Application of arachidonic acid in delaying reproductive aging

Abstract

The invention provides application of arachidonic acid in delaying reproductive aging, and particularly relates to the technical field of reproductive health. The invention provides application of arachidonic acid in preparation of a product for delaying reproductive aging and / or prolonging the reproductive period. By taking caenorhabditis elegans and mice as examples, the arachidonic acid can obviously activate reproductive aging gonad lysosome, protect oocyte quality and prolong reproductive cycle, thereby delaying reproductive aging. The invention provides a new application prospect of arachidonic acid in the aspects of delaying ovarian aging and protecting fertility.

Description

Technical Field

[0001] The invention belongs to the technical field of reproductive health, and in particular relates to the application of arachidonic acid in delaying reproductive aging. Background Art

[0002] The female reproductive system mainly includes the uterus, fallopian tubes and ovaries. The uterus is the place where menstruation occurs and the fetus is conceived; the fallopian tubes are muscular tubes that transport egg cells into the uterus; and the ovaries are organs that secrete hormones and produce eggs. As pregnant women age, humans face more and more reproductive health challenges. Reproductive aging or damage not only causes a decline in reproductive organ function, affecting fertility and reproductive quality, but also causes endocrine dysfunction, which in turn increases the risk of systemic multi-system diseases, such as cardiovascular disease, osteoporosis, obesity, tumors, diabetes, etc., and its harm cannot be underestimated. Therefore, in-depth research on the cellular and molecular mechanisms of reproductive aging can not only provide key theoretical support for the field of reproductive health, but also provide an important technical basis for research on delaying the human aging process and preventing senile diseases.

[0003] If reproductive organ aging and damage are not treated in time, it will seriously affect women's reproductive health and quality of life. At present, there is no effective drug to alleviate the aging or damage of the uterus and fallopian tubes. The strategies for preventing and treating ovarian dysfunction or aging include gene therapy, in vitro ovarian activation, artificial ovaries, mitochondrial transplantation, stem cell therapy, and drug intervention. Compared with other methods, there are problems such as ethics and less exploration. Drug intervention has the advantages of high safety, convenient operation, and economy. It is currently one of the most widely used and effective methods. At present, drugs that have been found to protect reproductive functions in various model animals include rapamycin, melatonin, resveratrol, coenzyme Q10, fisetin, dasatinib, and quercetin. As modern chemical drugs have poor efficacy in treating some diseases, the adverse reactions and drug resistance of some drugs are becoming more and more prominent. Therefore, there is an urgent need to find safer drugs to fight reproductive aging. Summary of the invention

[0004] The invention provides application of arachidonic acid in delaying reproductive senescence. The arachidonic acid can prolong the reproductive period and delay reproductive senescence.

[0005] The invention provides application of arachidonic acid in preparing products for delaying reproductive senescence and / or prolonging reproductive period.

[0006] In a preferred embodiment of the present invention, the arachidonic acid includes ω-3 arachidonic acid and / or ω-6 arachidonic acid.

[0007] In a preferred embodiment of the present invention, the reproductive aging includes physiological reproductive aging and / or pathological reproductive aging.

[0008] In a preferred embodiment of the present invention, the delaying of reproductive aging and / or the extension of the reproductive period includes at least one of the following: (1) activating the lysosomal activity of the ovary;

[0009] (2) regulating the development and ovulation of growing follicles;

[0010] (3) protecting the female reproductive reserve.

[0011] In a preferred embodiment of the present invention, the types of the products include at least one of the following: drugs, foods, cosmetics, and supplements.

[0012] The present invention also provides a drug for delaying reproductive aging and / or extending the reproductive period, which uses arachidonic acid as the active ingredient and further includes pharmaceutically acceptable excipients.

[0013] In a preferred embodiment of the present invention, the concentration of arachidonic acid in the drug is not less than 10 μM.

[0014] The present invention also provides a pharmaceutical composition for anti-reproductive aging, and the active ingredients include arachidonic acid and an active substance for extending the reproductive period;

[0015] The active substance for extending the reproductive period includes at least one of the following: coenzyme Q10, dehydroepiandrosterone, metformin, resveratrol, rapamycin, quercetin, etanercept, curcumin, arbutin, and astragalin.

[0016] In a preferred embodiment of the present invention, the dosage form of the pharmaceutical composition includes oral administration and / or injection.

[0017] In a preferred embodiment of the present invention, the dosage form of the pharmaceutical composition includes at least one of the following: tablets, capsules, granules, suspensions, pills, solutions, syrups, and injections.

[0018] Beneficial effects: The present invention provides the application of arachidonic acid in the preparation of products for delaying reproductive aging and / or extending the reproductive period. In an embodiment of the present invention, taking Caenorhabditis elegans as an example, 10 μM of arachidonic acid can significantly activate the lysosomes of the reproductive glands during reproductive aging, protect the quality of oocytes, extend the reproductive cycle of Caenorhabditis elegans, and thus delay reproductive aging. In another embodiment of the present invention, taking 12-month-old mice as an example, 12-month-old female mice were gavaged with 36.5, 75, and 150 mg / kg / day of arachidonic acid for two months. Compared with the aging control, the supplementation of arachidonic acid significantly increased the number of offspring, delayed the weight reduction caused by ovarian atrophy during aging, improved ovarian fibrosis, and activated the expression of ovarian lysosome-related genes, thus delaying reproductive aging. The present invention provides a new application prospect of arachidonic acid in delaying ovarian aging and protecting fertility. Description of the Drawings

[0019] Figure 1 Effect of administering ω-3 arachidonic acid or ω-6 arachidonic acid starting from the L4 stage on the daily egg production of wild-type nematodes;

[0020] Figure 2 Effect of administering ω-6 arachidonic acid starting from the L4 stage on oocytes on the 5th day of reproduction;

[0021] Figure 3 Effect of administering ω-6 arachidonic acid starting from the L4 stage and mating with male worms on the unhatched rate of offspring;

[0022] Figure 4 Statistical chart of the number of offspring of male mice mated at 13 months after administering ω-6 arachidonic acid starting from 12 months of age;

[0023] Figure 5 Statistical chart of the ovarian weight of mice administered ω-6 arachidonic acid starting from 12 months of age;

[0024] Figure 6 Masson staining diagram of the ovaries of mice administered ω-6 arachidonic acid starting from 12 months of age; Figure 7 Expression diagram of lysosome-related genes in the ovaries of mice administered ω-6 arachidonic acid starting from 12 months of age. Detailed implementation manner

[0025] The present invention provides the application of arachidonic acid in the preparation of products for delaying reproductive aging and / or extending the reproductive period.

[0026] The arachidonic acid (AA) described in the present invention is a natural polyunsaturated fatty acid, mainly present in some animal foods, such as animal offal and deep-sea fish, etc. In addition, it is also distributed in a certain amount in vegetable oils, such as safflower oil, sunflower oil, corn oil, and soybean oil, etc.; it has been found through research that AA is also an important essential fatty acid for the human body, and it is also the polyunsaturated fatty acid with the highest content and the widest distribution in the human body, and it has important significance in maintaining the structure and function of the cell membrane of the body.

[0027] The arachidonic acid described in the present invention includes ω-3 arachidonic acid (ω-3 Arachidonic acid) and / or ω-6 arachidonic acid (ω-6 Arachidonic acid), wherein ω-6 arachidonic acid is all-cis-5,8,11,14-eicosatetraenoic acid; ω-3 arachidonic acid is all-cis-8,11,14,17-eicosatetraenoic acid, and the chemical formula of both is C 20 H 32 O2.

[0028] The reproductive senescence described in the present invention includes physiological reproductive senescence and / or pathological reproductive senescence. Among them, in physiological reproductive senescence, with the increase of age, the quality and quantity of oocytes decrease, resulting in a decrease in the number of live offspring and postpartum generations.

[0029] In one embodiment of the present invention, taking Caenorhabditis elegans as an example, the arachidonic acid was administered at the L4 stage. It was found that when the arachidonic acid was administered starting from the L4 stage, the reproductive lifespan curve of wild-type nematodes could be significantly shifted to the right, the reproductive lifespan was extended, the defective oocytes were reduced, and the percentage of unhatched embryos produced by mating nematodes was decreased.

[0030] In another embodiment of the present invention, taking mice as an example, 12-month-old mice were given intragastric administration of arachidonic acid for two months. Compared with the aging control, the supplementation of arachidonic acid significantly increased the number of offspring, delayed the weight reduction caused by ovarian atrophy with aging, improved ovarian fibrosis, and activated the expression of ovarian lysosome-related genes, thereby delaying reproductive senescence.

[0031] The delaying of reproductive senescence and / or the extension of the reproductive period described in the present invention includes at least one of the following: (1) activating the activity of ovarian lysosomes;

[0032] (2) regulating the development and ovulation of growing follicles;

[0033] (3) protecting the female reproductive reserve.

[0034] The present invention proves that the arachidonic acid has a delaying effect on reproductive senescence and can extend the reproductive period. The arachidonic acid can be prepared into a product with the function of delaying reproductive senescence and / or extending the reproductive period. The types of the product include at least one of the following: drugs, foods, cosmetics, and supplements.

[0035] The present invention also provides a drug for delaying reproductive senescence and / or extending the reproductive period, which takes arachidonic acid as an active ingredient and also includes pharmaceutically acceptable excipients.

[0036] In one embodiment of the present invention, taking Caenorhabditis elegans as an example, it was found that when the concentration of arachidonic acid was not less than 10 μM, the effect was significant. In another embodiment of the present invention, taking mice as an example, it was found that when the concentration of arachidonic acid was not less than 36.5 mg / kg / day, the supplementation of arachidonic acid could significantly increase the ovarian weight, increase the number of offspring, and partially rescue the number of offspring caused by aging; and the supplementation of 75 mg / kg / day of arachidonic acid could significantly reduce ovarian fibrosis. The present invention does not have a special limitation on the dosage form of the drug, which can be oral administration or injection. The dosage form can be at least one of the following: tablets, capsules, granules, suspensions, pills, solutions, syrups, and injections.

[0037] The present invention also provides a pharmaceutical composition for anti-reproductive aging, and the active ingredients include arachidonic acid and reproductive period-extending active substances;

[0038] The reproductive period-extending active substances include at least one of the following: coenzyme Q10, dehydroepiandrosterone, metformin, resveratrol, rapamycin, quercetin, etanercept, curcumin, arbutin, and astragalin.

[0039] In the pharmaceutical composition of the present invention, the pharmaceutical active ingredients include arachidonic acid and other substances with reproductive period-extending activity. The pharmaceutical composition of the present invention can be prepared into oral medications and / or injectable medications, and the dosage forms can be at least one of the following: tablets, capsules, granules, suspensions, pills, solutions, syrups, and injections.

[0040] To further illustrate the present invention, the application of arachidonic acid provided by the present invention in delaying reproductive aging will be described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

[0041] In the present invention, the human reproductive system is the last system to develop and complete, and the development and maturity of the reproductive system symbolize the overall maturity of a person. The aging of the reproductive system is the first to start in a person's life. The physiological functions of women reach their peak at around 30 years old and then gradually decline, mainly manifested in the reduction of the quantity and quality of oocytes. Women enter menopause at around 50 years old and thus lose their reproductive ability.

[0042] In the present invention, the reproductive lifespan of Caenorhabditis elegans refers to the number of days from the start of egg-laying to the complete loss of reproductive ability. The larval stage of Caenorhabditis elegans is divided into four stages: L1, L2, L3, and L4:

[0043] L1 stage: Just hatched from the egg, the worm body is relatively small at this time, with only about 558 cells.

[0044] L2 stage: After a certain period of growth and development, the L1-stage larvae molt into the L2 stage.

[0045] L3 stage: If the environmental conditions are suitable, the L2-stage larvae will molt into the L3 stage. At this stage, the body of the nematode continues to grow, and the reproductive organs begin to develop. In hermaphrodite nematodes, the differentiation of germ cells begins in the L3 stage.

[0046] L4 stage: The L3-stage larvae further develop and molt into the L4 stage. Hermaphrodite individuals produce sperm in the L4 stage and lay eggs in the adult stage. If it is a male nematode, it has the ability to mate with hermaphrodites six days after the last larval molt (i.e., the L4 molt).

[0047] Example 1 Experiment on the egg-laying amount of Caenorhabditis elegans administered at the L4 stage

[0048] A control group (a control group without ω-3 arachidonic acid or ω-6 arachidonic acid) and a drug administration group (10 μM ω-3 arachidonic acid or 10 μM ω-6 arachidonic acid) were set up to conduct an experiment on the egg-laying amount of Caenorhabditis elegans during the L4 stage of drug administration.

[0049] Synchronized N2 nematodes at the L1 stage were placed in NGM medium and cultured at 20 °C for about 40 hours, and then cultured to the L4 stage and divided into 3 groups:

[0050] Control group: without arachidonic acid;

[0051] Experimental group 1: Add 10 μM ω-3 arachidonic acid;

[0052] Experimental group 2: Add 10 μM ω-6 arachidonic acid.

[0053] The nematodes cultured separately in the 3 groups were uniformly placed in an incubator at a constant temperature of 20 °C for culture, which was recorded as the 0th day of the experiment. Every day, the nematodes were picked into new corresponding media and transferred to a new plate once a day until their reproductive ability was lost.

[0054] After all the egg-laying plates were placed at 20 °C for 48 hours, the number of offspring was counted. If nematodes were observed not to lay eggs for two consecutive days, the last day of egg-laying was recorded as the end day of the reproductive cycle. Nematodes that died before the end of the reproductive period, burrowed into the medium, hatched eggs in the body, and had vulva bursting were recorded as lost.

[0055] The reproductive life curve is as Figure 1 shown. Administering ω-3 arachidonic acid or ω-6 arachidonic acid starting from the L4 stage can significantly shift the reproductive life curve of wild-type nematodes to the right; the reproductive life is extended, and the effect is obvious: * indicates P < 0.05, ** indicates P < 0.01, **** indicates P < 0.0001.

[0056] Every day, the remaining parental nematodes were washed with M9 (prepared by dissolving 3 g of potassium dihydrogen phosphate, 6 g of disodium hydrogen phosphate dihydrate, 5 g of sodium chloride and 1 mL of 1 M magnesium sulfate solution in deionized water and making up to 1 L), the offspring nematodes were removed, and the parental nematodes were transferred to a new NGM medium. Cultured until the 5th day of reproduction, the remaining parental nematodes were washed with M9, and the reproductive glands of the nematodes were photographed under a microscope, and the defects of the oocytes in the reproductive glands were scored and statistically analyzed.

[0057] For each oocyte image, the deterioration of oocytes, including hollow, deformed oocytes, small oocytes, and intrauterine oocytes, was scored and evaluated according to the criteria disclosed in the article (Luo S, Kleemann GA, Ashraf JM, Shaw WM, Murphy CT. TGF-beta and insulin signaling regulate reproductive aging via oocyte and germline quality maintenance. Cell. 2010;143(2):299-312.), and scored at three severity levels: mild, moderate, and severe. The results are as Figure 2 shown. On the 5th day of reproductive aging, the deterioration of oocytes was scored and evaluated, and the supplementation of arachidonic acid significantly improved the deterioration of oocytes.

[0058] Male wild-type nematodes were added for mating, and the daily egg production and the number of unhatched embryos were counted. The results are as Figure 3 shown. Administering ω-3 arachidonic acid starting from the L4 stage significantly reduced the percentage of unhatched embryos compared with the control.

[0059] Example 2

[0060] 12-month-old female C57 / BL6 mice were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences. The mice were housed under a 12-hour light / dark cycle at a temperature maintained at 22-25 °C. The mice were randomly divided into 4 groups: one group served as the control and was gavaged with a 1% aqueous solution of sodium carboxymethylcellulose; the other three groups were supplemented with arachidonic acid (Macklin) at concentrations of 36.5, 75, and 150 mg / kg / day mM by gavage.

[0061] The three concentrations of arachidonic acid were prepared with a 1% aqueous solution of sodium carboxymethylcellulose and gavaged every other day for 2 consecutive months. After 2 months of treatment, some mice were randomly selected for breeding, and the number of pups per litter was recorded to evaluate fertility. The other mice were used to evaluate the ovarian condition. The mice were euthanized at 14 months of age, and the ovaries were dissected and weighed. Some ovaries were fixed with 4% paraformaldehyde, and the other ovaries were quickly placed in liquid nitrogen for freezing. 8-week-old mice served as the young control group.

[0062] Masson staining: After fixation with 4% paraformaldehyde for 3 days, the sections were paraffin-embedded and stained according to the kit procedure (Solarbio, Modified Masson Trichrome Staining Kit, G1346). After sealing with neutral resin and allowing it to air-dry naturally, the sections could be scanned.

[0063] q-PCR: Ovarian tissues snap-frozen in liquid nitrogen were added with Trizol lysis buffer and extracted according to the RNA extraction procedure. The extracted RNA was stored at -80°C. RNA was reverse-transcribed into cDNA according to the reverse transcription kit protocol (PrimeScript RT reagent Kit, takara); Samples were loaded according to the qPCR kit (SYBR Primx Ex Taq II, takara) and run on a Real-Time PCR instrument (BD) according to the program.

[0064] Table 1 Primers used in q-PCR

[0065]

[0066]

[0067] With reproductive aging, the number of offspring decreased. ω-6 arachidonic acid supplementation started at 12 months of age and the mice were mated with male mice at 13 months of age while arachidonic acid supplementation continued. The results Figure 4 showed that compared with the aging control, arachidonic acid supplementation at 36.5 and 75 mg / kg / day significantly increased the number of offspring and could partially rescue the decrease in the number of offspring caused by aging.

[0068] With the progression of reproductive aging, ovarian tissues atrophy, leading to a decrease in weight (Yang Q, Li H, Wang H, Chen W, Zeng X, Luo X, et al. Deletion of enzymes for de novo NAD(+) biosynthesis accelerated ovarian aging. Aging Cell. 2023;22(9):e13904.). ω-6 arachidonic acid supplementation started at 12 months of age and continued for two months. The results Figure 5 showed that compared with the aging control, arachidonic acid supplementation at 36.5 and 75 mg / kg / day significantly increased ovarian weight.

[0069] Fibrosis is a hallmark of ovarian aging and affects the release of oocytes in the ovarian stromal region. Masson staining is one of the staining methods for showing tissue fibrosis. During staining, collagen fibers appear blue (Umehara T, Winstanley YE, Andreas E, Morimoto A, Williams EJ, Smith KM, et al. Female reproductive life span is extended by targeted removal of fibrotic collagen from the mouse ovary. Sci Adv. 2022;8(24):eabn4564.). Starting from 12 months of age, ω-6 arachidonic acid was supplemented for two months. The results are as Figure 6 shown. Compared with the aging control, the supplementation of 75 mg / kg / day of arachidonic acid significantly reduced ovarian fibrosis.

[0070] The expression of lysosome-related genes in the ovary was detected by q-PCR. Starting from 12 months of age, 75 mg / kg / day of ω-6 arachidonic acid was supplemented for two months. The results are as Figure 7 shown. Compared with the aging control, the supplementation of 75 mg / kg / day of ω-6 arachidonic acid significantly activated the expression of lysosome-related genes.

[0071] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of arachidonic acid in the preparation of a product for delaying reproductive aging and / or prolonging the reproductive period.

2. The application according to claim 1, wherein The arachidonic acid includes ω-3 arachidonic acid and / or ω-6 arachidonic acid.

3. The application according to claim 1, characterized in that The reproductive aging includes physiological reproductive aging and / or pathological reproductive aging.

4. The application according to claim 1 or 3, characterized in that, The delaying of reproductive aging and / or prolonging of the reproductive period includes at least one of the following: (1) activating the activity of ovarian lysosomes; (2) regulating the development and ovulation of growing follicles; (3) protecting female reproductive reserves.

5. The application according to claim 1, wherein The types of the product include at least one of the following: drugs, foods, cosmetics, and supplements.

6. A drug for delaying reproductive aging and / or prolonging the reproductive period, characterized in that, Taking arachidonic acid as the active ingredient, it also includes pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that, The concentration of arachidonic acid in the drug is not less than 10 μM.

8. A pharmaceutical composition for resisting reproductive senescence, characterized in that, The active ingredient includes arachidonic acid and an active substance for prolonging the reproductive period; The active substance for prolonging the reproductive period includes at least one of the following: coenzyme Q10, dehydroepiandrosterone, metformin, resveratrol, rapamycin, quercetin, etanercept, curcumin, arbutin, and astragalin.

9. The pharmaceutical composition according to claim 8, wherein, The dosage form of the pharmaceutical composition includes oral administration and / or injection.

10. The pharmaceutical composition according to claim 9, wherein The dosage form of the pharmaceutical composition includes at least one of the following: tablets, capsules, granules, suspensions, pills, solutions, syrups, and injections.

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