Application of alpha-ketoglutaric acid in ovarian granular cells

Abstract

The invention discloses an application of alpha-ketoglutaric acid in ovarian granular cells. According to the invention, alpha-ketoglutaric acid is taken as a research object, and the application of alpha-ketoglutaric acid in ovarian granular cells is researched at living body and cellular level: HE and TUNEL discover that alpha-ketoglutaric acid promotes development and oestrus of ovarian follicles of mice; through q-PCR and Western Blot, it is found that proliferation and activity of human ovarian granular cells can be promoted and the oxidative stress level of the human ovarian granular cells can be reduced by increasing AKG.

Description

Technical Field

[0001] The present invention belongs to the technical fields of cell engineering and genetic engineering, and particularly relates to the application of α-ketoglutaric acid in ovarian granulosa cells. Background Art

[0002] The effect of oxidative stress on the ovaries of female mammals is an area worthy of attention. When the balance between oxidants and antioxidants in the body is disrupted, resulting in an oxidative stress state, it will have multiple negative impacts on ovarian function. First of all, oxidative stress will damage the quality of oocytes, leading to problems such as DNA damage and mitochondrial dysfunction, and further affecting the fertilization ability of eggs and embryo development. Secondly, oxidative stress will also affect the normal development of follicles, leading to follicular atresia or hypoplasia and reducing the number of ovulations. Female mammals in a long-term oxidative stress state may even experience accelerated depletion of ovarian reserve, resulting in premature menopause. Therefore, maintaining a good antioxidant status is crucial for maintaining ovarian health, which can be achieved by consuming antioxidant-rich foods and appropriate exercise.

[0003] Granulosa cells (GCs) play a crucial role in the ovary and are essential for the reproductive health of female mammals. GCs play a key role in follicular development. They can respond to the stimulation of gonadotropins, promote follicular growth and development, and ultimately promote egg maturation. However, when GCs encounter oxidative stress, their functions will be severely affected. Oxidative stress refers to the disruption of the balance between reactive oxygen species (ROS) and antioxidants in the body. In this case, the physiological functions of GCs are disturbed, which may lead to abnormal follicular development and affect the quality of eggs. This will weaken the supporting role of GCs and further affect follicular growth and development. Therefore, maintaining a good antioxidant status is crucial for protecting GCs from oxidative stress damage and helps to ensure the overall health of the ovary.

[0004] As an intermediate metabolite of the tricarboxylic acid cycle, α-ketoglutaric acid (AKG) plays a crucial role in cell energy metabolism, protein synthesis, epigenetic regulation, stemness and differentiation, fertility and reproductive health, and cancer cell behavior. In addition to its known metabolic functions, AKG is also a potential anti-aging agent and antioxidant. It shows multiple effects against oxidative stress, helps to reduce cellular oxidative damage and maintain cell health. Some studies have found that AKG was detected in human follicular fluid, and its level decreased with age; while in porcine apoptotic follicles, the concentration of AKG was significantly lower than that in healthy follicles. However, the connection between AKG and the oxidative stress state and growth and development of human ovarian granulosa cells has not been reported yet. Summary of the Invention

[0005] To overcome the drawbacks and deficiencies of the prior art, the first object of the present invention is to provide the use of α-ketoglutaric acid and / or its pharmaceutically acceptable salts in promoting estrus in mammals.

[0006] The second object of the present invention is to provide the use of α-ketoglutaric acid and / or its pharmaceutically acceptable salts in promoting follicular development in mammals.

[0007] The third object of the present invention is to provide the use of α-ketoglutaric acid and / or its pharmaceutically acceptable salts in the culture of ovarian granulosa cells.

[0008] The object of the present invention is achieved by the following technical solutions:

[0009] The use of α-ketoglutaric acid and / or its pharmaceutically acceptable salts in the estrus of mammals is at least one of the following Applications 1 and 2:

[0010] Application 1: The use of α-ketoglutaric acid and / or its pharmaceutically acceptable salts in promoting estrus in mammals;

[0011] Application 2: The use of α-ketoglutaric acid and / or its pharmaceutically acceptable salts in the preparation of products for promoting estrus in mammals.

[0012] Further, the promotion of estrus in mammals is to shorten the estrus age of mammals.

[0013] Further, the product is any one of drugs, reagents, feeds, and feed additives.

[0014] Further, the dosage form of the drug or reagent is any one of oral dosage forms and injection dosage forms.

[0015] Further, the drug or reagent also contains pharmaceutically acceptable excipients or carriers.

[0016] Further, the mammal is any one of humans and mice.

[0017] The use of α-ketoglutaric acid and / or its pharmaceutically acceptable salts in follicular development is at least one of the following Applications 3 and 4:

[0018] Application 3: The use of α-ketoglutaric acid and / or its pharmaceutically acceptable salts in promoting follicular development in mammals;

[0019] Application 4: The use of α-ketoglutaric acid and / or its pharmaceutically acceptable salts in the preparation of products for promoting follicular development in mammals.

[0020] Furthermore, the promotion of mammalian follicular development is to increase the number of corpora lutea and / or antral follicles and reduce the number of preantral follicles.

[0021] Furthermore, the product is any one of a drug and a reagent.

[0022] Furthermore, the drug or reagent further comprises a pharmaceutically acceptable excipient or carrier.

[0023] Furthermore, the mammal is any one of a human and a mouse.

[0024] The application of α-ketoglutaric acid and / or its pharmaceutically acceptable salt in the culture of ovarian granulosa cells is at least one of the following applications 5 and 6:

[0025] Application 5: The application of α-ketoglutaric acid and / or its pharmaceutically acceptable salt in the in vitro culture of ovarian granulosa cells;

[0026] Application 6: The application of α-ketoglutaric acid and / or its pharmaceutically acceptable salt in the preparation of an additive for the in vitro culture of ovarian granulosa cells.

[0027] Furthermore, the Application 5 is: Under in vitro conditions, ovarian granulosa cells are cultured with a medium containing α-ketoglutaric acid to promote the proliferation and viability of ovarian granulosa cells and reduce the oxidative stress level of ovarian granulosa cells.

[0028] Furthermore, the concentration of the α-ketoglutaric acid is 100 μM to 2 mM; preferably 1 mM.

[0029] Furthermore, the culture time is 12 ± 2 h.

[0030] Furthermore, the in vitro culture additive is at least one of a proliferation promoter, a viability promoter, and an oxidative stress inhibitor.

[0031] Furthermore, the ovarian granulosa cells are any one of human and mouse ovarian granulosa cells.

[0032] Furthermore, the pharmaceutically acceptable salt can be: sodium salt, potassium salt, ammonium salt, amino acid salt, lactate, hydrochloride, phosphate, acetate, malate, citrate, aspartate, etc.

[0033] The verification results of the present invention are as follows:

[0034] 1. Human ovarian granulosa cells were treated with AKG at a concentration of 100 μM to 2 mM, and the cell viability was detected by CCK8 at 12 h, 24 h, 36 h, and 48 h. When the AKG concentration was 1 mM and the action time was 12 h, the cell viability of ovarian granulosa cells was the highest ( Figure 1 ).

[0035] 2. Treat human ovarian granulosa cells with AKG, and use EdU, reactive oxygen species detection kits, qRT-PCR, and Western Blot to detect the effects of AKG on the proliferation and oxidative stress of human ovarian granulosa cells respectively. The results show that the cell proliferation rate in the AKG group is significantly higher than that in the blank control group ( Figure 2 ). The detection results of oxidative stress show that the level of reactive oxygen species in the AKG group is significantly lower than that in the blank control group ( Figure 3 ).

[0036] 3. Inject different concentrations of AKG into the peritoneal cavity of C57BL / 6J mice, and count the age of first estrus of the mice. The results show that, compared with the control group, the age of first estrus of the mice in the 5 mg / kg and 10 mg / kg AKG treatment groups is significantly advanced. Since the error of the estrus age of the mice in the 10 mg / kg AKG group is relatively large, the 5 mg / kg AKG group is finally selected as the optimal concentration for advancing the age of first estrus of the mice ( Figure 4 ).

[0037] 4. Quantify the analysis of the number of different follicles in the ovaries by observing the HE sections of the mouse ovaries. The results show that, compared with the control group, the proportion of preantral follicles in the ovaries of the mice in the 5 mg / kg AKG group is significantly reduced, and the proportion of corpora lutea is significantly increased (P<0.01); this indicates that 5 mg / kg AKG has the most significant effect on promoting the development of ovarian follicles in mice. In summary, 5 mg / kg AKG is selected for subsequent experiments ( Figure 5 ).

[0038] 5. Detect the apoptosis of GCs in mouse ovarian follicles by TUNEL assay. The results show that, compared with the control group, there are fewer apoptotic GCs in the ovaries of the mice in the AKG group, which indicates that AKG can inhibit the apoptosis of GCs in the mouse ovaries ( Figure 6 ).

[0039] 6. Use qRT-PCR and Western Blot to detect the expression of key genes in the proliferation and oxidative stress pathways of mouse ovaries. The experimental results show that, compared with the control group, AKG significantly promotes the expression of key genes in the proliferation pathway; and inhibits the expression of key genes in the oxidative stress pathway ( Figure 7 ).

[0040] The present invention has the following advantages and effects compared with the prior art:

[0041] 1. The present invention takes AKG as the research object, and uses in vivo and cell-level studies to study its application in human and mouse ovarian granulosa cells. By treating cells with AKG, it is found that AKG can significantly relieve the oxidative stress level of ovarian granulosa cells and promote cell proliferation.

[0042] 2. The present invention uses mouse and human ovarian granulosa cells as experimental materials, and HE sections and TUNEL are used to detect the development of ovarian follicles and the apoptosis level of ovarian granulosa cells in mice. By increasing the level of AKG in human ovarian granulosa cells, it is found that compared with the control group, AKG significantly promotes the proliferation of human ovarian granulosa cells and reduces the level of oxidative stress.

[0043] 3. The cells are treated with AKG at a concentration of 100 μM to 2 mM, and the optimal treatment concentration and duration are screened out as follows: when the AKG concentration is 1 mM and the action duration is 12 h, the viability of human ovarian granulosa cells is the best.

[0044] 4. Mice are intraperitoneally injected with AKG at 5 mg / kg, 10 mg / kg, 15 mg / kg, and 30 mg / kg, and the optimal concentration of AKG to promote the initiation of puberty and ovarian development is screened out as 5 mg / kg.

[0045] 5. The technical solution of the present invention is carefully designed and the results are reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is a diagram showing the effects of different concentrations of AKG and different treatment times on the viability of human ovarian granulosa cells.

[0047] Figure 2 It is a diagram showing the effects of AKG on the proliferation of human ovarian granulosa cells detected by EdU method, qRT-PCR and Western Blot.

[0048] Figure 3 It is a diagram showing the effects of AKG on the oxidative stress of human ovarian granulosa cells detected by using a reactive oxygen species detection kit, qRT-PCR and Western Blot.

[0049] Figure 4 It is a diagram showing the effects of different concentrations of AKG on the estrus age of mice.

[0050] Figure 5 It is a diagram showing the development of ovarian follicles in mice treated with different concentrations of AKG observed by HE-stained sections.

[0051] Figure 6 It is a diagram showing the apoptosis of GCs in ovarian follicles of mice detected by TUNEL experiment.

[0052] Figure 7 It is a diagram showing the effects of AKG on the proliferation and oxidative stress of mouse ovarian granulosa cells detected by qRT-PCR and Western Blot. DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto. For the experimental methods without specific conditions noted in the following embodiments, they are generally in accordance with conventional conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0054] In the present invention, statistical methods are applied to analyze the results of 3 independent experiments in each embodiment, calculate "mean ± standard deviation" respectively, and use one-way analysis of variance for significant difference analysis (in the figure, "*" indicates P < 0.05, and "**" indicates P < 0.01).

[0055] The AKG (α-ketoglutaric acid) used in the following embodiments was purchased from SIGMA Corporation.

[0056] Example 1: Detection of ovarian granulosa cell viability

[0057] The CCK8 method was used to detect the proliferation of ovarian granulosa cells. The experimental steps refer to the CCK8 kit instructions:

[0058] (1) Granulosa cells were seeded in a 96-well plate. When the cell confluence reached 80%, they were treated with different concentrations of AKG, and at least 3 replicates were set for each group;

[0059] (2) At 12 h, 24 h, 36 h, and 48 h of culture, 100 μL of the prepared CCK8 solution was added to each well, a blank control was set, and the cells were incubated in a cell culture incubator for 1 - 4 h.

[0060] (3) The OD value was detected at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader (in the dark).

[0061] Example 2: RNA extraction and reverse transcription

[0062] (1) RNA extraction

[0063] ① Cells were lysed by adding TRIzol in proportion;

[0064] ② Left standing on ice for 10 min, centrifuged at 12000 rpm for 5 min, and then the supernatant was transferred;

[0065] ③ Chloroform was added, shaken well, left standing on ice for 15 min, and centrifuged at 12000 rpm at 4℃ for 15 min;

[0066] ④ The upper aqueous phase was transferred to a new 1.5 mL centrifuge tube free of enzymes, isopropanol was added, gently mixed, left standing on ice for 15 min, centrifuged at 12000 rpm at 4℃ for 15 min, and the supernatant was discarded to retain the RNA precipitate;

[0067] ⑤ Add pre-cooled 75% ethanol to resuspend and wash the RNA precipitate, centrifuge at 12,000 rpm for 15 min at 4°C, discard the supernatant, and retain the precipitate;

[0068] ⑥ Add DEPC water to resuspend and dissolve the precipitate, measure the RNA concentration, and store at -80°C.

[0069] (2) RNA reverse transcription. Refer to the PrimeScript RT Master Mix instruction manual. The reaction system is shown in Table 1:

[0070] Table 1 RNA reverse transcription system

[0071]

[0072] Note: The reaction conditions are 15 min at 37°C and 5 s at 85°C.

[0073] Example 3: qRT-PCR

[0074] The qRT-PCR detection in the present invention uses qPCR SYBR Green Master Mix kit. The comparative Ct value method is used to detect the content of sample genes in the experiment. The specific calculation formula is as follows:

[0075] Relative gene expression = 2 -{〈﹙实验组目的基因Ct值﹚-﹙实验组内参基因Ct值﹚〉-〈﹙对照组目的基因Ct值﹚-﹙对照组内参基因Ct值﹚〉}

[0076] GAPDH is used as an internal reference for the detected gene. The qRT-PCR primers used in the present invention are:

[0077] qRT-PCR-GAPDH Forward: 5′-TGTTCGTCATGGGTGTGAAC-3′;

[0078] Reverse: 5′-ATGGCATGGACTGTGGTCAT-3′.

[0079] Example 4: Culture of ovarian granulosa cells

[0080] Resuscitation of KGN cell line: Take out the cryopreserved cells from liquid nitrogen, thaw them in a 37°C water bath, add the suspension to an appropriate amount of complete medium, centrifuge at 1000 rpm for 5 min, and discard the supernatant; then resuspend the cells with 5 mL of complete medium, transfer them to a small culture flask, and mix well by the cross method. Incubate in a 37°C, 5% CO2 cell culture incubator, change the medium after about 48 h, and continue to culture for 24 h before subculturing.

[0081] Cell passage and transfection: When the cell confluence reaches 80%, discard the culture medium and wash twice with PBS; add 0.25% trypsin and digest at 37°C for 5 min, then terminate the digestion with complete medium; pipette the cells, transfer them to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells with PBS and wash twice; resuspend the cells with an appropriate amount of complete medium, evenly transfer them to a culture flask or culture plate, and culture in a cell incubator. Observe the cell status. When the cell confluence reaches 80%, perform transfection referring to the Lipofectamine@3000 kit instructions.

[0082] Example 5: Detection of ovarian granulosa cell proliferation

[0083] The EdU method was used to detect the proliferation of ovarian granulosa cells. The experimental steps refer to the instructions of the Ribobio EdU kit:

[0084] (1) Seed granulosa cells in a 48-well plate. When the cell confluence reaches 80%, perform drug treatment, and set at least 3 replicates for each group;

[0085] (2) After 24 h, add 200 μL of pre-prepared 50 μM EdU medium to each well, incubate in a cell incubator for 2 h, discard the culture medium, and wash twice with PBS;

[0086] (3) Add 200 μL of cell fixative (80% acetone diluted with PBS) to each well, incubate at room temperature for 30 min, and wash twice with PBS;

[0087] (4) Add 200 μL of permeabilization solution (PBS containing 0.5% Triton X-100) to each well, incubate on a shaker for 5 min for permeabilization, and wash twice with PBS;

[0088] (5) Add 200 μL of pre-prepared 1× Apollo staining solution to each well, incubate in the dark at room temperature for 30 min, and wash twice with PBS;

[0089] (6) Re-permeabilize the cells, repeat step (4);

[0090] (7) Add 200 μL of pre-prepared DAPI reaction solution to each well, incubate in the dark at room temperature for 30 min, and wash twice with PBS;

[0091] (8) Add 100 μL of PBS to each well for preservation, take pictures under a fluorescence microscope, and save the pictures.

[0092] Example 6: Detection of oxidative stress level in ovarian granulosa cells

[0093] The experimental steps refer to the instructions of the Beyotime Reactive Oxygen Species Assay Kit:

[0094] (1) Inoculate granulosa cells in a 96-well plate. When the cell confluence reaches 80%, perform drug treatment, and set at least 3 replicates for each group.

[0095] (2) After culturing for 24 h, discard the culture medium, wash the cells 3 times with PBS, add 100 μl of the prepared DCFH-DA reaction solution (diluted 1:1000 with serum-free medium) to each well, and incubate in a cell culture incubator for 20 min.

[0096] (3) Wash 3 times with serum-free medium, and use an ELISA reader to detect the OD value at a wavelength of 488 nm (protected from light).

[0097] (4) Observe the cells under a fluorescence microscope and take pictures for preservation.

[0098] Example 7: Mouse feeding and drug injection

[0099] (1) AKG treatment of mice experiment: Purchase 21-day-old C57BL / 6J female black mice from Guangdong Medicilon. Set up a blank control group, a normal saline group (0.9% NaCl), and an AKG experimental group (divided into four subgroups, with each injection concentration of 5 mg / kg, 10 mg / kg, 15 mg / kg, and 30 mg / kg for each group). After adaptively feeding the mice for 3 days after toe clipping, start intraperitoneal injection of the drug once every other day, with an injection volume of 0.1 mL per mouse, for a total of 10 injections. Check the estrus status of the mice every day and make records.

[0100] Example 8: HE staining of mouse ovaries

[0101] (1) Sacrifice the mice at 42 days of age, take the ovaries, and fix them with 4% paraformaldehyde.

[0102] (2) Refer to the HE staining instruction manual of Servier for the specific experimental steps: dehydration, infiltration, embedding, and sectioning; dewax the paraffin sections to water, stain with hematoxylin and eosin, and finally dehydrate and mount the sections.

[0103] (3) Place the sections under a bright-field microscope and take pictures for preservation.

[0104] Example 9: Detection of apoptosis of mouse ovarian granulosa cells

[0105] (1) Select paraffin blocks of mouse ovaries embedded with HE staining and make paraffin sections.

[0106] (2) Refer to the instruction manual of the TUNEL kit of Servier.

[0107] (3) Place the sections under an inverted fluorescence microscope for photography and save the pictures.

[0108] Result analysis:

[0109] 1. Treat cells with AKG at a concentration of 100 μM to 2 mM, and detect cell viability using CCK8 at 12 h, 24 h, 36 h, and 48 h. When the AKG concentration is 1 mM and the action time is 12 h, the cell viability of human ovarian granulosa cells is the highest ( Figure 1 ).

[0110] 2. Use AKG to act on ovarian granulosa cells, and use EdU, reactive oxygen species detection kit, qRT-PCR, and Western Blot to detect the effects of AKG on the proliferation and oxidative stress of ovarian granulosa cells respectively. The results show that the proliferation rate of the AKG group is significantly higher than that of the control group ( Figure 2 ). The detection results of oxidative stress show that the reactive oxygen species level in the AKG group is significantly lower than that in the control group ( Figure 3 ). In summary, AKG can promote the proliferation of ovarian granulosa cells and inhibit cell oxidative stress.

[0111] 3. Inject AKG lentivirus into the abdominal cavity of mice, and count the changes in the estrus age of mice. The results show that AKG at 5 mg / kg has the most significant effect on advancing the estrus age of mice ( Figure 4 ).

[0112] 4. Observe the development of follicles in the ovaries of mice by HE staining. The results show that the proportion of preantral follicles in the ovaries of mice in the AKG group is significantly decreased, and the proportion of corpora lutea is significantly increased (P < 0.01), which indicates that AKG can promote the development of ovarian follicles in mice ( Figure 5 ).

[0113] 5. Detect the apoptosis of GCs in ovarian follicles of mice by TUNEL assay. The results show that the apoptosis of GCs in the AKG group is reduced, indicating that AKG can inhibit the apoptosis of GCs in the ovaries of mice ( Figure 6 ).

[0114] 6. Detect the effects of AKG on the expression levels of genes related to the proliferation and oxidative stress of mouse ovarian granulosa cells by qRT-PCR and Western Blot. The results show that the expression levels of genes related to proliferation in the AKG treatment group are up-regulated; while the expression levels of genes related to oxidative stress are decreased ( Figure 7 ).

[0115] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of α-ketoglutaric acid and / or its pharmaceutically acceptable salts in mammalian estrus, characterized in that: For at least one of the following applications 1 and 2: Application 1: The use of α-ketoglutaric acid and / or a pharmaceutically acceptable salt thereof in promoting estrus in mammals; Application 2: The use of α-ketoglutaric acid and / or a pharmaceutically acceptable salt thereof in the preparation of a product for promoting estrus in mammals.

2. The application according to claim 1, wherein: The promotion of estrus in mammals is to shorten the estrus age of mammals.

3. The application according to claim 1, wherein: The product is any one of a drug, a reagent, a feed, and a feed additive; The mammal is any one of a human and a mouse.

4. The application according to claim 3, wherein: The dosage form of the drug or reagent is any one of an oral dosage form and an injection dosage form; The drug or reagent further comprises a pharmaceutically acceptable excipient or carrier.

5. Use of α-ketoglutaric acid and / or its pharmaceutically acceptable salts in follicular development, characterized in that For at least one of the following applications 3 and 4: Application 3: The use of α-ketoglutaric acid and / or a pharmaceutically acceptable salt thereof in promoting follicular development in mammals; Application 4: The use of α-ketoglutaric acid and / or a pharmaceutically acceptable salt thereof in the preparation of a product for promoting follicular development in mammals.

6. The application according to claim 5, wherein: The promotion of follicular development in mammals is to increase the number of corpora lutea and / or antral follicles and reduce the number of preantral follicles.

7. The application according to claim 5, wherein: The product is any one of a drug and a reagent; The drug or reagent further comprises a pharmaceutically acceptable excipient or carrier; The mammal is any one of a human and a mouse.

8. Use of α-ketoglutaric acid and / or its pharmaceutically acceptable salts in ovarian granulosa cell culture, characterized in that: For at least one of the following applications 5 and 6: Application 5: The use of α-ketoglutaric acid and / or a pharmaceutically acceptable salt thereof in the in vitro culture of ovarian granulosa cells; Application 6: The use of α-ketoglutaric acid and / or a pharmaceutically acceptable salt thereof in the preparation of an additive for the in vitro culture of ovarian granulosa cells.

9. The application according to claim 8, wherein: Application 5 is: In an in vitro environment, culturing ovarian granulosa cells with a medium containing α-ketoglutaric acid to promote the proliferation and viability of ovarian granulosa cells and reduce the oxidative stress level of ovarian granulosa cells; The in vitro culture additive is at least one of a proliferation promoter, a viability promoter, and an oxidative stress inhibitor.

10. The application according to claim 9, wherein: The concentration of α-ketoglutaric acid is 100 μM to 2 mM; The culture time is 12 ± 2 h; The ovarian granulosa cells are any one of human and mouse ovarian granulosa cells.

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