Use of pyruvate carboxylase in diagnosis and treatment of endometriosis

Abstract

The application discloses pyruvate carboxylase (PC) as a biomarker for detecting endometriosis and application of the biomarker in treating endometriosis. The biomarker can be used for detecting the expression level of PC in an endometrial tissue sample by an immunohistochemical method, and when the IHC score is greater than or equal to 4, the subject is determined to be at high risk of or suffering from endometriosis. Meanwhile, the application provides application of a drug (including specific siRNA, ZY-444, octyl gallate and the like) for inhibiting the expression of PC in treating endometriosis and a corresponding treatment method. The application provides a new biomarker, target and scheme for detecting and treating endometriosis, and has important clinical application value.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to diagnostic biomarkers and targeted treatment methods for endometriosis, and particularly to the application of pyruvate carboxylase (PC) in the detection and treatment of endometriosis. Background Technology

[0002] Endometriosis is a gynecological disease characterized by the colonization, growth, and infiltration of endometrial-like tissue outside the uterine cavity. Current theories regarding the pathogenesis of endometriosis mainly include the retrograde menstruation theory, endometrial stem cell implantation, and coelomic metaplasia theory. In addition, inflammatory factors, immune regulation and hormonal abnormalities, environmental factors, and genetic and epigenetic factors are also involved. Treatment for endometriosis currently includes traditional and emerging drugs. Traditional drugs are based on inhibiting ovulation, blocking menstruation, and maintaining a stable endocrine hormonal environment, including: ① gonadotropin-releasing hormone agonists that create a low-estrogenic environment; ② danazol and gestrinone that create a high-androgen environment; ③ oral contraceptives and progesterone that create a high-progesterone environment. However, these drugs only temporarily relieve symptoms; the lesions regain metabolic activity after treatment is stopped, and these drugs are not suitable for women who wish to conceive naturally. Emerging drugs mainly include: ① anti-angiogenic drugs: such as TNP-470; ② cell proliferation inhibitors: such as statins; ③ demethylating agents and histone deacetylase inhibitors; ④ anti-inflammatory drugs and immunomodulators, etc. Most emerging drugs are still in the exploratory stage and lack clearly defined therapeutic targets. Therefore, it is urgent to explore the pathogenesis and potential targets in existing technologies to provide direction for the development of emerging drugs. Summary of the Invention

[0003] Pyruvate carboxylase (PC) is an important metabolic enzyme that is abnormally expressed in many diseases, but its role in endometriosis has not been studied. This application aims to provide a new target for the development of innovative drugs for endometriosis by studying the expression of pyruvate carboxylase in endometriosis, thereby addressing the shortcomings of existing technologies.

[0004] Therefore, in a first aspect, this application provides a biomarker for the detection of endometriosis, said biomarker being pyruvate carboxylase (PC).

[0005] A second aspect of this application provides a method for detecting endometriosis using the aforementioned biomarker, namely pyruvate carboxylase (PC).

[0006] Furthermore, the method includes the following steps:

[0007] S1: Collect endometrial tissue samples from the subject;

[0008] S2: Immunohistochemistry (IHC) was used to detect the expression level of pyruvate carboxylase in the samples;

[0009] S3: Based on the immunohistochemical (IHC) score of pyruvate carboxylase protein obtained from S1, determine whether the sample is endometriosis.

[0010] Furthermore, in step S3, when the immunohistochemical staining intensity score of pyruvate carboxylase protein is ≥4, it indicates that the subject has a high risk of endometriosis or has endometriosis.

[0011] Furthermore, for the subjects who were at high risk of endometriosis or who had endometriosis, pyruvate carboxylase expression inhibitors were administered to treat endometriosis.

[0012] A third aspect of this application provides the use of a reagent for detecting pyruvate carboxylase expression levels in the preparation of a kit for diagnosing endometriosis.

[0013] A fourth aspect of this application provides the use of an active ingredient that inhibits pyruvate carboxylase or reduces the expression level of pyruvate carboxylase in the preparation of a medicament for treating endometriosis.

[0014] Furthermore, the active ingredient is a nucleic acid molecule, protein, and / or compound.

[0015] Furthermore, the compound is ZY-444 and / or octyl gallate.

[0016] Furthermore, the nucleic acid molecule is RNAi, siRNA, and / or shRNA.

[0017] Furthermore, the nucleic acid molecule is siRNA, and the siRNA is siRNA#1 or siRNA#2.

[0018] The positive strand sequence of siRNA#1 is: 5'-CACCGGCAGAAAGCAGAUGAATT-3' (SEQ ID NO.1);

[0019] The antisense strand sequence of siRNA#1 is: 5'-UUCAUCUGCUUUCUGCCGGUGTT-3' (SEQ ID NO.2);

[0020] The positive strand sequence of siRNA#2 is: 5'-UGGGAACAUCCUGCACCUGUATT-3' (SEQ ID NO.3);

[0021] The antisense strand sequence of siRNA#2 is: 5'-UACAGGUGCAGGAUGUUCCCATT-3' (SEQ ID NO.4).

[0022] In some embodiments, siRNA#1 is used to reduce the expression level of pyruvate carboxylase (PC).

[0023] In some embodiments, siRNA#2 is used to reduce the expression level of pyruvate carboxylase (PC).

[0024] In some embodiments, siRNA#1 and siRNA#2 are used simultaneously to reduce the expression level of pyruvate carboxylase (PC).

[0025] In some embodiments, a medicine containing the above-described active ingredient is administered to the patient.

[0026] Furthermore, the administration method includes intraperitoneal injection, oral administration, or local administration, preferably oral administration.

[0027] Furthermore, the drug is compound ZY-444 and / or Octyl gallate.

[0028] In some embodiments, the drug is compound ZY-444, the administration concentration of ZY-444 is 0.5-1 μM, the administration dose for mouse models is 2.5-5 mg / kg, and the recommended administration dose for patients is 10-20 mg / time, administered orally once daily.

[0029] In some embodiments, the drug is octyl gallate, the administration concentration of which is 5-10 μM, the administration dose in mouse models is 25-50 mg / kg, and the recommended administration dose for patients is 250-500 mg / time, administered orally once daily.

[0030] In some embodiments, the drug is a nucleic acid molecule siRNA, wherein the siRNA is siRNA#1 and / or siRNA#2.

[0031] The positive strand sequence of siRNA#1 is: 5'-CACCGGCAGAAAGCAGAUGAATT-3' (SEQ ID NO.1);

[0032] The antisense strand sequence of siRNA#1 is: 5'-UUCAUCUGCUUUCUGCCGGUGTT-3' (SEQ ID NO.2);

[0033] The positive strand sequence of siRNA#2 is: 5'-UGGGAACAUCCUGCACCUGUATT-3' (SEQ ID NO.3);

[0034] The antisense strand sequence of siRNA#2 is: 5'-UACAGGUGCAGGAUGUUCCCATT-3' (SEQ ID NO.4).

[0035] The pyruvate carboxylase (PC) provided in this application, as a biomarker for endometriosis and its related detection and treatment applications, has the following beneficial effects:

[0036] 1. Significantly Improved Diagnostic Accuracy and Specificity: This application is the first to discover that pyruvate carboxylase (PC) can serve as a specific biomarker for endometriosis, filling a gap in the molecular diagnostics of this disease. By detecting the expression level of PC in endometrial tissue using immunohistochemistry (IHC) and employing a clear IHC score (≥4 points) as the diagnostic criterion, objective and quantitative diagnosis of endometriosis is achieved. Compared to existing diagnostic methods that rely on symptom observation or imaging examinations, this method can identify lesions earlier and more accurately, reducing misdiagnosis and missed diagnosis, and buying valuable time for clinical intervention.

[0037] 2. Simple and easy-to-perform detection method: The detection method of this application only requires the collection of endometrial tissue samples from the subject, and the detection can be completed using mature immunohistochemical technology. The operation process is simple and low-cost, requiring no complex equipment or high-end technology platform, making it easy to promote and apply in medical institutions at all levels. At the same time, the quantitative scoring standard avoids subjective judgment differences, improves the consistency and reliability of test results, and is conducive to the establishment of a standardized diagnosis and treatment system.

[0038] 3. More Significant Efficacy of Targeted Therapy: This application is the first to identify PC as a therapeutic target for endometriosis. Inhibitory drugs developed for PC (including specific siRNAs, ZY-444, octyl gallate, etc.) can specifically block PC expression or activity, inhibiting the colonization, growth, and invasion of ectopic endometrial tissue at the molecular level, fundamentally improving the metabolic activity of the lesions rather than merely alleviating symptoms. Compared with traditional drugs, this targeted therapy can more effectively shrink or eliminate lesions, reduce the recurrence rate after drug withdrawal, and has less impact on ovarian function, making it more suitable for female patients who wish to conceive naturally.

[0039] This application combines the diagnostic value and therapeutic application of pyruvate carboxylase, forming a complete closed loop of "diagnosis-targeted therapy." After a definitive diagnosis is made by detecting PC expression levels, treatment can be directly initiated with PC-inhibiting drugs, achieving personalized and precise treatment. This integrated approach not only improves treatment efficiency but also reduces the side effects of indiscriminate medication, providing clinicians with a more systematic and efficient treatment strategy, and is of great significance for improving the overall level of diagnosis and treatment of endometriosis. Attached Figure Description

[0040] To more clearly illustrate the embodiments of this application, the accompanying drawings used in this application will be briefly described below. Unless otherwise specified, the asterisks representing statistical significance levels in the accompanying drawings of this invention have the following meanings: *P<0.05, **P<0.01, ***P<0.001.

[0041] Figure 1 Immunohistochemical staining revealed the expression levels of pyruvate carboxylase in normal endometrial tissue, endometrial tissue from patients with endometriosis, and ectopic ovarian tissue.

[0042] Figure 2. Statistical analysis of pyruvate carboxylase histochemical staining scores in three tissues.

[0043] Figure 3 Based on the expression of pyruvate carboxylase in the endometrial tissues of normal controls and endometriosis patients, receiver operating characteristic (ROC) curves were plotted to test the diagnostic value of pyruvate carboxylase for endometriosis.

[0044] Figure 4 The expression levels of pyruvate carboxylase in the endometrium of normal endometrium and endometriosis patients were compared in a centralized transcriptometrial dataset (GSE51981).

[0045] Figure 5 The expression levels of pyruvate carboxylase were compared in a centralized transcriptome dataset between normal endometrial stromal cells and ectopic stromal cells (GSE58178).

[0046] Figure 6 Pyruvate carboxylase promotes the progression of endometriosis in vitro. Small interfering RNA (siRNA) was transfected into immortalized endometriotic stromal cells (ihESCs), and the knockdown efficiency was verified at both RNA and protein levels using RT-qPCR and Western blotting.

[0047] Figure 7 The CCK-8 assay was used to detect the effect of knockdown of pyruvate carboxylase on the proliferation of ihESCs.

[0048] Figure 8The clone formation assay was used to examine the effect of knockdown of pyruvate carboxylase on the clone formation ability of ihESCs.

[0049] Figure 9 Statistical analysis of clone formation experimental results.

[0050] Figure 10 The wound healing assay was used to investigate the effect of knockdown of pyruvate carboxylase on the migration of ihESCs.

[0051] Figure 11 Statistical analysis of wound healing experiment results.

[0052] Figure 12 Transwell migration assays were used to investigate the effect of knockdown of pyruvate carboxylase on the migration of ihESCs.

[0053] Figure 13 Statistical analysis of Transwell migration experiment results.

[0054] Figure 14 Principal component analysis reveals the overall distribution trend of metabolomics test results in the PC knockdown group and the control group.

[0055] Figure 15 Volcano plots show the differential metabolites between the PC knockdown group and the control group, with VIP > 1.0, FC > 1.5 or FC < 0.667 and P < 0.05 as the cutoff points.

[0056] Figure 16 KEGG analysis revealed the pathways to which differentially metabolites were primarily enriched.

[0057] Figure 17 Comparison of the abundance of major metabolites in the glycolysis pathway between the PC knockdown group and the control group.

[0058] Figure 18 Pyruvate carboxylase was knocked down in ihESCs, and Western blotting was used to detect changes in the expression of glycolytic metabolic enzymes.

[0059] Figure 19 IHC staining was performed using ectopic ovarian tissue from patients to compare the expression of glycolytic metabolic enzymes in the high PC expression group and the low PC expression group.

[0060] Figure 20 Using 2-deoxy-D-glucose to inhibit glycolysis, the CCK-8 assay compared the effect of pyruvate carboxylase overexpression on ihESC proliferation in the treated and untreated groups.

[0061] Figure 21Using 2-deoxy-D-glucose to inhibit glycolysis, a colony formation assay was conducted to compare the effect of pyruvate carboxylase overexpression on ihESC proliferation in the treated and untreated groups.

[0062] Figure 22 Statistical analysis of clone formation experimental results.

[0063] Figure 23 Using 2-deoxy-D-glucose to inhibit glycolysis, a wound healing experiment was conducted to compare the effect of pyruvate carboxylase overexpression on ihESC migration in the treated and untreated groups.

[0064] Figure 24 Statistical analysis of wound healing experiment results.

[0065] Figure 25 Using 2-deoxy-D-glucose to inhibit glycolysis, Transwell migration assays were performed to compare the effect of pyruvate carboxylase overexpression on ihESC migration in the treated and untreated groups.

[0066] Figure 26 Statistical analysis of Transwell migration experiment results.

[0067] Figure 27 Adeno-associated virus (AAV) was used to overexpress pyruvate carboxylase in mouse endometrial cells to construct an allogeneic endometriosis model. The mice were sacrificed 3 weeks after model construction, and the endometriotic foci were removed.

[0068] Figure 28 Images of intramyolesions in mice with AAV overexpression of PC (n=5) and AAV control (n=5).

[0069] Figure 29 Comparison of the volume of ectopic foci in the two groups of mice.

[0070] Figure 30 Comparison of the quality of intralesional foci between the two groups of mice.

[0071] Figure 31 Immunohistochemical results of heterofocal pyruvate carboxylase, HK2, PFKFB3 and PKM2 in two groups of mice.

[0072] Figure 32 After treating ihESCs with different concentrations of octyl gallate for 48 hours, cell viability was detected using a CCK-8 assay.

[0073] Figure 33 After treating ihESCs with different concentrations of ZY-444 for 48 hours, cell viability was detected using a CCK-8 assay.

[0074] Figure 34 The colony formation assay was used to examine the effects of octyl gallate (5 μM) and ZY-444 (0.5 μM) treatment on the proliferation capacity of ihESCs.

[0075] Figure 35 Scratch assay and Transwell migration assay were used to detect the effect of octyl gallate (5 μM) on the migration ability of ihESCs.

[0076] Figure 36 Scratch and Transwell migration assays were used to examine the effect of ZY-444 (0.5 μM) on the migration ability of ihESCs.

[0077] Figure 37 A mouse model of allogeneic endometriosis was established (n=10). After 2 weeks, the mice were randomly divided into a treatment group and a control group. The treatment group received intraperitoneal injections of 2.5 mg / kg ZY-444 every 2 days, while the control group received an equal volume of the solvent. Two weeks after administration, the mice were euthanized, and the endometriotic lesions were removed and photographed.

[0078] Figure 38 Photos of abnormal lesions in the treatment group and the control group.

[0079] Figure 39 Comparison of the volume of ectopic foci in the two groups of mice.

[0080] Figure 40 Comparison of the quality of intralesional foci between the two groups of mice. Detailed Implementation

[0081] This application utilizes immunohistochemistry of patient tissue specimens and public database analysis to investigate the expression of pyruvate carboxylase and glycolytic metabolic enzymes in endometriosis, and to explore the diagnostic value of pyruvate carboxylase. Pyruvate carboxylase was knocked down or overexpressed in immortalized endometriotic stromal cells (ihESCs), and changes in proliferation were investigated using CCK-8 and colony formation assays, while changes in migration were investigated using wound healing and Transwell migration assays. Subsequently, targeted metabolomics and enrichment analysis were used to investigate the effect of pyruvate carboxylase knockdown on ihESC metabolism, and Western blotting was used to investigate the effect of pyruvate carboxylase on the expression of glycolytic metabolic enzymes. Glycolysis in ihESCs was inhibited using 2-deoxy-D-glucose, combined with pyruvate carboxylase overexpression, to investigate the role of glycolysis in pyruvate carboxylase-mediated endometriosis progression. Pyruvate carboxylase was overexpressed in the endometrium of C57BL / 6J mice using AAV, and the effect of pyruvate carboxylase on endometriosis in vivo was investigated using an allogeneic endometriosis model. The effects of pyruvate carboxylase inhibition on the progression of endometriosis in vitro and in vivo were investigated using the pyruvate carboxylase inhibitors ZY-444 and octyl gallate.

[0082] First, this application found that pyruvate carboxylase is elevated in both situ and ectopic tissues of patients with endometriosis and exhibits good diagnostic efficacy. Next, in vitro cell experiments showed that pyruvate carboxylase promotes the proliferation and migration of ihESCs, and in vivo animal experiments demonstrated that pyruvate carboxylase promotes the growth of endometriotic lesions. Then, targeted metabolomics and enrichment analysis revealed that pyruvate carboxylase promotes glycolysis of ihESCs, and the expression of pyruvate carboxylase is positively correlated with the expression of glycolytic metabolic enzymes HK2, PFKFB3, and PKM2. Using 2-deoxy-D-glucose to inhibit glycolysis, this application found that overexpression of pyruvate carboxylase no longer promotes the proliferation and migration of ihESCs.

[0083] Finally, pyruvate carboxylase inhibitors ZY-444 and octyl gallate inhibited the proliferation and migration of ihESCs, and ZY-444 also inhibited the growth of endometriotic lesions in mice. Therefore, pyruvate carboxylase is elevated in endometriosis and promotes endometriosis progression by enhancing glycolysis, making it a potential biomarker and therapeutic target.

[0084] Example 1

[0085] The purpose of this embodiment is to verify that the expression level of pyruvate carboxylase is significantly increased in patients with endometriosis and that it has good diagnostic efficacy.

[0086] To investigate the expression of pyruvate carboxylase in endometriosis patients, this study used immunohistochemistry (IHC) to stain 40 normal endometrial tissues, 40 endometrial tissues from endometriosis patients, and 30 ectopic ovarian tissues.

[0087] The staining method was as follows: tissue sections were dewaxed with xylene and dehydrated by a gradient of alcohols. Subsequently, the sections were immersed in antigen retrieval buffer (EDTA, pH 9.0) and heated to boiling for antigen retrieval. The sections were then incubated with 3% hydrogen peroxide for 30 minutes to block endogenous peroxidase activity, followed by 30 minutes of blockade with 5% bovine serum albumin. Next, diluted primary antibody was applied to the sections and incubated overnight at 4°C. The next day, horseradish peroxidase-conjugated secondary antibody was added and incubated at room temperature for 1 hour. Finally, staining was performed using DAB substrate solution (Proteintech, Cat#PR30010), and the cell nuclei were counterstained with hematoxylin (HaoKe, Cat#HK2053). IHC score = staining intensity (0-3) × percentage of positive cells (1-4).

[0088] Staining results showed that pyruvate carboxylase expression was elevated in the endometrial tissue of patients with endometriosis compared to normal endometrial tissue, and further increased in ovarian ectopic tissue (see Figures 1-2). Furthermore, the area under the receiver operating characteristic (AUC) curve for diagnosing endometriosis using pyruvate carboxylase expression in endometrial tissue reached 0.845. When the immunohistochemical score cutoff value was ≥4 points, the diagnostic sensitivity reached 77.5%, and the specificity reached 75% (see Figure 3).

[0089] Furthermore, this embodiment used the GSE51981 transcriptome dataset of endometrial tissue for analysis and found that the expression of pyruvate carboxylase in the endometrium of patients with endometriosis was higher than that in normal endometrium (see results). Figure 4 In addition, GSE58178 compared the RNA expression profiles of normal endometrial stromal cells and ectopic ovarian stromal cells, showing that pyruvate carboxylase expression was higher in the latter (see results). Figure 5 ).

[0090] Example 2

[0091] The purpose of this embodiment is to verify that pyruvate carboxylase can promote the progression of endometriosis in vitro.

[0092] To investigate the effect of pyruvate carboxylase on the progression of endometriosis, this study designed a siRNA targeting pyruvate carboxylase and transfected it into ihESCs. The knockdown efficiency was verified at both the RNA and protein levels (see results). Figure 6Two siRNAs were designed, siRNA#1 and siRNA#2, and their specific sequences are shown in the table below:

[0093] Table 1. Sequences of siRNAs that inhibit pyruvate carboxylase

[0094]

[0095] Next, the CCK-8 assay showed that knocking down pyruvate carboxylase significantly inhibited the proliferation of ihESCs (see results). Figure 7 The colony formation experiment showed that knocking down pyruvate carboxylase reduced colony formation (see results). Figure 8-9 ).

[0096] Furthermore, wound healing and Transwell migration assays revealed that knocking down pyruvate carboxylase weakened the migration ability of ihESCs (see results). Figure 10-13 ).

[0097] Example 3

[0098] This embodiment demonstrates through metabolic experiments that pyruvate carboxylase can enhance the glycolysis of ihESCs.

[0099] Since pyruvate carboxylase is an important metabolic enzyme, this example uses targeted metabolomics to investigate the effect of knocking down pyruvate carboxylase on the metabolism of ihESCs (using siRNA#1 to knock down the expression of pyruvate carboxylase, the siRNA#1 sequence is shown in Example 2), and 887 metabolites were identified.

[0100] The results for pyruvate carboxylase A showed a significant difference between the PC knockdown group (i.e., the group using siRNA #1 sequence) and the control group (see results). Figure 14 This indicates a significant difference in metabolic patterns between the two groups. Using VIP>1, FC>1 or <0.667, and P<0.05 as cutoffs, the PC knockdown group showed increased expression of 37 metabolites and decreased expression of 124 metabolites (see results). Figure 15 ).

[0101] KEGG enrichment analysis revealed that these changes were significantly associated with pathways such as arginine biosynthesis, purine metabolism, and glycolysis / gluconeogenesis (see results). Figure 16 ).

[0102] Since glycolysis / gluconeogenesis is an important metabolic pathway mediated by pyruvate carboxylase, this example analyzed the changes in metabolites in this pathway. Interestingly, this example found that knocking down pyruvate carboxylase significantly reduced the levels of many glycolytic intermediates, including glucose-6-phosphate, fructose-6-phosphate, and 3-phosphoglycerate (see results). Figure 17In addition, the glucose content in cells increased (see results). Figure 17 The decrease in glycolysis levels may lead to glucose accumulation. This result suggests that pyruvate carboxylase can enhance glycolysis in ihESCs.

[0103] Example 4

[0104] The purpose of this embodiment is to verify that pyruvate carboxylase can promote the expression levels of glycolysis-related metabolic enzymes and to further explore the effect of pyruvate carboxylase on glycolysis in endometriosis. To this end, this embodiment used siRNA (sequences see siRNA#1 and siRNA#2 in Example 2) to knock down pyruvate carboxylase in ihESCs, and found that the protein expression of glycolysis-related metabolic enzymes HK2, PFKFB3, and PKM2 was reduced (results see [link to example]). Figure 18 In this embodiment, IHC staining was performed on these metabolic enzymes in ectopic ovarian tissue specimens from patients, and it was found that the expression of pyruvate carboxylase, HK2, PFKFB3, and PKM2 were all positively correlated (see results). Figure 19 ).

[0105] Example 5

[0106] This embodiment uses lentiviral infection to overexpress pyruvate carboxylase in ihESCs; specifically, lentivirus (Genepharma) is used to overexpress PC in cells. (Total 1×10⁻⁶) 5 Cells were seeded into 6 cm culture dishes. After 24 hours, the culture medium was replaced with fresh medium and lentivirus (MOI=5) and HitransGP infection reagent (Gene Chem, Cat#REVG005) were added. Fresh medium was replaced again 24 hours after infection. PC overexpression efficiency was analyzed 96 hours after infection.

[0107] The purpose of this embodiment is to verify that pyruvate carboxylase promotes the progression of endometriosis in a glycolysis-dependent manner. To investigate whether pyruvate carboxylase's effect on endometriosis progression depends on glycolysis, this embodiment used 2-deoxy-D-glucose to block glycolysis in ihESCs, and simultaneously overexpressed pyruvate carboxylase using the aforementioned lentiviral infection method, observing the effect of pyruvate carboxylase overexpression on the phenotype. CCK-8 and colony formation experiments showed that even after overexpression of pyruvate carboxylase following the use of 2-deoxy-D-glucose, the proliferative capacity of ihESCs did not change (see results). Figure 20-22 Similarly, scratch assays and Transwell migration assays also showed that 2-deoxy-D-glucose treatment inhibited the effect of pyruvate carboxylase overexpression on the migration ability of ihESCs (see results). Figure 23-26 ).

[0108] Example 6

[0109] The purpose of this embodiment is to investigate the effect of pyruvate carboxylase on the progression of endometriosis through in vivo experiments.

[0110] To verify the effect of pyruvate carboxylase on the progression of endometriosis in an animal model, this study used female c57BL / 6J mice to construct an allogeneic endometriosis model. The mouse allogeneic endometriosis model was constructed as follows: 16 female C57BL / 6J mice (6 weeks old) were purchased from Hangzhou Medical College. Six mice were randomly selected and divided into two groups of three each. The PC-overexpressing group received a tail vein injection of 200 μL of PC-containing AAV9 per mouse, at a concentration of 5 × 10⁻⁶ μL. 12 Vg / mL; the control group received an equal volume of blank AAV via tail vein injection. One week after injection, both groups of mice received intramuscular injections of estradiol benzoate (150 μg / kg, every 2 days) for one week. Then, both groups of mice were euthanized, and the uteruses were removed and weighed to ensure a consistent total uterine mass between the two groups. Next, the uteruses of each group were cut into <1 mm fragments in warm saline, mixed thoroughly, and injected evenly into 5 recipient mice. Recipient mice received intramuscular injections of estradiol benzoate (150 μg / kg every 3 days) for one week starting on day 2 after model establishment. Three weeks after model establishment, mice were euthanized, and the mass and volume of endometriosis lesions were dissected and measured. Volume was calculated as length × width × width / 2.

[0111] The results showed that the volume and mass of endometriosis in the AAV-overexpressing PC group were significantly higher than those in the control group (see results). Figure 27-30 Furthermore, IHC results showed that PC, HK2, PFKFB3, and PKM2 were all elevated in the AAV-overexpressing PC group (see results). Figure 31 ).

[0112] Example 7

[0113] The above examples verified that pyruvate carboxylase can promote the progression of endometriosis. This example investigates the effects of two pyruvate carboxylase inhibitors, octyl gallate and ZY-444, on endometriosis. Octyl gallate (catalog number HY-N2011) and ZY-444 (catalog number HY-142870) were purchased from MedChemExpress.

[0114] Cells were seeded into 96-well plates at a density of 2000 cells / 100 μL. After the cells adhered, different concentrations of octyl gallate or ZY-444 were added. After incubation for 48 h, 10 μL of CCK-8 reagent was added to the wells, and the absorbance was measured at 450 nm using a microplate reader.

[0115] Forty-eight hours after the above treatment, 10 μM octyl gallate significantly induced the death of ihESCs (see results). Figure 32 ), while 1 μM ZY-444 can cause death in almost all ihESCs with octyl gallate and ZY-444 (see results). Figure 33 Colony formation experiments showed that both octyl gallate and ZY-444 significantly inhibited the proliferation of ihESCs, with almost no colony formation (see results). Figure 34 ).

[0116] The scratch assay method involves placing cells at a density of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 6-well plates. After 24 hours, cell density was observed. When the density approached 100%, the cell layer was vertically scraped off from the center of each well using a 200 μL pipette tip. The medium was then replaced with serum-free medium, and 5 μM octyl gallate or 0.5 μM ZY-444 was added. The initial scratch area (0 h) was photographed under a microscope, and the field of view was marked. After culturing in a cell incubator for 24 hours, the scratch area was photographed from the same field of view. Migration rate (%) = [(0 h scratch area - 24 h scratch area) / 0 h scratch area] × 100%.

[0117] The Transwell migration experiment method is as follows: 10 4 Cells were seeded into the upper chamber of a Trasnwell microplate chamber and cultured in FBS-free medium with 5 μM octyl gallate or 0.5 μM ZY-444 added. The lower chamber was cultured in medium containing 10% FBS. After 24 hours, the chambers were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. The remaining cells in the upper chamber were wiped off, and the cells that had passed through the chamber were photographed under a microscope and counted.

[0118] Scratch and transwell migration assays showed that both octyl gallate and ZY-444 could inhibit the migration ability of ihESCs (see results). Figures 35-36 Furthermore, in vivo experiments showed that ZY-444 significantly inhibited the growth of endometriosis lesions in mice (see results). Figure 37-40 ).

Claims

1. Use of a compound ZY-444 for inhibiting expression level of pyruvate carboxylase in the preparation of a drug for treating endometriosis.

2. Use according to claim 1, characterized in that, The compound ZY-444 is administered to a patient by intraperitoneal injection or orally.

3. Use according to claim 1, characterized in that, The compound ZY-444 is administered to a patient by topical administration.

4. Use according to claim 2, characterized in that, The compound ZY-444 is administered to a patient orally.

5. Use according to claim 2, characterized in that, The concentration of the ZY-444 administered is 0.5-1 μM.

6. Use of a pyruvate carboxylase biomarker in the preparation of a reagent for diagnosing endometriosis.

Images