Application of PON3 protein in preparation of medicine for preventing and treating polycystic ovarian syndrome

Abstract

The invention provides application of PON3 protein in preparation of a medicine for preventing and treating polycystic ovarian syndrome, and belongs to the technical field of biological medicine. It is found that the secretion amount of PON3 in follicular fluid of an obese PCOS patient is remarkably reduced, and it is found through further research that the expression level of endoplasmic reticulum stress-related protein in PCOS granular cells under the high fat condition is remarkably increased, and the PON3 protein level is remarkably reduced. The PON3 can be used for inhibiting the occurrence of endoplasmic reticulum stress of PCOS granular cells under a high fat condition through GRP78 / IRE1alpha and GRP78 / PERK pathways. The invention provides a new potential target for the occurrence and development of PCOS diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of PON3 protein in the preparation of drugs for the prevention and treatment of polycystic ovary syndrome. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is a reproductive endocrine and metabolic disorder characterized by anovulation or oligoovulation, hyperandrogenism, and polycystic ovarian changes, affecting 4%–21% of women of reproductive age worldwide. While the underlying pathogenesis of PCOS remains unclear, the disease is thought to be associated with oxidative stress, chronic low-grade inflammation, mitochondrial dysfunction, and metabolic disorders, ultimately leading to impaired ovarian function. Endoplasmic reticulum stress is caused by the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum, which activates three sensor proteins: inositol demand enzyme 1α (IRE1α), double-stranded RNA activated protein kinase-like endoplasmic reticulum kinase (PERK), and activated transcription factor 6 (ATF6), representing three branches of the unfolded protein response (UPR). Prolonged and persistent high androgen exposure leads to endoplasmic reticulum stress and induces granulosa cell autophagy or apoptosis, ultimately resulting in the accumulation of small follicles around the ovary and polycystic ovarian changes.

[0003] Although free radicals and reactive oxygen species are essential in various steps of female reproduction, high concentrations of these substances are associated with reproductive disorders and infertility. To maintain physiologically relevant stable concentrations of these substances, enzymatic and non-enzymatic antioxidants work in coordination within the body. Antioxidant enzymes include phosphoryloxygenases (PONs). The PON gene family consists of three genes: PON1, PON2, and PON3, all of which possess antioxidant properties. PON3, as an intracellular enzyme, is located in the plasma membrane, endoplasmic reticulum, nuclear membrane, and inner mitochondrial membrane, and can also be secreted extracellularly to exert its effects. PON3 has been reported in various human oxidative stress-related diseases. Numerous studies have shown that mouse PON3 may exert its antioxidant effects locally in various epithelial cells and cells. PON3 also has the potential to metabolize drugs and endogenous compounds; therefore, it may serve as a potential biomarker for oxidative stress-related diseases. However, there are currently no reports on the correlation between PON3 and PCOS under high-fat conditions. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the application of PON3 protein in the preparation of drugs for the prevention and treatment of polycystic ovary syndrome.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: Application of PON3 protein in the preparation of drugs for the prevention and treatment of polycystic ovary syndrome.

[0006] Preferably, the polycystic ovary syndrome is polycystic ovary syndrome under high-fat conditions.

[0007] Preferably, the drug increases PON3 protein levels.

[0008] Preferably, the drug inhibits endoplasmic reticulum stress in granulosa cells of polycystic ovary syndrome under high-fat conditions.

[0009] Preferably, the drug improves oocyte quality by inhibiting the activation of the GRP78 / IRE1α and GRP78 / PERK pathways in PCOS granulosa cells under high-lipid conditions due to endoplasmic reticulum stress.

[0010] Preferably, the drug reduces the expression levels of GRP78, p-IRE1α, and p-PERK proteins.

[0011] Preferably, the drug comprises PON3 protein.

[0012] Another object of the present invention is to provide a drug for the prevention and treatment of polycystic ovary syndrome under hyperlipidemia, wherein the active ingredient of the drug includes PON3 protein.

[0013] Preferably, the drug further includes pharmaceutical excipients.

[0014] Preferably, the dosage form of the drug includes oral dosage form and injectable dosage form.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of PON3 protein in the preparation of drugs for the prevention and treatment of polycystic ovary syndrome (PCOS). The study found that PON3 secretion was significantly reduced in the follicular fluid of obese PCOS patients. By constructing a DHEA (dehydroepiandrosterone)-induced PCOS mouse model and a DHT (dihydrotestosterone)-stimulated cell model under high-fat conditions, the expression levels of endoplasmic reticulum stress-related proteins in granulosa cells were detected using Western blotting and immunofluorescence. The results showed that under high-fat conditions, the expression levels of endoplasmic reticulum stress-related proteins in PCOS granulosa cells were significantly increased, while the PON3 protein level was significantly decreased. PON3 supplementation inhibited the activation of the GRP78 / IRE1α and GRP78 / PERK pathways in PCOS granulosa cells under high-fat conditions. This invention provides a new potential target for the development and progression of PCOS. Attached Figure Description

[0016] Figure 1Annotations for differentially expressed proteins; A) Volcano plot: the horizontal axis represents the Ratio value of differential expression change in the comparison group after Log2 transformation; the vertical axis represents the P-value of the statistical test after -Log10 transformation; red dots indicate differentially expressed proteins upregulated, blue dots indicate differentially expressed proteins downregulated, and gray dots indicate proteins with no significant difference; B) Clustering heatmap: each row represents a differentially expressed protein, and each column represents a sample; red represents high expression, and blue represents low expression; C) Violin plot: the vertical axis represents the protein quantification value after Log2 transformation. P <0.05); Figure 2 This section provides GO functional annotation and subcellular localization. A is the GO enrichment plot, with the horizontal axis representing the enrichment significance P-value of the -Log10 transformation and the vertical axis representing the corresponding GO functional description. The length of the bars indicates the enrichment significance, with longer bars indicating stronger enrichment significance. B shows the subcellular structural information of upregulated differentially regulated proteins, with the legend displaying the names and proportions of the corresponding species. C shows the subcellular structural information of downregulated differentially regulated proteins, with the legend displaying the names and proportions of the corresponding species. Figure 3 This study investigated the PON3-mediated endoplasmic reticulum stress in ovarian granulosa cells of PCOS mice. In the data, A-D represent the serum levels of TG, TC, LDL-C, and HDL-C detected using a biochemical assay kit; E represents the protein expression levels of PON3, GRP78, p-IRE1α, p-ATF6, and p-PERK in ovarian granulosa cells of PCOS mice. All data are expressed as mean ± SD. p <0.05,** p <0.01; ns, no significant difference; Figure 4 This study investigated PON3-mediated DHT-induced endoplasmic reticulum stress in granulosa cells under high-fat conditions. A represents cell viability of KGN cells treated with 200 μM and 300 μM PA for 24 h and 48 h, respectively; B represents lipid accumulation in KGN cells after 24 h of PA treatment using Oil Red O staining (scale bar: 50 μm); C represents the protein expression levels of PON3, GRP78, p-IRE1α, p-ATF6, and p-PERK in each treatment group of KGN cells; D represents the protein expression level of p-IRE1α in each treatment group of KGN cells detected by immunofluorescence (scale bar: 40 μm). All data are expressed as mean ± SD. * p <0.05,** p <0.01, *** p <0.001; ns, no significant difference; Figure 5To regenerate PON3's ability to inhibit DHT-induced endoplasmic reticulum stress in granulosa cells under high-fat conditions; A represents the protein expression levels of GRP78, p-IRE1α, and p-PERK in each treatment group of KGN cells; B represents the p-IRE1α protein expression level in each treatment group of KGN cells detected by immunofluorescence, with a scale bar of 40 μm; all data are expressed as mean ± SD; * p <0.05,** p <0.01, *** p <0.001; ns, no significant difference. Detailed Implementation

[0017] This invention provides the application of PON3 protein in the preparation of drugs for the prevention and treatment of polycystic ovary syndrome (PCOS), preferably PCOS under high-fat conditions.

[0018] Polycystic ovary syndrome (PCOS) is a syndrome of endocrine and metabolic disorders that has become very common in women of reproductive age. This invention identified 12 differentially expressed proteins in the follicular fluid (FF) of obese PCOS patients and non-PCOS patients. Two differentially expressed proteins were upregulated, and ten were downregulated. Notably, the expression level of PON3 protein in the FF of obese PCOS patients was significantly lower than that in non-PCOS patients. The PON enzyme family (PON-1, PON-2, and PON-3) is encoded by three adjacent genes located on human chromosome 7q21.3 and exhibits similar activities. All three PON genes are preserved in mammals, indicating the important physiological role of these antioxidant enzymes. Higher PON enzyme activity was observed in FF compared to serum, which was attributed to the expression and secretion of PON by granulosa cells in FF.

[0019] In this invention, the drug preferably increases the level of PON3 protein. The drug of this invention inhibits endoplasmic reticulum stress in granulosa cells of PCOS patients under high-fat conditions; the drug preferably inhibits the activation of the GRP78 / IRE1α and GRP78 / PERK pathways in PCOS granulosa cells under high-fat conditions; the drug preferably reduces the expression levels of GRP78, p-IRE1α, and p-PERK proteins; the preferred drug includes PON3 protein.

[0020] Endoplasmic reticulum stress (ERS) is triggered by the accumulation of unfolded / misfolded proteins in the endoplasmic reticulum (ER). ER stress activation has been widely observed in ovarian granulosa cells of PCOS patients and androgen-induced PCOS mice. IRE1α is involved in maintaining ER homeostasis; when IRE1α dissociates from ER chaperone-binding immunoglobulin (BIP), it initiates UPR (unfolded protein response) signaling and induces OS (oxidative stress), inflammation, and cell death. This invention constructs a DHEA-induced PCOS mouse model and a DHT-induced KGN cell model under high-fat conditions. Animal-level results showed that the expression levels of ERS-related proteins in ovarian tissue cells of PCOS mice were significantly increased compared to control mice, and were negatively correlated with PON3 protein expression levels. Cell-level results showed that the expression levels of ERS-related proteins in the DHT+PA (palmitic acid) treatment group were significantly upregulated compared to the control group, while PON3 protein levels were significantly downregulated, suggesting that PON3 mediates ERS in PCOS granulosa cells under high-fat conditions. The accumulation of misfolded and unfolded proteins in the ER can replace the binding of BiP to PERK, ATF6, and IRE1, thereby activating them. This study found that PON3 inhibits endoplasmic reticulum stress in PCOS granulosa cells under high-fat conditions through the GRP78 / IRE1α and GPR78 / PERK signaling pathways.

[0021] The present invention also provides a drug for the prevention and treatment of polycystic ovary syndrome under hyperlipidemia, wherein the active ingredient of the drug includes PON3 protein.

[0022] In this invention, the drug preferably further includes pharmaceutical excipients, and the dosage form of the drug can be selected from either oral dosage forms or injectable dosage forms.

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1 Animal experiments and cell experiments 1. Materials and Methods 1.1 Ethical Statement All participants provided written informed consent, donated their biological waste materials, and underwent follicular fluid collection and related de-identified clinical information, including age, fertility diagnosis, body mass index, and treatment regimen (Table 1). The request for and use of samples from this invention were approved by the hospital's ethics committee.

[0025] Table 1 Comparison of basic clinical information

[0026] 1.2 Follicular fluid collection and clinical biochemical analysis Follicular fluid was collected from the dominant follicles of each participant via transvaginal ultrasound guidance. Only clear follicular fluid free from blood contamination was included. Samples were centrifuged at 2500 rpm for 10 min, and the supernatant was collected and stored in cryopreservation tubes. All clinical information, such as age and BMI (Table 1), was collected at enrollment. Participants' biochemical characteristics were examined at Yinchuan Maternal and Child Health Hospital.

[0027] 1.3 KGN cell culture KGN cells were purchased from Shanghai Binsui Biotechnology Co., Ltd. Cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a CO2 incubator. Cells were treated with dihydrotestosterone (DHT) (Selleck, USA) at a concentration of 20 μM; palmitic acid (PA) (MCE, USA) was used to treat cells in a high-lipid state at concentrations of 200 μM and 300 μM for 24 h and 48 h, respectively, followed by CCK8 and Oil Red O staining; PON3 (MCE, USA) was used to treat cells at a concentration of 10 μg / mL.

[0028] KGN cells were cultured until the cell density reached 70%, at which point the drug addition experiment began.

[0029] DHT group: Add 1 μL of DHT solution (DHT dissolved in DMSO, working concentration is 20 µM), and incubate at 37℃ for 24 h; DHT+PA group: 1 μL of DHT solution was added along with 1 μL of LPA solution (PA dissolved in anhydrous ethanol solution, working concentration of 200 µM), and the mixture was incubated at 37℃ for 24 h. DHT+PON3 group: 1 μL of DHT solution was added along with 1 μL of PON3 solution (PON3 ​​dissolved in PBS solution, working concentration 10 μg / mL), and the mixture was incubated at 37℃ for 24 h. DHT+PA+PON3 group: 1 μL of DHT solution and 1 μL of PA solution were added simultaneously, along with 1 μL of PON3 solution, and the mixture was incubated at 37℃ for 24 h.

[0030] 1.4 Establishment of a mouse model of polycystic ovary syndrome Five- to six-week-old C57BL / 6J mice were purchased from Ningxia Medical University, and their use was approved by the Animal Welfare and Ethics Committee of Ningxia University (Approval No.: NXU-2020-012). In this study, female C57BL / 6J mice were subcutaneously injected with 100 μL of the androgen DHEA at a concentration of 12 mg / mL, and fed a high-fat diet to establish an obese PCOS mouse model. The control group was injected with 100 μL of castor oil using the same method. Serum and ovarian tissue were collected from the mice on day 21 after subcutaneous DHEA injection for subsequent analysis.

[0031] 1.5 Proteomics The DIA proteomics study was completed by Hangzhou Jingjie Biotechnology Co., Ltd.

[0032] 1.6 Detection of serum TG (triglycerides), TC (total cholesterol), LDL-C (low-density cholesterol), and HDL-C (high-density lipoprotein cholesterol) levels Blood samples from mice were obtained from ocular veins and centrifuged at 4500×g for 20 min at 4°C. Serum was collected. Serum levels of TG, TC, LDL-C, and HDL-C were measured using a biochemical analysis kit (Elabscience, China) according to the manufacturer's instructions.

[0033] 1.7 Western blotting After receiving the protein samples, the procedure was performed according to the manufacturer's instructions. Protein concentration was measured using a BCA kit (KeyGen, China), separated by SDS-PAGE, and wet-transferred to a PVDF membrane (Millipore, USA). The membrane was then incubated overnight with PON3 antibody (Affinity, China), GRP78 antibody (Affinity, China), p-IRE1α antibody (Affinity, China), IREα antibody (CST, USA), p-ATF6 antibody (Affinity, China), ATF6 antibody (Affinity, China), p-PERK antibody (Proteintech, China), PERK antibody (Proteintech, China), and GAPDH antibody (Proteintech, China). The next day, the samples were incubated with peroxidase-conjugated secondary antibody (ZSGB-BIO, China) and visualized using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, USA).

[0034] 1.8 CCK8 Detection Cells with different treatments were seeded into 96-well plates. After treatment, 10% CCK8 (Vazyme, China) medium was added to each well. The 96-well plates were then placed back into the cell culture incubator and incubated for 1.5 h. Finally, the absorbance was measured at 450 nm using a microplate reader (Perkin Elmer, USA).

[0035] 1.9 Oil Red O staining After seeding cells into 6-well plates and treating them, the culture medium was discarded, and 4% paraformaldehyde (Solarbio, China) was added for fixation for 20 min. Then, an appropriate volume of Oil Red O working solution was added, and the cells were incubated at room temperature in the dark for 1 h. The Oil Red O working solution was removed, and the cells were washed with PBS until no background color was visible. Each well was then soaked with PBS and observed and photographed under a microscope (Olympus, JPN).

[0036] 1.10 Cellular Immunofluorescence Assay After cell treatment, the culture medium was aspirated and the cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 for 15 min, and then blocked with 10% donkey serum for 30 min. The blocking solution was removed, and the cells were incubated overnight at 4°C with the primary antibody p-IRE1α. The next day, the cells were incubated with the secondary antibody (Yeasen, China) conjugated with Alexa Flour 488. Finally, the cell nuclei were stained with DAPI (Beyotime, China) for 5 min. The cells were then observed and imaged using a DMI 8 (Leica stellaris 5, GER) microscope.

[0037] 1.11 Statistical analysis of protein expression Protein absorbance (A) values ​​were analyzed using ImageJ software. All data were statistically analyzed using T-test or One-way ANOVA in GraphPadPrism 8.0 software. Protein expression levels are expressed as mean ± SD, and P < 0.05 was considered statistically significant.

[0038] 2 Results 2.1 Differential Protein Annotation This invention collected follicular fluid from 9 non-PCOS patients and 9 obese PCOS patients for proteomics sequencing, identifying a total of 414 proteins (Table 2). Differentially enriched proteins were screened according to the criteria of fold change (FC) > 1.5 or FC < 1 / 1.5 with a P value < 0.05. Two differentially regulated proteins were found to be upregulated, and 10 differentially regulated proteins were found to be downregulated. Figure 1(A~B in Table 3). Violin plots show that the PXDN protein level was significantly higher in the obese PCOS group compared to the non-PCOS group, while the protein levels of PON3, LAMB2, GANAB, SHBG, and CYP11A1 were significantly lower. Figure 1 (C in the middle).

[0039] Table 2. Statistical Table of Protein Identification

[0040] Table 3 The number of differential proteins

[0041] 2.2 GO Functional Annotation and Subcellular Localization This invention performs GO functional annotation on the enriched differentially expressed proteins. Molecular function analysis shows that the differentially expressed proteins are mainly related to extracellular matrix structural constituent, heme binding, and tetrapyrrole binding functions. Biological processes show that differentially expressed proteins are mainly involved in basement membrane organization, neuron projection guidance, and cellular response to tumor necrosis factor. Figure 2 (A in the original text). Subcellular localization results showed that the two upregulated differentially expressed proteins were located in the extracellular space, while the downregulated differentially expressed proteins were located in the extracellular space, mitochondria, and endoplasmic reticulum. Figure 2 (B~C in the middle).

[0042] 2.3 PON3-mediated endoplasmic reticulum stress in PCOS ovarian granulosa cells under high-fat conditions Previous omics data revealed a significant downregulation of PON3 protein levels in the follicular fluid of obese PCOS patients. PON3, as an oxygen phosphatase, participates in endoplasmic reticulum stress in cells. To further investigate whether PON3 mediates endoplasmic reticulum stress in PCOS granulosa cells under high-fat conditions, a PCOS mouse model was established by subcutaneously injecting 60 mg / kg DHEA into 5-week-old C57 mice for 21 consecutive days while simultaneously feeding them a high-fat diet. The control group received the same dose of adjuvant. After modeling, the body weight of the two groups of mice was measured. The body weight of the PCOS mouse group was 24.13 ± 2.56 g, while the body weight of the control group was 20.78 ± 1.95 g. The body weight of the PCOS mice was significantly higher than that of the control group (P < 0.05), indicating a statistically significant difference.

[0043] Lipid-related markers revealed that serum triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels in PCOS mice were significantly increased compared to the control group, while high-density lipoprotein cholesterol (HDL-C) levels were significantly decreased. Figure 3 (A~D in the table) indicates that androgen-induced elevated cholesterol levels and abnormal lipid metabolism are observed in PCOS mice. Subsequently, this invention used Western blotting to detect the levels of PON3 and endoplasmic reticulum stress-related proteins in PCOS ovarian granulosa cells. The results showed that the levels of GRP78, p-IRE1α, and p-PERK proteins in PCOS mouse granulosa cells were significantly upregulated compared to the control group, while the level of PON3 protein was significantly downregulated (…). Figure 3 (E in the text).

[0044] A DHT-induced granulocyte cell model under high-fat conditions was established by co-stimulating KGN cells with DHT and PA. To determine the optimal concentration and duration of PA stimulation, the survival rate of KGN cells treated with 200 μM and 300 μM PA for 24 h and 48 h was detected using a CCK8 assay. The results showed that 200 μM PA significantly reduced cell viability. Figure 4 In the A section, Oil Red O staining results showed increased lipid accumulation in KGN cells under the same conditions. When the PA concentration was 200 μM and the treatment time was 24 h, increased lipid accumulation was observed in KGN cells, and the cell viability was significantly higher than that at 300 μM. Figure 4 (B in the text). Subsequently, Western blotting and immunofluorescence were used to detect the expression levels of PON3 and endoplasmic reticulum stress-related proteins in cells. The results showed that the levels of GRP78, p-IRE1α, and p-PERK proteins were significantly increased and the level of PON3 protein was significantly decreased in the DHT+PA group, consistent with the results in the animal group. Figure 4 (C~D in the middle).

[0045] 2.4 Exogenous PON3 supplementation inhibits DHT-induced endoplasmic reticulum stress in granular cells under high-fat conditions. To further investigate the regulatory role of PON3 on DHT-induced endoplasmic reticulum stress in granulosa cells under high-fat conditions, a complementation experiment was conducted by adding recombinant PON3 protein exogenously. The experiment was divided into 5 groups: Ctrl group, DHT group, DHT+PA group, DHT+PON3 group and DHT+PA+PON3 group.

[0046] Western blotting and immunofluorescence results showed that exogenous PON3 significantly reduced the levels of DHT-induced endoplasmic reticulum stress-related proteins GRP78, p-IRE1α, and p-PERK in granulocytes under high-fat conditions. Figure 5 (A~B in the original text).

[0047] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of PON3 protein in the preparation of drugs for the prevention and treatment of polycystic ovary syndrome.

2. The application according to claim 1, characterized in that, The polycystic ovary syndrome mentioned refers to polycystic ovary syndrome under high-fat conditions.

3. The application according to claim 1, characterized in that, The drug increases PON3 protein levels.

4. The application according to claim 1, characterized in that, The drug inhibits endoplasmic reticulum stress in granulosa cells of polycystic ovary syndrome under high-fat conditions.

5. The application according to claim 4, characterized in that, The drug improves oocyte quality by inhibiting the activation of the GRP78 / IRE1α and GRP78 / PERK pathways in PCOS granulosa cells under endoplasmic reticulum stress under high-lipid conditions.

6. The application according to claim 5, characterized in that, The drug reduces the expression levels of GRP78, p-IRE1α, and p-PERK proteins.

7. The application according to claim 1, characterized in that, The drug includes the PON3 protein.

8. A drug for the prevention and treatment of polycystic ovary syndrome under hyperlipidemia, characterized in that, The active pharmaceutical ingredient includes PON3 protein.

9. The medicament according to claim 8, characterized in that, The drug also includes pharmaceutical excipients.

10. The medicament according to claim 8, characterized in that, The dosage forms of the drug include oral dosage forms and injectable dosage forms.

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