Application of IRF1 expression inhibitor in preparation of medicine for treating ubiquitous apoptosis of granulosa cells of polycystic ovarian syndrome

Abstract

The invention provides an application of an expression inhibitor of IRF1 (Interferon Regulating Factor 1) in preparation of a medicine for treating PANoptosis of granular cells of PCOS (Polycystic Ovarian Syndrome). The research finds that IRF1 is highly expressed in granulosa cells and ovary of a PCOS patient, and the expression of the IRF1 is knocked down by si-IRF1, so that the PCOS granulosa cell PANoptosis can be inhibited. The results show that the IRF1 can induce the granulosa cell PANoptosis, and is a new target spot of the PCOS granulosa cell PANoptosis drug therapy. It is indicated that the IRF1 knockout / knockdown reagent is a potential novel medicine used for PCOS granular cell PANoptosis medicine treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of an IRF1 expression inhibitor in the preparation of a drug for treating PANoptosis of granulosa cells in polycystic ovary syndrome. Background Art

[0002] Polycystic ovary syndrome (PCOS) is a common reproductive endocrine and metabolic disease, with a prevalence rate of 8%-13% in women of reproductive age. It often presents as ovulation disorder, insulin resistance, hyperinsulinemia, obesity, hyperandrogenism, infertility, polycystic ovarian changes, and increased cardiovascular risk. Since its pathogenesis is unclear, the clinical treatment of PCOS mainly focuses on symptomatic treatment, adopting an individualized treatment plan. Ovaries of PCOS patients are often accompanied by a chronic low-grade inflammatory state, and the elevated level of reactive oxygen species in their granulosa cells will induce apoptosis of granulosa cells.

[0003] PANoptosis is a novel form of programmed cell death, which includes three forms: apoptosis, pyroptosis, and necroptosis. It is mediated by PANoptosome complexes, which are multimolecular platforms that can simultaneously regulate and activate multiple programmed cell death mechanisms. PANoptosis is an inflammatory programmed cell death mode, involving multiple inflammatory signaling pathways, and its activation will release inflammatory factors, such as Caspase1, Caspase3, NLRP3, IL-1β, etc., exacerbating the chronic low-grade inflammatory state. Currently, a number of studies have shown that PCOS is related to apoptosis, pyroptosis, and necroptosis of granulosa cells. The expression levels of apoptosis-related factors such as Caspase7, Caspase8, Bcl2, and Bax in PCOS granulosa cells are increased, the expression levels of pyroptosis-related factors such as NLRP3, Caspase1, GSDMD, and IL-1β are increased, and the expression levels of necroptosis-related factors such as RIPK1 and RIPK3 are increased. In addition, a number of studies have confirmed that INF-γ induces the activation of the JAK / STAT pathway, thereby inducing an increase in IRF1 expression and inducing PANoptosis. Our latest research found that PCOS patients have granulosa cell PANoptosis and an increased INF-γ expression level, and INF-γ can induce granulosa cell PANoptosis. However, there is currently no research on its downstream factor IRF1 and PCOS granulosa cells, and the PANoptosis mechanism and pathway related to PCOS have not been studied or reported. Therefore, clarifying the pathogenesis and pathway of PANoptosis in PCOS is crucial for improving the pathological changes of PCOS.

[0004] Granulosa cells (GCs) are the most important cell type in the ovary except for oocytes and play an important role in maintaining ovarian function. Granulosa cells (GCs), cumulus cells (CCs), oocytes, macrophages, and follicular fluid form a follicular microenvironment that promotes oocyte maturation and ovulation. Studies have confirmed that apoptosis of granulosa cells can lead to poor follicular development, ovulation disorders, and abnormal sex hormone secretion in patients with PCOS, ultimately resulting in follicular atresia. In granulosa cells, certain pathological stimuli such as oxidative stress and infection can activate the pyroptosis pathway, disrupt the homeostasis of the follicular microenvironment, and affect oocyte development and maturation. Our previous study found that PANoptosis exists in granulosa cells of PCOS patients, which is one of the important pathological mechanisms of PCOS. However, relevant research is still scarce. Therefore, it is of great significance to study granulosa cell PANoptosis and its relationship with the pathogenesis of PCOS, and it is urgent to develop effective drug treatment targets for granulosa cell pan-apoptosis in polycystic ovary syndrome and drugs for treating granulosa cell pan-apoptosis in polycystic ovary syndrome. Summary of the Invention

[0005] The object of the present invention is to provide the application of IRF1 as a target in the preparation of drugs for treating granulosa cell pan-apoptosis in polycystic ovary syndrome. Through experiments, the present invention found that IRF1 is highly expressed in granulosa cell pan-apoptosis in polycystic ovary syndrome. Further, it was found that by targeting and inhibiting the expression of IRF1, the PANoptosis of PCOS granulosa cells can be inhibited. Therefore, an expression inhibitor of IRF1 can be used to prepare drugs for treating granulosa cell pan-apoptosis in polycystic ovary syndrome.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, there is provided the application of IRF1 as a target in screening drugs for preventing, alleviating, and / or treating granulosa cell pan-apoptosis in polycystic ovary syndrome, and the screening method includes screening substances that can inhibit the expression of the IRF1 gene.

[0008] In the second aspect of the present invention, there is provided the application of an expression inhibitor of IRF1 in the preparation of drugs for treating granulosa cell pan-apoptosis in polycystic ovary syndrome.

[0009] Furthermore, the expression inhibitor of IRF1 includes at least one of the following components:

[0010] IRF1 inhibitor;

[0011] IRF1 knockout reagent, IRF1 knockdown reagent.

[0012] Further, the IRF1 inhibitor includes at least one of a protein that specifically binds to IRF1, a small interfering molecule that specifically interferes with the expression and processing of the IRF1 gene, or a recombinant vector containing the small interfering molecule that specifically interferes with the expression and processing of the IRF1 gene.

[0013] Further, the IRF1 knockdown reagent includes interferon regulatory factor 1.

[0014] In the third aspect of the present invention, a drug for treating granulosa cell pan-apoptosis in polycystic ovary syndrome is provided, and the drug includes at least one of a knockout reagent for IRF1 and a knockdown reagent for IRF1.

[0015] The IRF1 knockout reagent includes: shRNA and / or gRNA targeting the target gene and CRISPR / Cas9.

[0016] The IRF1 knockdown reagent includes: si-IRF1, as shown in SEQ ID NO.17 - SEQ ID NO.18.

[0017] The drug further includes a pharmaceutically acceptable excipient. The excipient includes at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetening agent, or a coloring agent. The dosage form of the drug includes at least one of a granule, a tablet, a pill, a capsule, an injection, or a dispersant.

[0018] In the fourth aspect of the present invention, the application of IRF1 as a molecular marker in the preparation of a diagnostic and / or prognostic product for granulosa cell pan-apoptosis in polycystic ovary syndrome is provided.

[0019] Further, the application of a detection reagent for IRF1 in the preparation of a diagnostic and / or prognostic product for granulosa cell pan-apoptosis in polycystic ovary syndrome.

[0020] Further, the detection reagent for IRF1 includes a detection reagent for IRF1 protein (such as an ELISA detection kit). In other embodiments, the detection reagent for IRF1 further includes a detection primer for IRF1 or an immunohistochemical detection reagent.

[0021] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0022] The present invention provides the use of IRF1 as a target in the preparation of a drug for treating granulosa cell pan - apoptosis in polycystic ovary syndrome. Through research, the present invention discovers that IRF1 is highly expressed in granulosa cells and ovaries of PCOS patients, and inhibiting the expression of IRF1 using si - IRF1 can inhibit PANoptosis of PCOS granulosa cells. These results indicate that IRF1 is a new target for drug treatment of PANoptosis in PCOS granulosa cells. It shows that the reagent for knocking out / down IRF1 is a potential new drug for drug treatment of PANoptosis in PCOS granulosa cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 : Schematic diagram of the IRF1 - induced PANoptosis signaling pathway.

[0025] Figure 2 : The expression levels of PANoptosis - related genes, IRF1 mRNA and protein in granulosa cells of PCOS patients increase.

[0026] (A) mRNA levels of PANoptosis - related molecules (including NLRP3, IL - 1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis) in granulosa cells of PCOS and control patients. ** indicates P < 0.01, *** indicates P < 0.001.

[0027] (B) Protein levels of PANoptosis - related molecules (including Caspase1, GSDMD related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis) in granulosa cells of PCOS and control patients.

[0028] (C) Secretion levels of pyroptosis - related IL - 1β in the culture media of granulosa cells of PCOS and control patients.

[0029] (D) IRF1 mRNA expression levels in granulosa cells of PCOS and control patients. ** indicates P < 0.01.

[0030] (E) IRF1 protein expression levels in granulosa cells of PCOS and control patients.

[0031] Figure 3: The expression level of IFN-γ in granulosa cells of PCOS patients increased, and the expression levels of PANoptosis-related genes, IRF1 mRNA and protein in KGN cells treated with LPS and IFN-γ increased.

[0032] (A) IFN-γ mRNA levels in granulosa cells of PCOS and control patients, ** indicates P < 0.01.

[0033] (B) IFN-γ mRNA levels in the culture medium of granulosa cells of PCOS and control patients, *** indicates P < 0.001.

[0034] (C) mRNA levels of PANoptosis-related molecules (including NLRP3, IL-1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis) in KGN cells treated with LPS and IFN-γ, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0035] (D) Protein levels of PANoptosis-related molecules (including Caspase1, GSDMD related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis) in KGN cells treated with LPS and IFN-γ.

[0036] (E) Secretion level of IL-1β related to pyroptosis in the culture medium of KGN cells treated with LPS and IFN-γ, *** indicates P < 0.001.

[0037] (F) IRF1 mRNA expression level in KGN cells treated with LPS and IFN-γ, ** indicates P < 0.01, *** indicates P < 0.001.

[0038] (G) IRF1 protein expression level in KGN cells treated with LPS and IFN-γ.

[0039] Figure 4 : Knockdown of IRF1 expression can inhibit the expression of PANoptosis-related gene mRNA and protein levels in KGN cells co-treated with LPS + IFN-γ.

[0040] (A) IRF1 mRNA and protein expression levels in KGN cells co-treated with LPS + IFN-γ after intervention with the IRF1 knockdown reagent si-IRF1, *** indicates P < 0.001.

[0041] (B) After intervening in KGN cells co-treated with LPS + IFN-γ using the IRF1 knockdown reagent si-IRF1, the mRNA levels of PANoptosis-related molecules (including NLRP3, IL-1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis), * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0042] (C) After intervening in KGN cells co-treated with LPS + IFN-γ using the IRF1 knockdown reagent si-IRF1, the protein levels of PANoptosis-related molecules (including Caspase1, GSDMD related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis).

[0043] (D) After intervening in KGN cells co-treated with LPS + IFN-γ using the IRF1 knockdown reagent si-IRF1, the secretion level of IL-1β related to pyroptosis in the medium, *** indicates P < 0.001. Detailed implementation manners

[0044] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than limiting the present invention.

[0045] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail.

[0046] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or by existing methods.

[0047] The application of HAS2 of the present application as a target in the preparation of a drug for treating granulosa cell pan-apoptosis in polycystic ovary syndrome will be described in detail below in combination with examples and experimental data.

[0048] Example 1 Increased expression level of IRF1 and presence of PANoptosis in granulosa cells of PCOS patients

[0049] I. Research objects and materials

[0050] 1. Research subjects: PCOS patients (n = 37) and non-PCOS patients (n = 42) who visited the Reproductive Center of Renmin Hospital of Wuhan University from March to June 2022 were recruited. The PCOS group was diagnosed according to the Rotterdam criteria in 2003 and met at least two of the following three symptoms: reduced or absent ovulation, clinical and / or biochemical signs of hyperandrogenism, and polycystic ovaries after excluding other etiologies. The control group consisted of women with infertility mainly due to male factors alone. This study has been approved by the Ethics Committee of Renmin Hospital of Wuhan University, and all patients were informed and signed the informed consent form.

[0051] 2. Collection of follicular fluid (FF): Follicular fluid from 76 patients was obtained from the follicular aspirate collected during oocyte retrieval after the superovulation stimulation protocol.

[0052] II. Experimental methods

[0053] 1. Extraction and culture of primary human granulosa cells

[0054] (1) Extraction of GC: The FF was centrifuged at 1500 rpm for 10 minutes, and then the supernatant was discarded. The precipitate was suspended in PBS (Gibco, Invitrogen Life Technologies), and the cell suspension was slowly added to Ficoll (Sigma) at a ratio of 1:1 and centrifuged at 1800 rpm for 20 minutes. The middle white granulosa cell layer was aspirated with a Pasteur pipette, incubated with red blood cell lysis buffer (Biosharp) for 5 minutes, and washed with PBS.

[0055] (2) Culture of GC: The washed GC was cultured in a 6-well plate (5×105 cells / well) of DMEM / F12 supplemented with 10% FBS, penicillin / streptomycin sulfate (100 U / mL and 100 μg / mL respectively) at 37°C in 5% CO2 for 24 h.

[0056] 4. The cultured granulosa cells were collected, RNA and proteins were extracted, and the cell culture medium was collected for the following detections:

[0057] (1) RT-qPCR was used to detect the mRNA levels of PANoptosis-related molecules (including NLRP3, IL-1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis) in granulosa cells.

[0058] (2) Western Blot was used to detect the protein levels of PANoptosis-related molecules (including Caspase1, GSDMD related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis).

[0059] (3) Detect the secretion level of pyroptosis-related IL-1β in cell culture medium by ELISA method.

[0060] (4) Detect the mRNA and protein levels of IRF1 in granulosa cells by RT-qPCR and Western Blot respectively.

[0061] III. Experimental Results

[0062] The results are as Figure 2 shown in (A). The expression levels of NLRP3, IL-1β, Caspase1, Caspase3, and MLKL mRNA in granulosa cells of the PCOS group were significantly higher than those of the control group.

[0063] As Figure 2 shown in (B). The protein expression levels of Cleaved-Caspase1, N-GSDMD, Cleaved-Caspase3, and p-MLKL in granulosa cells of the PCOS group were significantly higher than those of the control group.

[0064] As Figure 2 shown in (C). The secretion level of IL-1β in the cell culture medium of granulosa cells in the PCOS group was significantly higher than that of the control group.

[0065] As Figure 2 shown in (D) and (E). The expression levels of IRF1 mRNA (D) and protein (E) in granulosa cells of the PCOS group were significantly higher than those of the control group.

[0066] In summary, the above results confirmed that the expression level of IRF1 in granulosa cells of PCOS patients was increased, and PANoptosis (including pyroptosis, apoptosis, and necroptosis) existed. Specifically, the expression levels of NLRP3, IL-1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL mRNA related to necroptosis in granulosa cells increased, the protein levels of Cleaved-Caspase1, N-GSDMD related to pyroptosis, Cleaved-Caspase3 related to apoptosis, and p-MLKL related to necroptosis increased, and the secretion level of pyroptosis-related IL-1β in the culture medium increased.

[0067] Example 2 Construction and verification of an in vitro PANoptosis model of human ovarian granulosa cells

[0068] I. Preparation of human ovarian granulosa cell cancer cell line (KGN cell line)

[0069] 1. Source of KGN cells: Purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

[0070] 2. KGN cell culture: The culture method is the same as that of primary granulosa cell culture. Cells are cultured in a 6-well plate of DMEM / F12 supplemented with 10% FBS, penicillin / streptomycin (100 U / mL and 100 μg / mL respectively) at 37°C in 5% CO2 (5×105 cells / well).

[0071] 3. Cell passage: When the KGN cell density reaches 80%-90%, cells are digested routinely with trypsin for passage and then inoculated into a six-well plate, with 1.2×106 cells inoculated per well.

[0072] 4. Starvation treatment: When the cell density in the six-well plate reaches 50%-60%, the culture medium is replaced with DMEM / F12 complete medium containing 2% fetal bovine serum for starvation treatment, and the cells are cultured for another 24 h.

[0073] II. Experimental methods

[0074] 1. Treatment of KGN cells: LPS and IFN-γ drug powders are both prepared into drug solutions using 1×PBS. After starving KGN cells in DMEM / 12 complete medium containing 2% fetal bovine serum for 24 h, the well-grown KGN cells in the six-well plate are divided into a control group, an LPS group, an IFN-γ group, and an LPS + IFN-γ group. PBS, 1 ng / μL LPS, 100 ng / mL IFN-γ, and 1 ng / μL LPS + 100 ng / mL IFN-γ are used to treat the starved KGN cells according to the above method for 24 h to obtain a human ovarian granulosa cell PANoptosis model.

[0075] 2. Collect the primary granulosa cells after culture and the KGN cells in each group after treatment, extract RNA and proteins, and collect the cell culture medium for the following detections:

[0076] (1) Detect the IFN-γ mRNA level in primary granulosa cells by RT-qPCR and detect the IFN-γ secretion level in the granulosa cell culture medium by ELISA.

[0077] (2) Detect the mRNA expression levels of PANoptosis-related molecules in KGN cells of each group by RT-qPCR, including NLRP3, IL-1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis.

[0078] (3) Detect the protein levels of PANoptosis-related molecules in KGN cells of each group by Western Blot, including Caspase1, GSDMD related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis.

[0079] (4) The secretion level of IL-1β in the cell culture medium of each group was detected by ELISA method.

[0080] (5) The mRNA and protein levels of IRF1 in the cells of each group were detected by RT-qPCR and Western Blot respectively.

[0081] III. Experimental Results

[0082] The results were as Figure 3 (A) and (B) showed that, compared with the control group, the expression level of IFN-γ mRNA in granulosa cells of the PCOS group was significantly increased, and the secretion level of IFN-γ in the cell culture medium was also significantly increased.

[0083] The results were as Figure 3 (C) showed that the expression levels of NLRP3, IL-1β, Caspase1, Caspase3, and MLKL mRNA in the LPS+IFN-γ group were significantly higher than those in the control group.

[0084] As Figure 3 (D) showed that the protein expression levels of Cleaved-Caspase1, N-GSDMD, Cleaved-Caspase3, and p-MLKL in the LPS+IFN-γ group were significantly higher than those in the control group.

[0085] As Figure 3 (E) showed that the secretion level of IL-1β in the cell culture medium of the LPS+IFN-γ group was significantly higher than that in the control group.

[0086] As Figure 3 (F) and (G) showed that the expression levels of IRF1 mRNA (F) and protein (G) in the LPS+IFN-γ group were significantly higher than those in the control group.

[0087] The above results confirmed that the expression level of IFN-γ in primary granulosa cells of PCOS patients was increased, and PANoptosis (including pyroptosis, apoptosis, and necroptosis) occurred in KGN cells treated with LPS combined with IFN-γ. Specifically, the mRNA expression levels of NLRP3, IL-1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis increased in KGN cells treated with LPS combined with IFN-γ, the protein levels of Cleaved-Caspase1, N-GSDMD related to pyroptosis, Cleaved-Caspase3 related to apoptosis, and p-MLKL related to necroptosis increased, the secretion level of IL-1β related to pyroptosis in the cell culture medium increased, and the mRNA and protein expression levels of IRF1 in KGN cells treated with LPS combined with IFN-γ increased, which confirmed the successful construction of the KGN cell PANoptosis model.

[0088] Example 3 Knockdown of IRF1 expression can inhibit PANoptosis in human ovarian granulosa cells

[0089] I. Knockdown of IRF1 expression using si-IRF1

[0090] KGN cells were induced with LPS and IFN-γ and divided into a control group, an LPS group, an IFN-γ group, and an LPS + IFN-γ group. The cells were treated with PBS, 1 ng / μl LPS, 100 ng / ml IFN-γ, and 1 ng / μl LPS + 100 ng / ml IFN-γ for 24 h, respectively. Before co-inducing KGN cells with LPS + IFN-γ, KGN cells were pretreated with si-IRF1 (20 μM) for 1 hour, denoted as the LPS + IFN-γ + si-IRF1 group. The forward sequence of si-IRF1 used in this invention is (5’-3’): GGAUGAGGAAGGGAAAUUATT (SEQ ID NO.17); the reverse sequence is (5’-3’): UAAUUUCCCUUCCUCAUCCTT (SEQ ID NO.18).

[0091] II. Experimental methods

[0092] The treated KGN cells in each group were collected, RNA and proteins were extracted, and the cell culture medium was collected for the following detections:

[0093] 1. RT-qPCR and Western Blot were used to detect the IRF1 mRNA and protein levels in each group of cells, respectively.

[0094] 2. RT-qPCR was used to detect the mRNA expression levels of PANoptosis-related molecules in each group of KGN cells, including NLRP3, IL-1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis.

[0095] 3. Western Blot was used to detect the protein levels of PANoptosis-related molecules in each group, including Caspase1, GSDMD related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis.

[0096] 4. ELISA was used to detect the secretion level of IL-1β in the cell culture medium of each group.

[0097] Table 1 Primer sequences used in RT-qPCR in the above examples

[0098]

[0099] III. Experimental results

[0100] The results are as Figure 4 shown in (A). Treatment with LPS + IFN-γ in KGN cells increased the expression levels of IRF1 mRNA and protein, while treatment with the IRF1 knockdown reagent si-IRF1 inhibited the high expression of IRF1 induced by LPS + IFN-γ.

[0101] As Figure 4 shown in (B). Treatment with LPS + IFN-γ in KGN cells increased the expression levels of NLRP3, IL-1β, Caspase1, Caspase3, and MLKL mRNA, while treatment with the IRF1 knockdown reagent si-IRF1 inhibited the high expression induced by LPS + IFN-γ.

[0102] As Figure 4 shown in (C). Treatment with LPS + IFN-γ in KGN cells increased the expression levels of Cleaved-Caspase1, N-GSDMD, Cleaved-Caspase3, and p-MLKL proteins, while treatment with the IRF1 knockdown reagent si-IRF1 inhibited the high expression induced by LPS + IFN-γ.

[0103] The results are as Figure 4 shown in (D). Treatment with LPS + IFN-γ in KGN cells increased the secretion level of IL-1β in the medium, while treatment with the IRF1 knockdown reagent si-IRF1 inhibited the IL-1β secretion induced by LPS + IFN-γ.

[0104] In summary, the above results showed that knocking down the expression of IRF1 significantly reduced the expression of PANoptosis-related genes mRNA and protein in the PANoptosis model of KGN cells induced by LPS + IFN-γ. Additionally, as a form of cell-level programmed cell death, the mechanism of PANoptosis is highly conserved in vivo and in vitro, and the in vitro model is sufficient to verify the effectiveness of drug targets. Thus, it was confirmed that knocking down the expression of IRF1 using si-IRF1 could inhibit PANoptosis of KGN cells induced by LPS + IFN-γ.

[0105] Finally, it should also be noted that the term "including", "comprising", or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or device.

[0106] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made to these embodiments by those skilled in the art once they learn the basic creative concept. Therefore, the appended claims are intended to be interpreted to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0107] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Use of IRF1 as a target in screening drugs for preventing, alleviating and / or treating pan-apoptosis of granulosa cells in polycystic ovary syndrome, characterized in that: The screening method includes screening substances that can inhibit the expression of IRF1 gene.

2. Application of IRF1 expression inhibitor in the preparation of drugs for treating pan-apoptosis of granulosa cells in polycystic ovary syndrome.

3. The use according to claim 2, characterized in that: The IRF1 expression inhibitor comprises at least one of the following components: IRF1 knockout reagent, IRF1 knockdown reagent.

4. The use according to claim 3, characterized in that: The IRF1 inhibitor includes at least one of a protein that specifically binds to IRF1, a small interfering molecule that specifically interferes with the expression and processing of the IRF1 gene, or a recombinant vector containing the small interfering molecule that specifically interferes with the expression and processing of the IRF1 gene.

5. The use according to claim 3, characterized in that: The IRF1 knockdown reagent includes interferon regulatory factor 1.

6. The use according to claim 2, characterized in that: The drug also includes pharmaceutically acceptable excipients.

7. A drug for treating pan-apoptosis of granulosa cells in polycystic ovary syndrome, characterized in that: The drug includes at least one of an IRF1 knockout agent and an IRF1 knockdown agent.

8. Application of IRF1 as a molecular marker in the preparation of diagnostic and / or prognostic products for pan-apoptosis of granulosa cells in polycystic ovary syndrome.

9. Use of IRF1 detection reagents in the preparation of diagnostic and / or prognostic products for pan-apoptosis of granulosa cells in polycystic ovary syndrome.

10. The use according to claim 9, characterized in that: The IRF1 detection reagent includes an IRF1 detection primer or an immunohistochemical detection reagent.

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