A method for constructing an experimental autoimmune prostatitis rat model

Abstract

The application relates to a method for constructing an experimental autoimmune prostatitis rat model, which comprises the following steps: S1, preparing a prostate specific antigen mixture; S2, selecting male SD rats as experimental rats and dividing the rats into a physiological saline control group, a low-dose model group and a high-dose model group; S3, continuously injecting for 7 days, wherein the physiological saline control group receives 0.2ml of physiological saline injection every day, the low-dose model group receives 0.1ml of the prostate specific antigen mixture injection every day, and the high-dose model group receives 0.2ml of the prostate specific antigen mixture injection every day; and S4, continuing to feed all the experimental rats in the groups until the 28th day, wherein the experimental rats in the low-dose model group and the high-dose model group are induced by the prostate specific antigen mixture to cause experimental autoimmune prostatitis, and an experimental autoimmune prostatitis rat model is obtained. The method can not only reduce the use of animals and the cost, but also improve the modeling efficiency and stability.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically to a method for constructing an experimental autoimmune prostatitis rat model. Background Technology

[0002] Chronic non-bacterial prostatitis (CNP) is one of the most common diseases of the urinary system, accounting for more than 90% of all prostatitis cases. Due to the unique anatomical and physiological characteristics of the prostate, CNP patients experience a variety of different clinical symptoms, have a high recurrence rate, and poor treatment response. Therefore, it is the most common and complex clinical syndrome among prostatitis subtypes. Currently, the etiology and pathophysiology of CNP are still not fully understood, and developing reliable animal models that can accurately reproduce the complex pathophysiological characteristics of human chronic non-bacterial prostatitis (CNP) remains a major challenge in this field.

[0003] Currently, immune-mediated CNP models mainly include castration combined with estrogen induction, autoimmune response induction, and spontaneous models. Although spontaneous models have high clinical relevance and stable pathological manifestations, their application in modern research is limited due to strict age and species requirements, long modeling cycles, high costs, and poor reproducibility. Previous studies have provided increasing evidence that multiple factors are associated with the occurrence of prostatitis, including pathogen infection, urinary reflux, neuroendocrine disorders, and oxidative stress. Among the various pathogenic hypotheses of CNP, autoimmune mechanisms are considered to play a central role. Therefore, experimental autoimmune prostatitis (EAP) animal models have become important tools for studying the pathogenesis of CNP and evaluating new therapies. Currently, commonly used immune-related EAP models mainly rely on induction using homologous prostate tissue protein homogenates. While this method can induce autoimmune prostatitis, it has some limitations: (1) it requires a large number of animals for homogenate preparation, and the batch-to-batch differences in protein concentration and activity lead to high costs and ethical concerns; (2) significant differences in protein concentrations across different studies result in poor model reproducibility, large variations in modeling success rates, and hinder direct comparison of results between different laboratories. Therefore, developing a new, standardized, and efficient EAP model construction method to reduce the use of animals is of significant practical importance. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method for constructing an experimental autoimmune prostatitis rat model, which can construct a highly reliable and effective experimental autoimmune prostatitis rat model. This method not only reduces the use of animals and lowers costs, but also improves modeling efficiency and stability, providing a reliable experimental tool for studying the pathogenesis of CNP and validating novel treatment strategies. The technical solution adopted is as follows:

[0005] A method for constructing an experimental autoimmune prostatitis rat model, characterized by comprising the following steps:

[0006] S1. Prepare a prostate-specific antigen mixture, which is made from the following proportions of raw materials. For every 10 mL of prostate-specific antigen mixture prepared, the raw materials used are: a bacterial count of not less than 1 x 10⁻⁶ per 10 mL. 6 Inactive Mycobacterium tuberculosis, 100ug human prostate-specific antigen, 100mg bovine serum albumin, 50mg mitogenic pentapeptide, 100ug protamine sulfate, 10mL vaccine adjuvant;

[0007] S2. Select several healthy male SD rats of similar weight as experimental rats, and randomly divide these experimental rats into a saline control group, a low-dose model group, and a high-dose model group.

[0008] S3. The groin area of ​​all groups of experimental mice was regularly injected subcutaneously for 7 consecutive days. Among them, the experimental mice in the saline control group received 0.2 mL of saline injection per day, the experimental mice in the low-dose model group received 0.1 mL of prostate-specific antigen mixture per day, and the experimental mice in the high-dose model group received 0.2 mL of prostate-specific antigen mixture per day.

[0009] S4. All groups of experimental mice continued to be fed until day 28. The mice in the low-dose model group and the high-dose model group were induced to develop experimental autoimmune prostatitis by the prostate-specific antigen mixture, thereby obtaining the experimental autoimmune prostatitis rat model.

[0010] In step S1 above, the raw material used to prepare the prostate-specific antigen mixture includes protamine sulfate, a 5-kDa cationic polypeptide derived from salmon sperm. It can bind to negatively charged unfractionated heparin (UFH) and downregulate thrombin production by inhibiting factor V activation. It has an inhibitory effect on thrombin in the conversion of fibrinogen to fibrin, and this inhibition is concentration-dependent, partial, and reversible. Human prostate-specific antigen (human PSA antigen (PSA Ag protein)) can be obtained from Wuhan Boote Biotechnology Co., Ltd., product model orb98816; bovine serum albumin can be obtained from Wuhan Boote Biotechnology Co., Ltd., product model orb80188; mitogenic pentapeptide is a synthetic E. coli outer membrane lipoprotein N-terminal analog (cysteyl-serine-serine-aspartic-alanine) and can be used for immune adjuvant research; vaccine adjuvant contains an oil phase, emulsifier and water, and uses MedChemExpress product model HY-16019.

[0011] Because prostate-specific antigen (PSA) is secreted by prostate epithelial cells and belongs to the kininase family of proteins, it is present in prostate tissue and semen. Its main physiological function is to prevent semen coagulation, and it has extremely high tissue and organ specificity. While xenogeneic PSA is highly similar in amino acid sequence, it also has subtle differences. When xenogeneic PSA is injected into the body, the immune system recognizes it as a foreign antigen and initiates a strong immune response, producing antibodies and activating T lymphocytes. These activated immune systems and antibodies not only attack the xenogeneic PSA, but also, due to the similarity of their molecular structures, they mistakenly recognize and attack the PSA produced by the body's own prostate, thereby attacking the prostate tissue that expresses PSA. This invention actively breaks the body's immune tolerance to PSA by injecting a mixture of prostate-specific antigen (PSA) (containing human PSA and immune adjuvants, such as inactive Bacillus thuringiensis, albumin, etc.) into male SD rats in step S3, thereby inducing specific autoimmune inflammation against prostate tissue. Other immune adjuvants in the antigen mixture can non-specifically enhance the immune response, making the specific immune response against PSA stronger and more persistent, thus more effectively breaking immune tolerance.

[0012] As a preferred embodiment of the present invention, in step S1, the preparation process of the prostate-specific antigen mixture is as follows: first, the bacterial count is not less than 1×10⁻⁶ per 10 mL. 6One sample of Mycobacterium tuberculosis is added to 10 mL of vaccine adjuvant, mixed and dissolved, and then subjected to dry heat autoclaving to obtain a vaccine adjuvant containing the inactivated Mycobacterium tuberculosis. Then, 100 μg of human prostate-specific antigen, 100 mg of bovine serum albumin, 50 mg of mitogenic pentapeptide, and 100 μg of protamine sulfate are added, and they are thoroughly mixed until completely dissolved to obtain the prostate-specific antigen mixture. Specifically, it can be thoroughly mixed until completely dissolved by rotary mixing, and then stored in a sealed container at 3-5°C.

[0013] In a preferred embodiment of the present invention, in step S2, the selected male SD rats (i.e., SPF-grade male Sprague-Dawley rats) are 6-8 weeks old and weigh 200±20g. These male SD rats can be housed in a barrier system, with bedding changed regularly, humidity controlled between 40% and 70%, and ambient temperature maintained at (23±1)℃. The housing follows a 12-hour light-12-hour dark cycle. Before the experiment, the male SD rats are allowed to acclimatize to this environment for one week, thus obtaining several healthy male SD rats of similar weight. These male SD rats are then randomly divided into three groups: a saline control group (n=6), a high-dose model group (n=6), and a low-dose model group (n=6).

[0014] As a preferred embodiment of the present invention, in step S3, during the process of regularly injecting the groin area of ​​all groups of experimental mice subcutaneously for 7 consecutive days, the injections are alternately performed in the left groin area and the right groin area of ​​the experimental mice.

[0015] As a preferred embodiment of the present invention, from the injection of the experimental mice on day 1 to day 28, the weight, food and water intake, fur condition and activity of the experimental mice in all groups were monitored daily: the experimental mice in the saline control group showed no obvious abnormalities in any of the indicators; compared with the saline control group, the experimental mice in the low-dose model group and the high-dose model group showed a significant decrease in weight and activity, and exhibited pain-related behaviors such as arching their backs and licking their lower abdomen, accompanied by yellowish fur and hair loss, confirming that the EAP rat model was successfully induced and was not affected by non-specific stress.

[0016] As a further preferred embodiment of the present invention, the construction method further includes step S5, which involves first collecting blood samples from experimental mice, then euthanizing the mice by carbon dioxide asphyxiation, and then dissecting and collecting tissues from their prostate, bladder, testes, heart, liver, spleen, lungs, and kidneys. The collected blood and tissues are then observed, monitored, and analyzed comparatively to verify the effectiveness and specificity of the experimental autoimmune prostatitis rat model. Specifically, the collected blood and tissues can be observed, monitored, and analyzed comparatively using methods such as hematoxylin and eosin (HE) staining, immunohistochemical analysis (IHC), and enzyme-linked immunosorbent assay (ELISA) to verify the effectiveness and specificity of the experimental autoimmune prostatitis rat model.

[0017] To validate the effectiveness of the newly established EAP rat model induced by a mixture of prostate-specific antigens, we first monitored the overall health of male SD rats. All groups had similar initial body weights. Compared to the saline control group, rats in both the low-dose and high-dose model groups showed significant and progressively decreasing weight gain over the 28-day experimental period, indicating that the induced autoimmune response had systemic effects. Histopathological evaluation of the prostate tissue provided clear evidence of successful model establishment. Rats in the saline control group maintained intact prostate tissue structure, normal glandular structure, and no signs of inflammation. In contrast, rats in the low-dose model group developed moderate prostatitis, characterized by glandular structural damage, luminal dilation, and mild inflammatory cell infiltration. The high-dose model group exhibited the most severe pathological condition, characterized by extensive glandular destruction, loss of tissue integrity, and dense inflammatory cell infiltration within the stroma. These monitoring results demonstrate that our novel method successfully established an EAP rat model, and that its effect on pathological severity is clearly dose-dependent. Next, we analyzed the immune and inflammatory characteristics of the EAP rat model. Serum analysis showed a significant humoral immune response. The levels of immunoglobulins (IgA, IgG, IgM) were significantly elevated in both the low-dose and high-dose model groups, with the most pronounced changes in the high-dose group. Both model groups also elicited a strong cytokine response: serum levels of the pro-inflammatory cytokine IL-1β increased dramatically, while the levels of the anti-inflammatory cytokine IL-10 decreased, with the most significant changes observed in the high-dose group. Immunohistochemical analysis of prostate tissue confirmed the local inflammatory environment, and the dose-dependent upregulation of key pro-inflammatory mediators TNF-α and CRP in the prostate stroma of the model group rats was confirmed. This synergistic enhancement of systemic and local inflammatory responses highlights the immunogenicity of our modeling method. Then, given the link between androgens and prostate health, we assessed hormone levels. Enzyme-linked immunosorbent assay (ELISA) results showed that, compared with the saline control group, the local testosterone and serum androgen levels in the EAP rat model group were significantly reduced. This inhibition was more pronounced in the high-dose model group, consistent with the dose-dependent aggravation of prostatitis, and supports the physiological relevance of the model. Finally, to assess the safety of the method for constructing the experimental autoimmune prostatitis rat model, we tested systemic parameters of the mice. Serum biochemical analysis showed no significant differences in liver function parameters (alanine aminotransferase, albumin) or kidney function parameters (creatinine, urea) among the three groups. Furthermore, histopathological examination of major organs such as the heart, liver, spleen, lungs, and kidneys, as well as the seminal vesicles, bladder, and testes, revealed no signs of pathological damage or impairment in any of the model groups.These results indicate that the EAP rat model induced by the prostate-specific antigen mixture is organ-specific and does not cause significant systemic toxicity.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) This invention uses male SD rats as experimental mice and divides them into a saline control group, a low-dose model group and a high-dose model group. Then, the prepared PSA mixture is regularly injected subcutaneously into the groin area of ​​the experimental mice in the low-dose model group and the high-dose model group. The PSA mixture is used to induce the experimental mice in the low-dose model group and the high-dose model group to construct an EAP rat model with high reliability and effectiveness. This not only reduces the use of animals and lowers costs, but also avoids the need to sacrifice a large number of animals to prepare prostate protein homogenate and the batch-to-batch differences in the concentration and activity of the prepared prostate protein homogenate. The standardized operation provides modeling efficiency and overcomes the limitations of the application of immune-mediated CNP models in modern research. More importantly, it induces pathological changes that are very similar to the histopathological features (such as glandular structure destruction, extensive inflammatory cell infiltration and interstitial edema) observed in clinical CNP patients, thereby verifying the effectiveness and clinical relevance of this construction method and providing a reliable experimental tool for studying the pathogenesis of CNP and verifying new treatment strategies.

[0020] (2) The EAP rat model constructed in this invention showed a significant dose-dependent effect. Low-dose antigen mixture successfully induced prostate inflammation, while high doses led to more severe glandular damage and denser inflammatory cell infiltration. At the immunological level, we observed significantly elevated serum immunoglobulin levels (IgA, IgG, IgM) and significantly upregulated local pro-inflammatory factors (TNF-α, CRP) in the model group, while the anti-inflammatory factor IL-10 decreased. Existing studies have confirmed that pro-inflammatory cytokines can induce local tissue damage by enhancing the expression of adhesion molecules and the synthesis of prostaglandins, while the decrease in IL-10 levels exacerbates the inflammatory response. The systemic and local prostate immune inflammatory status observed in the EAP rat model is consistent with the results of previous multi-omics studies, which indicate that CNP can drive immune imbalance through epigenetic regulation of the expression of immune-related genes. It is particularly noteworthy that these changes in immune indicators were most significant in the high-dose model group, which is consistent with the most severe histopathological damage observed, thus further demonstrating the reliability and effectiveness of this EAP rat model.

[0021] (3) The EAP rat model constructed in this invention also showed a significant reduction in hormone levels, which is another important finding consistent with clinical observations. Clinical studies have shown that CNP patients often experience endocrine disorders, and the reduction in androgen levels may exacerbate the inflammatory process by regulating the local immune microenvironment within the prostate. The successful replication of this function in our constructed EAP rat model demonstrates that it can not only simulate morphological changes but also key physiological changes that occur during the disease process, providing an ideal platform for studying the role of the interaction between hormones and immunity in prostatitis. Attached Figure Description

[0022] Figure 1 This is a schematic diagram showing the dose-dependent pathological severity of the EAP rat model provided by the preferred embodiment of the present invention: (a) the weight changes of each group of experimental mice over 28 days; (b) the comparison of prostate tissue pathology of each group of experimental mice using the hematoxylin-eosin (HE) staining method.

[0023] Figure 2 This is a schematic diagram illustrating the strong and dose-dependent immune inflammatory response induced by the EAP rat model provided in the preferred embodiment of the present invention: (a) the serum immunoglobulin expression levels of each group of experimental mice were detected and compared by enzyme-linked immunosorbent assay (ELISA); (b) the expression levels of IL-1β and IL-10 of each group of experimental mice were detected and compared by enzyme-linked immunosorbent assay (ELISA); and (c) the expression levels of TNFα and CRP of each group of experimental mice were compared by immunohistochemical analysis (IHC).

[0024] Figure 3 The following is a schematic diagram of the EAP rat model provided by the preferred embodiment of the present invention, showing a dose-dependent reduction in testosterone and androgen levels: (a) comparing the testosterone expression levels of each group of experimental mice using enzyme-linked immunosorbent assay (ELISA); (b) comparing the androgen expression levels of each group of experimental mice using enzyme-linked immunosorbent assay (ELISA).

[0025] Figure 4 This is a schematic diagram of the EAP rat model provided by the preferred embodiment of the present invention, showing that no systemic toxicity reaction occurred after induction: (a) comparison of biochemical indicators of each group of experimental mice; (b) comparison of pathological tissues of the heart, liver, lungs and kidneys of each group of experimental mice; (c) comparison of histopathological tissues of the seminal vesicles, bladder and testes of each group of experimental mice using the hematoxylin-eosin (HE) staining method. Detailed Implementation

[0026] The method for constructing this experimental autoimmune prostatitis rat model includes the following steps:

[0027] S1. Prepare a prostate-specific antigen (PSA) mixture, which is made from the following proportions of raw materials. For every 10 mL of PSA mixture prepared, the raw materials used must have a bacterial count of not less than 1 x 10⁻⁶ per 10 mL. 6 Inactive Mycobacterium tuberculosis, 100ug human prostate-specific antigen, 100mg bovine serum albumin, 50mg mitogenic pentapeptide, 100ug protamine sulfate, 10mL vaccine adjuvant;

[0028] S2. Select several healthy male SD rats of similar weight as experimental rats, and randomly divide these experimental rats into a saline control group, a low-dose model group, and a high-dose model group.

[0029] S3. The prostate-specific antigen mixture was regularly injected subcutaneously into the groin area of ​​all groups of experimental mice. The mice in the saline control group received 0.2 mL of saline solution per day, the mice in the low-dose model group received 0.1 mL of prostate-specific antigen mixture per day, and the mice in the high-dose model group received 0.2 mL of prostate-specific antigen mixture per day for 7 consecutive days.

[0030] S4. All groups of experimental mice continued to be fed until day 28 (i.e., day 28 from day 1 of injection). The experimental mice in the low-dose model group and the high-dose model group were induced to develop experimental autoimmune prostatitis by the prostate-specific antigen mixture, thereby obtaining the experimental autoimmune prostatitis rat model.

[0031] In this embodiment, in step S2, the male SD rats (i.e., SPF-grade male Sprague-Dawley rats) are 6-8 weeks old, and their weight is (200±20) grams. These male SD rats can be housed in a barrier system, with bedding changed regularly, humidity controlled between 40% and 70%, and the ambient temperature maintained at (23±1)℃. The housing follows a 12-hour light-12-hour dark cycle. Before the experiment, the male SD rats are allowed to acclimatize to this environment for one week, thus obtaining several healthy male SD rats of similar weight. These male SD rats are then randomly divided into three groups: a saline control group (n=6), a high-dose model group (n=6), and a low-dose model group (n=6).

[0032] In this embodiment, the preparation process of the prostate-specific antigen mixture in step S1 is as follows: first, the bacterial count is not less than 1×10⁻⁶ per 10 mL.6 One sample of Mycobacterium tuberculosis is added to 10 mL of vaccine adjuvant, mixed and dissolved, and then subjected to dry heat autoclaving to obtain a vaccine adjuvant containing the inactivated Mycobacterium tuberculosis. Then, 100 μg of human prostate-specific antigen, 100 mg of bovine serum albumin, 50 mg of mitogenic pentapeptide, and 100 μg of protamine sulfate are added, and they are thoroughly mixed until completely dissolved to obtain the prostate-specific antigen mixture. Specifically, it can be thoroughly mixed until completely dissolved by rotary mixing, and then stored in a sealed container at 3-5°C.

[0033] In this embodiment, during step S3, the injections are administered to the experimental mice alternately in the left and right groin areas over seven consecutive days. For example, the injection is given in the left groin area on the first day, the right groin area on the second day, and the left groin area on the third day, and so on.

[0034] In this embodiment, from the injection of the experimental mice on day 1 to day 28, the weight, food and water intake, fur condition and activity of the experimental mice in all groups were monitored daily: the experimental mice in the saline control group showed no significant abnormalities in any of the indicators; compared with the saline control group, the experimental mice in the low-dose model group and the high-dose model group showed a significant decrease in weight and activity, and exhibited pain-related behaviors such as arching their backs and licking their lower abdomen, accompanied by yellowish fur and hair loss, confirming that the EAP rat model was successfully induced and was not affected by non-specific stress.

[0035] In this embodiment, the construction method further includes step S5, which involves first collecting blood samples from experimental mice, then euthanizing the mice by carbon dioxide asphyxiation, and then dissecting and collecting tissues from their prostate, bladder, testes, heart, liver, spleen, lungs, and kidneys. The collected blood and tissues are then observed, monitored, and analyzed using methods such as hematoxylin and eosin staining (HE), immunohistochemical analysis (IHC), and enzyme-linked immunosorbent assay (ELISA) to verify the effectiveness and specificity of the experimental autoimmune prostatitis rat model.

[0036] A. Hematoxylin-eosin staining (HE)

[0037] The method of hematoxylin-eosin (HE) staining includes: (1) After the experimental mice are euthanized, tissues of the prostate, heart, liver, spleen, lung and kidney are collected and the surrounding membranes and adipose tissue are carefully removed. Then the tissues are rinsed with physiological saline three times. After the tissues are dried with filter paper, their weight is measured using an electronic balance. Tissues of the prostate, heart, liver, spleen, lung and kidney are collected from each group of experimental mice and fixed with 4% formaldehyde for 72 hours. The tissues are rinsed, dehydrated, cleared, paraffin-embedded, embedded, sectioned and baked at room temperature. (2) The sections are stained with hematoxylin for 1 minute, rinsed with water, differentiated with 1% hydrochloric acid ethanol solution for a few seconds, rinsed with running water for 10 minutes, and then stained with eosin dye for a few seconds. (3) After staining, the sections are sealed and the local pathological changes of each organ are observed under an optical microscope (Olympus, Japan).

[0038] B. Immunohistochemistry (IHC)

[0039] Immunohistochemistry (IHC) is used to assess the expression levels of TNF-α and hs-CRP in tissues. The method of immunohistochemistry (IHC) includes: (1) After the experimental mice are sacrificed, tissues of the prostate, heart, liver, spleen, lung and kidney are collected and the surrounding membranes and adipose tissue are carefully removed. The tissues are then rinsed three times with physiological saline. After the tissues are dried with filter paper, their weight is measured using an electronic balance. Tissues of the prostate, heart, liver, spleen, lung and kidney are collected from each group of experimental mice and fixed with 4% formaldehyde for 72 hours. The tissues are rinsed, dehydrated, cleared, paraffin-embedded, embedded, sectioned and baked at room temperature. (2) Paraffin sections are antigen-retrieved with 1×EDTA antigen retrieval solution and exposed to antigen sites according to the conventional method. The sections are placed in 3% hydrogen peroxide solution and incubated at room temperature. (2) Incubate for 10 minutes to block endogenous peroxidase; (3) Cover the tissue with 5% BSA solution and place at room temperature for 20 minutes, then remove the BSA solution and add diluted primary antibody containing TNF-α and hs-CRP (dilution ratio: 1:250) dropwise to each slice to cover the tissue and incubate overnight at 4°C; after removing the slices from 4°C, add 50-100 μL of diluted secondary antibody to each slice and incubate at room temperature for 30 minutes; add 50-100 μL of DAB to each slice and observe under a microscope; (4) Add hematoxylin dropwise to the slices, dehydrate with (B-6) graded ethanol and clear in xylene; after mounting, observe under an optical microscope.

[0040] C. Serological and biochemical detection methods

[0041] Serological and biochemical detection methods included: (1) collecting blood samples from the abdominal aorta after anesthetizing experimental mice; (2) centrifuging the samples at 3000 rpm for 10 minutes after the blood had been allowed to stand for one hour; the separated serum was stored at -80℃; (3) homogenizing prostate tissue to determine the prostate index. The levels of IgA, IgM, IgG, albumin, transaminase, IL-1β, IL-10, and androgen and testosterone (T) levels in rat serum were analyzed according to the instructions using an ELISA kit.

[0042] (4) creatinine levels were measured by creatine oxidase method and urea nitrogen levels were determined by urease method; absorbance values ​​were measured at appropriate wavelengths using an enzyme-linked immunosorbent assay (ELISA) reader, and the results were calculated accordingly.

[0043] D. Enzyme-linked immunosorbent assay (ELISA)

[0044] Serum immunoglobulins IgA, IgG, IgM and cytokines IL-1β and IL-10 were detected by enzyme-linked immunosorbent assay (ELISA), while TNF-α and CRP in tissues were detected by immunohistochemistry. Testosterone, androgens and liver / kidney function indicators were also analyzed.

[0045] To validate the effectiveness of the newly established EAP rat model induced by a mixture of prostate-specific antigens, we first monitored the overall health of male SD rats. All groups had similar initial body weights. Compared to the saline control group, rats in both the low-dose and high-dose model groups showed significant and progressive weight gain during the 28-day experimental period (e.g., ...). Figure 1 a) This indicates that the induced autoimmune response had systemic effects. Histopathological evaluation of the prostate tissue provided clear evidence of the successful establishment of the model (e.g. Figure 1 (b) In the saline control group, the prostate tissue structure of the mice remained intact, the glandular structure was normal, and there were no signs of inflammation. In contrast, the mice in the low-dose model group developed moderate prostatitis, characterized by glandular structural damage, luminal dilation, and mild inflammatory cell infiltration. The mice in the high-dose model group had the most severe pathological condition, characterized by extensive glandular destruction, loss of tissue integrity, and dense inflammatory cell infiltration in the stroma. These monitoring results indicate that our new method can successfully construct an EAP rat model, and its effect on pathological severity is significantly dose-dependent.

[0046] Next, we analyzed the immune and inflammatory characteristics of the EAP rat model. Serum analysis showed a significant humoral immune response. The levels of immunoglobulins (IgA, IgG, and IgM) in both the low-dose and high-dose model groups were significantly elevated, with the most pronounced changes in the high-dose model group (Figure 2a). Furthermore, both model groups elicited a strong cytokine response: the level of the pro-inflammatory cytokine IL-1β in serum increased dramatically, while the level of the anti-inflammatory cytokine IL-10 decreased, with the most significant changes observed in the high-dose model group (Figure 2b). Immunohistochemical analysis of the prostate tissue confirmed the local inflammatory environment. The dose-dependent upregulation of key pro-inflammatory mediators TNF-α and CRP in the prostate stroma of both the high-dose and low-dose model groups was confirmed (Figure 2c). This synergistic enhancement of systemic and local inflammatory responses highlights the immunogenicity of our modeling method.

[0047] Then, given the link between androgens and prostate health, we assessed hormone levels, and enzyme-linked immunosorbent assay (ELISA) results showed that, compared with the saline control group, the EAP rat model group had significantly higher levels of local testosterone (…). Figure 3 a) and serum androgens ( Figure 3 The levels of b) were significantly reduced. This inhibition was more pronounced in the high-dose model group, which is consistent with the dose-dependent aggravation of prostatitis and supports the physiological relevance of the model.

[0048] Finally, to assess the safety of the experimental autoimmune prostatitis rat model construction method, we tested the systemic parameters of the mice. Serum biochemical analysis showed no significant differences in liver function indicators (alanine aminotransferase, albumin) or kidney function indicators (creatinine, urea) among the three groups (Figure 4a). Furthermore, histopathological examination of major organs such as the heart, liver, spleen, lungs, and kidneys (Figure 4b), as well as the seminal vesicles, bladder, and testes (Figure 4c), revealed no signs of pathological damage or impairment in any of the model groups.

[0049] These results indicate that the EAP rat model induced by the prostate-specific antigen mixture is organ-specific and does not cause significant systemic toxicity.

[0050] Furthermore, it should be noted that the names of the various parts of the specific embodiments described in this specification may differ. All equivalent or simple variations made to the structure, features, and principles described in this invention are included within the scope of protection of this invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not deviate from the structure of this invention or exceed the scope defined in these claims, all of which should fall within the scope of protection of this invention.

Claims

1. A method for constructing an experimental autoimmune prostatitis rat model, characterized in that... Includes the following steps: S1. Prepare a prostate-specific antigen mixture, which is made from the following proportions of raw materials. For every 10 mL of prostate-specific antigen mixture prepared, the raw materials used are: a bacterial count of not less than 1 x 10⁻⁶ per 10 mL. 6 The vaccine contained inactive Mycobacterium tuberculosis, 100 μg human prostate-specific antigen, 100 mg bovine serum albumin, 50 mg mitogenic pentapeptide, 100 μg protamine sulfate, and 10 mL of vaccine adjuvant. Among them, mitogenic pentapeptide is a synthetic N-terminal analog of Escherichia coli outer membrane lipoprotein, namely cysteyl-serine-serine-aspartic-alanine. S2. Select several healthy male SD rats of similar weight as experimental rats, and randomly divide these experimental rats into a saline control group, a low-dose model group, and a high-dose model group. S3. The groin area of ​​all groups of experimental mice was regularly injected subcutaneously for 7 consecutive days. Among them, the experimental mice in the saline control group received 0.2 mL of saline injection per day, the experimental mice in the low-dose model group received 0.1 mL of prostate-specific antigen mixture per day, and the experimental mice in the high-dose model group received 0.2 mL of prostate-specific antigen mixture per day. S4. All groups of experimental mice continued to be fed until day 28. The mice in the low-dose model group and the high-dose model group were induced to develop experimental autoimmune prostatitis by the prostate-specific antigen mixture, thereby obtaining the experimental autoimmune prostatitis rat model.

2. The method for constructing an experimental autoimmune prostatitis rat model according to claim 1, characterized in that: In step S1, the preparation process of the prostate-specific antigen mixture is as follows: first, the bacterial count is not less than 1×10⁻⁶ per 10 mL. 6 One tubercle bacillus was added to 10 mL of vaccine adjuvant, mixed and dissolved, and then subjected to dry heat and high pressure inactivation to obtain a vaccine adjuvant containing the inactive tubercle bacillus; then 100 μg of human prostate-specific antigen, 100 mg of bovine serum albumin, 50 mg of mitogenic pentapeptide, and 100 μg of protamine sulfate were added, and they were mixed thoroughly until completely dissolved to obtain the prostate-specific antigen mixture.

3. The method for constructing an experimental autoimmune prostatitis rat model according to claim 1, characterized in that: In step S2, the selected male SD rats are 6-8 weeks old and weigh 200±20 grams.

4. The method for constructing an experimental autoimmune prostatitis rat model according to claim 1, characterized in that: In step S3, during the process of regularly injecting the groin area of ​​all groups of experimental mice subcutaneously for 7 consecutive days, the injections were alternately performed in the left and right groin areas of the experimental mice.

5. A method for constructing an experimental autoimmune prostatitis rat model according to any one of claims 1-4, characterized in that: From day 1 to day 28 after injection into the mice, the weight, food and water intake, fur condition, and activity levels of all groups of mice were monitored daily. The mice in the saline control group showed no significant abnormalities in any of the indicators. Compared with the saline control group, the mice in the low-dose and high-dose model groups showed a significant decrease in weight and activity, exhibited pain-related behaviors such as arching their backs and licking their lower abdomen, and had yellowish fur and hair loss. This confirmed that the experimental autoimmune prostatitis rat model was successfully induced and was not affected by nonspecific stress.

6. A method for constructing an experimental autoimmune prostatitis rat model according to any one of claims 1-4, characterized in that: The construction method further includes step S5, which involves first collecting blood samples from the experimental mice, then euthanizing the mice by carbon dioxide asphyxiation, and then dissecting and collecting tissues from the prostate, bladder, testes, heart, liver, spleen, lungs, and kidneys. The collected blood and tissues are then observed, monitored, and analyzed to verify the effectiveness and specificity of the experimental autoimmune prostatitis rat model.

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