Use of glycine in alleviating damage to bovine ovarian granulosa cells caused by microplastics

Abstract

The application discloses application of glycine in relieving bovine ovarian granulosa cell damage. The glycine is applied to relieving bovine ovarian granulosa cell damage caused by microplastics. The glycine has a good protection effect on ovarian damage caused by microplastics, improves cell viability, reduces active oxygen, inhibits ferroptosis, and improves bovine ovarian granulosa cells after microplastic damage. The application not only discloses that the glycine is a new target for treating bovine ovarian granulosa cell damage caused by microplastics, but also clarifies the effect of the glycine in treating bovine ovarian granulosa cell damage caused by microplastics, and has important theoretical significance and application value.

Description

Technical Field

[0001] This invention relates to the field of applications in alleviating ovarian granulosa cell damage, specifically to the use of glycine in alleviating microplastic-induced damage to bovine ovarian granulosa cells. Background Technology

[0002] Microplastics are plastic particles smaller than 5 mm in size. As an emerging environmental pollutant, they possess physicochemical properties such as being difficult to degrade, having strong adsorption capacity, low density, easy floating, and small size. Microplastics can enter the body through ingestion, respiration, and skin contact, accumulating in organs such as the liver, intestines, and lungs. The ovary is one of the organs most susceptible to environmental pollutants. Microplastics can cause steroid hormone secretion disorders and disrupt the arrangement of ovarian granulosa cell layers, resulting in ovarian damage, especially damage to ovarian granulosa cells. Therefore, it is essential to find a strategy to mitigate ovarian granulosa cell damage caused by microplastics.

[0003] Glycine, a constituent amino acid of the antioxidant glutathione, possesses biological functions such as cell protection, anti-inflammation, and metabolic regulation. Glycine has been proven to play an important role in the female reproductive system. Exogenous glycine supplementation can improve the efficiency of oocyte in vitro maturation and in vitro fertilization, reduce cell apoptosis, and improve mitochondrial and endoplasmic reticulum dysfunction. Furthermore, glycine has a significant alleviating effect on phthalic acid monobutyl ester-induced tissue or cell damage, suggesting it may be a potential therapeutic agent for microplastic-related damage. Therefore, this invention investigates the protective effect of glycine supplementation using a microplastic-induced bovine ovarian granulosa cell damage model, aiming to discover safe and effective substances against bovine ovarian granulosa cell damage. Summary of the Invention

[0004] To alleviate microplastic-induced damage to bovine ovarian granulosa cells, this invention provides the application of glycine in reducing bovine ovarian granulosa cell damage.

[0005] The technical solution of the present invention is as follows: This invention provides a preferred application of glycine in alleviating damage to bovine ovarian granulosa cells.

[0006] Furthermore, the ovarian granulosa cell damage mentioned above refers to bovine ovarian granulosa cell damage caused by microplastics.

[0007] Furthermore, the microplastics used are 500 nm polystyrene microplastics.

[0008] Furthermore, the concentration of the 500 nm polystyrene microplastics is 100 μg / mL.

[0009] Furthermore, the granulosa cells are bovine ovarian granulosa cells.

[0010] Furthermore, the 500 nm polystyrene microplastics were used to treat the granular cells for 12 hours.

[0011] Furthermore, the concentration of glycine is 2 mM.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention alleviates ovarian granulosa cell damage caused by microplastics through the following methods: It enhances cell viability; reduces reactive oxygen species, lowers ferrous ion concentration, and alleviates lipid peroxidation.

[0013] Therefore, glycine has important application value in the treatment of ovarian granulosa cell damage, especially in the treatment of ovarian granulosa cell damage caused by microplastics. Detailed Implementation

[0014] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Any modifications or substitutions made to the present invention without departing from its spirit and essence are within the scope of the present invention.

[0015] In the following examples, the microplastics were purchased from TransYuan Biotechnology. The microplastics were prepared by dissolving 20 mL of microplastics with a concentration of 25 mg / mL in 30 mL of distilled water, and then treating bovine ovarian granulosa cells with the microplastics at a concentration of 100 μg / mL for 12 h.

[0016] In the following examples, glycine was purchased from Sigma. The glycine was prepared by dissolving 30 mg of glycine in 2 mL of distilled water and treating bovine ovarian granulosa cells with glycine at a concentration of 2 mM for 12 h.

[0017] Example 1 verifies the effects of microplastics and glycine on the viability of bovine ovarian granulosa cells, including the following steps: First, primary bovine ovarian granulosa cells were cultured in vitro. Ovarian samples from healthy dairy cows were collected from a local slaughterhouse in Xianyang City, Shaanxi Province. Immediately after collection, the ovaries were placed in PBS preheated to 37°C with a 1% penicillin-streptomycin solution and transported to the laboratory within 1 hour. The ovarian surface was disinfected and cleaned with 75% ethanol. Granulosa cells and follicular fluid were extracted from the bovine follicles using a 25-gauge needle. The obtained cell suspension was centrifuged at 1500 r / min for 10 min to separate the granulosa cells and follicular fluid. After centrifugation, the supernatant was discarded, and the precipitated granulosa cells were retained. Next, the granulosa cells were washed twice with DMEM / HIGH medium containing 1% penicillin-streptomycin solution. A cell culture medium consisting of 89% DMEN / HIGH GLUCOSE medium, 10% FBS, and 1% penicillin-streptomycin was added, and the cells were cultured in a cell culture incubator at 38.5°C and 5% CO2. When the cell confluence reached 70%, the medium was replaced with fresh medium and treated with microplastics and glycine for subsequent experiments.

[0018] Adjust the density of bovine primary ovarian granulosa cells to 1×10⁻⁶. 6 Cells were seeded at a density of 70% (100 μg / mL polystyrene microplastics and 2 mM glycine) into 96-well cell culture plates. Six replicates were then added to each treatment, with wells containing only complete culture medium as a blank control. After 12 h of treatment, 100 μL of serum-free culture medium and 10 μL of cell proliferation assay (CCK-8) solution were added to both the cell wells and the blank wells. The plates were then wrapped in aluminum foil to prevent light exposure and incubated for 3 h. The absorbance of each well was then measured at 450 nm using a microplate reader, and cell viability was calculated according to the manufacturer's instructions. Figure 1 As shown, microplastics at 100 μg / mL can significantly reduce cell viability, and glycine supplementation can alleviate the reduction in cell viability caused by microplastics.

[0019] Adjust the density of bovine primary ovarian granulosa cells to 1×10⁻⁶. 6 Cells were seeded at a density of 70% (100 μg / mL polystyrene microplastics and 2 mM glycine) into 24-well cell culture plates. Cell culture medium containing 100 μg / mL polystyrene microplastics and 2 mM glycine was added, with three replicates per treatment. After 12 h of treatment, the cell culture medium was removed, and diluted Calcein AM / PI working solution was added. The plates were incubated at 37°C in the dark for 30 min. The staining solution was carefully removed from the wells, and the cells were washed once with PBS along the well walls. The medium was then replaced with complete culture medium. The cells were observed under an inverted fluorescence microscope, and the average fluorescence intensity was analyzed using ImageJ 1.8.0 software. Figure 2 and 3As shown, this further demonstrates that glycine alleviates the decrease in ovarian granulosa cell viability caused by microplastics.

[0020] Example 2 verifies the effects of microplastics and glycine on ferroptosis in bovine ovarian granulosa cells, including the following steps: Adjust the density of bovine primary ovarian granulosa cells to 1×10⁻⁶. 6 Cells were seeded at a density of 70% (100 μg / mL polystyrene microplastics and 2 mM glycine) into 24-well cell culture plates. Cell culture medium containing 100 μg / mL polystyrene microplastics and 2 mM glycine was added, with three replicates per treatment. After 12 h of treatment, the cell culture medium was removed, and diluted 2,7-dichlorofluorescein diacetate (DCFH-DA) working solution was added. DCFH-DA was diluted to a final concentration of 10 μM with serum-free medium. After incubation at 37°C in the dark for 20 min, the cells were washed three times with serum-free medium. Images were acquired using a fluorescence inverted microscope, and the average fluorescence intensity was analyzed using ImageJ 1.8.0 software. Figure 4 and 5 It was found that, compared with the control group, 100 μg / mL polystyrene microplastics caused an increase in reactive oxygen species (ROS) levels in bovine ovarian granulosa cells, while glycine supplementation could significantly alleviate the increase in ROS caused by microplastics.

[0021] Adjust the density of bovine primary ovarian granulosa cells to 1×10⁻⁶. 6 Cells were seeded at a density of 70% (100 μg / mL polystyrene microplastics and 2 mM glycine) into 24-well cell culture plates. Cell culture medium containing 100 μg / mL polystyrene microplastics and 2 mM glycine was added, with three replicates per treatment. After 12 h of treatment, the cell culture medium was removed, and diluted FerroOrange working solution was added. FerroOrange solution was diluted to a final concentration of 1 μmol / L with HBSS. After incubation at 37°C in the dark for 30 min, images were acquired using a fluorescence inverted microscope, and the average fluorescence intensity was analyzed using ImageJ 1.8.0 software. Results are as follows: Figure 6 and 7 As shown, compared with the microplastic-only treatment group, glycine supplementation significantly reduced microplastic-induced Fe2+ in bovine ovarian granulosa cells. 2+ An increase in levels.

[0022] Adjust the density of bovine primary ovarian granulosa cells to 1×10⁻⁶. 6Cells were seeded at a density of 70% (100 μg / mL polystyrene microplastics) into 24-well cell culture plates. Cell culture medium containing 100 μg / mL polystyrene microplastics and 2 mM glycine was added, with three replicates per treatment. After 12 h of treatment, the cell culture medium was removed, and diluted C11 BODIPY 581 / 591 working solution was added. The C11 BODIPY 581 / 591 solution was diluted to a final concentration of 5 μM with serum-free medium. After incubation at 37°C in the dark for 30 min, images were acquired using a fluorescence inverted microscope, and the average fluorescence intensity was analyzed using ImageJ 1.8.0 software. Figure 8 and 9 It can be seen that, compared with the microplastic-only treatment group, glycine supplementation can significantly reduce the increase in lipid peroxidation level induced by microplastics in bovine ovarian granulosa cells.

[0023] In summary, this invention demonstrates that glycine improves ferrodeogenesis in granulosa cells by regulating ferrous ion and reactive oxygen species content, effectively alleviating microplastic-induced decrease in ovarian granulosa cell viability. This indicates that glycine has significant research and application prospects in the field of reproductive health. This invention not only reveals the role of glycine in treating microplastic-induced ovarian granulosa cell damage, but also has important theoretical significance and application value.

[0024] The above embodiments, which describe the specific features of the present invention, are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made to the present invention by those skilled in the art based on the above description of the invention shall fall within the scope of protection of the present invention. Attached Figure Description

[0025] A patent application entitled "Application of Glycine in Alleviating Microplastic-Induced Damage to Bovine Ovarian Granulosa Cells" includes nine figures in its specification, the descriptions of which are as follows: Figure 1 In one embodiment of this application, the effects of microplastics and glycine on the viability of bovine ovarian granulosa cells are described.

[0026] Figure 2 This image shows the fluorescence intensity of Calcein and PI after treating bovine ovarian granulosa cells with microplastics and glycine in one embodiment of this application.

[0027] Figure 3 This is a quantitative analysis of the average fluorescence intensity of Calcein and PI after treating bovine ovarian granulosa cells with microplastics and glycine in one embodiment of this application.

[0028] Figure 4This is an image showing the fluorescence intensity of ROS after treating bovine ovarian granulosa cells with microplastics and glycine in one embodiment of this application.

[0029] Figure 5 This is a quantitative analysis of the average ROS fluorescence intensity after treating bovine ovarian granulosa cells with microplastics and glycine in one embodiment of this application.

[0030] Figure 6 In one embodiment of this application, after treating bovine ovarian granulosa cells with microplastics and glycine, Fe... 2+ Images of fluorescence intensity.

[0031] Figure 7 In one embodiment of this application, after treating bovine ovarian granulosa cells with microplastics and glycine, Fe... 2+ Quantitative analysis of average fluorescence intensity.

[0032] Figure 8 This is an image showing the fluorescence intensity of C11 BODIPY cells after treatment with microplastics and glycine in one embodiment of this application.

[0033] Figure 9 This is a quantitative analysis of the average fluorescence intensity of C11 BODIPY cells after treatment with microplastics and glycine in one embodiment of this application.

Claims

1. Application of glycine in alleviating microplastic-induced damage to bovine ovarian granulosa cells.

2. The application according to claim 1, wherein the ovarian granulosa cell damage is bovine ovarian granulosa cell damage caused by microplastics.

3. In the application according to claim 2, the microplastic is a 500 nm polystyrene microplastic.

4. The application according to claim 3, characterized in that the concentration of polystyrene microplastics is 100 μg / mL.

5. The application according to claim 2, wherein the ovarian granulosa cells are primary bovine ovarian granulosa cells.

6. In the application according to claim 2, the polystyrene microplastics are used to treat the granular cells for 12 hours.

7. In the application according to claim 2, the damage to the ovarian granulosa cells is manifested as decreased cell viability, increased reactive oxygen species, and ferroptosis.

8. The application according to claim 1, characterized in that, The glycine can alleviate ovarian granulosa cell damage caused by microplastics.

9. The application according to claim 8, characterized in that, The concentration of glycine is 2 mM.

10. In the application according to claim 8, the glycine alleviates ovarian granulosa cell damage caused by microplastics by increasing cell viability, reducing reactive oxygen species, and inhibiting ferroptosis.

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