Application of triacetylglycerol in the preparation of drugs for the prevention or treatment of infertility

Abstract

This invention belongs to the field of biomedicine and discloses the application of triacetylglycerol (GTA) in the preparation of drugs for the prevention or treatment of infertility. This invention bypasses ACLY defects by supplementing with acetyl donor substances, restoring histone acetylation levels and thereby improving decidualization function. It can be effectively applied to the prevention and treatment of recurrent miscarriage (RSA) and repeated implantation failure, expanding the new applications of GTA in the pharmaceutical field.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of triacetylglycerol in the preparation of drugs for the prevention or treatment of infertility. Background Technology

[0002] Decidualization of the uterine lining in women refers to the process in early embryo implantation where endometrial stromal cells differentiate into decidual cells under the influence of progesterone and other hormones, secreting various factors to support embryonic growth. Adequate decidualization is crucial for maintaining pregnancy. Studies show that approximately 1%–2% of women of reproductive age experience recurrent miscarriage (RSA), and there is evidence suggesting that abnormal decidualization is one of the underlying causes of miscarriage in these cases.

[0003] In terms of molecular mechanisms, decidualization is accompanied by extensive gene expression reprogramming and changes in chromatin state. Among these, epigenetic regulation, particularly histone acetylation, has attracted considerable attention. Histone acetylation is typically associated with gene activation and is an important modification marker at the promoters of various genes required for stromal cell differentiation. For example, during human decidualization, the promoter regions of typical functional genes prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP1) show increased levels of histone H3 lysine acetylation (H3K27ac), which is closely related to gene transcriptional activation. Correspondingly, PRL and IGFBP1 are secreted at high levels in decidual cells, serving as important markers of decidualization degree. Conversely, dysregulation of histone acetylation regulation can lead to abnormal expression of decidual-related genes, thereby inducing decidualization defects.

[0004] Acetyl-CoA is the direct substrate for histone acetylation, and an adequate intracellular supply is crucial for maintaining normal acetylation levels. ATP-citrate lyase (ACLY) is a key enzyme in the synthesis of acetyl-CoA from carbon sources such as glucose. In recent years, increasing evidence suggests that cellular metabolic state influences epigenetic modifications such as histone acetylation by altering the levels of intermediate metabolites like acetyl-CoA, thereby participating in gene expression regulation. ACLY converts mitochondrial citrate into acetyl-CoA and transports it to the cytoplasm and nucleus for use by histone acetyltransferases, thus promoting histone acetylation.

[0005] Current interventions for RSA or implantation failure primarily focus on empirical measures such as endocrine support, immunotherapy, and anticoagulation therapy. However, these therapies do not address the defects in decidual function itself, resulting in limited efficacy. For epigenetic abnormalities, histone deacetylase inhibitors can theoretically be used to increase histone acetylation levels. However, HDAC inhibitors have broad effects and may cause side effects. For example, studies applying the HDAC inhibitor TSA to human endometrial stromal cells showed a certain degree of enhanced expression of decidualization markers, but simultaneously disrupted the balance of expression of matrix metalloproteinases and their inhibitors, adversely affecting the invasive ability of embryonic trophoblast cells. In other words, a global increase in histone acetylation may excessively inhibit the degradation of extracellular matrix required for embryonic invasion, thus affecting the implantation process. Therefore, directly using HDAC inhibitors is not an ideal strategy.

[0006] On the other hand, supplementing metabolic substrates is another possible intervention approach. For example, Tang et al. reported the role of the glutamine-α-ketoglutarate (αKG) metabolic axis in decidualization: glutamine metabolism is impaired in the endometrium of RSA patients, leading to poor decidual development; in mouse models, glutamine deficiency reduces αKG levels, impairs decidualization, and increases embryo loss rate, while αKG supplementation shows promise in correcting this decidual defect. This study suggests that specific metabolite supplementation could be a potential strategy for correcting decidualization abnormalities. However, metabolic supplementation therapies targeting the acetyl-CoA-histone acetylation axis have not yet been reported. Summary of the Invention

[0007] The purpose of this invention is to provide the application of triacetylglycerol (GTA) in the preparation of drugs for the prevention or treatment of infertility. By supplementing acetyl donor substances to bypass ACLY defects, histone acetylation levels are restored, thereby improving decidualization function. It can be effectively applied to the prevention and treatment of recurrent miscarriage (RSA) and repeated implantation failure, expanding the new applications of GTA in the pharmaceutical field.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] The application of a triacetate (Glycerol Triacetate, abbreviated as GTA) in the preparation of drugs for the prevention or treatment of infertility.

[0010] This invention discovers that decreased expression of ATP-citrate lyase (ACLY) leads to impaired decidualization function of human endometrial stromal cells (hESCs). GTA, as an intervention, can correct the decidualization defect of hESCs caused by decreased ACLY expression, thus being applied to the prevention and treatment of infertility (such as RSA and recurrent implantation failure), expanding the new applications of GTA in the medical field. Specifically, after entering the cell, GTA can be hydrolyzed to release acetic acid, which is then rapidly converted into acetyl-CoA by acetyl-CoA synthase (ACSS2) in the cytoplasm and nucleus. This process skips the step of ACLY converting citrate to acetyl-CoA, directly replenishing the intracellular acetyl-CoA pool, thus providing a substrate for histone acetylation. Under nutrient-limited or hypoxic conditions, cells rely on ACSS2 to reuse acetyl groups from exogenous acetic acid to maintain histone acetylation levels. Therefore, when ACLY function is insufficient, supplementing acetyl-CoA through the acetic acid production pathway via GTA hydrolysis is feasible and effective. Furthermore, GTA can be synthesized by esterification of glycerol and acetic acid under acid catalysis, with the chemical formula C9H10H2O. 14 O6 is a colorless, transparent, oily liquid at room temperature. It has good thermal stability and biocompatibility. GTA is widely available and inexpensive. It is an FDA-approved food additive used in the food and pharmaceutical fields. It has good safety and low adverse reactions.

[0011] Furthermore, the use of the triacetylglycerol in the preparation of drugs for the prevention or treatment of recurrent miscarriage.

[0012] Furthermore, the application of the triacetylglycerol in the preparation of drugs for the prevention or treatment of recurrent implantation failure.

[0013] Furthermore, the use of the triacetylglycerol in the preparation of drugs for the prevention or treatment of infertility related to decidualization defects caused by low ACLY expression.

[0014] Furthermore, the triacetylglycerol restores the expression of decidual-related genes by enhancing histone acetylation, thereby restoring the normal epigenetic markers and functions of decidual tissue.

[0015] This invention discovered that ACLY expression was significantly reduced in the decidual tissue of RSA patients during early pregnancy. Further cell function experiments demonstrated that interfering with ACLY inhibited the decidualization process of human endometrial stromal cells (hESCs), accompanied by a significant decrease in histone H3K27ac levels. In other words, when cells cannot effectively produce acetyl-CoA, insufficient histone acetylation labeling leads to restricted transcription of key decidualization genes such as PRL and IGFBP1, thus impairing decidual differentiation. Therefore, in this invention, adding GTA to primary human endometrial stromal cells with ACLY knockdown significantly restored intracellular histone acetylation levels such as H3K27ac, and restored the expression of impaired decidualization marker genes. This indicates that GTA can correct the metabolic-epigenetic imbalance caused by ACLY deficiency, improve abnormal decidualization, and thus potentially increase embryo implantation rates and reduce the incidence of recurrent miscarriage.

[0016] Furthermore, the drug includes a pharmaceutically acceptable carrier.

[0017] Furthermore, the pharmaceutically acceptable carrier is a filler, wetting agent, binder, disintegrant, or lubricant.

[0018] Furthermore, the dosage form of the pharmaceutical preparation is oral tablets, granules, or capsules.

[0019] This invention bypasses ACLY deficiency by supplementing acetyl donor substances, restoring histone acetylation levels and thereby improving decidualization function. This strategy is drastically different from traditional methods and has a novel mechanism of action. In summary, GTA, as a metabolic substrate supplement, provides a novel intervention pathway for the pathogenesis of infertility (such as RSA and recurrent implantation failure). Compared with traditional treatments, its advantages lie in its direct and targeted action: restoring histone acetylation and gene expression regulation by supplementing acetyl substrates, without altering endocrine levels or broadly suppressing immune function, resulting in fewer side effects and high specificity. Furthermore, GTA is a small molecule compound that can be administered orally and is easily absorbed by tissues, eliminating the need for complex gene therapy methods, thus demonstrating good clinical feasibility and innovation.

[0020] This invention provides a new approach and method for solving the problem of endometrial decidualization defects caused by low ACLY expression. This invention focuses on the correction of metabolic-epigenetic imbalance, aiming to restore the normal epigenetic markers and functions of decidual tissue, thereby improving the embryo implantation environment and preventing infertility caused by this, such as recurrent miscarriage and repeated implantation failure. Attached Figure Description

[0021] Figure 1 The expression of ACLY in the uterine epithelium of patients with recurrent miscarriage;

[0022] Figure 2 The dynamic expression pattern of ACLY during the decidualization process of hESC;

[0023] Figure 3 Knockdown of ACLY leads to impaired decidualization process;

[0024] Figure 4 To supplement GTA, it can restore decidualization abnormalities caused by ACLY knockdown. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1: Detection of ACLY in patient decidual tissue

[0027] This embodiment verifies the correlation between low ACLY expression and decidual dysfunction by detecting the expression of ACLY in the decidual tissue of RSA patients and normal pregnant women, providing a clinicopathological basis for GTA intervention strategies.

[0028] The specific method was as follows: decidual tissue was collected from four healthy women in early pregnancy who underwent induced abortion at 6–8 weeks of gestation as the control group, and decidual tissue was collected from five patients with recurrent miscarriage (≥2 consecutive spontaneous abortions) at 6–8 weeks of gestation as the RSA group. All specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and prepared into 5 μm thick tissue sections. Immunohistochemistry (IHC) was used to detect the expression and localization of ACLY protein. IHC staining was performed using enzyme-linked immunosorbent assay (ELISA). The sections were incubated with ACLY primary antibody overnight, and the secondary antibody was the corresponding HRP-labeled antibody followed by DAB staining. The results were observed and photographed under a microscope. In addition, some freshly frozen decidual tissue was extracted for Western blotting (WB) to quantitatively compare the difference in ACLY levels between the RSA group and the control group.

[0029] This study collected decidual tissue from normal controls in early pregnancy and patients with recurrent spontaneous abortion (RSA). The expression and localization of ACLY were preliminarily detected by IHC, real-time quantitative PCR (RT-qPCR), and Western blotting. Specifically, immunohistochemical analysis revealed ACLY expression in decidual tissues of both control and RSA patients: GE, glandular epithelium; S, stroma (…). Figure 1 (A); the protein level of ACLY in the decidual tissue of CON and RSA was detected by Western blotting (WB). Figure 1 B in the text); quantitative analysis of ACLY protein WB results ( Figure 1 (C in the text); **p<0.01.

[0030] like Figure 1 As shown in Figure AC, the results indicated that ACLY expression was significantly reduced in the decidual tissue of RSA patients compared to normal decidual tissue. This suggests that low ACLY expression exists in the decidual tissue of RSA patients.

[0031] Example 2: Dynamic expression patterns of ACLY and acetyl-CoA during the decidualization process of hESCs

[0032] This embodiment utilizes an endometrial stromal cell decidualization model to clarify the dynamic expression pattern of ACLY and the changes in acetyl-CoA before and after decidualization at the cellular level.

[0033] The specific method was as follows: hESCs were isolated from the endometrial tissue of healthy donors and cultured in DMEM / F12 medium containing 10% FBS. In in vitro decidualization induction, after 2 days of cell culture, the medium was replaced with induction medium supplemented with estrogen (E2, 10 nM), medroxyprogesterone acetate (MPA, 1 μM), and bis(butyryl) cyclic adenosine monophosphate (cAMP, 0.5 mM). Induction was performed for 2, 4, and 6 days, and cells were collected at different time points for experimental analysis.

[0034] In this embodiment, the mRNA levels of IGFBP1, PRL, and ACLY in hESCs at 0, 2, 4, and 6 days of decidualization were detected by RT-qPCR. Figure 2 In the A section, Western blotting was used to detect the protein levels of ACLY and p-ACLY in hESCs at 0, 2, 4, and 6 days of decidualization. Figure 2 (B in the text) Immunofluorescence detection of ACLY localization and protein content changes in hESCs decidualization at 0, 2, 4, and 6 days (in the text). Figure 2 The levels of ACLY protein in hESCs at 0 and 4 days of decidualization were detected by nuclear-cytoplasmic separation Western blotting (C). Figure 2 The concentration of acetyl-CoA in hESCs was detected by ELISA on days 0 and 2 of decidualization. Figure 2 E in

[0035] like Figure 2 As shown in AC, the results of RT-qPCR, WB, and immunofluorescence assays showed that ACLY expression was significantly increased after decidualization compared to before decidualization. Figure 2 As shown in D, nuclear-cytoplasmic separation WB results indicate that the increase in ACLY after decidualization of hESCs is mainly concentrated in the nucleus. Figure 2As shown in E, the ELISA results showed that the content of acetyl-CoA was significantly increased after decidualization compared with that of undecidualized hESCs.

[0036] Example 3: Interference with ACLY leads to impaired decidualization process in primary hTSCs

[0037] This embodiment utilizes the hESCs decidualization model to simulate a low-expression state of ACLY by interfering with the ACLY gene, and observes the changes in gene expression and epigenetic indicators during decidualization, further demonstrating the key role of ACLY in decidualization and the functional defects caused by knocking down ACLY.

[0038] The specific method was as follows: The hESCs isolated in Example 2 were divided into two groups: (1) negative control group (shCtrl / siCtrl); (2) ACLY knockdown group (shACLY and siACLY). Each group was set up with 3 replicates, and the knockdown efficiency was confirmed by RT-qPCR and WB detection of ACLY expression. Two days after decidualization induction, the transcriptional levels of ACLY and decidualization-related genes were detected by RT-qPCR, and the expression of ACLY, total H3 and its acetylation modification, and decidual secretory protein IGFBP1 were analyzed by WB.

[0039] In this embodiment, the mRNA expression levels of ACLY, ACSS2, IGFBP1, PRL, and FOXO1 in shCtrl and shACLY were detected by RT-qPCR 2 days after decidualization. Figure 3 A in the text). The protein levels of ACLY, ACSS2, and IGFBP1 in shCtrl and shACLY were detected by Western blotting (WB). Figure 3 In the B), Western blot analysis of histone acetylation modifications (H3K27ac, H3K9ac, H3ac) was performed 2 days after decidualization. shCtrl and sh ACLY Levels in the group ( Figure 3 (C) Western blot analysis was performed on the protein levels of ACLY, ACSS2, and IGFBP1 in siCtrl and siACLY 2 days after decidualization. Figure 3 In the D), Western blot analysis of histone acetylation modifications (H3K27ac, H3K9ac, H3ac) 2 days after decidualization showed siCtrl and si ACLY Levels in the group ( Figure 3 (E in the text).

[0040] like Figure 3As shown in Figure A, RT-qPCR quantification results indicated that the ACLY mRNA level in the shACLY group was lower than that in the shCtrl group, indicating a significant interference effect. Regarding functional genes, ACLY knockdown led to a significant decrease in the mRNA levels of classic decidualization markers PRL, IGFBP1, and FOXO1.

[0041] like Figure 3 As shown in the BE diagram, the Western blot results further validated the above findings: on day 2 of decidualization, compared with the control group, the protein level of IGFBP1 in the shACLY group was significantly reduced, and the level of H3 acetylation (H3ac) in the whole group was significantly reduced, with the decrease in H3K27ac being the most prominent. Similar effects were obtained by specifically knocking down ACLY cells using siRNA.

[0042] The above data fully demonstrate that ACLY plays a crucial role in decidualization, and its low expression leads to reduced histone acetylation and impaired expression of functional genes, thereby hindering the normal decidual differentiation process. This lays the foundation for further verification of the effectiveness of GTA intervention.

[0043] Example 4: GTA intervention experiment on decidualization defects caused by ACLY knockdown

[0044] Based on the in vitro ACLY knockdown decidualization model in Example 3 above, this embodiment introduces GTA as an exogenous acetyl donor for intervention, and observes its effects on histone acetylation levels and decidualization-related functional indicators to evaluate the effect of GTA in correcting decidualization abnormalities.

[0045] The specific method was as follows: using a primary hESC decidualization system, after ACLY interference treatment (siACLY), the cells were divided into two groups: one group was treated with 4 mM GTA, and the other group was treated without GTA as a control. Cell samples were collected at the end of day 2 of induction for various tests, including qPCR and Western blotting.

[0046] In this embodiment, the mRNA expression levels of ACLY, ACSS2, and IGFBP1 in siCtrl, siACLY, and siACLY with added GTA were detected by RT-qPCR 2 days after decidualization. Figure 4 The levels of AC in siCtrl, siACLY, IGFBP1, and histones H3K27ac and H3ac were detected by Western blotting 2 days after decidualization, and the protein levels of AC were measured by Western blotting after the addition of GTA. Figure 4 (D in the middle).

[0047] like Figure 4As shown in AC, RT-qPCR results indicated that GTA treatment had no significant effect on the mRNA levels of ACLY and ACSS2 genes themselves, suggesting that exogenous acetic acid did not provide feedback regulation of the expression of these enzymes. However, in terms of functional genes, GTA significantly increased the expression of decidualization markers: after adding GTA in the siACLY background, the mRNA level of IGFBP1 was significantly upregulated compared to the untreated group.

[0048] like Figure 4 As shown in Figure D, the intervention effect of GTA is more intuitive at the protein level. Western blotting revealed that the band intensities of IGFBP1, histone H3K27ac, and whole H3ac were significantly enhanced in the siACLY+GTA group compared to the siACLY group. Notably, the ACSS2 protein level did not differ significantly from the untreated group after GTA treatment, indicating that GTA mainly works by providing substrates rather than altering ACSS2 expression. Therefore, GTA can partially restore decidualization abnormalities caused by ACLY knockdown.

[0049] The results of this invention provide crucial functional validation: in a model of impaired ACLY activity, GTA supplementation can bypass metabolic bottlenecks and restore the expression of decidual-related genes by enhancing histone acetylation. This demonstrates the feasibility of using metabolic substrate supplementation to improve epigenetic status. The GTA application strategy proposed in this invention has achieved the expected results at the cellular level, supporting its use as a novel intervention for the prevention and treatment of diseases such as recurrent miscarriage associated with low ACLY expression.

Claims

1. The use of a triacetylglycerol in the preparation of a drug for the prevention or treatment of recurrent miscarriage.

2. The application according to claim 1, characterized in that, The triacetylglycerol restores the expression of decidual-related genes by enhancing histone acetylation, thereby restoring normal epigenetic markers and functions of decidual tissue.

3. The application according to claim 1, characterized in that, The drug includes a pharmaceutically acceptable carrier.

4. The application according to claim 3, characterized in that, The pharmaceutically acceptable carrier is a filler, wetting agent, binder, disintegrant, or lubricant.

5. The application according to claim 1, characterized in that, The dosage form of the pharmaceutical preparation is oral tablets, granules, or capsules.

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