectomies were performed under pentobarbital sodium anesthesia, and testosterone propionate, dissolved in olive oil, was subcutaneously injected with 1.0 mg/kg for a duration of four weeks. The negative control group underwent the same surgical procedure without testicular removal. Concurrently, the treatment groups received sildenafil citrate (5.0–20.0 mg/kg) via intragastric administration, dissolved in 5% sodium carboxymethyl cellulose (CMC-Na) as a solvent, over a four-week period. The negative control group, castration group, and model control group received an equivalent volume of the solvent. Twenty-four hours after the final administration, animals were euthanized under pentobarbital sodium anesthesia, blood samples were collected, and the prostate was dissected and measured. The weights of the ventral and dorsal lobes of the prostate were recorded, and the total wet weight of the prostate was computed. Part of the tissue was fixed in paraformaldehyde solution for subsequent pathological analyses, and the other part was frozen in liquid nitrogen at -80°C for protein detection. The evaluation of rat prostate weight gain was conducted by determining the ratio of 100 times the prostate weight to the terminal body weight.

Pathological Assessment

The prostate tissue fixed by paraformaldehyde for 48 hours was removed and trimmed, dehydrated, cleaned, waxed and embedded in paraffin, and cut into 4 µ m thick slices with a microtome (Leica, China). Following dewaxing with xylene, immersion in 100% ethanol, and rehydration in 75% ethanol, sections were stained with hematoxylin and eosin (H&E) for 3–5 minutes and sealed with neutral gum. The histological and morphological changes of each group were observed under inverted microscope (Nikon Eclipse 50i, Japan). Ten of the largest prostatic cavities were systematically chosen from each animal tissue section, and their respective areas were quantified. The total prostatic lumen area was computed based on the average values obtained. A total of 10 epithelial samples per animal and 120 epithelial samples per experimental group were selected for analysis. The determination and subsequent analysis were conducted using Nikon NISElements BR 3.1 software (Japan).

Hormone Level Detection

Blood samples were collected, and subsequent serum isolation was achieved through centrifugation lasting 15 minutes (3000 rpm/min, 4°C). The serum was promptly preserved at -80°C to facilitate subsequent concentration assessments. Quantification of serum E2, T, PRL, and PBP concentrations ensued in accordance with the instructions of the corresponding enzyme-linked immunosorbent assay kit (NovaTeinBio, Inc., Cambridge, USA), and detected by an enzyme reader (Zenyth200st, Austria).

Evaluation of EMT Gene Expression by a Microarray Analysis

Total RNA was extracted from four samples derived from model control and 5 mg/kg sildenafil citrate group using a RNeasy Microarray Tissue Mini Kit (SABiosciences, Qiagen, Maryland 21703, USA), including the optional on-column DNase digestion step provided in the manual. Subsequent to extraction, the concentration and purity of the obtained RNA specimens were ascertained through ultraviolet spectrophotometry and denaturing gel electrophoresis. Consecutively, in adherence to the manufacturer's protocol, procedures encompassing amplification, array hybridization, washing, and scanning were systematically executed. Data were analyzed by using the ΔΔCT method and microarray data analysis software.

Study 2: Toxic Effects of Sildenafil Citrate on Aged Rats

Animal Treatment

Thirty-two SPF male SD rats, weighing 200–220 g, aged 5–7 weeks, were purchased from Shanghai Bikaiyi Biotechnology Co., Ltd. (Shanghai, China). Maintained in a controlled environment with a 12h:12h light/dark cycle, all animals, with free feeding (Shanghai Shilin Science & Tech Co., Ltd., Shanghai China) and drinking, were housed under conditions of 20–26°C and 40–70% humidity. The animals were placed on sawdust bedding in standard polypropylene cages till the age of 1.5 years. The reporting of all animal experiments in the manuscript follows the recommendations in the ARRIVE guidelines. This study and included experimental protocols were approved by the institutional animal care and use committee of Shanghai lnstitute for Biomedical and Pharmaceutical Technologies.

After 5-day adaptation period, all the rats were randomly divided into 4 groups (n = 8) according to their body weight: the vehicle, sildenafil citrate (5 mg/kg), sildenafil citrate (10 mg/kg) and sildenafil citrate (20 mg/kg). A 5% CMC-Na served as the solvent, sildenafil citrate (5–20 mg/kg) was administered via intragastric route over a duration of ten weeks, while the control group was given an equivalent volume of the solvent. Daily monitoring of the animals' general behavior and weekly weight assessments were conducted throughout the experimental period. Twenty-four hours after the final administration, animals were euthanized under pentobarbital sodium anesthesia, blood samples were collected, and the prostate was dissected and measured. The weights of the ventral and dorsal lobes of the prostate were recorded, and the total wet weight of the prostate was computed. Part of the tissue was fixed in paraformaldehyde solution for subsequent pathological analyses, and the other part was frozen in liquid nitrogen at -80°C for protein detection. The evaluation of rat prostate weight gain was conducted by determining the ratio of 1000 times the prostate weight to the terminal body weight.

Pathological Assessment

The prostate tissue fixed by paraformaldehyde for 48 hours was removed and trimmed, dehydrated, cleaned, waxed and embedded in paraffin, and cut into 4 µm thick slices with a microtome (Leica, China). Following dewaxing with xylene, immersion in 100% ethanol, and rehydration in 75% ethanol, sections were stained with hematoxylin and eosin (H&E) for 3–5 minutes and sealed with neutral gum. The histological and morphological changes of each group were observed under inverted microscope (Nikon Eclipse 50i, Japan). Ten of the largest prostatic cavities were systematically chosen from each animal tissue section, and their respective areas were quantified. The total prostatic lumen area was computed based on the average values obtained. A total of 10 epithelial samples per animal and 120 epithelial samples per experimental group were selected for analysis. The determination and subsequent analysis were conducted using Nikon NISElements BR 3.1 software (Japan).

Detection of Serum Hormone Levels

Blood samples were collected, and subsequent serum isolation was achieved through centrifugation lasting 15 minutes (3000 rpm/min, 4°C). The serum was promptly preserved at -80°C to facilitate subsequent concentration assessments. Quantification of Serum E2, T and insulin concentrations ensued in accordance with the instructions of the corresponding enzyme-linked immunosorbent assay kit (NovaTeinBio, Inc., Cambridge, USA), and detected by an enzyme reader (Zenyth200st, Austria).

Detection of Hormone Levels Based on Prostate Tissue

The tissues of DLP were cut into pieces, weighed, ground in phosphate-buffered saline (PBS) (w:v = 1:10) and centrifuged for homogenate preparation. Quantification of E2, T and insulin concentrations in the DLP ensued in accordance with the instructions of the corresponding enzyme-linked immunosorbent assay kit (NovaTeinBio, Inc., Cambridge, USA), and detected by an enzyme reader (Zenyth200st, Austria).

Statistical Analysis

All the test data were expressed in the form of mean ± SD, statistically analyzed using IBM SPSS Statistics 26.0 software (SPSS Inc., Chicago, IL, USA). p < 0.05 was considered as the statistically significant level. After meeting the criteria of normality test and homogeneity of variance test, ANOVA was applied for statistical comparisons. If the difference between groups was statistically significant, LSD method was used to analyze the post hoc multiple comparisons. Finally, the statistical analysis results are displayed visually by using GraphpadPrism10.1.0 software (GraphPad Software, San Diego, CA, USA).

Ethical Standards

All rats in this study were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals of Shanghai lnstitute for Biomedical and Pharmaceutical Technologies Animal Care and Use Committee.

Author Contribution

Conceptualization and design, J.L., J.W. and S.H.; formal analysis, S.H. and D.H.; investigation, S.H., D.H., J.J. and C.S.; methodology, S.H., D.H., X.S., R.Y., J.L., and J.W.; supervision, J.W.; validation, D.H., X.S., R.Y., C.S., J.J., J.L. and J.W.; visualization, S.H.; writing—original draft, S.H.; writing—review and editing, J.L. and J.W.; funding acquisition, J.W. All authors have read and agreed to the published version of the manuscript.

Data Availability

Research data shall be made available upon reasonable request to the corresponding author.

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No competing interests reported.

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Sildenafil Citrate Induces Prostatic Hyperplasia in BPH Model Rats and Aged Rats

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Abstract

Figures

Introduction

Results

Study 1: Toxic Effects of Sildenafil Citrate on BPH Model Rats

Anatomical Analysis of Prostate in BPH Model Rats Treated with Sildenafil Citrate

Histopathological Analysis of Prostates in BPH Model Rats Induced by Sildenafil Citrate

Analysis of Serum Hormone levels in BPH Model Rats Induced by Sildenafil Citrate

Study 2: Toxic Effects of Sildenafil Citrate on Aged Rats

Anatomical Analysis of Prostate in Aged Rats Treated with Sildenafil Citrate

Histopathological Analysis of Prostates in Aged Rats Induced by sildenafil Citrate

Discussion

Conclusion

Materials and Methods

Study 1: Toxic Effects of Sildenafil Citrate on BPH Model Rats

Animal Treatment

Pathological Assessment

Hormone Level Detection

Evaluation of EMT Gene Expression by a Microarray Analysis

Study 2: Toxic Effects of Sildenafil Citrate on Aged Rats

Animal Treatment

Pathological Assessment

Detection of Serum Hormone Levels

Detection of Hormone Levels Based on Prostate Tissue

Statistical Analysis

Declarations

Ethical Standards

Author Contribution

Data Availability

References

Additional Declarations

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