Application of minocycline in preparation of medicine for treating benign prostatic hyperplasia

Abstract

The invention provides application of minocycline in preparation of a medicine for treating benign prostatic hyperplasia, and belongs to the technical field of biological medicine. The invention provides application of minocycline in preparation of a medicine for treating benign prostatic hyperplasia, and provides a safe, convenient and efficient oral medicine treatment scheme for clinically treating benign prostatic hyperplasia, especially fibrosis pathological changes accompanied by benign prostatic hyperplasia. According to the scheme, improvement and volume retraction of a gland tissue structure can be realized directly aiming at a core pathological link of a disease, so that the urination obstruction symptom of a patient is effectively relieved, wounds and risks caused by surgical treatment are avoided, the accessibility of treatment and the compliance of the patient are greatly improved, and remarkable clinical application value and market prospect are achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of minocycline in the preparation of a medicament for the treatment of benign prostatic hyperplasia. Background Technology

[0002] Benign prostatic hyperplasia (BPH) is a common urinary system disease in middle-aged and elderly men, with its incidence increasing significantly with age. The disease is mainly characterized by the proliferation of prostatic epithelial and stromal components, leading to an enlarged prostate and subsequent compression of the urethra, causing a series of lower urinary tract symptoms such as urinary frequency, urgency, difficulty urinating, and a weak urine stream, severely impacting the patient's quality of life. Pathologically, a significant proportion of BPH patients not only exhibit an increased number of cells in the proliferating tissue but also experience the occurrence and development of interstitial fibrosis. Fibrosis refers to the excessive deposition of extracellular matrix in tissues, resulting in loss of tissue elasticity and increased rigidity. In the prostate, interstitial fibrosis is considered one of the important factors leading to decreased glandular elasticity, increased urethral mechanical obstruction, and poor response to drug treatment. Therefore, reversing or inhibiting prostatic interstitial fibrosis has become an important target in BPH treatment.

[0003] Currently, the clinical treatment of benign prostatic hyperplasia (BPH) mainly follows a stepwise approach. For mild to moderate patients, the first-line treatment is medication, commonly including alpha-blockers and 5α-reductase inhibitors. The former primarily improves symptoms by relaxing the smooth muscles of the prostate and bladder neck, while the latter reduces prostate volume by lowering androgen levels. However, both classes of drugs have very limited effectiveness in improving existing interstitial fibrosis. For severe patients who do not respond well to medication or experience complications, surgical procedures such as transurethral resection of the prostate (TURP) remain the gold standard. Although the surgery is effective, it is an invasive procedure with risks such as bleeding, infection, urethral stricture, urinary incontinence, and impaired sexual function, and not all patients can tolerate it.

[0004] Therefore, developing a therapeutic drug that can effectively intervene in the process of prostate fibrosis and can be administered in a non-invasive manner is of urgent need and great significance for filling the current gap in clinical treatment of BPH and improving patient prognosis. Summary of the Invention

[0005] The purpose of this invention is to provide the application of minocycline in the preparation of a drug for treating benign prostatic hyperplasia, thereby solving the technical problem in the prior art of lacking an effective non-invasive oral drug treatment for benign prostatic hyperplasia with interstitial fibrosis.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the use of minocycline or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of benign prostatic hyperplasia.

[0008] Preferably, in the above applications, benign prostatic hyperplasia is accompanied by interstitial fibrosis.

[0009] Preferably, in the above applications, the drug is prepared into an oral dosage form.

[0010] Preferably, in the above applications, the oral dosage form is selected from tablets, capsules, granules, powders, pills, lozenges, oral solutions, syrups, oral suspensions, or oral emulsions.

[0011] The present invention also provides the use of a pharmaceutical composition in the preparation of a medicament for treating benign prostatic hyperplasia, said pharmaceutical composition containing minocycline or a pharmaceutically acceptable salt thereof as an active ingredient, and at least one pharmaceutically acceptable carrier or excipient.

[0012] Preferably, in the above applications, benign prostatic hyperplasia is accompanied by interstitial fibrosis.

[0013] Preferably, in the above applications, the excipients include fillers, binders, disintegrants, or lubricants.

[0014] Preferably, in any of the above applications, the daily dose of the drug, calculated as minocycline, is 50 mg to 200 mg.

[0015] The present invention also provides a method for screening or evaluating candidate drugs for treating benign prostatic hyperplasia, comprising establishing an in vitro or in vivo screening model based on the ZC3H12A-Th1-IFNγ-JAK / STAT signaling axis, and using the model to test the activity of the candidate drug; the establishment of the screening model includes constructing a prostate cell model with downregulated ZC3H12A expression or loss of function; the prostate cell model comprises prostate basal cells; the testing of the activity of the candidate drug includes detecting the effect of the candidate drug on the expression or activity of at least one molecule among ZC3H12A, IFNγ, KRT14, ATF3, GPX3, STAT1, or STAT3 in the signaling axis.

[0016] The present invention also provides a kit for screening or evaluating candidate drugs for the treatment of benign prostatic hyperplasia, the kit comprising reagents for detecting the expression or activity of at least one molecule selected from ZC3H12A, IFNγ, KRT14, ATF3, GPX3, STAT1, or STAT3.

[0017] The beneficial effects of this invention are: This invention provides the application of minocycline in the preparation of a drug for treating benign prostatic hyperplasia (BPH), offering a safe, convenient, and effective oral medication regimen for the clinical treatment of BPH, particularly its accompanying fibrotic pathological changes. This regimen directly targets the core pathological aspects of the disease, improving glandular tissue structure and reducing its volume, thereby effectively relieving patients' urinary obstruction symptoms. It avoids the trauma and risks associated with surgical treatment, greatly improving treatment accessibility and patient compliance, and possesses significant clinical application value and market potential. Attached Figure Description

[0018] Figure 1 Results of single-cell transcriptome sequencing and Th1 cell validation of BPH tissue: Figure 1 A: Schematic diagram of single-cell transcriptome sequencing (scRNA-seq) experimental workflow and bioinformatics analysis strategy; Figure 1 B: t-SNE dimensionality reduction clustering diagram of cell populations in normal prostate tissue and BPH tissue, showing the distribution of different cell populations; Figure 1 C: Relative abundance of cell populations in the normal group and the BPH group; Figure 1 D: Bubble diagram of KEGG pathway enrichment analysis of differentially expressed genes on T cells, revealing the activation of inflammation-related pathways; Figure 1 E: Scatter plot of flow cytometry detection of Th1 cell (CD4+IFNγ) infiltration in clinical prostate tissue; Figure 1 F: Statistical comparison of the proportion of Th1 cell infiltration between the normal group and the BPH group (P<0.001). Figure 2 Mechanism analysis of the Th1 / IFNγ-JAK / STAT axis driving epithelial-mesenchymal transition (EMT): Figure 2 A: Bubble graph showing the expression levels of IFNγ receptors (IFNGR1 / 2) in epithelial cells; Figure 2 B: Diagram of the signaling interaction communication network between Th1 cells and epithelial cells constructed based on CellChat; Figure 2 C: GSEA enrichment analysis curve of EMT-related gene set in BPH epithelial cells; Figure 2 D: Volcano diagram of differentially expressed genes (DEGs) in epithelial cells between the normal group and the BPH group; Figure 2 E: Bubble plot of expression abundance of epithelial marker (CDH1) and interstitial marker (VIM); Figure 2 F: Immunohistochemical (IHC) staining images of E-cadherin and Vimentin in prostate tissue sections, showing protein localization and expression; Figure 2 G: GSEA enrichment analysis of the JAK-STAT signaling pathway in BPH epithelial cells; Figure 3Lineage tracing analysis for the transdifferentiation of basal cells into fibroblasts: Figure 3 A: t-SNE reclustering diagram of prostate epithelial cell subsets, clarifying the basal cell population; Figure 3 B: Immunofluorescence (IF) staining image of KRT14, a basal cell marker, in BPH tissue; Figure 3 C: A pseudo-temporal developmental trajectory of basal cell differentiation into fibroblasts constructed based on the Monocle2 algorithm; Figure 3 D: Density distribution map of cells along the pseudo-temporal trajectory, showing the differentiation trend; Figure 4 Screening and clinical validation of the core target ZC3H12A: Figure 4 A: Volcano plot of differentially expressed genes in the BPH transcriptome; Figure 4 B: Heatmap of the association between WGCNA co-expression module and clinical traits (BPH vs Normal); Figure 4 C: Venn diagram of screening key candidate genes by integrating LASSO, SVM-RFE and XGBoost algorithms; Figure 4 D: ROC curve (AUC value assessment) of the diagnostic efficacy of the core gene ZC3H12A for BPH. Figure 4 E: Immunofluorescence double staining colocalization map of ZC3H12A (red) and CD4 (green) in tissue; Figure 4 F: Statistical graph of qRT-PCR detection of ZC3H12A mRNA expression level in clinical samples; Figure 5 In vivo efficacy validation of minocycline in treating BPH rats: Figure 5 A: A timeline diagram illustrating the construction, drug administration, and detection of the rat BPH model; Figure 5 B: Comparison of the gross morphology of prostate tissues from rats in each experimental group (Normal, BPH, Mino); Figure 5 C: Statistical graph of prostate index (PI) of rats in each group to evaluate the effect of glandular atrophy; Figure 5 D: Statistical graph of the recovery of ZC3H12A mRNA expression level in prostate tissue of rats in each group; Figure 5 E: Statistical graph of changes in serum IFNγ concentration in each group of rats detected by ELISA; Figure 5 F: Quantitative statistical graph of the proportion of Th1 cell infiltration in prostate tissue of rats in each group. Detailed Implementation

[0019] This invention provides the use of minocycline or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating benign prostatic hyperplasia (BPH). In this invention, "minocycline" refers to a tetracycline antibiotic, and its "pharmaceutically acceptable salt" includes, but is not limited to, hydrochloride, sulfate, and phosphate, with minocycline hydrochloride being the most commonly used form in clinical practice. "Treatment" refers to alleviating disease symptoms, slowing disease progression, improving pathological indicators, or normalizing the disease state. "Benign prostatic hyperplasia" is a non-cancerous lesion commonly seen in middle-aged and elderly men, characterized by hyperplasia of prostatic epithelial and stromal cells.

[0020] Preferably, in the above applications, benign prostatic hyperplasia (BPH) is accompanied by interstitial fibrosis. Interstitial fibrosis is an important pathological process in the progression of BPH, characterized by excessive deposition and remodeling of extracellular matrix components such as collagen in the prostatic stroma, leading to decreased tissue elasticity and increased rigidity. BPH with interstitial fibrosis is usually insensitive to traditional drug treatment and is associated with more severe lower urinary tract symptoms. This invention addresses this clinical challenge by providing a new treatment strategy.

[0021] Preferably, in the above applications, the drug is prepared into an oral dosage form. An oral dosage form refers to a drug preparation administered orally, offering advantages such as convenient administration, high patient compliance, and no need for specialized medical procedures, making it a core component of achieving "non-invasive treatment." In some embodiments of the present invention, the oral dosage form may be, but is not limited to, solid dosage forms such as tablets, capsules, granules, powders, pills, and lozenges, or liquid dosage forms such as oral solutions, syrups, oral suspensions, and oral emulsions.

[0022] Preferably, in the above applications, the oral dosage form is selected from tablets, capsules, granules, powders, pills, lozenges, oral solutions, syrups, oral suspensions, or oral emulsions. Tablets are solid dosage forms made by mixing and compressing drugs and excipients; they can be further processed into film-coated tablets, enteric-coated tablets, sustained-release tablets, or controlled-release tablets to improve stability, taste, or regulate release behavior. Capsules are dosage forms in which drugs are filled into empty capsules or sealed in soft capsule materials, which can mask unpleasant drug odors. Granules and powders are dry granular or powdered preparations made from drugs and excipients, facilitating dosage dispensing and swallowing. Pills and lozenges are traditional forms of solid dosage forms. Oral solutions are clear liquid preparations formed by dissolving drugs in suitable solvents; syrups are concentrated oral solutions containing high concentrations of sucrose or sweeteners; oral suspensions are heterogeneous systems formed by dispersing poorly soluble solid drugs in a liquid medium; and oral emulsions are oral preparations formed by emulsifying two immiscible liquids.

[0023] The present invention also provides the use of a pharmaceutical composition in the preparation of a medicament for treating benign prostatic hyperplasia, said pharmaceutical composition containing minocycline or a pharmaceutically acceptable salt thereof as an active ingredient, and at least one pharmaceutically acceptable carrier or excipient.

[0024] The term "pharmaceutical composition" refers to a formulation containing an active ingredient and typically mixed with one or more inert, non-toxic carriers or excipients. Its purpose is to provide a pharmaceutically elegant and easily administered form. The term "carrier or excipient" is a collective term for all other components in a pharmaceutical formulation besides the active ingredient; these components themselves typically do not possess therapeutic activity but are crucial for imparting the necessary physicochemical properties to the formulation, ensuring manufacturing feasibility, stability, and in vivo behavior.

[0025] Preferably, in the above applications, benign prostatic hyperplasia is accompanied by interstitial fibrosis. Preferably, in the above applications, the excipients include fillers, binders, disintegrants, or lubricants. In the pharmaceutical compositions of the present invention, particularly in solid oral dosage forms, commonly used excipients include, but are not limited to, the following categories: Fillers (or diluents): These are used to increase the weight of tablets or the volume of capsule contents to ensure the appropriate size for easy production and accurate single-dose formulation. Common fillers include, but are not limited to: sugars such as lactose, sucrose, and mannitol; cellulose derivatives such as microcrystalline cellulose; starches and their derivatives such as pregelatinized starch and corn starch; and inorganic salts such as dicalcium phosphate.

[0026] Binders: Used to impart viscosity to powders during granulation, promoting particle formation, or to enhance powder cohesion in direct tableting. Common binders include, but are not limited to: cellulose derivatives such as hydroxypropyl methylcellulose and hydroxypropyl cellulose; polymers such as polyvinylpyrrolidone; and natural products such as starch paste and gelatin.

[0027] Disintegrants: These are substances used to rapidly disintegrate tablets or capsules into small particles upon contact with water after oral administration, promoting drug dissolution and absorption. Common disintegrants include, but are not limited to: croscarmellose sodium, carboxymethyl starch sodium, low-substituted hydroxypropyl cellulose, croscarmellose (croscarmellose), and natural starch.

[0028] Lubricants: Used to reduce friction between granules or powders and the mold, ensuring smooth tableting or filling processes and preventing tablet sticking. Common lubricants include, but are not limited to: magnesium stearate, sodium stearate fumarate, talc, and colloidal silica.

[0029] Preferably, in any of the above applications, the daily dose of the drug, calculated as minocycline, is 50 mg to 200 mg. Dosage range is a key parameter to ensure drug safety and efficacy. In this invention, considering the need for long-term medication for treating chronic diseases, the daily dose, calculated as the active ingredient minocycline, is 50 mg to 200 mg. More preferably, the daily dose may be 60 mg to 150 mg, for example, about 100 mg. Even more preferably, the daily dose is 100 mg, which provides good therapeutic effect within a safe range. The daily dose may be given as a single dose, or divided into two doses (e.g., 50 mg each time), or given multiple times.

[0030] The present invention also provides a method for screening or evaluating candidate drugs for treating benign prostatic hyperplasia, comprising establishing an in vitro or in vivo screening model based on the ZC3H12A-Th1-IFNγ-JAK / STAT signaling axis, and using the model to test the activity of the candidate drug; the establishment of the screening model includes constructing a prostate cell model with downregulated ZC3H12A expression or loss of function; the prostate cell model comprises prostate basal cells; the testing of the activity of the candidate drug includes detecting the effect of the candidate drug on the expression or activity of at least one molecule among ZC3H12A, IFNγ, KRT14, ATF3, GPX3, STAT1, or STAT3 in the signaling axis.

[0031] This method provides a drug discovery platform based on a specific pathological mechanism (ZC3H12A-Th1-IFNγ-JAK / STAT axis). Establishing a "screening model" is a standard technique in this field; in vitro models can use cultured cell lines, while in vivo models can use genetically engineered animals or chemically induced disease animal models. "Constructing prostate cell models with downregulated or absent ZC3H12A expression" can be achieved through RNA interference techniques (such as transfection with shRNA or siRNA), gene editing techniques (such as CRISPR / Cas9 knockout), or the use of specific inhibitors. "Prostate basal cells" are stem cell / progenitor cell populations in the prostate epithelium, which can be isolated and cultured from prostate tissue using primary culture, or using immortalized human prostate epithelial cell lines (such as BPH-1 or RWPE-1). The aforementioned "detection of the effect on expression or activity" is a routine method in pharmacological research. For example, changes in gene or protein expression levels can be detected by quantitative polymerase chain reaction (qPCR) or Western blotting; the concentration of cytokines (such as IFNγ) in cell culture supernatant or serum can be detected by enzyme-linked immunosorbent assay (ELISA); and the activation (phosphorylation) status of signaling proteins (such as STAT1 and STAT3) can be detected by phosphorylation-specific antibodies.

[0032] This invention also provides a kit for screening or evaluating candidate drugs for the treatment of benign prostatic hyperplasia, the kit comprising reagents for detecting the expression or activity of at least one molecule, ZC3H12A, IFNγ, KRT14, ATF3, GPX3, STAT1, or STAT3. The "kit" is a product pre-packaged with the main reagents necessary for the above-mentioned detection, and may include specific primers (for qPCR), specific antibodies (for Western blotting, ELISA, or immunohistochemistry), detection substrates, buffers, standards, and instructions for use. For example, a kit for detecting ZC3H12Am RNA expression may contain qPCR primer pairs targeting the ZC3H12A gene and an internal reference gene (such as GAPDH), reverse transcription reagents, and qPCR premix. The use of this kit facilitates standardized procedures and promotes the widespread adoption and application of this screening method in laboratories.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1. Single-cell transcriptome sequencing (scRNA-seq) and analysis: Sample source: With the approval of the ethics committee, fresh surgical resection tissue samples were collected from 6 patients with clinically and pathologically confirmed benign prostatic hyperplasia (BPH) (derived from transurethral resection of the prostate, TURP) and 3 normal prostate tissue samples (derived from normal prostate tissue from brain-dead patients) as controls.

[0035] Sequencing platform and reagents: After the tissue was prepared into a single-cell suspension by enzymatic digestion, a single-cell sequencing library was constructed using the 10x Genomics Chromium platform, and sequencing was performed using an Illumina NovaSeq 6000 sequencer.

[0036] Data analysis standard: Use the Seurat package in R language for data dimensionality reduction and cluster analysis.

[0037] Quality control standard: Filter out low-quality cells with mitochondrial gene content >10% or gene count <200.

[0038] Key gene screening: The screening threshold for differentially expressed genes (DEGs) was set to |log₂FC|>1 and adjusted P<0.05. Cell subpopulation definition: Identifying cell types based on classical markers, with particular attention to subpopulations in epithelial cells and the infiltration ratio of Th1 cells (CD4+ / IFNγ+).

[0039] 2. Tissue transcriptome sequencing (RNA-seq): With the approval of the ethics committee, fresh surgically removed tissue samples (derived from transurethral resection of the prostate (TURP)) from 8 patients with clinically and pathologically confirmed benign prostatic hyperplasia (BPH) and 8 normal prostate tissue samples (derived from normal prostate tissue adjacent to radical cystectomy) were collected as controls. The raw sequencing data were first quality assessed using FastQC, followed by trimmomatic removal of adapters and low-quality bases. Next, clean reads were mapped to the human reference genome GRCh38 (hg38) using STAR alignment software, and gene annotation was performed based on the Ensembl database. Gene quantification was performed using featureCounts to obtain read counts, followed by differential expression analysis using the R package DESeq2. The selection criteria for differentially expressed genes (DEGs) were p-value < 0.05 and |log2FC| > 0.5. Finally, the clusterProfiler package was used to perform Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on the DEGs to explore their potential biological functions. Genes that also appeared in the results of Weighted Gene Co-expression Network (WGCNA) analysis were identified as candidate DEGs. Subsequently, the glmnet, caret, and xgboost packages in R were used to analyze these genes using three different machine learning algorithms: Least Absolute Shrinkage and Selection Operator (LASSO), Support Vector Machine-Recursive Feature Elimination (SVM-RFE), and XGBoost (selected based on the top 5 gain values). All three algorithms ultimately identified ZC3H12A as a common candidate differentially expressed gene.

[0040] 3. Expression detection methods: qRT-PCR detection: Total RNA was extracted from prostate tissue, reverse transcribed, and amplified using the SYBR Green method. GAPDH was used as an internal reference gene, and the relative expression level of ZC3H12A mRNA was calculated using the 2^ method.

[0041] Immunofluorescence (IF) and immunohistochemistry (IHC): Antibody information: ZC3H12A (dilution ratio 1:200, brand: Invitrogen, catalog number: PA5-22137), KRT14 (dilution ratio 1:100, brand: Invitrogen, catalog number: MA5-32214), Vimentin (dilution ratio 1:200, brand: Invitrogen, catalog number: OMA1-06001), E-cadherin (dilution ratio 1:100, brand: Invitrogen, catalog number: PA5-32178), CD4 (dilution ratio 1:100, brand: Invitrogen, catalog number: 14-2444-80), IFNγ (dilution ratio 1:200, brand: Invitrogen, catalog number: PA5-95560).

[0042] Secondary Antibody Information: Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 - A-11001 (Dilution ratio 1:1000, Brand: Invitrogen, Catalog Number: A11001), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568 - A-11011 (Dilution ratio 1:1000, Brand: Invitrogen, Catalog Number: A11011) Quantitative analysis: Five high-power fields (×400) were selected from each group, and the number of positive cells was counted using ImageJ software.

[0043] Sample groups: Normal control group (Normal, n=4), Benign prostatic hyperplasia group (BPH, n=4), Minocycline treatment group (Mino N=4) 4. Rat model of benign prostatic hyperplasia and drug intervention: Animal model establishment: Eight-week-old male SD rats were randomly divided into groups. The model group was induced by testosterone propionate (TP) by subcutaneous injection of testosterone propionate (dose: 20 mg / kg) daily for 4 consecutive weeks to establish a stable prostatic hyperplasia model.

[0044] Dosage regimen: Groups: sham surgery group (Normal), BPH model group (BPH), and minocycline treatment group (Mino).

[0045] Dosage and frequency: The treatment group received minocycline via gavage daily at a dose of 20 mg / kg / day for 4 consecutive weeks concurrently with model establishment. The model group received an equal volume of corn oil, as detailed below: Experimental materials Test drug: Minocycline hydrochloride, purchased from MedChemExpress (MCE), product number HY-17412, purity >98%.

[0046] Solvent: Medical-grade corn oil Dissolution aid: CNC ultrasonic cleaner (200W power, 40kHz frequency).

[0047] Drug preparation Weighing in the dark: Before each experiment, in a dark environment, accurately calculate and weigh the required amount of minocycline hydrochloride powder based on the total weight of the rats that day, and place it in a light-protected centrifuge tube.

[0048] Oil phase mixing: Add a predetermined volume of medical-grade corn oil to the centrifuge tubes to make the final mixture contain a concentration of minocycline hydrochloride of 4 mg / mL.

[0049] Ultrasonic dissolution and dispersion: Centrifuge tubes were placed in an ultrasonic cleaner and ultrasonically treated at 30-35°C for 30 minutes, with each tube removed and vibrated for 10 seconds every 10 minutes. Ultrasonic energy was used to fully refine the minocycline hydrochloride particles and uniformly disperse them in corn oil, resulting in a homogeneous drug suspension.

[0050] State maintenance: Administer the drug immediately after preparation, and during administration (such as when changing the rat interval), manually shake or vortex for 30 seconds every 5 minutes to prevent drug particles from settling.

[0051] Dosing regimen Dosage: Administer 20 mg / kg / day based on the rat's body weight.

[0052] Administration route and volume: Daily intraperitoneal injection. The administration volume is 5 mL / kg (i.e., 1 mL of drug suspension is injected daily into a 200 g rat).

[0053] Dosing cycle: Dosing was administered daily for a total of 28 days (4 weeks) from the date of model establishment.

[0054] Control group treatment: The model group (and the blank control group) were given the same volume (5 mL / kg) of medical-grade corn oil daily.

[0055] Prostate volume measurement: After the experiment, the rats were sacrificed and the prostate tissue was completely dissected.

[0056] Wet weight measurement: The wet weight of the prostate was measured using a precision balance.

[0057] Prostate Index (PI): The formula is PI = wet weight of prostate (mg) / body weight of rat (g).

[0058] Experimental results: (1) Characterization of pathological mechanisms ( Figure 1 and Figure 2 ): Flow cytometry detection of Th1 cell infiltration: Single-cell suspension preparation: Fresh human or rat prostate tissue was collected, washed with pre-cooled PBS to remove blood accumulation, and cut into 1 mm³ pieces. The tissue was placed in a digestion solution containing type I collagenase (1 mg / mL) and DNase I (0.1 mg / mL) and digested in a shaker at 37°C for 45–60 minutes. After digestion, the cells were filtered through a 70 μm cell sieve, centrifuged, and the supernatant was discarded. Red blood cells were lysed and resuspended in PBS to prepare a single-cell suspension. Antibody staining: Cells were collected, and surface marker antibodies (anti-CD45, anti-CD3, anti-CD4) were added and incubated at 4°C in the dark for 30 minutes. After washing, fixation / permeabilization solution was added and incubated at room temperature for 20 minutes for intracellular penetration. Finally, intracellular cytokine antibody (anti-IFN-γ) was added and incubated at room temperature in the dark for 30 minutes. Analytical instrument: Navios flow cytometer (Beckman Coulter). Data processing and analysis were performed using Kaluza analysis software version 1.4 (Beckman Coulter) for data acquisition.

[0059] Pseudo-temporal developmental trajectory analysis: To investigate the transdifferentiation of prostate basal cells into fibroblasts, single-cell trajectories were constructed using the Monocle 2 package in R. First, Seurat objects were converted to CellDataSet objects, and the differentialGeneTest function was used to filter for differentially ordered genes defining the cell process. Then, the DDRTree algorithm was used for dimensionality reduction, constructing a minimum spanning tree and visualizing the pseudo-temporal trajectory of the cells to analyze the evolutionary path of cell states with changes in pseudotime values.

[0060] Gene set enrichment analysis (GSEA): To reveal functional differences among different groups or cell subpopulations, GSEA analysis was performed using the clusterProfiler package in R. The list of differentially expressed genes was sorted from highest to lowest log2 FC value and compared with Hallmark gene sets (e.g., EMT-related gene sets) or KEGG pathway gene sets (e.g., JAK-STAT signaling pathway) in the MSigDB database. An adjusted p-value (p.adjust) < 0.05 and a false detection rate (FDR) < 0.25 were used as criteria for significant pathway enrichment.

[0061] Histopathological staining (IHC and IF): Prostate tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (4 μm thick). After dewaxing in xylene and rehydration with graded ethanol, the sections were placed in antigen retrieval solution (pH 6.0 or pH=9.0) for high-temperature and high-pressure antigen retrieval. After natural cooling, the sections were blocked with 5% goat serum or BSA at room temperature for 30 minutes.

[0062] (1) Immunohistochemistry (IHC): The slides were incubated overnight at 4°C with primary antibodies (such as E-cadherin or Vimentin). The next day, HRP-labeled secondary antibodies were added and incubated at room temperature for 1 hour. The slides were then developed using a DAB staining kit, and the cell nuclei were counterstained with hematoxylin. After dehydration and clearing, the slides were mounted and examined under a microscope.

[0063] (2) Immunofluorescence (IF): Sections were incubated overnight at 4°C with primary antibodies (such as ZC3H12A, Vimentin, CD4, KRT14, etc.). The next day, secondary antibodies with fluorescent labels (Alexa Fluor 488 and 568) were added and incubated at room temperature in the dark for 1 hour. The sections were mounted using anti-fluorescence quenching mounting medium containing DAPI. Images were acquired using a Niko fluorescence microscope, and colocalization analysis or fluorescence intensity quantification was performed using ImageJ software.

[0064] The results showed that flow cytometry was used to validate clinical samples ( Figure 1 EF), the results confirmed that the proportion of CD4+IFNγ (Th1) cell infiltration in the prostate tissue of BPH patients was significantly higher than that in the normal group (P<0.001), indicating that the Th1 immune microenvironment is a key factor driving BPH. Mechanistic analysis showed ( Figure 2 AG), Th1 cells activate the JAK-STAT signaling pathway by secreting IFNγ, which acts on epithelial cell receptors. Immunohistochemistry ( Figure 2F) Visually confirmed that the epithelial marker E-cadherin was absent in BPH tissue, while the mesenchymal marker Vimentin was upregulated, in conjunction with GSEA analysis ( Figure 2 G) clarified the process by which the "immune-epithelial" interaction induces EMT.

[0065] (2) Cell transdifferentiation trajectory ( Figure 3 ): Pseudo-time-series trajectory analysis ( Figure 3 The CD (corticotropic fibroblast) dynamics revealed the evolution of basal cell fate. With increasing pseudotime values, the expression of basal cell markers KRT7 and KRT14 gradually decreased, while the expression of profibrotic fibroblast markers ATF3 and GPX3 significantly increased. This result confirms at the single-cell level that prostate basal cells undergo trans-lineage transdifferentiation under inflammatory stress, forming pathogenic fibroblasts.

[0066] (3) Target screening and validation Figure 4 ): By integrating three machine learning algorithms ( Figure 4 C), precisely identifying ZC3H12A as the core gene from key modules of WGCNA. ROC analysis showed a diagnostic AUC as high as 0.875 ( Figure 4 D), possessing excellent biomarker potential. qRT-PCR ( Figure 4 F) Further confirmation showed that ZC3H12A was significantly underexpressed in BPH tissue (P<0.01), and its expression level was negatively correlated with the degree of Th1 cell infiltration, suggesting that its absence as a negative regulatory factor is the key to the pathological process.

[0067] (4) In vivo drug efficacy and volume reduction ( Figure 5 ): In rat in vivo experiments ( Figure 5 In the BC group, the prostate volume was significantly increased and congestion was severe; while in the minocycline intervention group, the prostate volume showed a visible and significant shrinkage. Statistical data showed ( Figure 5 C), minocycline significantly reduced the prostate index (P<0.01). Molecular level validation showed that ( Figure 5 Minocycline effectively upregulated the expression of ZC3H12A and simultaneously inhibited serum IFNγ levels and the infiltration of Th1 cells in tissues, fully demonstrating its efficacy in reversing prostate fibrosis and reducing glandular volume by regulating the "ZC3H12A-Th1 axis".

[0068] Molecular and pathological improvement: such as Figure 5As shown in D and 5F, minocycline treatment significantly restored ZC3H12A mRNA expression in prostate tissue (P<0.05), accompanied by a significant decrease in the proportion of Th1 cell infiltration in the tissue (P<0.01). These results confirm that minocycline effectively reverses the progression of benign prostatic hyperplasia through the mechanism of "upregulating ZC3H12A and inhibiting Th1 infiltration".

[0069] As demonstrated by the above embodiments, this invention provides complete experimental evidence, from the analysis of clinical pathological mechanisms to the verification of animal efficacy, proving that minocycline can significantly reverse the core pathological phenotype of benign prostatic hyperplasia. These experiments not only reveal a key immune-epithelial interaction axis and its core regulatory target in disease progression, but more importantly, they demonstrate in animal models that minocycline can effectively improve disease characteristics by regulating this target, thus providing a solid foundation for its therapeutic application.

[0070] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of minocycline or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of benign prostatic hyperplasia.

2. The application according to claim 1, characterized in that, The benign prostatic hyperplasia was accompanied by interstitial fibrosis.

3. The application according to claim 1 or 2, characterized in that, The drug is prepared into an oral dosage form.

4. The application according to claim 3, characterized in that, The oral dosage form is selected from tablets, capsules, granules, powders, pills, lozenges, oral solutions, syrups, oral suspensions, or oral emulsions.

5. The use of a pharmaceutical composition in the preparation of a medicament for treating benign prostatic hyperplasia, characterized in that, The pharmaceutical composition contains minocycline or a pharmaceutically acceptable salt thereof as the active ingredient, and at least one pharmaceutically acceptable carrier or excipient.

6. The application according to claim 5, characterized in that, The benign prostatic hyperplasia was accompanied by interstitial fibrosis.

7. The application according to claim 5, characterized in that, The excipients include fillers, binders, disintegrants, or lubricants.

8. The application according to any one of claims 1 to 7, characterized in that, The daily dose of the drug, calculated as minocycline, is 50 mg to 200 mg.

9. A method for screening or evaluating candidate drugs for the treatment of benign prostatic hyperplasia, characterized in that, This includes establishing an in vitro or in vivo screening model based on the ZC3H12A-Th1-IFNγ-JAK / STAT signaling axis, and using the model to test the activity of the candidate drugs; The establishment of the screening model includes constructing a prostate cell model with downregulated ZC3H12A expression or loss of function; The prostate cell model includes prostate basal cells; The testing of the candidate drug activity includes detecting the effect of the candidate drug on the expression or activity of at least one molecule among ZC3H12A, IFNγ, KRT14, ATF3, GPX3, STAT1, or STAT3 in the signal axis.

10. A kit for screening or evaluating candidate drugs for the treatment of benign prostatic hyperplasia, characterized in that, The kit contains reagents for detecting the expression or activity of at least one molecule selected from ZC3H12A, IFNγ, KRT14, ATF3, GPX3, STAT1, or STAT3.

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