Introduction
Benign prostate hyperplasia (BPH) is a common benign disease in old men. The incidence of the gross enlargement of the prostate gland has been reported to be 40% in 70-year-old men, and microscopic foci of the prostate gland are present in up to 80% of these men [
1]. BPH is a multifactorial disease; androgens, estrogens, stromal–epithelial interactions, growth factors, and neurotransmitters may play a role, either singly or in combination, in the etiology of the hyperplastic process [
2]. In recent years, it has been considered that inflammation is not just an occasional concomitant occurrence accompanying BPH, but instead, plays a key role in its etiology [
3]. This is manifested by the high incidence of inflammation that has been found in excised BPH samples [
4], and the degree of inflammation has shown a significant correlation with the severity of BPH [
5-
7]. BPH has been hypothesized to be an immune-mediated inflammatory disease, and inflammation may directly contribute to prostate growth [
8].
At present, the hormone-induced animal model of BPH is mainly used for BPH-related studies. Indeed, testosterone plays an essential role in the development of BPH. In 1976, Walsh and Wilson [
9] first reported the successful use of hormones in castrated dogs to induce a BPH model. Based on their research, the animals currently used as models in many BPH drug therapy experiments are predominantly rats, and the model method is based on large doses of androgen, with castration or no castration, and androgen alone or a combination of estrogen and androgen [
10‐
13]. However, the serum testosterone level of humans decreases with age [
14]; hence, large exogenous doses of testosterone contradict the natural evolution of BPH. The hormone-induced models may thus, not fully represent the true nature of BPH.
To expand the scope of the model methods of BPH, other causes of BPH, such as inflammation, should be considered for the induction of BPH models. To mimic the appearance of inflammation with BPH, which is consistent with its natural evolution, we attempted to induce chronic inflammation in rats to investigate the pathological changes of the prostate gland and the related underlying mechanisms.
Material and methods
Animals and experimental design
A total of 36 male Sprague–Dawley rats (9 weeks old, 250–300 g) were supplied by the Laboratory Animal Center of the Tongji Medical College, Huazhong University of Science and Technology. The rats were housed in individual cages in a room maintained at 20–26 °C with a relative humidity of 35–75%; they were subjected to an alternating 12-h light–dark cycle (8:00 a.m. and 8:00 p.m.). The animals had free access to food and water. All procedures involving the animals were approved by the Institutional Animal Care and Use Committee of the Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Hubei, China). After 7 days of acclimatization, the rats were randomly divided into four groups (n = 9 per group): (1) normal control group (NC), (2) testosterone-induced BPH model group (TI), (3) EAP group, and (4) prostate antigen supply group (they were sacrificed to obtain antigens for injecting the rats in the EAP group).
The induction of EAP was performed according to Zhou’s [
15] method. The nine rats in group 4 were sacrificed by cervical dislocation and the prostate tissues were obtained and homogenized in 0.5% TritonX-100 (Beyotime Biotechnology, Shanghai, China); the homogenates were maintained in an ice water bath. They were then centrifuged at 10,000
g for 30 min at 4 ℃ and the supernatants were separated. The protein concentrations in the supernatants were assayed by the BCA (bicinchoninic acid) assay (Multisciences Biotechnology Co. Ltd., Hangzhou, China) and diluted to concentrations of 10 mg/ml with 0.1 mol/L PBS buffer (pH 7.2). The nine rats in the EAP group were anesthetized by the intraperitoneal injection of pentobarbital (40 mg/kg). Then, they were intraperitoneally injected with 0.5 ml of Pertussis–Diphtheria–Tetanus vaccine (Wuhan Institute of Biological Products, Wuhan, Hubei, China). Next, a total of 1.0 ml of rat prostate protein extract and FAC (Freund’s Adjuvant Complete; Sigma-Aldrich, MO, USA) emulsion (1:1) was injected subcutaneously into their necks, inner thighs, backs, or hind limbs. The rats in the EAP group were immunized twice, i.e., on day 0 and day 30. The rats in the TI group were injected subcutaneously with 5 mg/kg testosterone propionate (Solarbio Science & Technology Co. Ltd., Beijing, China) (dissolved in corn oil) daily for 45 days. The rats in the NC group did not undergo any treatment. After 45 days, the rats were sacrificed, and the ventral lobes of their prostate glands were quickly removed. One-third of the bilateral ventral lobes were fixed with 4% paraformaldehyde (Beyotime Biotechnology), and the remaining tissues were frozen at − 80 °C.
H&E staining, Masson’s trichrome staining, hexamine silver staining, immunohistochemical (IHC) staining, and immunofluorescence (IF) staining
The 4% paraformaldehyde-fixed prostate tissue samples from rats in each group were subjected to paraffin embedding and sectioned at a thickness of 5 μm. The tissue sections were subjected to hematoxylin–eosin (H&E) staining, Masson’s trichrome staining, and hexamine silver staining using standard procedures, and examined under a light microscope. IHC staining was performed as previously described [
16]. The sections were incubated with primary antibodies against Ki-67 (1:500; Affinity Biosciences, OH, USA) and transforming growth factor β1 (TGF-β1) (1:400; Proteintech Group, Wuhan, China) at 37 °C for 1 h. Then, they were treated with a biotinylated secondary antibody according to the standard protocol. For IF staining, the sections were washed and incubated with antibodies specific for alpha smooth muscle actin (α-SMA) (1:200; Affinity Biosciences) overnight at 4 °C, and then with donkey anti-rabbit IgG (Life Technologies; Thermo Fisher Scientific, Rockford, IL, USA) for 1 h in the dark at room temperature. The slides were mounted by adding DAPI-Fluoromount-G (Southern Biotech) and examined using a fluorescence microscope (Olympus, Tokyo, Japan).
Grading of the inflammation degree
The prostate samples were graded according to the degree of inflammation: mild, inflammatory foci constituted by 7–14 mononuclear cells; moderate, inflammatory foci constituted by 15–29 mononuclear cells; severe, inflammatory foci constituted by 30 or more mononuclear cells.
Semi-quantitative assessment of the histopathologic findings in the prostate samples
We used a chart score protocol that was established by Scolnik [
17] and then improved by Golomb [
18] to perform a better evaluation of the ventral prostate hyperplastic conditions in rats. This protocol considered the acinar morphology, i.e., features such as crowding, intraluminal villosities, loss of basal nuclear polarity, and hyperplastic nodules, which were scored according to their degree of severity and distribution pattern. The examination, description, and scoring of the slides were performed in a blinded manner. Histoscores were expressed in arbitrary units and were obtained from a thorough examination of two H&E-stained sections and one hexamine silver-stained section, considering six different fields of vision for samples from each animal.
Quantitative real-time PCR (q-PCR)
Total RNA from the samples was extracted using the Trizol reagent (Invitrogen). The purified RNA samples were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, DE, USA), and 1 μg of each total RNA sample was reverse transcribed using a PrimeScript™ RT Master Mix (TaKaRa, Dalian, China). The cDNA was subjected to quantitative real-time PCR (q-PCR) analysis to detect the mRNA levels using the SYBR reagent kit (TaKaRa) and QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific). All reactions were customized for the specific PCR products and performed in triplicate. The mRNA expression levels of the examined genes were normalized to that of β actin; the relative mRNA expression levels were calculated using the 2−ΔΔCt method.
Western blotting analysis
The prostatic tissue was ground in liquid nitrogen and the total protein was extracted using a protein extraction kit. Equal amounts (40 μg/lane) of proteins were separated by SDS–polyacrylamide gel electrophoresis; the resultant bands were transferred onto PVDF membranes. After being blocked in 5% bovine serum albumin for 1 h at room temperature, the membranes were incubated with antibodies against TGF-β1 (1:500; Proteintech Group), RhoA (1:5000; Proteintech Group), ROCK1 (1:500; Proteintech Group), and β-actin (1:1000; Affinity Biosciences) overnight at 4 °C. The membranes were washed thrice in TBST for 30 min, and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; Affinity Biosciences) for 1 h followed, by a further washing step for 30 min. Finally, the bands were analyzed using an enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific). The data were normalized using β actin as an internal control. All samples were analyzed independently via three repetitions and the mean values were determined.
Assessment of NO, malondialdehyde (MDA), superoxide dismutase (SOD), and ROS
The SOD activity, NO levels, and MDA levels in the prostate tissues were assessed using a superoxide dismutase (SOD) assay kit (WST-1 method), nitric oxide (NO) assay kit (microwell plate method), and malondialdehyde (MDA) assay kit (TBA method), respectively, according to the manufacturer’s instructions. These commercial assay kits were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). The assays were performed in triplicate, and the total protein concentrations were detected to normalize the data. The reactive oxygen species (ROS) generated in situ in the prostate tissues was assessed using DHE-ROS Assay Kit (BestBio Science, Shanghai, China), according to the manufacturer’s instructions. DAPI was used for staining the nuclei. The stained tissues were visualized using a fluorescence microscope.
Statistical analysis
The results were analyzed using GraphPad Prism version 7.04 (GraphPad Software, San Diego, CA, USA) and expressed as the means ± standard deviations (SDs). Differences of the mean values between the three groups were analyzed using one-way ANOVA followed by Tukey’s post hoc multiple comparisons test. For all statistical tests, P values < 0.05 were considered statistically significant.
Discussion
The BPH nodules of individual patients are composed of distinct cell types, and pathological heterogeneity is an underappreciated confounder to identifying novel drug target [
19]. Causative molecular pathways involved in a specific BPH phenotype require in-depth research. Our experimental study found that chronic prostatitis induced by autoimmune method in rats showed heterogeneous prostatic hyperplasia from conventional hormone model methods. The EAP rats showed increased stroma compartments and reactive epithelial hyperplasia, while testosterone caused severe epithelial hyperplasia or even atypical hyperplasia.
Many lines of evidence have supported that androgens are permissive, but insufficient for the induction of BPH [
20]. The incidence of BPH increases with age in human, but the serum testosterone levels decrease [
14]. Androgen supplementation in men does not appear to increase the risk of incident BPH or LUTS (lower urinary tract symptoms) [
21,
22]. In the past nearly 50 years, researchers are accustomed to using large doses of testosterone to induce BPH in animal experiments as hormones can quickly and easily induce significant prostate epithelial hyperplasia in young animals. However, it has limitations: (1) the theoretical basis of the hormone-induced model is that the proportion of estrogen and androgen increases with age [
20], while disregarding the role of other factors such as inflammation in BPH. (2) Exogenous hormones strongly destroy the hormonal balance of young animals, affecting the immune state of the prostate, as the prostate is also a potential immune organ [
23]. (3) Because of the inconsistency of the hormone model induction method, the final pathological status of BPH is not uniform. In particular, there are no consistent reports on whether significant proliferation in the prostatic stroma is observed. For example, some studies reported stromal lesions [
11,
12,
24], whereas others only reported epithelial lesions [
10,
13,
25‐
28]. Taken together, the traditional method of hormone-induced model has some limitations in the study of BPH-related mechanism and treatment. Therefore, a new method of induction of hormone-induced model is worthy exploring.
There is already numerous clinical evidence supporting the correlation between BPH and inflammation [
5-
7]. Many in vitro and in vivo studies have indicated that the activation and dysregulation of prostatic immune cells, and the release of several cytokines and growth factors including IL-2, IL-4, IL-6, IL-17, fibroblast growth factor 2 (FGF-2), were both associated with the initiation and progression of BPH [
23]. We found a sharp increase for the size of the stromal compartment in the prostates from EAP rats, accompanied by elevated mRNA levels of several cytokines that stimulated the growth of stromal cells, such as IL-2, IL-4, and IL-18 [
23]. Franiel et al. [
29] reported that areas of chronic prostatitis tissues had a larger stromal volume than areas of normal prostate tissues. The mean stroma-to-epithelium ratio in the prostate adenomas from men with symptomatic BPH is also higher than that in those from asymptomatic patients [
30]. Therefore, inflammation may be a key factor affecting the stromal compartment in both humans and rats, even further promote symptomatic BPH. Unlike rats with chronic prostate inflammation, our research found that large dose of hormone strongly cause epithelial hyperplasia, even atypical hyperplasia in rats. This may be due to the DNA damage induced by serious oxidative stress in the prostate tissues of TI rats. Oxidative stress is commonly observed in hormone-induced BPH models [
13,
25,
26], and elevated HIF-1α means significant hypoxia in the model group [
24]. However, there was no increase in the MDA levels in the EAP group, because the elevated SOD limited the detrimental effect from ROS. Correspondingly only moderate epithelial hyperplasia was observed in the EAP group. Overall, our results suggested that inflammation and hormones induced heterogeneous BPH in the rat model through different mechanisms.
Recent studies have revealed inflammation implicated as a cause of prostatic fibrosis that contributes to bladder outlet obstruction [
31]. Fibrosis in BPH is promoted by the activation of excessive α-SMA-expressing myofibroblasts, which are differentiated from fibroblasts during chronic inflammation and deposit ECM components [
32,
33]. TGF-β1 can upregulate the activation of the RhoA/ROCK pathway, promoting cytoskeletal rearrangement and ultimately inducing fibroblast-to-myofibroblast differentiation [
34]. In our experiments, elevated TGF-β1 in EAP rats led to activation of RhoA/ROCK pathway and fibrosis of the prostate, while rats under hormonal intervention did not undergo this process.
Autoimmune response, one of the main causes of chronic prostatitis [
35-
39], is also considered to be as a trigger for the dysregulation of the prostatic immune system and results in the development of BPH nodules [
23]. Activated by autoantigens, such as prostate steroid-binding protein, T lymphocytes play a key role as effector and helper cells in the cell-mediated immune response [
38,
40]. Besides, prostate cells also released cytokines forming a prostatic network that affect the prostatic proliferation [
41]. For example, IL17 mainly from T lymphocytes, activates the NFκB pathway, regulates the expression of IL-6, IL-8, and IL-1 in epithelial and stromal cells, and upregulates the expression of COX2 in macrophages and epithelial cells [
23]. COX-2 suppresses the apoptosis by enhancing the expression of anti-death proteins [
24]. IL-2, IL-4, and IL18 stimulate the proliferation of stromal cells. IL-8 induces paracrine of FGF-2 in stromal cells. In our experiment, the inflammatory factor network was examined initially by q-PCR, but further experiments were needed to explore the detailed associations.
Notably, the rats with testosterone administration also showed evidence of inflammatory infiltration compared with the NC group, which might be due to two reasons different from the EAP group. On the one hand, hypoxia and oxidative stress are both observed in hormone-induced BPH rat models and oxidative stress can activate the NF-κB signaling pathway, known as an important inflammatory transcriptional regulator [
42]. Abnormal ROS affect the activities of inflammatory mediators and cellular processes involved in the initiation, promotion, and progression of human neoplasms, including BPH and prostate cancer [
43]. On the other hand, androgens can be metabolized to estrogens locally within the prostate via the aromatase enzyme [
44]. Over-activated aromatase in transgenic mice leads to obstructive voiding [
11]. Aromatase inhibitor administered to beagles treated with testosterone and androstenedione, inhibited hormone-induced prostate growth [
20]. Testosterone also reduces ER-β and enhances ER-α expression [
45], and ER-α stimulation in the prostate results in hyperplasia, inflammation, and dysplasia [
20]. In addition, estrogens activate an inflammatory response through the activation of the nonclassical G-protein-coupled estrogen receptor 1, increasing expression of IL-8 [
46]. The development of inflammation in response to estrogen is independent of androgens and is the direct response of the prostate to estrogens [
44]. Thus, although testosterone alone was administered in our study, the prostates in the TI rats were still affected by endogenous estrogen and developed inflammatory infiltration. Further, the complex metabolism of sex hormones in the prostate makes it difficult to explain the detail mechanism for drug experiment using a hormone-induced BPH model.
In summary, the induction of inflammation can promote the proliferation of prostatic epithelia and stroma, and the effect of inflammation on the stroma is more significant than that of testosterone-induced epithelial changes in rats. In the inflammatory state, increased TGF-β levels contribute to the transdifferentiation of prostate fibroblasts into myofibroblasts, and deposition of new ECM, promoting fibrosis in the prostate gland, in which RhoA/ROCK signaling plays an important role. In study of the therapeutic effects of drugs on stroma components during BPH, we recommend the use of the EAP model, which qualifies as a model of BPH and aids the understanding of the mechanisms underlying inflammation in BPH.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.