Application of ginsenoside CK in preparation of porcine oocyte damage inhibitor

Abstract

The present application belongs to the field of biological medicine, and relates to application of ginsenoside CK in preparation of a porcine oocyte damage inhibitor. The present application aims to explore the influence of CK on in-vitro maturation of porcine oocytes induced by BaP and the embryo development capacity thereof. The research of the present application finds that adding CK can improve in-vitro maturation of porcine oocytes and subsequent embryo development after damage induced by BaP by increasing the antioxidant activity of oocytes and improving the function of mitochondria.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically, relates to the application of ginsenoside CK in the preparation of a porcine oocyte damage inhibitor. Background Technology

[0002] Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon containing a benzene ring. It is widely present in the environment, primarily from two sources: First, waste gases from the incomplete combustion of fuels such as coal, oil, and natural gas during industrial production and daily life, including vehicle exhaust, rubber production, and cigarette smoke, pollute water, air, and soil, eventually entering food products such as vegetables, fruits, grains, seafood, and meat. Second, during the smoking, baking, and frying processes, fats, cholesterol, proteins, and carbohydrates undergo a series of reactions under high temperatures, forming polycyclic aromatic hydrocarbons, including benzo[a]pyrene, which are highly carcinogenic. Studies have found high concentrations of BaP in the follicular fluid of smoking women. Because BaP is a toxic component, it can lead to ovarian dysfunction, including premature ovarian failure and ovarian hypoplasia. BaP can also alter steroid balance, particularly ovarian estrogen receptors, resulting in reduced pregnancy rates. Early embryonic exposure to BaP can also have permanent effects on fertility. BaP metabolites cause persistent DNA damage and induce mutagenesis in many somatic cells. Mounting evidence suggests that BaP metabolites impair follicle growth by inducing apoptosis and increasing the activation of primordial follicles. They can also reduce ovarian function and cause immaturity by inducing apoptosis. This type of ovotoxicant induces early apoptosis, leading to follicular atresia, follicular growth failure, and consequently infertility. Due to its ovotoxicity and long-term persistence, it can cause significant damage to the human reproductive system.

[0003] Ginseng, a perennial herb of the Araliaceae family, is a traditional Chinese medicine with a history of use in Asia for over a thousand years. It is commonly used as a nutritional food and traditional medicine supplement, and is frequently used as a nutritional tonic during preconception and pregnancy. Ginseng's medicinal value has been confirmed to include regulating the central nervous system; improving bodily adaptability; regulating the cardiovascular system; and being suitable for adjusting blood pressure, restoring heart and liver function, and treating neurasthenia and general weakness. Studies have shown that ginsenosides are the main bioactive components of ginseng, possessing various functions such as anti-oxidation, anti-apoptosis, and immune stimulation. Ginsenoside compound K (CK) is the main intestinal metabolite of ginsenoside Rb1, a proto-naphthyl glycol saponin, and enters the circulatory system via the bloodstream. In the reproductive system, studies have shown that ginsenosides can promote the proliferation of chicken germ cells and improve ovarian dysfunction in young rats. Ginsenoside Rg1 has estrogen-like activity and may have estrogen-like functions during oocyte development. Ginsenosides have a structure similar to steroids, suggesting that ginsenosides may have steroid-like biological effects on the reproductive system. Summary of the Invention

[0004] This invention aims to investigate the effects of CK on BaP-induced in vitro maturation of porcine oocytes and their embryonic development capacity.

[0005] To achieve the above objectives, the present invention provides the application of ginsenoside CK in the preparation of a porcine oocyte damage inhibitor.

[0006] Furthermore, the damage is benzo[a]pyrene-induced apoptosis of porcine oocytes.

[0007] Furthermore, the damage refers to the failure of porcine oocytes to mature through meiosis induced by benzo[a]pyrene.

[0008] The ginsenoside CK of the present invention is commercially available, for example, from Nanjing Guangrun Biological Products Co., Ltd. (analytical grade).

[0009] The study of this invention found that adding CK can improve the in vitro maturation of porcine oocytes damaged by BaP and subsequent embryonic development by increasing the antioxidant activity of oocytes and improving mitochondrial function.

[0010] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0011] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0012] Figure 1A-1B The study demonstrated that different concentrations of benzo[a]pyrene reduced oocyte quality and in vitro maturation rate. Among them, Figure 1A The proportion of the average diameter of the cumulus-oocyte complex is shown. In each group, the columns from left to right represent the Con group, H2O2 group, BaP40 group, and BaP80 group, respectively. Figure 1B The expulsion rate of oocyte polar bodies during the MII stage is shown.

[0013] Figure 2A-2C This study demonstrates how different concentrations of ginsenoside CK treatment improved oocyte quality and in vitro maturation rate. Among these, Figure 2A The proportion of the average diameter of the cumulus-oocyte complex is shown. In each group, the columns from left to right represent the BaP40 group, the B+CK2 group, the B+CK10 group, and the B+CK50 group, respectively. Figure 2B The expulsion rate of oocyte polar bodies during the MII stage is shown. Figure 2C The expression levels of genes related to cumulus cell expansion are shown. In each group, the columns from left to right represent the Con group, BaP40 group, and B+CK10 group, respectively.

[0014] Figure 3A The level of ROS in MII oocytes was shown by immunofluorescence staining. Figure 3B The ATP content of oocytes in the MII stage is shown by immunofluorescence staining. Figure 3C The immunofluorescence staining of mitochondrial activity in MII stage oocytes was shown.

[0015] Figure 4A The expression levels of apoptosis factors in parthenogenetic activated embryos during the blastocyst stage were shown. Figure 4B The expression levels of pluripotency genes during the blastocyst stage of parthenogenetic activated embryos were shown.

[0016] Figure 5A The level of ROS in the 2-cell stage of parthenogenetic activated embryos was shown by immunofluorescence staining. Figure 5B The immunofluorescence staining of mitochondrial activity in parthenogenetic activated embryos at the 2-cell stage was shown.

[0017] Figure 6 A schematic diagram illustrating the protective effect of ginsenoside CK against benzo[a]pyrene-induced oocyte damage. Detailed Implementation

[0018] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0019] Unless otherwise specified, all conditions in the examples were performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers were not specified were all commercially available products. The ginsenoside CK used in the examples was purchased from Nanjing Guangrun Biological Products Co., Ltd. (analytical grade).

[0020] 1. Detect the degree of expansion and maturation rate of the cumulus-oocyte complex.

[0021] The expansion of cumulus-oocyte complexes after 44 hours of culture was observed under a microscope (Olympus, Japan). The diameter of the cumulus-oocyte complex was measured, and the expansion was categorized into three grades: less than 500 μm, between 500 μm and 1000 μm, and 1000 μm and above. Cumulus cells were dissected in NCSU37 medium containing 0.1% hyaluronidase. The naked oocytes were washed three times with PBS containing 0.1% polyvinyl alcohol (PVA) and treated with 25 μg / mL... -1 Hoechest 33342 staining. Stained oocytes were imaged and their maturation rate assessed using fluorescence microscopy.

[0022] 2. Real-time quantitative polymerase chain reaction (PCR) detection of gene expression

[0023] Total mRNA was extracted from oocytes, embryos, and cumulus cells using the Dynabeads mRNA Direct Extraction Kit (Invitrogen). mRNA concentration was measured using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). Complementary DNA was synthesized using the SuperScript III First-Strand Synthesis Kit (Invitrogen). PCR reactions were performed using an Mx3005P system (Agilent Technologies). Each 20 μL reaction volume contained 1 μL cDNA and 1 μL 10 pmol μL... -1 Forward and reverse primers, 10 μL of SYBR premix Taq (Roche), and 7 μL of nuclease-free water. The amplification steps included: 95°C denaturation for 10 min; 95°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 1 min, for 40 cycles. Relative gene expression levels were measured using 2... -ΔΔCT Method calculation.

[0024] 3. Detection of reactive oxygen species (ROS) and ATP levels

[0025] Oocytes or embryos were washed with PBS containing 0.1% PVA and stained with 2′,7′-dichlorodihydrofluorescein to detect ROS. Oocytes were stained with PBS-PVA diluted to 10 μM 2′,7′-dichlorodihydrofluorescein in the dark for 30 minutes. After washing three times with PBS, the oocytes or embryos were placed on a slide and imaged using an Olympus fluorescence microscope.

[0026] ATP levels in oocytes or embryos were stained using the BODIPY FL ATP (BODIPY-ATP, A12410) molecular probe. Oocytes or embryos were fixed in 4% paraformaldehyde for 1 hour, then washed three times with PBS containing 0.1% PVA. Oocytes or embryos were then placed in 500 nM BODIPY FL ATP and stained in a CO2 incubator for 30 minutes. After washing three times with PBS, oocytes or embryos were placed on slides and imaged using an Olympus fluorescence microscope.

[0027] 4. Detection of mitochondrial membrane potential (MMP)

[0028] Mitochondrial staining (MMP) in oocytes and embryos was determined using a mitochondrial staining kit (JC-1, Invitrogen). Oocytes and embryos were stained with 10 μg of mitochondrial solution per mL. -1 JC-1 cells were cultured in vitro at 38°C in a 5% CO2 incubator for 30 minutes. They were then washed three times with pre-chilled 1× staining buffer and placed on ice for imaging within 30 minutes. Oocytes and embryos were placed on slides and imaged using a fluorescence microscope (Olympus). MMPs were assessed based on the ratio of red to green staining.

[0029] 5. Detection of in vitro development of parthenogenetic activated (PA) embryos

[0030] Parthenogenetic activation of oocytes was performed in a 0.28 mol mannitol activation solution containing 0.1 mmol MgSO4 and 0.05 mmol CaCl2, using a single 1.5 kV cm-wavelength pulse. -1 A direct current pulse was applied to activate oocytes that had clearly expelled the first polar body for 60 μs. The activated oocytes were cultured in NCSU37 medium containing 2 mmol of 6-dimethylaminopurine for 4 h, and then transferred to in vitro culture (IVC) medium for 7 days. Embryo development rate was observed and calculated.

[0031] Test Results

[0032] Figure 1A-1B The study demonstrated that different concentrations of benzo[a]pyrene reduced oocyte quality and in vitro maturation rate. Among them, Figure 1A The proportion of the average diameter of the cumulus-oocyte complex is shown. In each group, the columns from left to right represent the Con group, H2O2 group, BaP40 group, and BaP80 group, respectively. Figure 1B The results show the polar body extrusion rate of oocytes in the MII stage. Compared with the control group, treatment with 40 μM, 80 μM benzo[a]pyrene, and H2O2 resulted in a decrease in the average diameter of the cumulus-oocyte complex, a decrease in the proportion of large-diameter cumulus-oocyte complexes, an increase in the proportion of small-diameter cumulus-oocyte complexes, and a significant decrease in the polar body extrusion rate, indicating that benzo[a]pyrene treatment reduced oocyte quality and maturation rate.

[0033] Figure 2A-2C This study demonstrates how different concentrations of ginsenoside CK treatment improved oocyte quality and in vitro maturation rate. Among these, Figure 2A The proportion of the average diameter of the cumulus-oocyte complex is shown. In each group, the columns from left to right represent the BaP40 group, the B+CK2 group, the B+CK10 group, and the B+CK50 group, respectively. Figure 2B The expulsion rate of oocyte polar bodies during the MII stage is shown. Figure 2C The expression levels of genes related to cumulus cell expansion are shown. In each group, the columns from left to right represent the Con group, BaP40 group, and B+CK10 group, respectively. Results showed that treatment with 10 μg / ml ginsenoside CK increased the average diameter of the cumulus-oocyte complex, increased the proportion of large-diameter cumulus-oocyte complexes, significantly increased the polar body extrusion rate, and significantly improved the expression levels of genes related to cumulus cell expansion. This indicates that treatment with 10 μg / ml ginsenoside CK significantly improved oocyte quality and maturation rate.

[0034] Figure 3A The level of ROS in MII oocytes was shown by immunofluorescence staining. Figure 3B The ATP content of oocytes in the MII stage is shown by immunofluorescence staining. Figure 3C The results showed that mitochondrial activity in MII stage oocytes was inhibited by immunofluorescence staining. Treatment with ginsenoside CK inhibited the increase in ROS levels in MII stage oocytes induced by benzo[a]pyrene, improved the decrease in ATP content in MII stage oocytes induced by benzo[a]pyrene, and improved the decrease in mitochondrial membrane potential in MII stage oocytes induced by benzo[a]pyrene, indicating that ginsenoside CK can improve the decline in oocyte quality in MII stage oocytes caused by benzo[a]pyrene.

[0035] Figure 4A The study showed the level of apoptosis in parthenogenetic activated embryonic blastocysts. Figure 4B The study showed the expression levels of pluripotency genes in the blastocyst stage of parthenogenetic activated embryos. The results indicated that ginsenoside CK inhibited the increased apoptosis in benzo[a]pyrene-induced parthenogenetic activated embryos during the blastocyst stage and improved the decreased expression of pluripotency genes in the blastocyst stage induced by benzo[a]pyrene, suggesting that ginsenoside CK can improve the decreased blastocyst quality caused by benzo[a]pyrene-induced parthenogenetic activated embryos.

[0036] Figure 5A The level of ROS in the 2-cell stage of parthenogenetic activated embryos was shown by immunofluorescence staining. Figure 5BThe results showed that immunofluorescence staining of mitochondrial activity in the 2-cell stage of parthenogenetic activated embryos inhibited the increase in ROS levels in the 2-cell stage of benzo[a]pyrene-induced parthenogenetic activated embryos and improved the decrease in mitochondrial membrane potential in the 2-cell stage of benzo[a]pyrene-induced parthenogenetic activated embryos, indicating that ginsenoside CK can improve the quality decline of parthenogenetic activated embryos caused by benzo[a]pyrene.

[0037] Table 1 shows the effects of benzo[a]pyrene and ginsenoside CK treatment on the in vitro development of parthenogenetic activated embryos. The results showed that 40 μM benzo[a]pyrene treatment significantly reduced the number of blastocysts in parthenogenetic activated embryos, and 10 μg / ml ginsenoside CK treatment significantly improved the decrease in blastocyst rate caused by benzo[a]pyrene.

[0038] Table 1. Effects of benzo[a]pyrene and ginsenoside supplementation (CK) treatment on in vitro development of parthenogenetic activated embryos.

[0039]

[0040] The results above show that adding 10 μg mL to the in vitro maturation medium... -1 CK reduced oocyte damage induced by 40 μM BaP and significantly increased oocyte maturation rate and expression levels of maturation-related genes (P < 0.05). After CK addition, reactive oxygen species (ROS) levels in oocytes were significantly reduced, while ATP content and mitochondrial membrane potential (MMP) were significantly increased (P < 0.05). Furthermore, adding CK to BaP-treated oocytes improved embryonic development by inducing pluripotency gene expression and inhibiting oocyte apoptosis. A schematic diagram illustrating the protective effect of ginsenoside CK against benzo[a]pyrene-induced oocyte damage is shown below. Figure 6 As shown.

[0041] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. Application of ginsenoside CK in the preparation of inhibitors for benzo[a]pyrene-induced porcine oocyte damage, wherein, The concentration of ginsenoside CK is 10 μg / ml.

2. The application according to claim 1, wherein, The damage was benzo[a]pyrene-induced apoptosis of porcine oocytes.

3. The application according to claim 1, wherein, The damage refers to the failure of porcine oocytes to mature through meiosis induced by benzo[a]pyrene.

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