Model for inducing benign prostatic hyperplasia of rats by bisphenol A and construction method

Abstract

The invention provides a bisphenol A induced rat benign prostatic hyperplasia model and a construction method, and relates to the technical field of animal models. The method comprises the following steps: S1, selecting rats which are 8-9 months old and 9-10 weeks old, and adaptively feeding the rats in a suitable environment for 7 days or more; s2, jointly applying high-fat feed and bisphenol A to the adaptively fed rats to induce benign prostatic hyperplasia; the induction time is 60-80 days, and performing biological index evaluation after the induction is completed. The exposure level of low-dose BPA adopted by the invention is closer to the actual environment exposure level of people, and a main exposure way is simulated through oral gavage. The model constructed by the method can better simulate the real process of chronic and low-dose exposure pathopoiesis of the environmental endocrine disrupter in etiology, and provides an experimental tool with more practical significance for related risk evaluation.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and more specifically, to a bisphenol A-induced benign prostatic hyperplasia model in rats and a method for constructing it. Background Technology

[0002] Benign prostatic hyperplasia (BPH) is an age-related degenerative disease of the urinary system characterized by enlarged prostate and urethral obstruction. It has become one of the major threats to the health of middle-aged and elderly men worldwide, and its epidemiological characteristics and public health significance are increasingly prominent with the aging population. BPH not only leads to complications such as lower urinary tract symptoms (LUTS) and urinary retention, but also has a high comorbidity with chronic diseases such as metabolic syndrome, cardiovascular disease, and depressive symptoms, significantly reducing patients' quality of life and increasing the disease burden.

[0003] Epidemiological data shows that approximately half of men over 60 years of age worldwide have benign prostatic hyperplasia (BPH), with a prevalence exceeding 80% in those over 80. In China, the prevalence of BPH among men over 60 years of age has reached 50%–83%, and this disease burden will further intensify with the accelerating aging of the population. However, current prevention and control of BPH still face bottlenecks such as unclear etiologies and insufficient intervention measures.

[0004] The link between environmental / food endocrine disrupting chemicals (EDCs) and prostate disease has attracted much attention. They can participate in the pathological process of benign prostatic hyperplasia (BPH) and even cancer through mechanisms such as interfering with androgen / estrogen signaling pathways, inducing oxidative stress, and epigenetic modifications. Bisphenol A (BPA), as one of the most widely used EDCs globally, is almost unavoidably exposed to due to its prevalent use in plastic products, food packaging, and heat-sensitive materials. BPA can be detected in the urine of over 90% of the general population. Epidemiological studies show a positive correlation between exposure levels and prostate histological changes and the severity of low-grade prostate disease (LUTS). Animal experiments further confirm that low-dose BPA exposure during prenatal or adulthood can induce prostate stromal hyperplasia and the formation of an inflammatory microenvironment.

[0005] Currently, the establishment of BPH rat models mainly includes testosterone induction, estrogens-androgen synergistic induction urogenital sinus implantation, and metabolic syndrome models. However, there is no unified modeling method for inducing BPH and related benign prostatic hyperplasia (BPH), nor are there any effective prevention and control strategies. Therefore, establishing a stable, economical animal model of benign prostatic hyperplasia that can simulate environmental exposure factors is of great significance for revealing the etiology of BPH and developing prevention strategies. Summary of the Invention

[0006] The purpose of this invention is to provide a model and method for constructing bisphenol A-induced benign prostatic hyperplasia (BPH) in rats, which can solve the problems of existing models being unable to simulate BPA environmental exposure factors, provide etiological information on BPH, and develop prevention strategies.

[0007] The technical problem solved by this invention is achieved by the following technical solution.

[0008] On the one hand, embodiments of this application provide a method for constructing a bisphenol A-induced benign prostatic hyperplasia model in rats, comprising the following steps: S1: Select rats aged 8-9 months and 9-10 weeks and acclimatize them in a suitable environment for 7 days or more; S2: Benign prostatic hyperplasia was induced in adaptively fed rats by combined administration of a high-fat diet and bisphenol A; the induction period was 60-80 days, and biological indicators were evaluated after the induction was completed.

[0009] Furthermore, in step S1, the rats are healthy SPF-grade male SD rats; the suitable environment includes: ventilation in the culture room, temperature of 20-26℃, humidity of 40%-70%, and a light-dark alternation cycle of 12 hours each.

[0010] Furthermore, in step S2, the high-fat feed consists of: 20.0% sucrose, 15% lard, 1.2% cholesterol, 0.2% sodium cholate, 8% casein, 1.8% dicalcium phosphate, with the remainder being maintenance feed.

[0011] Furthermore, the concentration of bisphenol A is 30-1000 μg / kg.bw.

[0012] Furthermore, biological indicators include physiological indicators, serum hormone indicators, tissue structure, and molecular markers.

[0013] Further physiological indicators include body mass index, organ index, prostate coefficient, and testicular coefficient; serum hormone indicators include serum testosterone and dihydrotestosterone.

[0014] On the other hand, embodiments of the present invention provide a bisphenol A-induced benign prostatic hyperplasia model in rats, which is constructed by the above method.

[0015] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: 1. The low-dose BPA exposure used in this invention is closer to the actual environmental exposure level in the population, and the main exposure route is simulated through oral gavage. The model constructed in this way can better simulate the real process of pathogenesis caused by chronic, low-dose exposure to environmental endocrine disruptors from an etiological perspective, providing a more practical experimental tool for related risk assessment.

[0016] 2. This invention explores the different modes of action of bisphenol A and high-fat diet in benign prostatic hyperplasia (BPH). In addition to exposure to a single environmental pollutant, dietary background intervention was added to explore the interaction between the two in the construction of the BPH model. This provides a clear decision-making basis for subsequent researchers to select experimental animals and design exposure conditions, avoiding experimental failures or biases caused by inappropriate condition selection. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a graph showing the changes in body weight of different groups of 8-9 month old rats in Example 1 of the present invention; Figure 2 This is a statistical graph showing the testicular coefficient and prostate coefficient of different groups of 8-9 month old rats in Example 1 of the present invention; Figure 3 This is a comparison of serum testosterone and dihydrotestosterone levels in different groups of 8-9 month old rats in Example 1 of this invention; Figure 4 The images show the pathological measurement results of prostate tissue from various groups of 8-9 month old rats in Example 1 of this invention. Figure 5 The image shows the pathological staining of prostate tissue from various groups of 8-9 month old rats in Example 1 of this invention (×50). Figure 6 The image shows the histopathological staining of prostate tissue from each group of 8-9 month old rats in Example 1 of this invention (×200). Figure 7 The images show the immunohistochemical staining results of 8-9 month old rats in Example 1 of this invention. Figure 8 Immunohistochemical staining images of 8-9 month old rats in Example 1 of this invention (×200, ×400). Figure 9 This is a graph showing the changes in body weight of different groups of 9-10 week old rats in Example 2 of the present invention; Figure 10 This is a statistical graph showing the testicular coefficient and prostate coefficient of different groups of 9-10 week old rats in Example 2 of the present invention; Figure 11 This is a comparison of serum testosterone and dihydrotestosterone levels in different groups of 9-10 week old rats in Example 2 of this invention; Figure 12The images show the pathological measurement results of prostate tissue from various groups of 9-10 week old rats in Example 2 of this invention. Figure 13 The image shows the pathological staining of prostate tissue from each group of 9-10 week old rats in Example 2 of this invention (×50). Figure 14 The image shows the histopathological staining of prostate tissue from each group of 9-10 week old rats in Example 2 of this invention (×200). Figure 15 The images show the immunohistochemical staining results of 9-10 week old rats in Example 2 of this invention. Figure 16 Immunohistochemical staining images (×200, ×400) of 9-10 week old rats in Example 2 of this invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0021] Example 1 This embodiment provides a detailed method for constructing a bisphenol A-induced benign prostatic hyperplasia model in rats, including the following steps: S1: Fifty healthy SPF-grade male SD rats aged 8-9 months were selected. The rats were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd., and weighed 600±50g. They were housed in standard polypropylene cages in an animal room (Barrier-grade (SPF) animal room of West China School of Public Health, Sichuan University), which was kept quiet, clean, well-ventilated, and with appropriate lighting. The temperature was 20-26℃, the humidity was 40%-70%, and the light-dark cycle was 12 hours. They were acclimatized for 7 days. During the rearing process, the animals were provided with free access to water and sufficient food. The drinking water was sterilized purified water.

[0022] S2: First, prepare the high-fat feed: Add 20.0% sucrose, 15% lard, 1.2% cholesterol, 0.2% sodium cholate, 8% casein, and 1.8% dicalcium phosphate to a sufficient amount of maintenance feed. After thorough mixing, the high-fat feed is ready for use.

[0023] The maintenance feed is formulated in accordance with GB14924.3—2010, with fishmeal and soybean meal as the main protein sources, corn and wheat as the carbohydrate sources, and soybean oil to supplement the fat deficiency.

[0024] After the acclimatization period, all rats were weighed and numbered. They were then randomly divided into three experimental groups and two control groups, with ten rats in each group, spaced 10g apart by weight (550g–560g, 560g–570g, and so on). The experimental groups consisted of a maintenance diet with high-dose BPA (ND-HBPA), a high-fat diet with low-dose BPA (HFD-LBPA), and a high-fat diet with high-dose BPA (HFD-HBPA). The control groups consisted of a maintenance diet group (ND) and a high-fat diet group (HFD). In the high-dose BPA groups, the BPA content was 1000 μg / kg bw; in the low-dose BPA groups, the BPA content was 30 μg / kg bw. The test substances were then administered orally to each group using corn oil as a solvent at a dose of 0.4 mL / 100 g bw. The control groups were administered the test substances orally using the same solvent. The rats were given the treatment for 80 consecutive days, and their weight was recorded twice a week during this period.

[0025] After feeding, biological indicators of rats in each group were measured, including: 1) Physiological indicators: After feeding, all rats were fasted for 24 hours, weighed, and then anesthetized with an intraperitoneal injection of sodium pentobarbital (45 mg / kg bw), at a dose of 1 mL / 100 g bw. After blood collection, the rats were euthanized by exsanguination through the abdominal aorta. The prostate was removed, rinsed thoroughly with pre-cooled physiological saline (4°C), and dried with filter paper. The wet weight of the organ was measured. The organ index was calculated using the following formula:

[0026] After calculation, IBM SPSS Statistics 26.0 statistical software was used to analyze the experimental data, which are expressed as mean ± standard deviation. Normality and homogeneity of variance were first tested. Data that followed a normal distribution and had homogeneity of variance were compared between groups using one-way ANOVA, followed by pairwise comparisons using the LSD test. Data that did not conform to homogeneity of variance were analyzed using Tamhane's T² test. All tests were two-tailed, with P < 0.05 considered statistically significant.

[0027] The calculation results are as follows Figure 1 and Figure 2 As shown, different letters A and B indicate statistically significant differences between the 8-9 month old rat groups. Among them, Figure 1The table shows the differences in body weight among the groups of rats. It can be seen that from day 56, the body weight of all HFD-fed rats was significantly higher than that of the ND-fed group. There were no statistically significant differences in body weight among the remaining groups, indicating that a high-fat diet significantly increases the body weight of rats, but BPA had no significant effect on rat body weight. Figure 2 The differences in testicular and prostate coefficients among the rat groups (different letters A and B indicate statistically significant differences between 8-9 month old rat groups) show that the testicular coefficient of the ND-HBPA group was significantly higher than that of other groups, while there was no statistically significant difference in prostate coefficients among the groups.

[0028] Therefore, it can be concluded that a maintenance diet combined with high doses of BPA can affect the testicular index of 8-9 month old rats, but its effect on other physiological indicators is relatively low. Low doses of BPA or high doses of BPA combined with a high-fat diet have relatively low effects on the physiological indicators of 8-9 month old rats. This is attributed to the fact that the internal organs of 8-9 month old rats have matured, hence the relatively small impact of BPA on physiological indicators.

[0029] 2) Serum Hormone Indicators: Serum testosterone (T, CSB-E05100r) and dihydrotestosterone (DHT, CSB-E07879r) were measured using an enzyme-linked immunosorbent assay (ELISA) kit based on the blood samples collected in the above experiments. A SpectraMAX Plus 384 microplate reader was used, and absorbance was measured at an appropriate wavelength according to the ELISA kit instructions. The experimental results are as follows: Figure 3 As shown, there was no statistically significant difference in serum testosterone and dihydrotestosterone among the groups of rats, indicating that BPA and high-fat diets had no significant effect on serum hormones in rats.

[0030] 3) Tissue Structure: This experiment mainly observed the structure of the rat prostate gland, measured the height of the prostate epithelium, and the area of ​​the glandular lumen. Prostate tissue obtained after euthanizing the rats was fixed in 4% paraformaldehyde for at least 24 hours, followed by dehydration, paraffin embedding, and sectioning. After baking at 60℃, the sections were dewaxed in xylene and gradually hydrated using a gradient of ethanol solutions. The sections were then stained sequentially in hematoxylin and eosin solutions, dehydrated, mounted with neutral resin, and examined and measured under a microscope.

[0031] Six prostate tissue samples were randomly selected from each group of rats for HE staining. Pathological measurements were as follows: Figure 4 As shown in the figure, compared with the ND group, the HFD group showed a significant increase in prostatic epithelial height and a significant decrease in glandular lumen area; compared with the ND group, the ND-HBPA group showed a significant increase in prostatic epithelial height; and the HFD-LBPA group showed a significant increase in prostatic epithelial height compared with the HFD group.

[0032] Histopathological images of prostate tissue from each group of rats are shown below. Figure 5 and Figure 6 As shown (magnification: ×50 and ×200; scale bar = 500μm & 100μm, red arrows indicate epithelial cell hyperplasia, green arrows indicate inflammatory cell infiltration, and blue arrows indicate glandular lumen narrowing. A is the ND group, B is the ND-HBPA group, C is the HFD group, D is the HFD-LBPA group, and E is the HFD-HBPA group), the prostate tissue of rats in each group has a relatively clear structure, with intact and continuous glandular epithelium and glandular lumen tissue, and varying degrees of epithelial cell hyperplasia and inflammatory cell infiltration. HFD combined with low-dose BPA exposure induced the most severe benign prostatic hyperplasia lesions, with a significant increase in glandular epithelial height and a reduction in glandular lumen area, while the lesion severity was relatively mild in the HFD alone or HFD combined with high-dose BPA intervention groups. BPA intervention in the ND group significantly increased epithelial height and significantly decreased glandular lumen area. Compared with the ND group, the prostate epithelial height of HFD rats was significantly increased and the glandular lumen area was significantly decreased. Therefore, it can be concluded that for 8-9 month old rats, both high-fat diet and BPA can promote the proliferation of prostate epithelial cells, and the effect of low concentration of BPA combined with high-fat diet is greater.

[0033] 4) Molecular markers: This experiment used immunohistochemistry to detect the expression level of PCNA protein in prostate tissue, including: Six rat prostate tissues were randomly selected from each group, and the procedures included paraffin embedding, sectioning, and dewaxing (same as Experiment 3). After microwave antigen retrieval, the dewaxed sections were incubated overnight at 4°C with primary antibody (PCNA) and primary antibody (Ki67), followed by incubation at 37°C for 45 minutes with secondary antibody (HRP-labeled goat anti-rabbit). After washing with TBST, DAB staining was performed, followed by drying, mounting, and microscopic examination. The expression of PCNA in prostate tissue was analyzed using QuPath 3.2 image acquisition. The immunohistochemical staining results for each group are shown below. Figure 7 As shown, there was no statistically significant difference in PCNA expression levels among the groups.

[0034] Immunohistochemical staining images as shown Figure 8 As shown (magnification: ×200 and ×400; scale bar = 100μm & 50μm; hematoxylin staining of cell nuclei appears blue, and positive PCNA expression appears dark brown (red arrow). A is the ND group, B is the ND-HBPA group, C is the HFD group, D is the HFD-LBPA group, and E is the HFD-HBPA group), prostate cell nuclei appear blue, and positive PCNA expression appears dark brown. Adult rats have a higher basal prostate proliferation level, which may be related to age-related tissue degeneration or spontaneous hyperplasia. However, neither HFD nor BPA significantly altered PCNA expression, suggesting that the proliferative regulation mechanism of prostate tissue tends to stabilize with age.

[0035] Example 2 The methods in this embodiment are largely the same as those in Embodiment 1, except that this embodiment additionally includes subcutaneous injection of testosterone propionate as a positive control group to evaluate the difference in efficacy between the model of this invention and existing models. The steps include: S1: Sixty healthy SPF-grade male SD rats aged 9–10 weeks were selected. The rats were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd., and weighed 300±30g. They were housed in standard polypropylene cages in an animal facility (the barrier-grade (SPF) animal facility of West China School of Public Health, Sichuan University), maintaining a quiet, clean, well-ventilated environment with appropriate lighting. The temperature was 20–26℃, humidity 40%–70%, and the light-dark cycle was 12 hours. They underwent 7 days of acclimatization feeding. During the rearing process, the animals were provided with free access to water and sufficient food, using sterilized purified water.

[0036] S2: First, prepare the high-fat feed: Add 20.0% sucrose, 15% lard, 1.2% cholesterol, 0.2% sodium cholate, 8% casein, and 1.8% dicalcium phosphate to a sufficient amount of maintenance feed. After thorough mixing, the high-fat feed is ready for use.

[0037] The maintenance feed is formulated in accordance with GB14924.3—2010, with fishmeal and soybean meal as the main protein sources, corn and wheat as the carbohydrate sources, and soybean oil to supplement the fat deficiency.

[0038] After the acclimatization period, all rats were weighed and numbered. They were then randomly divided into groups of 10 rats each, spaced 12g apart (270g–282g, 282g–294g, and so on), forming two experimental groups and three control groups. The experimental groups consisted of a maintenance diet with high-dose BPA (Nd-Lbpa) and a high-fat diet with low-dose BPA (Hfd-Lbpa). The control groups consisted of a maintenance diet (Nd), a high-fat diet (Hfd), and a maintenance diet with testosterone (Nd-T). In the low-dose BPA group, the BPA content was 30 μg / kg bw. The test substances were then administered orally to each group via gavage using corn oil as a solvent, at a dose of 0.4 mL / 100 g bw. The control groups received the test substances orally via gavage using the same solvent. Testosterone propionate was administered subcutaneously at a dose of 5 mg / kg bw. The rats were given the treatment for 60 consecutive days, and their weight was recorded twice a week during this period.

[0039] After feeding, biological indicators of rats in each group were measured, including: 1) Physiological indicators: After feeding, all rats were fasted for 24 hours, weighed, and then anesthetized with an intraperitoneal injection of sodium pentobarbital (45 mg / kg bw), at a dose of 1 mL / 100 g bw. After blood collection, the rats were euthanized by exsanguination through the abdominal aorta. The prostate was removed, rinsed thoroughly with pre-cooled physiological saline (4°C), and dried with filter paper. The wet weight of the organ was measured. The organ index was calculated using the following formula:

[0040] After calculation, IBM SPSS Statistics 26.0 statistical software was used to analyze the experimental data, which are expressed as mean ± standard deviation. Normality and homogeneity of variance were first tested. Data that followed a normal distribution and had homogeneity of variance were compared between groups using one-way ANOVA, followed by pairwise comparisons using the LSD test. Data that did not conform to homogeneity of variance were analyzed using Tamhane's T² test. All tests were two-tailed, with P < 0.05 considered statistically significant.

[0041] The calculation results are as follows Figure 9 and Figure 10 As shown, different letters A and B indicate statistically significant differences between the 8-9 month old rat groups. Among them, Figure 9 The differences in body weight among the groups of rats are shown. It can be seen that from day 7, the Nd-T group rats had significantly lower body weight and the least weight gain compared to the other groups, while the Hfd group rats had the heaviest body weight and the greatest weight gain. There were no statistically significant differences in body weight and 60-day weight gain between the Nd and Nd-Lbpa groups, and there were no statistically significant differences in body weight and 60-day weight gain between the Hfd-Lbpa group and the Hfd group.

[0042] Figure 10 The differences in testicular and prostate coefficients among the rat groups (different letters a, b, c indicate statistically significant differences between 9-10 week old rat groups) show that the testicular and prostate coefficients of the Nd-T group were significantly higher than those of other groups; the prostate coefficient of the Hfd-Lbpa group was significantly higher than that of the Hfd group; and the prostate coefficient of the Nd-Lbpa group was also significantly higher than that of the Nd group.

[0043] Therefore, it can be seen that testosterone propionate can significantly affect the testicular index and prostate index of growing rats, and low doses of BPA can affect the prostate index of rats. Among them, the effect of low doses of BPA combined with maintenance diet is more significant than that of low doses of BPA combined with high-fat diet.

[0044] 2) Serum Hormone Indicators: Serum testosterone (T, CSB-E05100r) and dihydrotestosterone (DHT, CSB-E07879r) were measured using an enzyme-linked immunosorbent assay (ELISA) kit based on the blood samples collected in the above experiments. A SpectraMAX Plus 384 microplate reader was used, and absorbance was measured at an appropriate wavelength according to the ELISA kit instructions. The experimental results are as follows: Figure 11 As shown, compared with the Hfd group rats, the Hfd-Lbpa group rats had significantly higher levels of both testosterone and dihydrotestosterone, indicating that low-dose BPA can affect the levels of testosterone and dihydrotestosterone in growing rats.

[0045] 3) Tissue Structure: This experiment mainly observed the structure of the rat prostate gland, measured the height of the prostate epithelium, and the area of ​​the glandular lumen. Prostate tissue obtained after euthanizing the rats was fixed in 4% paraformaldehyde for at least 24 hours, followed by dehydration, paraffin embedding, and sectioning. After baking at 60℃, the sections were dewaxed in xylene and gradually hydrated using a gradient of ethanol solutions. The sections were then stained sequentially in hematoxylin and eosin solutions, dehydrated, mounted with neutral resin, and examined and measured under a microscope.

[0046] Six prostate tissue samples were randomly selected from each group of rats for HE staining. Pathological measurements were as follows: Figure 12 As shown in the figure, compared with the Nd group, the height of the prostate epithelium in all groups was significantly increased, and the area of ​​the prostate gland cavity was significantly decreased in all groups except the Hfd group. The Nd-T group rats had the highest prostate epithelium height and the smallest gland cavity area. The Nd-Lbpa group rats had a significantly higher prostate epithelium height than the Nd group and a significantly lower gland cavity area than the Nd group.

[0047] Histopathological images of prostate tissue from each group of rats are shown below. Figure 13 and Figure 14 As shown (magnification: ×50 and ×200; scale bar = 500μm & 100μm, red arrows indicate epithelial cell proliferation, green arrows indicate inflammatory cell infiltration, and blue arrows indicate glandular lumen stenosis. A is the Nd group, B is the Nd-Lbpa group, C is the Nd-T group, D is the Hfd group, and E is the Hfd-Lbpa group), the prostate tissue of rats in each group has a relatively clear structure, with intact and continuous glandular epithelium and glandular lumen tissue, and varying degrees of epithelial cell proliferation and inflammatory cell infiltration.

[0048] Rats aged 9–10 weeks showed a more pronounced sensitivity to BPA: severe glandular hyperplasia was observed in both the Nd-Lbpa group and the testosterone intervention group, indicating that BPA had a stronger endocrine disrupting effect on the prostate during the developmental stage. Further histological analysis revealed that BPA significantly increased glandular epithelial height and reduced glandular lumen area in both the normal diet and HFD groups, while HFD itself only independently increased glandular epithelial height, suggesting that BPA may directly induce glandular structural abnormalities, while HFD indirectly enhances its effect through metabolic pathways. In 9–10-week-old rats, both high-fat diets and BPA promoted prostate epithelial cell hyperplasia, with low-concentration BPA combined with a maintenance diet showing the most significant effect on hyperplasia.

[0049] 4) Molecular markers: This experiment used immunohistochemistry to detect the expression level of PCNA protein in prostate tissue, including: Six rat prostate tissues were randomly selected from each group, and the procedures included paraffin embedding, sectioning, and dewaxing (same as Experiment 3). After microwave antigen retrieval, the dewaxed sections were incubated overnight at 4°C with primary antibody (PCNA) and primary antibody (Ki67), followed by incubation at 37°C for 45 minutes with secondary antibody (HRP-labeled goat anti-rabbit). After washing with TBST, DAB staining was performed, followed by drying, mounting, and microscopic examination. The expression of PCNA in prostate tissue was analyzed using QuPath 3.2 image acquisition. The immunohistochemical staining results for each group are shown below. Figure 15 As shown in the figure, the expression level of PCNA in rats in the Nd-Lbpa group was significantly increased compared with that in the Nd group; the expression level of PCNA in rats in the Nd-T group was decreased compared with that in the Nd group, but the difference was not statistically significant, and was significantly decreased compared with that in the Nd-Lbpa group.

[0050] Immunohistochemical staining images as shown Figure 16 As shown (magnification: ×200 and ×400; scale bar = 100μm & 50μm, hematoxylin staining of cell nuclei appears blue, and positive PCNA expression appears dark brown (red arrow). A is the Nd group, B is the Nd-Lbpa group, C is the Nd-T group, D is the Hfd group, and E is the Hfd-Lbpa group), prostate cell nuclei appear blue, and positive PCNA expression appears dark brown.

[0051] It can be seen that the expression level of prostate PCNA in 9-10 week old rats is significantly regulated by BPA. BPA exposure in the Nd group significantly increased the proliferation markers, but the increase in PCNA under the background of high-fat diet was not statistically significant, suggesting that it may interfere with the direct activation effect of BPA on the cell cycle through metabolism, such as chronic inflammation or oxidative stress.

[0052] In summary, this invention provides a model and method for constructing a bisphenol A-induced benign prostatic hyperplasia (BPH) model in rats. The low-dose BPA exposure used in this invention more closely resembles the actual environmental exposure levels in humans, and the main exposure route is simulated via oral gavage. The resulting model, etiologically, better simulates the real process of chronic, low-dose exposure to environmental endocrine disruptors, providing a more practical experimental tool for related risk assessment.

[0053] This invention explores the different modes of action of bisphenol A and high-fat diet in benign prostatic hyperplasia (BPH). In addition to exposure to a single environmental pollutant, dietary background intervention was added to explore the interaction between the two in the construction of the BPH model. This provides a clear decision-making basis for subsequent researchers to select experimental animals and design exposure conditions, and avoids experimental failures or deviations caused by inappropriate condition selection.

[0054] This invention uses a maintenance diet combined with a low dose of BPA (30 μg / kg.bw) to intervene in sexually mature rats for 60 days, overcoming the systemic side effects that may be caused by traditional hormone induction methods, as well as the problem of impure models caused by spontaneous proliferation in older animals.

[0055] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for constructing a bisphenol A-induced benign prostatic hyperplasia model in rats, characterized in that, Includes the following steps: S1: Select rats aged 8-9 months and 9-10 weeks and acclimatize them in a suitable environment for 7 days or more; S2: Benign prostatic hyperplasia was induced in adaptively fed rats by combined administration of a high-fat diet and bisphenol A; the induction period was 60-80 days, and biological indicators were evaluated after the induction was completed.

2. The construction method according to claim 1, characterized in that, In step S1, the rats are healthy SPF-grade male SD rats; the suitable environment includes: ventilation in the culture room, temperature of 20-26℃, humidity of 40%-70%, and a light-dark alternation cycle of 12 hours each.

3. The construction method according to claim 1, characterized in that, In step S2, the high-fat feed consists of: 20.0% sucrose, 15% lard, 1.2% cholesterol, 0.2% sodium cholate, 8% casein, 1.8% dicalcium phosphate, with the remainder being maintenance feed.

4. The construction method according to claim 3, characterized in that, The concentration of bisphenol A is 30-1000 μg / kg.bw.

5. The construction method according to claim 4, characterized in that, The biological indicators include physiological indicators, serum hormone indicators, tissue structure, and molecular markers.

6. The construction method according to claim 5, characterized in that, The physiological indicators include body mass index, organ index, prostate coefficient, and testicular coefficient; the serum hormone indicators include serum testosterone and dihydrotestosterone.

7. A bisphenol A-induced benign prostatic hyperplasia model in rats constructed by the method described in claim 1.

Images