Application of expression inhibitor of HAS2 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 HAS2 in preparation of a medicine for treating PANoptosis of granulosa cells of polycystic ovarian syndrome (PCOS). The expression inhibitor of HAS2 can be used for preparing a medicine for treating PANoptosis of the granulosa cells of the PCOS. The research finds that the HAS2 is highly expressed in granulosa cells and ovary of a PCOS patient and can induce the granulosa cells PANoptosis, and the PANoptosis of the PCOS granulosa cells can be inhibited by using 4-MU to inhibit the expression of the HAS2. The results show that the HAS2 is a new target spot of PANoptosis drug therapy of the PCOS granular cells. It is shown that the HAS2 knockout reagent or / and the HAS2 inhibitor are / is a potential novel drug used for PCOS granular cell PANoptosis drug therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of a HAS2 expression inhibitor in preparing a medicament for treating pan-apoptosis of granulosa cells in polycystic ovary syndrome. Background Art

[0002] Polycystic ovary syndrome (PCOS) is a common reproductive endocrine and metabolic disorder, with a prevalence of 8%-13% among women of childbearing age. Common manifestations include ovulatory dysfunction, insulin resistance, hyperinsulinemia, obesity, hyperandrogenism, infertility, polycystic ovarian changes, and increased cardiovascular risk. Because its pathogenesis remains unclear, clinical treatment for PCOS is primarily symptomatic, employing individualized treatment plans. The ovaries of patients with PCOS are often accompanied by chronic low-grade inflammation, resulting in elevated levels of reactive oxygen species in granulosa cells, which can induce apoptosis.

[0003] Pan-apoptosis (PANoptosis) is a new type of programmed cell death that includes three forms: apoptosis, pyroptosis, and necroptosis. It is mediated by the PANoptosome complex, which is a multi-molecular platform that can simultaneously regulate and activate multiple programmed cell death mechanisms. PANoptosis is an inflammatory programmed cell death method that involves multiple inflammatory signaling pathways. Its activation releases inflammatory factors such as Caspase1, Caspase3, NLRP3, IL-1β, etc., exacerbating the chronic low-grade inflammatory state. Currently, many studies have shown that PCOS is associated with granulosa cell apoptosis, pyroptosis, and necroptosis. Our latest research found that PCOS patients have pan-apoptosis of granulosa cells and may be involved in mediating local chronic inflammatory responses in the ovary.

[0004] Granulosa cells (GCs) are the most important cell type in the ovary, besides the oocyte, and play a crucial role in maintaining ovarian function. Granulosa cells (GCs), cumulus cells (CCs), oocytes, macrophages, and follicular fluid form the follicular microenvironment that promotes oocyte maturation and ovulation. Studies have demonstrated that granulosa cell apoptosis can lead to follicular hypoplasia, ovulatory disorders, and abnormal sex hormone secretion in PCOS patients, ultimately leading to follicular atresia. In granulosa cells, certain pathological stimuli, such as oxidative stress and infection, can activate the pyroptosis pathway, disrupting follicular microenvironmental homeostasis and affecting oocyte maturation. Our previous studies have identified the presence of PANoptosis in granulosa cells in PCOS patients, which is a key pathological mechanism of PCOS. However, relevant research remains limited, emphasizing the importance of studying granulosa cell PANoptosis and its relationship to the pathogenesis of PCOS.

[0005] Therefore, there is an urgent need to develop effective drug treatment targets for the pan-apoptosis of granulosa cells in polycystic ovary syndrome and drugs for the treatment of pan-apoptosis of granulosa cells in polycystic ovary syndrome. Summary of the Invention

[0006] The present invention aims to provide the use of HAS2 as a target in the preparation of a drug for treating pan-apoptosis of granulosa cells in polycystic ovary syndrome. The present invention has found through experiments that HAS2 is highly expressed in pan-apoptosis of granulosa cells in polycystic ovary syndrome, and further found that by targeted inhibition of HAS2 expression, PANoptosis of PCOS granulosa cells is inhibited. Therefore, the expression inhibitor of HAS2 can be used to prepare a drug for treating pan-apoptosis of granulosa cells in polycystic ovary syndrome.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect of the present invention, there is provided the use of HAS2 as a target in screening drugs for preventing, alleviating and / or treating pan-apoptosis of granulosa cells in polycystic ovary syndrome, wherein the screening method comprises screening substances capable of inhibiting HAS2 gene expression.

[0009] In a second aspect of the present invention, provided is the use of an HAS2 expression inhibitor in the preparation of a medicament for treating pan-apoptosis of granulosa cells in polycystic ovary syndrome.

[0010] Furthermore, the HAS2 expression inhibitor includes at least one of the following components:

[0011] HAS2 inhibitors;

[0012] Knockout reagent for HAS2.

[0013] Furthermore, the HAS2 inhibitor includes a protein that specifically binds to HAS2, a small interfering molecule that specifically interferes with HAS2 gene expression and processing, or a recombinant vector containing the small interfering molecule that specifically interferes with HAS2 gene expression and processing.

[0014] Furthermore, the HAS2 inhibitor includes: hydroxymethylcoumarol.

[0015] In a third aspect of the present invention, a drug for treating pan-apoptosis of granulosa cells in polycystic ovary syndrome is provided, wherein the drug comprises at least one of a HAS2 inhibitor and a HAS2 knockout agent.

[0016] The HAS2 inhibitors include hydroxycoumarol.

[0017] The HAS2 knockout reagent includes: shRNA and / or gRNA targeting the target gene.

[0018] The drug also includes pharmaceutically acceptable excipients. The excipients include at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener, or a colorant. The drug may be in the form of a granule, tablet, pill, capsule, injection, or dispersant.

[0019] In a fourth aspect of the present invention, there is provided the use of HAS2 as a molecular marker in the preparation of a diagnostic and / or prognostic product for pan-apoptosis of granulosa cells in polycystic ovary syndrome.

[0020] Furthermore, the detection reagent of HAS2 is used in the preparation of diagnostic and / or prognostic products for pan-apoptosis of granulosa cells in polycystic ovary syndrome.

[0021] Furthermore, the HAS2 detection reagent includes a HAS2 protein detection reagent (such as an ELISA detection kit). In other embodiments, the HAS2 detection reagent also includes a HAS2 detection primer or an immunohistochemistry detection reagent.

[0022] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0023] The present invention provides the use of HAS2 as a target in the preparation of a drug for treating pan-apoptosis of granulosa cells in polycystic ovary syndrome. The present invention has found through research that HAS2 is highly expressed in granulosa cells and ovaries of PCOS patients and can induce granulosa cell PANoptosis, and the use of 4-MU (hydroxymethylcoumarol) to inhibit the expression of HAS2 can inhibit PCOS granulosa cell PANoptosis. These results indicate that HAS2 is a new target for drug treatment of PCOS granulosa cell PANoptosis. It shows that HAS2 knockout reagents and / or HAS2 inhibitors are potential new drugs for drug treatment of PCOS granulosa cell PANoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 : The expression levels of PANoptosis-related genes and HAS2 mRNA and protein are increased in granulosa cells of PCOS patients.

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

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

[0028] (C) Levels of pyroptosis-related IL-1β secretion in the culture medium of granulosa cells from PCOS patients and the control group.

[0029] (D) HAS2 mRNA expression levels in granulosa cells of PCOS patients and control group, ** indicates P < 0.01.

[0030] (E) HAS2 protein expression levels in granulosa cells of PCOS patients and control group.

[0031] Figure 2: The expression level of IFN-γ is increased in granulosa cells of PCOS patients, and the expression levels of PANoptosis-related genes and HAS2 mRNA and protein are increased in KGN cells treated with LPS and IFN-γ.

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

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

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

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

[0036] (E) The secretion level of pyroptosis-related IL-1β in the culture medium of KGN cells after LPS and IFN-γ treatment.

[0037] (F) HAS2 mRNA expression levels in KGN cells after LPS and IFN-γ treatment, **** indicates P < 0.0001.

[0038] (H) HAS2 protein expression levels in KGN cells after LPS and IFN-γ treatment.

[0039] Figure 3 : Inhibiting the expression of HAS2 can inhibit the expression of PANoptosis-related genes at the mRNA and protein levels in KGN cells co-treated with LPS+IFN-γ.

[0040] (A) HAS2 mRNA and protein expression levels in KGN cells co-treated with LPS and IFN-γ after intervention with the HAS2 inhibitor 4-MU. ** indicates P < 0.01.

[0041] (B) HAS2 inhibitor 4-MU was used to intervene in KGN cells co-treated with LPS+IFN-γ, and the mRNA levels of PANoptosis-related molecules (including pyroptosis-related NLRP3, IL-1β, and Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL). * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0042] (C) Protein levels of PANoptosis-related molecules (including pyroptosis-related Caspase1 and GSDMD, apoptosis-related Caspase3, and necroptosis-related MLKL) after intervention with the HAS2 inhibitor 4-MU in KGN cells co-treated with LPS+IFN-γ.

[0043] (D) After the HAS2 inhibitor 4-MU was used to intervene in the LPS+IFN-γ co-treated KGN cells, the secretion level of pyroptosis-related IL-1β in the culture medium was reduced. ** indicates P < 0.01, *** indicates P < 0.001.

[0044] Figure 4 : Inhibiting the expression of HAS2 can inhibit the expression of PANoptosis-related genes at the mRNA and protein levels in the ovaries of PCOS mice.

[0045] (A) Schematic diagram of mouse vaginal smear and estrous cycle of control mice, DHEA-induced PCOS mice, and 4-MU-treated PCOS mice.

[0046] (B) HE staining of ovarian sections of control mice, DHEA-induced PCOS mice, and 4-MU-treated PCOS mice.

[0047] (C) Serum levels of sex hormones such as T, LH, and FSH, as well as the LH / FSH ratio, in the control group, the DHEA-induced PCOS group, and the 4-MU-treated PCOS group. * indicates P < 0.05, ** indicates P < 0.01, and **** indicates P < 0.0001.

[0048] (D) HAS2 and PANoptosis-related molecules (including pyroptosis-related NLRP3, IL-1β, and Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL) mRNA levels in ovarian tissues of control mice, DHEA-induced PCOS mice, and 4-MU-treated PCOS mice. ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0049] (E) HAS2 protein levels in ovarian tissues of control mice, DHEA-induced PCOS mice, and 4-MU-treated PCOS mice.

[0050] (F) Protein levels of PANoptosis-related molecules (including pyroptosis-related Caspase1 and GSDMD, apoptosis-related Caspase3, and necroptosis-related MLKL) in ovarian tissues of control mice, DHEA-induced PCOS mice, and 4-MU-treated PCOS mice.

[0051] (G) Immunohistochemical staining of PANoptosis-related molecules GSDMD, Cleaved-Caspase3, and p-MLKL in the ovaries of control mice, DHEA-induced PCOS mice, and 4-MU-treated PCOS mice.

[0052] (H) Serum pyroptosis-related IL-1β secretion levels in the control group, DHEA-induced PCOS group, and 4-MU-treated PCOS group. * indicates P < 0.05, ** indicates P < 0.01. DETAILED DESCRIPTION

[0053] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0054] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0055] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained through existing methods.

[0056] The application of HAS2 as a target in the preparation of a drug for treating pan-apoptosis of granulosa cells in polycystic ovary syndrome will be described in detail below in combination with examples and experimental data.

[0057] Example 1: Increased HAS2 expression and PANoptosis in granulosa cells of PCOS patients

[0058] 1. Research Objects and Materials

[0059] 1. Study 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 collected. The PCOS group was diagnosed according to the 2003 Rotterdam criteria and met at least two of the following three symptoms: decreased ovulation or anovulation, clinical and / or biochemical signs of hyperandrogenism, and polycystic ovaries after excluding other causes. The control group consisted of women whose infertility was mainly caused by pure male factors. This study has been approved by the Ethics Committee of Renmin Hospital of Wuhan University, and all patients were informed and signed informed consent.

[0060] 2. Follicular fluid (FF) collection: Follicular fluid (FF) of 76 patients was obtained from follicular aspirates collected at the time of oocyte retrieval after superovulation stimulation protocol.

[0061] 2. Experimental Methods

[0062] 1. Extraction and Culture of Primary Human Granulosa Cells

[0063] (1) GC extraction: FF was centrifuged at 1500 rpm for 10 minutes, and the supernatant was discarded. The pellet was suspended in PBS (Gibco, Invitrogen Life Technologies), and Ficoll (Sigma) was slowly added to the cell suspension at a 1:1 ratio, and the cells were centrifuged at 1800 rpm for 20 minutes. The white granular cell layer in the middle was aspirated with a Pasteur pipette, incubated with red blood cell lysis buffer (Biosharp) for 5 minutes, and washed with PBS.

[0064] (2) Culture of GCs: The washed GCs were cultured (5 × 105 cells / well) in a 6-well plate containing DMEM / F12 supplemented with 10% FBS and penicillin / streptomycin sulfate (100 U / mL and 100 μg / mL, respectively) at 37°C in the presence of 5% CO2 for 24 h.

[0065] 4. Collect the cultured granulosa cells, extract RNA and protein, and collect the cell culture medium for the following tests:

[0066] (1) RT-qPCR was used to detect the mRNA levels of PANoptosis-related molecules (including pyroptosis-related NLRP3, IL-1β, Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL) in granulosa cells.

[0067] (2) Western Blot was used to detect the protein levels of PANoptosis-related molecules (including pyroptosis-related Caspase1 and GSDMD, apoptosis-related Caspase3, and necroptosis-related MLKL).

[0068] (3) The ELISA method was used to detect the secretion level of pyroptosis-related IL-1β in cell culture medium.

[0069] (4) RT-qPCR and Western Blot were used to detect the HAS2 mRNA and protein levels in granulosa cells, respectively.

[0070] 3. Experimental Results

[0071] The results are as follows Figure 1 As shown in (A), the mRNA expression levels of NLRP3, IL-1β, Caspase1, Caspase3, and MLKL in granulosa cells of the PCOS group were significantly higher than those in the control group.

[0072] like Figure 1 As shown in (B), the expression levels of Cleaved-Caspase1, N-GSDMD, Cleaved-Caspase3, and p-MLKL proteins in granulosa cells of the PCOS group were significantly higher than those in the control group.

[0073] like Figure 1 As shown in (C), the IL-1β secretion level in the culture medium of granulosa cells in the PCOS group was significantly higher than that in the control group.

[0074] like Figure 1 As shown in (D) and (E), the expression levels of HAS2 mRNA (D) and protein (E) in granulosa cells of the PCOS group were significantly higher than those in the control group.

[0075] In summary, the above results confirmed that HAS2 expression levels were elevated in granulosa cells of PCOS patients, and PANoptosis (including pyroptosis, apoptosis, and necroptosis) was present. Specifically, the expression levels of NLRP3, IL-1β, and Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis were increased in granulosa cells; the protein levels of cleaved-Caspase1 and N-GSDMD related to pyroptosis, cleaved-Caspase3 related to apoptosis, and p-MLKL related to necroptosis were increased; and the secretion level of IL-1β related to pyroptosis in the culture medium was increased.

[0076] Example 2 Construction and verification of an in vitro human ovarian granulosa cell PANoptosis model

[0077] 1. Preparation of Human Ovarian Granulosa Cell Carcinoma Cell Line (KGN Cell Line)

[0078] 1. KGN cells were purchased from Procell Life Science Co., Ltd. (Wuhan, China).

[0079] 2. KGN cell culture: The culture method was consistent with that of primary granulosa cells, and they were cultured in 6-well plates (5×105 cells / well) at 37°C in DMEM / F12 supplemented with 10% FBS and penicillin / streptomycin sulfate (100 U / mL and 100 μg / mL, respectively) in 5% CO2.

[0080] 3. Cell passaging: When the KGN cell density reaches 80%-90%, use trypsin to routinely digest and pass the cells, then inoculate them into six-well plates with 1.2×106 cells per well.

[0081] 4. Starvation treatment: When the cell density in the six-well plate reaches 50%-60%, replace the culture medium with DMEM / F12 complete medium containing 2% fetal bovine serum for starvation treatment and continue culturing for 24 hours.

[0082] 2. Experimental Methods

[0083] 1. Treatment of KGN cells: LPS and IFN-γ drug powders were prepared into drug solutions using 1×PBS. After starvation treatment of KGN cells with DMEM / 12 complete medium containing 2% fetal bovine serum for 24 hours, the KGN cells in six-well plates with good growth status were divided into control group, LPS group, IFN-γ group, and LPS+IFN-γ group. The starved KGN cells were treated with PBS, 1 ng / μL LPS, 100 ng / mL IFN-γ, and 1 ng / μL LPS+100 ng / mL IFN-γ according to the above method for 24 hours to obtain the human ovarian granulosa cell PANoptosis model.

[0084] 2. Collect the cultured primary granulosa cells and the treated KGN cells from each group, extract RNA and protein, and collect the cell culture medium for the following tests:

[0085] (1) RT-qPCR was used to detect the IFN-γ mRNA level in primary granulosa cells, and ELISA was used to detect the IFN-γ secretion level in the granulosa cell culture medium.

[0086] (2) RT-qPCR was used to detect the mRNA expression levels of PANoptosis-related molecules in KGN cells of each group, including pyroptosis-related NLRP3, IL-1β, and Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL.

[0087] (3) Western Blot was used to detect the protein levels of PANoptosis-related molecules in KGN cells of each group, including pyroptosis-related Caspase1 and GSDMD, apoptosis-related Caspase3, and necroptosis-related MLKL.

[0088] (4) The ELISA method was used to detect the secretion level of IL-1β in the cell culture medium of each group.

[0089] (5) RT-qPCR and Western Blot were used to detect the HAS2 mRNA and protein levels in each group of cells, respectively.

[0090] 3. Experimental Results

[0091] The results are as follows Figure 2 As shown in (A) and (B), compared with the control group, the expression level of IFN-γ mRNA in granulosa cells of the PCOS group was significantly increased, and the level of IFN-γ secretion in the cell culture medium was also significantly increased.

[0092] The results are as follows Figure 2 As shown in (C), the mRNA expression levels of NLRP3, IL-1β, Caspase1, Caspase3, and MLKL in the LPS+IFN-γ group were significantly higher than those in the control group.

[0093] like Figure 2 As shown in (D), the expression levels of Cleaved-Caspase1, N-GSDMD, Cleaved-Caspase3, and p-MLKL proteins in the LPS+IFN-γ group were significantly higher than those in the control group.

[0094] like Figure 2 As shown in (E), the IL-1β secretion level in the culture medium of the LPS+IFN-γ group was significantly higher than that of the control group.

[0095] like Figure 2 As shown in (F) and (G), the expression levels of HAS2 mRNA (F) and protein (G) in the LPS+IFN-γ group were significantly higher than those in the control group.

[0096] The above results confirmed that the expression level of IFN-γ was increased in primary granulosa cells of PCOS patients, and PANoptosis (including pyroptosis, apoptosis, and necroptosis) occurred in KGN cells treated with LPS combined with IFN-γ. Specifically, the mRNA expression levels of pyroptosis-related NLRP3, IL-1β, and Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL in KGN cells treated with LPS combined with IFN-γ increased, and the protein levels of pyroptosis-related Cleaved-Caspase1 and N-GSDMD, apoptosis-related Cleaved-Caspase3, and necroptosis-related p-MLKL increased. The secretion level of pyroptosis-related IL-1β in the culture medium increased, and the expression levels of HAS2 mRNA and protein increased in KGN cells treated with LPS combined with IFN-γ, confirming the successful establishment of the KGN cell PANoptosis model.

[0097] Example 3: Inhibiting HAS2 expression can inhibit PANoptosis in human ovarian granulosa cells

[0098] 1. Inhibition of HAS2 expression using hydroxymethylcoumarol

[0099] KGN cells were induced with LPS and IFN-γ and divided into control, LPS, IFN-γ, and LPS+IFN-γ groups. 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 hours. Prior to co-induction with LPS and IFN-γ, KGN cells were pretreated with hydroxymethylcoumarin (50 μM) for 1 hour, designated the LPS+IFN-γ+4-MU group.

[0100] 2. Experimental Methods

[0101] The KGN cells from each group were collected after treatment, RNA and protein were extracted, and the cell culture medium was collected for the following tests:

[0102] 1. RT-qPCR and Western Blot were used to detect the HAS2 mRNA and protein levels in each group of cells, respectively.

[0103] 2. RT-qPCR was used to detect the mRNA expression levels of PANoptosis-related molecules in KGN cells of each group, including pyroptosis-related NLRP3, IL-1β, Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL.

[0104] 3. Western Blot was used to detect the levels of PANoptosis-related molecular proteins in each group, including pyroptosis-related Caspase1 and GSDMD, apoptosis-related Caspase3, and necroptosis-related MLKL.

[0105] 4. ELISA was used to detect the secretion level of IL-1β in the cell culture medium of each group.

[0106] 3. Experimental Results

[0107] The results are as follows Figure 3 As shown in (A), LPS+IFN-γ treatment increased the expression of HAS2 mRNA and protein levels in KGN cells, while treatment with the HAS2 inhibitor 4-MU suppressed the LPS+IFN-γ-induced high expression of HAS2.

[0108] like Figure 3As shown in (B), LPS+IFN-γ treatment in KGN cells increased the mRNA expression levels of NLRP3, IL-1β, Caspase1, Caspase3, and MLKL, while treatment with the HAS2 inhibitor 4-MU suppressed the high expression induced by LPS+IFN-γ.

[0109] like Figure 3 As shown in (C), LPS+IFN-γ treatment in KGN cells increased the expression levels of Cleaved-Caspase1, N-GSDMD, Cleaved-Caspase3, and p-MLKL proteins, while treatment with the HAS2 inhibitor 4-MU suppressed the high expression induced by LPS+IFN-γ.

[0110] The results are as follows Figure 3 (D) As shown, LPS+IFN-γ treatment in KGN cells increased the level of IL-1β secretion in the culture medium, while treatment with the HAS2 inhibitor 4-MU suppressed LPS+IFN-γ-induced IL-1β secretion.

[0111] In summary, the above results show that inhibiting HAS2 expression significantly reduced the expression of PANoptosis-related genes mRNA and protein in the LPS+IFN-γ-induced KGN cell PANoptosis model. This confirms that using 4-MU to inhibit HAS2 expression can inhibit LPS+IFN-γ-induced KGN cell PANoptosis.

[0112] Example 4: Inhibiting HAS2 expression can inhibit ovarian PANoptosis in PCOS mice

[0113] 1. Construction of PCOS Mouse Model

[0114] 1. Forty 21-day-old female ICR mice were housed in a SPF animal facility under standard conditions (22-24°C and 60-70% relative humidity) with free access to food and water. The mice were randomly divided into three groups: a control group, a PCOS group, and a PCOS+4-MU group. The control group received daily injections of olive oil and intragastric administration of normal saline. The PCOS group received daily injections of dehydroepiandrosterone (DHEA) (240 mg DHEA dissolved in 1 ml olive oil, 40 μl / mouse / day) and intragastric administration of normal saline. The PCOS+4-MU group received injections of DHEA and intraperitoneal injections of 4-MU (dissolved in normal saline, 150 μl / mouse / day).

[0115] 2. During the treatment period, all animals were weighed once a day, and vaginal smears were collected daily starting on the 10th day after the first DHEA injection until the end of the experiment. Mice were sacrificed 21 days after the first injection. Blood was collected and centrifuged after an overnight fast, and serum samples were collected and stored at -80°C for further analysis. One ovary was dissected for PCR or Western blot analysis, while the other ovary was fixed in 10% paraformaldehyde solution for sectioning, staining, and other experiments. All protocols and experiments were approved by the Ethics Committee of Wuhan University Renmin Hospital (No.: WDRY2018-K013).

[0116] 2. After killing the mice, collect serum samples and ovarian tissue for the following analyses or tests:

[0117] 1. Vaginal smears were performed on mice in each group for 14 consecutive days and the estrous cycle was observed.

[0118] 2. The ovaries of mice in each group were taken for HE staining to detect the morphological changes of the ovaries of mice in each group.

[0119] 3. The ELISA method was used to detect the serum sex hormone levels of mice in each group.

[0120] 4. RT-qPCR was used to detect the mRNA levels of HAS2 and PANoptosis-related molecules in the ovarian tissues of mice in each group, including pyroptosis-related NLRP3, IL-1β, and Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL.

[0121] Table 1 Primer sequences used in RT-qPCR in the above examples

[0122]

[0123]

[0124] 5. Western blot was used to detect the levels of HAS2 and PANoptosis-related molecular proteins in the ovarian tissues of mice in each group, including pyroptosis-related Caspase1 and GSDMD, apoptosis-related Caspase3, and necroptosis-related MLKL.

[0125] 6. Immunohistochemical staining was used to detect the localization and expression of PANoptosis-related molecules GSDMD, Cleaved-Caspase3, and p-MLKL in the ovarian tissues of each group of mice.

[0126] 3. Experimental Results

[0127] The results are as follows Figure 4As shown in (A), compared with the control group, the estrous cycle of PCOS mice was disordered, while after treatment with 4-MU, the estrous cycle of PCOS mice returned to normal.

[0128] like Figure 4 As shown in (B), compared with the control group, the ovaries of PCOS mice showed polycystic changes, while after PCOS mice were treated with 4-MU, the polycystic changes in the ovaries of mice disappeared.

[0129] like Figure 4 As shown in (C), serum T and LH levels were significantly elevated in PCOS mice, and 4-MU treatment could partially reduce T levels but not LH levels; the LH / FSH ratio was slightly elevated in PCOS mice and was improved by 4-MU treatment.

[0130] The results are as follows Figure 4 As shown in (D), the expression levels of HAS2, NLRP3, IL-1β, Caspase1, Caspase3, and MLKL mRNA were increased in the ovarian tissues of PCOS mice, while treatment with the HAS2 inhibitor 4-MU suppressed the high expression in PCOS mice.

[0131] like Figure 4 As shown in (E), the HAS2 protein level in the ovarian tissue of PCOS mice was elevated, while treatment with the HAS2 inhibitor 4-MU suppressed the high expression in PCOS mice.

[0132] like Figure 4 As shown in (F), the protein levels of Cleaved-Caspase1, N-GSDMD, Cleaved-Caspase3, and p-MLKL were increased in the ovarian tissues of PCOS mice, while treatment with the HAS2 inhibitor 4-MU suppressed the high expression in PCOS mice.

[0133] The results are as follows Figure 4 As shown in (G), the expression levels of GSDMD, Cleaved-Caspase3, and p-MLKL in the ovarian granulosa cells of PCOS mice were increased, while the treatment with HAS2 inhibitor 4-MU suppressed the high expression in the granulosa cells of PCOS mice. Figure 4 As shown in (H), the serum IL-1β secretion level of PCOS group mice was increased, and the treatment with HAS2 inhibitor 4-MU suppressed the secretion of IL-1β.

[0134] In summary, the above results confirmed the presence of PANoptosis (including pyroptosis, apoptosis, and necroptosis) in the ovaries of PCOS mice induced by DHEA, and the increased expression level of HAS2. Treatment with the HAS2 inhibitor 4-MU can inhibit PANoptosis in the ovaries of PCOS mice. Specifically, the mRNA expression levels of pyroptosis-related NLRP3, IL-1β, and Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL in the ovarian tissues of PCOS mice increased. The protein levels of pyroptosis-related Cleaved-Caspase1 and N-GSDMD, apoptosis-related Cleaved-Caspase3, and necroptosis-related p-MLKL increased. The expression levels of HAS2 mRNA and protein increased. Treatment of PCOS mice with the HAS2 inhibitor 4-MU inhibited the expression of the above-mentioned PANoptosis-related molecules.

[0135] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0136] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0137] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. The use of HAS2 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 HAS2 gene.

2. Application of HAS2 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 HAS2 expression inhibitor comprises at least one of the following components: HAS2 inhibitors; Knockout reagent for HAS2.

4. The use according to claim 3, characterized in that: The HAS2 inhibitor includes a protein that specifically binds to HAS2, a small interfering molecule that specifically interferes with the expression and processing of the HAS2 gene, or a recombinant vector containing the small interfering molecule that specifically interferes with the expression and processing of the HAS2 gene.

5. The use according to claim 4, characterized in that: The HAS2 inhibitors include: hydroxymethylcoumarin.

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 a HAS2 inhibitor and a HAS2 knockout agent.

8. Application of HAS2 as a molecular marker in the preparation of diagnostic and / or prognostic products for pan-apoptosis of granulosa cells in polycystic ovary syndrome.

9. Application of HAS2 detection reagent 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 HAS2 detection reagent includes a HAS2 detection primer or an immunohistochemical detection reagent.

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