Dihydrotestosterone-induced in-vitro human ovarian granular cell pan-apoptosis model and construction method thereof

Abstract

The invention discloses a dihydrotestosterone-induced in-vitro human ovarian granular cell pan apoptosis model and a construction method thereof, and belongs to the field of reproductive medicine. According to the invention, hungry-treated human ovarian granulosa cells are cultured by using a dihydrotestosterone-containing culture medium, such that the human ovarian granulosa cell pan-apoptosis model is obtained. The constructed human ovarian granular cell pan-apoptosis model has key pan-apoptosis characteristics such as apoptosis, pyroptosis and necrotic apoptosis at the same time, and can be used as a cell model for researching a pathological mechanism of polycystic ovarian syndrome. The invention establishes a good foundation for researching the pathogenic mechanism of pan-apoptosis on polycystic ovarian syndrome.

Description

Technical Field

[0001] The present invention belongs to the field of reproductive medicine, and particularly relates to a dihydrotestosterone (DHT)-induced in vitro human ovarian granulosa cell pan-apoptosis model and a construction method thereof. Background Art

[0002] Polycystic ovarian syndrome (PCOS) is the most common reproductive endocrine disorder in women of childbearing age and is one of the main causes of anovulatory infertility, with an incidence rate as high as 15% in women of reproductive age. [1] It is a highly heterogeneous and complex endocrine metabolic disorder, clinically manifested as hyperandrogenism, irregular menstruation or amenorrhea, insulin resistance, and infertility, etc. Its pathogenesis remains unclear so far. Exploring its molecular mechanism and pathogenic mechanism is very necessary for accurately selecting individualized treatment plans in the future.

[0003] In 2019, American scholar Malireddi first proposed a new type of programmed cell death (PCD) method, namely pan-apoptosis. Pan-apoptosis has the key characteristics of pyroptosis, apoptosis, and necroptosis, but cannot be explained by any one of these three PCD pathways alone. [2] Pan-apoptosis is regulated by a cascade of upstream sensors and molecular signals. These sensors and cascade signals can provide a "molecular scaffold" to promote the coupling and binding of proteins and molecules required for pyroptosis (such as NLRP3, ASC, and Caspase1), apoptosis (Caspase3 / 8), and necroptosis (RIPK3, RIPK1, MLKL), and assemble into a multimeric complex, namely the pan-apoptosome. [3,4] It can serve as the "master switch" for the initiation of the three PCD pathways and the activation platform for downstream molecules. [5] So far, four upstream sensor molecules of pan-apoptosis have been identified, including Z-DNA binding protein 1 (ZBP1), receptor-interacting protein kinase 1 (RIPK1), absent in melanoma 2 (AIM2), and Nod-like receptor family pyrin domain-containing protein 12 (NLRP12). They can respond to specific stimuli and trigger the assembly of the corresponding pan-apoptosome, forming four types of pan-apoptosomes with different sensors and regulators, namely ZBP1-PANoptosome, AIM2-PANoptosome, RIPK1-PANoptosome, and NLRP12-PANoptosome. [6,7,8,9] .

[0004] Hyperandrogenemia is a typical pathological and biochemical manifestation of PCOS. Studies have found that increased local androgen levels in the ovaries can induce various ovarian dysfunction and pathological manifestations. The study by Huang et al. found that macrophage pyroptosis existed in the serum and ovaries of PCOS mice induced by dehydroepiandrosterone (DHEA), resulting in estrogen synthesis dysfunction and granulosa cell apoptosis.

[10] . Zhou et al. found that DHEA intervention in human T-cell leukemia cells could mediate pyroptosis and lead to granulosa cell apoptosis and follicular atresia in PCOS patients and animal models.

[11] , suggesting that hyperandrogenemia may play an important role in the pathogenesis of PCOS by mediating granulosa cell PCD through DHEA.

[0005] In previous studies by the applicant, it was first found that pan-apoptosis existed in the granulosa cells of PCOS patients, which is one of the important pathological mechanisms leading to chronic inflammation and ovulation disorders in PCOS. However, there is currently no report on the role of pan-apoptosis in the pathogenesis of PCOS. Therefore, establishing a disease research model of pan-apoptosis in PCOS is particularly important for clarifying the pathogenesis of PCOS and its precise intervention.

[0006] References:

[0007] 1. March WA, Moore VM, Willson KJ, Phillips DI, Norman RJ, Davies MJ. The prevalence of polycystic ovary syndrome in a community sample assessed under contrasting diagnostic criteria. Hum Reprod. 2010;25(2):544–551.

[0008] 2. Christgen S, Zheng M, Kesavardhana S, Karki R, Malireddi RKS, Banoth B, Place DE, Briard B, Sharma BR, Tuladhar S, Samir P, Burton A, Kanneganti TD. Identification of the PANoptosome: A Molecular Platform Triggering Pyroptosis, Apoptosis, and Necroptosis (PANoptosis). Front Cell Infect Microbiol. 2020;29;10:237.

[0009] 3. Place DE, Lee S, Kanneganti TD. PANoptosis in microbial infection. Curr Opin Microbiol. 2021;59:42 - 49.

[0010] 4. Malireddi R, Kesavardhana S, Karki R, Kancharana B, Burton AR, Kanneganti TD. RIPK1 distinctly regulates yersinia - induced inflammatory cell death, PANoptosis. Immunohorizons, 2020, 4(12):789 - 796.

[0011] 5. Samir P, Malireddi RKS, Kanneganti TD. The PANoptosome: a deadly protein complex driving pyroptosis, apoptosis, and necroptosis (PANoptosis). Front Cell Infect Microbiol, 2020, 10:238.

[0012] 6. Lee S, Karki R, Wang Y, Nguyen LN, Kalathur RC, Kanneganti TD. AIM2 forms a complex with pyrin and ZBP1 to drive PANoptosis and host defence. Nature. 2021;597(7876):415 - 419.

[0013] 7. Sundaram B, Pandian N, Mall R, Wang Y, Sarkar R, Kim HJ, Malireddi RKS, Karki R, Janke LJ, Vogel P, Kanneganti TD. NLRP12 - PANoptosome activates PANoptosis and pathology in response to heme and PAMPs. Cell. 2023;186(13):2783 - 2801.

[0014] 8. Rostamtabar M, Esmaeilzadeh S, Karkhah A, Amiri M, Rahmani A, Bakouei F, Nouri HR. Elevated expression of IL-18 but not IL-1β gene is associated with NALP3 and AIM2 inflammasome in Polycystic Ovary Syndrome. Gene. 2020;731:144352.

[0015] 9. Zheng M, Karki R, Vogel P, Kanneganti TD. Caspase-6 Is a Key Regulator of Innate Immunity, Inflammasome Activation, and Host Defense. Cell. 2020;181(3):674-687.

[0016] 10. Huang J, Chen P, Xiang Y, Liang Q, Wu T, Liu J, Zeng Y, Zeng H, Liang X, Zhou C. Gut microbiota dysbiosis-derived macrophage pyroptosis causes polycystic ovary syndrome via steroidogenesis disturbance and apoptosis of granulosa cells. Int Immunopharmacol. 2022 Jun;107:108717.

[0017] 11. Zhou J, Peng X, Mei S. Autophagy in Ovarian Follicular Development and Atresia. Int J Biol Sci. 2019;15(4):726-737 Summary of the Invention

[0018] Based on the fact that the pathogenesis of PCOS has not been fully elucidated yet, and pan - apoptosis is one of the important pathological mechanisms of PCOS. The purpose of the present invention is to provide a method for constructing a pan - apoptosis model of human ovarian granulosa cells induced by dihydrotestosterone (DHT) in vitro, so as to lay a good foundation for further in - depth study of the role of pan - apoptosis of ovarian granulosa cells in the pathogenesis of PCOS.

[0019] The purpose of the present invention is achieved by the following technical solutions:

[0020] In the first aspect of the present invention, a method for constructing a pan - apoptosis model of human ovarian granulosa cells induced by dihydrotestosterone in vitro is provided, which includes the following steps: culturing human ovarian granulosa cells treated by starvation with a medium containing dihydrotestosterone to obtain a pan - apoptosis model of human ovarian granulosa cells.

[0021] Furthermore, the method for constructing a pan - apoptosis model of human ovarian granulosa cells induced by dihydrotestosterone in vitro includes the following steps:

[0022] (1) Human ovarian granulosa cells in the logarithmic growth phase are starved - cultured with a medium containing 0.5% - 5% serum for 24 - 72 h;

[0023] (2) The human ovarian granulosa cells treated by starvation culture are cultured with a medium containing dihydrotestosterone and 10% - 20% serum for 12 - 24 h to obtain a pan - apoptosis model of human ovarian granulosa cells.

[0024] The human ovarian granulosa cells are preferably KGN cells. The medium is preferably DMEM / F12 complete medium. The concentration of dihydrotestosterone is preferably 100 nM - 1000 nM.

[0025] In step (1), the serum content is preferably 2%, and the culture time is preferably 24 h.

[0026] In step (2), the concentration of dihydrotestosterone is 500 nM, the serum content is preferably 10%, and the culture time is preferably 24 h.

[0027] In the second aspect of the present invention, a pan - apoptosis model of human ovarian granulosa cells constructed by the above - mentioned construction method is provided, and this cell model simultaneously exhibits key features of pan - apoptosis such as apoptosis, pyroptosis, and necroptosis.

[0028] In the third aspect of the present invention, an application of the above - mentioned pan - apoptosis model of human ovarian granulosa cells as a cell model for studying the pathological mechanism of PCOS is provided.

[0029] Advantages and beneficial effects of the present invention: The present invention provides for the first time an in vitro human ovarian granulosa cell pan-apoptosis model induced by dihydrotestosterone and a method for constructing the same. This cell model simultaneously exhibits key features of pan-apoptosis such as apoptosis, pyroptosis, and necroptosis. Specifically, in this cell model, the mRNA expression levels of NLRP3, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis increase, the protein levels of Cleaved-Caspase1, N-GSDMD related to pyroptosis, Cleaved-Caspase3 related to apoptosis, and p-MLKL related to necroptosis increase, and the secretion level of the pyroptosis-related factor IL-1β in the cell culture supernatant increases. This cell model lays a good foundation for further in-depth study of the pathogenic mechanism of pan-apoptosis in PCOS. Description of the Drawings

[0030] Figure 1 : The expression levels of pan-apoptosis-related genes mNRA and proteins in granulosa cells of PCOS patients increase.

[0031] (A) mRNA levels of pan-apoptosis-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. (B) Protein levels of pan-apoptosis-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. (C) Secretion level of pyroptosis-related IL-1β in the culture medium of granulosa cells of PCOS and control patients. ** indicates P < 0.01, *** indicates P < 0.001.

[0032] Figure 2 : The expression levels of pan-apoptosis-related genes mNRA and proteins in KGN cells treated with DHT increase.

[0033] (A) mRNA levels of pan-apoptosis-related molecules (including NLRP3, IL-1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis) after DHT treatment of KGN cells. (B) Protein levels of pan-apoptosis-related molecules (including Caspase1, GSDMD related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis) after DHT treatment of KGN cells. (C) Secretion level of IL-1β in the culture supernatant after DHT treatment of KGN cells. * indicates P < 0.05, *** indicates P < 0.001. Detailed Embodiments

[0034] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0035] Example 1: Pan-apoptosis exists in granulosa cells of PCOS patients

[0036] 1. Follicular fluid was collected from patients with PCOS (n = 10, PCOS group) and non-PCOS (n = 10, control group) who underwent IVF-assisted pregnancy at Renmin Hospital of Wuhan University from April 2022 to July 2022. The PCOS group was diagnosed according to the Rotterdam criteria in 2003, that is, after excluding other etiologies, at least two of the following three symptoms need to be met: oligo-ovulation or anovulation; clinical and / or biochemical signs of hyperandrogenism; polycystic ovaries. Non-PCOS patients were female patients with infertility caused by simple male factors (such as oligospermia or asthenospermia).

[0037] 2. Granulosa cells were extracted as follows: The follicular aspirate during oocyte retrieval from the patients was centrifuged at 300 g for 20 minutes, a part of the supernatant was collected, and granulosa cells were separated from the precipitate by density gradient centrifugation. The granulosa cells were cultured in DMEM / F12 complete medium containing 10% fetal bovine serum at 37 °C and 5% CO2 for 24 h.

[0038] 3. After culturing, the granulosa cells were collected to extract RNA and protein for RT-qPCR and Western Blot detection respectively, and the cell culture medium was collected for ELISA detection.

[0039] 4. The mRNA levels of pan-apoptosis-related molecules (including NLRP3, IL-1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis) in granulosa cells were detected by RT-qPCR.

[0040] The results are shown in Figure 1 (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.

[0041] 5. The protein levels of pan-apoptosis-related molecules (including Caspase1, GSDMD related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis) were detected by Western Blot.

[0042] The results are shown in Figure 1 (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 of the control group.

[0043] 6. Detection of the secretion level of pyroptosis-related IL-1β in cell culture medium by ELISA method.

[0044] The results are shown in Figure 1 (C). The secretion level of IL-1β in the granulosa cell culture medium of the PCOS group was significantly higher than that of the control group.

[0045] In summary, the above results confirmed the existence of pan-apoptosis (including pyroptosis, apoptosis, necroptosis) in the granulosa cells of PCOS patients. Specifically, the mRNA expression levels of NLRP3, IL-1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL 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.

[0046] Example 2 A method for constructing an in vitro human ovarian granulosa cell pan-apoptosis model induced by dihydrotestosterone

[0047] 1. Cell preparation

[0048] 1) Culturing cells in vitro: KGN cells in good growth state were routinely cultured in DMEM / F12 complete medium containing 10% fetal bovine serum at 37 °C and 5% CO2.

[0049] 2) Cell passage: When the density of KGN cells reached 80%-90%, they were routinely digested with trypsin and passaged, and then inoculated into six-well plates, with 1.2×10^6 cells inoculated in each well.

[0050] 3) Starvation treatment: When the cell density in the six-well plates reached 50%-60%, the medium was replaced with DMEM / F12 complete medium containing 2% fetal bovine serum for starvation treatment, and the cells were cultured for another 24 h.

[0051] 2. Drug intervention

[0052] 1) Drug preparation: Dihydrotestosterone (DHT) drug powder was dissolved in DMSO to prepare a drug solution as a stock solution, which was stored at -20 °C.

[0053] 2) Treating cells with drugs: The DHT stock solution was diluted with DMEM / 12 complete medium containing 10% fetal bovine serum to a working solution with a concentration of 500 nM. After starving KGN cells with DMEM / 12 complete medium containing 2% fetal bovine serum for 24 h, the medium was replaced with DMEM / F12 complete medium containing DHT and the cells were cultured for another 24 h to obtain an in vitro human ovarian granulosa cell pan-apoptosis model.

[0054] Example 3. Verification of a Dihydrotestosterone-Induced Pan-Apoptosis Model in Human Ovarian Granulosa Cells

[0055] 1. Treatment of KGN cells with DHT

[0056] The KGN cells in six-well plates with good growth status were divided into a control group and a DHT group. After treating the starved KGN cells with DMSO and 500 nM DHT respectively according to the method in Example 2 for 24 h, the treated KGN cells and the culture medium were collected for the following detections.

[0057] 1) Detection of the mRNA levels of pan-apoptosis-related molecules in each group by RT-qPCR, including NLRP3, IL-1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis.

[0058] The results are shown in Figure 2 (A). The mRNA expression levels of NLRP3, Caspase1, Caspase3, and MLKL in the DHT group were significantly higher than those in the control group.

[0059] 2) Detection of the protein levels of pan-apoptosis-related molecules in each group by Western Blot, including Caspase1, GSDMD related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis.

[0060] The results are shown in Figure 2 (B). The protein expression levels of Cleaved-Caspase1, N-GSDMD, Cleaved-Caspase3, and p-MLKL in the DHT group were significantly higher than those in the control group.

[0061] 3) Detection of the secretion level of IL-1β in the cell culture medium of each group by ELISA.

[0062] The results are shown in Figure 2 (C). The secretion level of IL-1β in the culture medium of the DHT group was significantly higher than that in the control group.

[0063] In summary, the above results confirmed that pan - apoptosis (including pyroptosis, apoptosis, necroptosis) occurred in DHT - treated KGN cells. Specifically, the mRNA expression levels of NLRP3, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis increased in DHT - treated KGN cells. 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 culture medium increased, which confirmed the successful construction of the KGN cell pan - apoptosis model.

[0064] Table 1. Primer sequences used in RT - qPCR in the above examples

[0065]

[0066]

[0067] The above examples are only used to help illustrate the present invention. The implementation modes of the present invention are not limited by the above examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for constructing a dihydrotestosterone-induced in vitro pan-apoptosis model of human ovarian granulosa cells, characterized in that, It includes the following steps: The starved human ovarian granulosa cells are cultured in a medium containing dihydrotestosterone to obtain a human ovarian granulosa cell pan-apoptosis model.

2. The method for constructing a dihydrotestosterone-induced in vitro human ovarian granulosa cell pan-apoptosis model according to claim 1, wherein It includes the following steps: (1) The human ovarian granulosa cells in the logarithmic growth phase are starved and cultured in a medium containing 0.5%-5% serum for 24-72 h; (2) The starved human ovarian granulosa cells are cultured in a medium containing dihydrotestosterone and 10%-20% serum for 12-24 h to obtain a human ovarian granulosa cell pan-apoptosis model.

3. The method for constructing a dihydrotestosterone-induced in vitro human ovarian granulosa cell pan-apoptosis model according to claim 1 or 2, characterized in that: The human ovarian granulosa cells described above are KGN cells.

4. The method for constructing a dihydrotestosterone-induced in vitro human ovarian granulosa cell pan-apoptosis model according to claim 1 or 2, characterized in that: The medium described above is DMEM / F12 complete medium.

5. The method for constructing a dihydrotestosterone-induced in vitro human ovarian granulosa cell pan-apoptosis model according to claim 1 or 2, characterized in that: The concentration of the dihydrotestosterone described above is 100 nM-1000 nM.

6. The method for constructing a dihydrotestosterone-induced in vitro human ovarian granulosa cell pan-apoptosis model according to claim 2, characterized in that: In step (1), the serum content is 2%.

7. The method for constructing a dihydrotestosterone-induced in vitro human ovarian granulosa cell pan-apoptosis model according to claim 2, wherein: In step (2), the concentration of the dihydrotestosterone is 500 nM.

8. The method for constructing a dihydrotestosterone-induced in vitro human ovarian granulosa cell pan-apoptosis model according to claim 2, wherein: In step (2), the serum content is 10%.

9. A human ovarian granulosa cell pan-apoptosis model, characterized in that: Obtained by the construction method according to any one of claims 1-8.

10. Use of the human ovarian granulosa cell pan-apoptosis model according to claim 9 as a cell model for studying the pathological mechanism of polycystic ovary syndrome.

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