Application of pea peptide in preparation of medicine for preventing and treating ovarian injury

Abstract

The invention relates to application of pea peptide in preparation of medicines for preventing and treating ovarian injury, and belongs to the technical field of biological medicines. It is found for the first time that the pea peptide can significantly relieve ovarian injury caused by decabromodiphenylethane, which indicates that the pea peptide has the purpose of preventing and / or treating ovarian injury. Experiments show that the pea peptide can reduce expression of key protein gamma-H2AX of an ovarian tissue DNA damage pathway, expression of key protein related to an ovarian tissue senescence pathway and expression of key protein related to ovarian tissue inflammasome, can significantly improve morphological damage of an ovarian tissue structure and significantly relieve senescence of ovarian tissue cells, and can be used for preparing the pea peptide. The invention provides a novel medicine for preventing and / or treating ovarian injury clinically, and has better clinical application value and wide application prospect.

Description

Technical Field

[0001] The present invention relates to the application of pea peptide in preparing medicine for preventing and treating ovarian damage, and belongs to the technical field of biomedicine. Background Art

[0002] The primary function of the ovaries is ovulation and the secretion of female hormones. With aging, ovarian function begins to decline around the ages of 45 to 50, with estrogen levels dropping, marking the onset of perimenopause. However, some women experience premature ovarian failure before the age of 40 due to environmental, dietary, lifestyle, psychological, or pathological factors. Ovarian aging can cause physical and physiological changes in women, such as menstrual changes, osteoporosis, vasomotor symptoms (hot flashes, night sweats), increased skin wrinkling, mood swings, and other menopausal symptoms. It can also weaken the body's immune system, accelerate aging, and severely impact women's quality of life.

[0003] Decabromodiphenyl ethane (DBDPE) is a new brominated flame retardant with advantages such as high bromine content, stable flame retardancy, and UV resistance. It is considered a promising alternative to the traditional flame retardant decabromodiphenyl ether (BDE-209). In recent years, my country's annual DBDPE production has grown at a rate of up to 80%, and total DBDPE production is expected to reach nearly 90 tons per year by 2026. DBDPE is widely used in plastics, electronic and electrical products, building materials, and textile coatings. It easily enters the environment through volatilization or abrasion, and is a major new brominated flame retardant component in environmental media such as water, sediment, atmosphere, and soil. DBDPE is one of the most widely used flame retardants and an emerging environmental pollutant. Human exposure to DBDPE in the environment is primarily through food. Data from the Fifth China Total Diet Study (TDS), conducted in 2011, and related studies have shown the presence of DBDPE in a variety of foods and in human samples such as blood, breast milk, hair, and nails.

[0004] Epidemiological studies have found that serum concentrations of four polybrominated diphenyl ether (PBDE) congeners (BDE-47, BDE-99, BDE-100, and BDE-153) during pregnancy are positively correlated with prolonged pregnancy. Although there is currently no epidemiological evidence linking DBDPE to female reproductive damage, DBDPE is structurally and physically similar to PBDEs, raising concerns about its potential impact on female reproductive health. Recently, Radović et al. found that exposure to mixtures including DBDPE in electronic waste recycling areas increases the risk of reproductive system problems. Laboratory studies have observed that exposure of female zebrafish to 1 nM and 100 nM DBDPE for 28 days resulted in DBDPE accumulation in gonadal tissue and affected estrogen levels. Oral exposure of male SD rats to 50 mg / kg / day and 500 mg / kg / day for 28 days resulted in male reproductive toxicity, including decreased sperm count and motility. The above studies suggest that exposure to the new endocrine disruptor DBDPE has a damaging effect on the reproductive systems of both sexes, and effective intervention measures are worth exploring.

[0005] Pea peptides are small, active oligopeptides derived from pea protein through the action of proteases and subsequent special processing. Research results both domestically and internationally have shown that pea peptides have numerous positive effects on the human body, including antioxidant activity, free radical scavenging, and the ability to lower blood pressure, blood lipids, and cholesterol. However, there are currently no reports of pea peptides being used in the preparation of drugs for the prevention and treatment of ovarian damage. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides the use of pea peptides in the preparation of drugs for preventing and treating ovarian damage.

[0007] The technical solutions of the present invention are as follows: Use of pea peptide in the preparation of drugs for preventing and / or treating ovarian damage.

[0008] Preferably, according to the present invention, the molecular weight of the pea peptide is 1000-10000 Daltons, and it is prepared from pea protein as raw material by extrusion, cooling, drying, crushing, and then enzymatic hydrolysis with neutral protease.

[0009] Preferably according to the present invention, the ovarian damage refers to ovarian damage caused by decabromodiphenylethane (DBDPE).

[0010] Preferably, according to the present invention, the drug for preventing and / or treating ovarian damage is a pharmaceutical composition with pea peptide as the main active ingredient.

[0011] Further preferably, the drug for preventing and / or treating ovarian damage further comprises a pharmaceutically acceptable excipient.

[0012] Preferably, according to the present invention, the dosage form of the drug for preventing and / or treating ovarian damage is any pharmaceutically approved dosage form.

[0013] Further preferably, the dosage form of the drug for preventing and / or treating ovarian damage includes granules, tablets, granules, capsules, pills or injections.

[0014] The present invention uses Wistar female rats as experimental subjects, and orally exposes them to different doses of DBDPE for 8 consecutive weeks. Pea peptide is also administered for intervention to observe the effects of DBDPE and pea peptide on the ovaries of female rats. It is found that pea peptide has a significant protective effect on the ovaries of female rats and can reduce ovarian damage.

[0015] The beneficial effects of the present invention are: This study, published in the journal Nature Communications, demonstrates for the first time that pea peptides can significantly mitigate ovarian damage caused by decabromodiphenylethane (DBDPE), demonstrating their potential for preventing and / or treating ovarian damage. Experiments have shown that pea peptides can reduce the expression of γ-H2AX, a key protein in the DNA damage pathway in ovarian tissue, as well as key proteins involved in the aging pathway and inflammasomes in ovarian tissue. This significantly improves morphological damage to ovarian tissue structure and significantly alleviates aging of ovarian tissue cells. This provides a new clinically useful and promising drug for the prevention and / or treatment of ovarian damage, demonstrating promising clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Figure 3 shows the hematoxylin-eosin (HE) staining results of ovarian tissues of rats in each group. The scale bar is 200 μm.

[0017] Figure 2 The results of β-galactosidase (SA-β-gal) staining of ovarian tissues of rats in each group after aging. The scale bar is 50 μm.

[0018] Figure 3 These are the detection results of the expression levels of aging-related proteins p53 and p21 in the ovarian tissue of rats in each group.

[0019] Figure 4 These are the detection results of the expression levels of aging-related protein p16 in the ovarian tissue of rats in each group.

[0020] Figure 5 These are the detection results of the expression levels of aging-related proteins Rb and p-Rb in the ovarian tissue of rats in each group.

[0021] Figure 6 The results of immunofluorescence staining of DNA damage protein γ-H2AX in ovarian tissues of rats in each group. The scale bar is 50 μm.

[0022] Figure 7 These are the detection results of the expression levels of inflammasome-related proteins NLRP3 and IL-1β in the ovarian tissue of rats in each group.

[0023] Figure 8 These are the detection results of the expression levels of inflammasome-related proteins Caspase1 and Cleave-Caspase1 in the ovarian tissue of rats in each group.

[0024] Figure 9 These are the detection results of the expression levels of inflammasome-related protein GSDMD in the ovarian tissue of rats in each group. DETAILED DESCRIPTION

[0025] In order to better understand the essence of the present invention, the present invention is further described below with reference to the embodiments, which however should not be construed as limiting the present invention.

[0026] The molecular weight of the pea peptide in the embodiment is 1000-10000 Daltons, the product batch number is 20230208, and the manufacturer is Yantai Shuangta Food Co., Ltd.

[0027] DBDPE was added to a sodium carboxymethyl cellulose solution with a mass concentration of 0.5% to prepare a DBDPE suspension.

[0028] Example 1 Forty Wistar female rats were adaptively fed for one week and randomly divided into four groups: control group (Ctrl), low-dose DBDPE group (L), high-dose DBDPE group (H), and high-dose DBDPE combined with pea peptide group (H+T), with 10 rats in each group.

[0029] Then, rats in the DBDPE high-dose group were given DBDPE suspension at a dose of 500 mg / kg•bw once a day for 8 consecutive weeks by gavage; rats in the DBDPE low-dose group were given DBDPE suspension at a dose of 100 mg / kg•bw once a day for 8 consecutive weeks by gavage; rats in the control group (Ctrl) were given 0.5% sodium carboxymethyl cellulose solution at a dose of 500 mg / kg•bw once a day for 8 consecutive weeks; rats in the DBDPE high-dose combined with pea peptide group were given DBDPE suspension at a dose of 500 mg / kg•bw once a day for 8 consecutive weeks by gavage, and pea peptide was administered at a dose of 50 mg / kg•bw through drinking water during the continuous gavage. The frequency was the same as that of DBDPE suspension.

[0030] Using the above method, four groups of rat models were constructed in this example. After anesthesia, blood was collected from the abdominal aorta and centrifuged at 1300×g for 10 minutes at 4°C. Serum was then collected and measured for estradiol using radioimmunoassay. The animals' abdominal cavities were then opened, and the ovaries were quickly removed, weighed, and the organ coefficient was calculated. The right ovary was fixed in 4% paraformaldehyde for morphological analysis, while the left ovary was quickly frozen in liquid nitrogen and transferred to -80°C for future storage.

[0031] Example 2 The ovarian tissues of the four rat models constructed in Example 1 were paraffin-embedded and sectioned, and then HE staining was performed. The results were as follows: Figure 1 HE staining is a conventional technique and can be performed according to existing methods.

[0032] Depend on Figure 1 As can be seen, in the control group (Ctrl), ovarian follicles of all levels and the corpus luteum structures distributed within the follicles were distinct. The granulosa cell layer within the follicles was thick, follicular fluid and cumulus ovaries were visible, and primary oocytes within the follicles were clearly visible. Compared with the control group (Ctrl), the low-dose DBDPE group (L) and the high-dose DBDPE group (H) showed a significant decrease in ovarian follicles, with atretic follicles observed. The granulosa cell layer within the follicles was thinner and the cells were disorganized, with the changes being most pronounced in the high-dose group. This demonstrates that the rat model was successfully established and that DBDPE causes significant damage to the rat ovaries and oocytes.

[0033] At the same time, compared with the high-dose DBDPE group (H), the high-dose DBDPE combined with pea peptide group (H+T) showed an increase in primordial and growing follicles, a decrease in atretic follicles, and significantly reduced ovarian and follicular damage. This suggests that pea peptide can effectively antagonize DBDPE-induced ovarian toxicity, reduce DBDPE-induced ovarian and follicular damage, and have a significant protective effect on the ovaries, reducing ovarian damage.

[0034] Example 3 During cellular aging, the activity of the enzyme senescence-associated-β-galactosidase (SA-β-gal) increases. β-gal is a lysosomal hydrolase that, in an acidic environment (pH = 6), catalyzes the substrate X-gal to produce a blue-green product with low water solubility. Therefore, SA-β-gal is a commonly used marker of cellular senescence.

[0035] The ovarian tissues of the four rat models constructed in Example 1 were taken and then embedded in OCT and frozen in sections. The SA-β-gal staining was performed. Figure 2Among them, OCT embedding, frozen sectioning and SA-β-gal staining are all existing conventional techniques and can be performed according to existing methods.

[0036] Depend on Figure 2 As can be seen, the blue and green staining was not obvious in the control group (Ctrl), while the low-dose DBDPE group (L) showed sporadic blue staining scattered throughout the ovarian stroma. The high-dose DBDPE group (H) showed distinct blue-positive areas in the granulosa cell layer. This demonstrates that the rat model was successfully established and that DBDPE induces significant aging of the granulosa cell layer.

[0037] Compared with the high-dose DBDPE group (H), the high-dose DBDPE combined with pea peptide group (H+T) showed a significant decrease in the blue-stained positive area of ​​the ovaries. This suggests that pea peptide can alleviate and repair ovarian aging caused by DBDPE.

[0038] Example 4 The total protein in the ovarian tissue of the four groups of rat models constructed in Example 1 was extracted, and then the expression level of key proteins related to the ovarian tissue aging pathway in the ovarian tissue of each group of rats was detected by Western blot, with β-actin as the internal reference. The results are as follows Figures 3-5 shown.

[0039] Among them, the key proteins related to the ovarian tissue aging pathway include p53 protein, p21 protein, p16 protein, Rb protein, and p-Rb protein.

[0040] The specific steps are as follows: RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors was added to the ovarian tissues of the four groups of rat models constructed in Example 1, and a homogenate was prepared using an electric homogenizer. After the homogenate was allowed to stand on ice for 30 min, it was centrifuged at 4 ° C and 12000 × g for 10 min. The supernatant was taken to complete protein extraction, and the protein concentration in the supernatant was determined by BCA method. 4× Buffer was added to the supernatant and heated at 100 ° C for 10 min to fully denature the protein. The denatured protein sample was subjected to SDS-PAGE electrophoresis, 140V electrophoresis for 1 h, and the protein in the gel was transferred to a PVDF membrane at 300mA constant current in an ice bath for 1.5 h, blocked with 5% skim milk powder for 1 h, incubated with the primary antibody at 4 ° C overnight, washed with TBST buffer the next day, incubated with the secondary antibody at room temperature for 1.5 h, washed with TBST, and chemiluminescence was performed by ECL developer. Images were collected using a chemiluminescence imaging system, and integrated optical density analysis was performed using imageJ.

[0041] The antibodies used were as follows: anti-p53 and p16 antibodies (Proteintech, Wuhan Tri-Tac), anti-p21 antibody (SantaCruz, USA), anti-Rb and p-Rb antibodies (CST, USA), and anti-β-actin antibody (Invitrogen, USA).

[0042] Depend on Figures 3-5 It can be seen that compared with the control group (Ctrl), the expression levels of p21 protein and p53 protein in the low-dose DBDPE group (L) were significantly decreased (P < 0.05), and the expression levels of p53 protein and p21 protein in the high-dose DBDPE group (H) were increased, among which the increase in p53 protein expression was statistically significant (P < 0.05).

[0043] Compared with the low-dose DBDPE group (L) and the high-dose DBDPE group (H), the expression level of p16 protein increased in a dose-dependent manner, and the difference was statistically significant (P<0.05).

[0044] Compared with the DBDPE low-dose (L) and DBDPE high-dose (H) groups, the expression level of p-Rb protein was decreased, accompanied by an increase in the expression level of Rb protein, and both changes were dose-dependent.

[0045] The above results indicate that the rat model of the present invention was successfully constructed, and DBDPE can cause changes in the expression levels of key proteins related to the ovarian tissue aging pathway in ovarian tissue, leading to ovarian aging.

[0046] At the same time, compared with the high-dose DBDPE group (H), the high-dose DBDPE combined with pea peptide group (H+T) showed significantly decreased expression levels of p53, p21, p16, and Rb proteins, while significantly increased expression levels of p-Rb protein. The changes in p53, p21, Rb, and p-Rb protein expression were statistically significant (P < 0.05). This suggests that pea peptide can reverse DBDPE-induced changes in the expression levels of key proteins associated with the ovarian tissue aging pathway, alleviating, repairing, and even reversing DBDPE-induced ovarian aging.

[0047] Example 5 Paraffin sections of ovarian tissues of the four groups of rat models constructed in Example 1 were prepared, and the expression levels of key proteins related to ovarian tissue DNA damage in the ovarian tissues of the rats in each group were detected by immunofluorescence staining. The results were as follows: Figure 6 Among them, the key protein associated with DNA damage in ovarian tissue is γ-H2AX. Preparation of paraffin sections is a conventional technique and can be performed according to existing methods.

[0048] The specific steps of immunofluorescence staining are as follows: Ovarian tissue was prepared into paraffin sections, dewaxed with xylene, rehydrated with gradient ethanol, and placed in a 3% hydrogen peroxide solution to remove endogenous peroxidase. The sections were then heated in a microwave for antigen retrieval, cooled to room temperature, and then permeabilized and blocked. The sections were then placed in a solution containing a γ-H2AX-specific primary antibody at 4°C overnight, washed with PBST buffer the next day, reacted with a fluorescent secondary antibody in the dark for 1.5 h at room temperature, washed with PBST buffer, and mounted with DAPI anti-fluorescence quenching mounting medium. Tissue fluorescence imaging was observed using a live cell imaging system.

[0049] The antibodies used were as follows: anti-γ-H2AX antibody (Abcam, USA), fluorescent-labeled goat anti-rabbit Alexa Fluor 488 (Zhongshan Jinqiao, Beijing).

[0050] Depend on Figure 6 As shown, γ-H2AX expression varied in the ovaries of rats in all groups, with red fluorescence signals visible in the granulosa cell layer, central oocyte, and peripheral stroma within all levels of follicles. While γ-H2AX expression was low in the control group, it was significantly elevated in the low- and high-dose DBDPE groups in a dose-dependent manner. In particular, oocytes with high γ-H2AX expression were observed in the central part of some follicles in the high-dose DBDPE group. Compared with the high-dose DBDPE group, the high-dose DBDPE combined with pea peptide group showed significantly reduced γ-H2AX-positive signals in the ovarian tissue. This suggests that DBDPE exposure induces double-strand breaks in the DNA of rat germ cells, and that pea peptide intervention can mitigate DBDPE-induced DNA damage in germ cells.

[0051] Example 6 The total protein in the ovarian tissue of the four groups of rat models constructed in Example 1 was extracted, and the expression levels of key proteins related to ovarian tissue inflammasomes in the ovarian tissue of each group of rats were detected by Western blot, with β-actin and GAPDH as internal references. Figures 7-9 shown.

[0052] Among them, the key proteins related to ovarian tissue inflammasomes include NLRP3 protein, IL-1β protein, Caspase1 protein, Cleave-Caspase1 protein, and GSDMD protein.

[0053] The specific Western blot method was the same as that in Example 4, except that the antibodies used were as follows: anti-GSDMD antibody (Proteintech, Wuhan Tri-Tek), anti-IL-1β, NLRP3, Caspase1, and Cleave-Caspase1 antibodies (CST, USA), anti-β-actin (Invitrogen, USA), and anti-GAPDH antibody (ABclonal, Wuhan).

[0054] Depend on Figures 7-9 Compared with the control group, the expression levels of NLRP3, IL-1β, Caspase1, Cleave-Caspase1, and GSDMD proteins in the ovarian tissues of DBDPE-exposed rats increased in a dose-dependent manner. The expression levels of NLRP3, IL-1β, Caspase1, and Cleave-Caspase1 in the low-dose DBDPE group and the high-dose DBDPE group were significantly higher than those in the control group (P < 0.05). Compared with the high-dose DBDPE group, the expression of NLRP3, IL-1β, Caspase1, and Cleave-Caspase1 proteins in the high-dose DBDPE combined with pea peptide group was significantly decreased (P < 0.05). The expression level of Cleave-Caspase1 protein showed a downward trend, but the difference was not statistically significant (P > 0.05). This suggests that DBDPE exposure induces an inflammatory response in rat ovarian tissue, and pea peptide can alleviate the inflammatory response induced by DBDPE exposure to a certain extent.

[0055] In summary, the present invention demonstrates for the first time the use of pea peptides in the preparation of drugs for the prevention and / or treatment of ovarian damage, demonstrating their ability to significantly mitigate ovarian damage caused by decabromodiphenylethane (DBDPE). Experiments have shown that pea peptides can reduce the expression of γ-H2AX, a key protein in the DNA damage pathway in ovarian tissue, as well as key proteins involved in the aging pathway and inflammasomes in ovarian tissue. This significantly improves morphological damage to ovarian tissue structure and significantly alleviates aging of ovarian tissue cells. This provides a new drug for the prevention and / or treatment of ovarian damage, demonstrating promising clinical applications and broad prospects.

[0056] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Use of pea peptide in the preparation of a drug for preventing and / or treating ovarian damage, characterized in that: The ovarian damage refers to the ovarian damage caused by decabromodiphenylethane.

2. The use of the pea peptide according to claim 1 in preparing a drug for preventing and / or treating ovarian damage, wherein: The pea peptide has a molecular weight of 1,000 to 10,000 Daltons and is prepared from pea protein by extrusion, cooling, drying, crushing, and then enzymatic hydrolysis with a neutral protease.

3. The use of the pea peptide according to claim 1 in preparing a drug for preventing and / or treating ovarian damage, wherein: The medicine for preventing and / or treating ovarian damage is a pharmaceutical composition with pea peptide as a main active ingredient.

4. The use of the pea peptide according to claim 3 in preparing a drug for preventing and / or treating ovarian damage, wherein: The drug for preventing and / or treating ovarian damage further comprises pharmaceutically acceptable excipients.

5. The use of the pea peptide according to claim 1 in preparing a drug for preventing and / or treating ovarian damage, wherein: The dosage form of the drug for preventing and / or treating ovarian damage is any pharmaceutically approved dosage form.

6. The use of the pea peptide according to claim 5 in preparing a drug for preventing and / or treating ovarian damage, wherein: The dosage forms of the drug for preventing and / or treating ovarian damage include granules, tablets, granules, capsules, pills or injections.

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