Application of caloric restricted diet scheme in preparation of product for improving female reproductive metabolism abnormality and heredity caused by hyperandrogen exposure

Abstract

The invention discloses application of a caloric restricted diet scheme in preparation of a product for improving female reproductive metabolism abnormality and heredity caused by hyperandrogen exposure, and belongs to the technical field of pharmacology. According to the invention, an IVF-ET means is utilized for the first time to clarify the key effect of the ovum in reproduction and metabolism disorder inheritance caused by mediated hyperandrogen exposure. In addition, a large number of experiments confirm that 20% calorie limited diet can improve reproductive metabolism phenotype abnormality of the contemporary and offspring exposed by high male and block female reproductive metabolism abnormality heredity caused by high male hormone exposure. 68 key effect genes which can play a role in the diet scheme are further obtained through ovum single cell whole genome methylation sequencing and bioinformatics analysis screening after calorie limitation, and the key effect genes are expected to become early intervention targets of phenotypic inheritance of female reproductive metabolism abnormality and offspring abnormality caused by hyperandrogen exposure in the future.

Description

Technical Field

[0001] The present invention belongs to the field of pharmacological technology, and specifically relates to the application of a calorie-restricted diet in the preparation of a product for improving female reproductive metabolic abnormalities and hereditary abnormalities caused by high androgen exposure. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Polycystic ovary syndrome (PCOS) is a female reproductive and metabolic interactive disease. Hyperandrogenism is one of the most important clinical features of PCOS, affecting about 40%-80% of PCOS women, causing complex metabolic and endocrine changes in the body, including hypothalamic-pituitary-ovarian axis dysfunction and energy metabolism disorders. Hyperandrogenism and pathophysiological changes such as obesity and insulin resistance promote each other and form a vicious cycle, which not only seriously threatens women's own health, but also a large number of studies have confirmed that when the mother has hyperandrogenemia, the fetus can also be exposed to an excessively high level of androgen environment, which is manifested as an increase in the anorectal distance of the offspring, an increase in the level of testosterone in the umbilical venous blood, or a more vigorous level of sebum secretion at birth, which in turn has a complex impact on growth and development, cardiovascular health, endocrine metabolic function, reproductive system, etc. Animal experiments have shown that mouse models exposed to intrauterine androgens can undergo transgenerational transmission of PCOS-like phenotypes, posing serious long-term health threats to their offspring and even grandchildren. As carriers of environmental epigenetic information, germ cells are key to the occurrence of intergenerational / transgenerational genetic effects. Although the role of eggs in mediating the inheritance of abnormal phenotypes caused by high androgen exposure is unknown, oocytes in the developmental stage are likely to be affected by long-term high androgen in the female body and undergo epigenetic remodeling, thereby mediating the occurrence of reproductive and metabolic disorders in offspring. Exposure to adverse factors in early life and even during gamete development often increases the risk of chronic diseases in adult individuals. These findings also remind us that the early developmental stage has a high degree of plasticity and may become a critical period for reshaping health and disease trajectories, preventing the development of adult diseases and transgenerational transmission. The concept of prevention and treatment of chronic diseases should be extended to the entire life cycle, including the pre-pregnancy period.

[0004] At present, androgen receptor antagonists and oral contraceptives are often used in clinic to improve the female internal environment disorder caused by high androgen. However, these intervention measures have a slow onset of effect in long-term treatment, poor patient compliance, and different individual health benefits. In addition, since hormones and related receptors are widely distributed in various systems throughout the body, they often bring many side effects. In the long run, there is a lack of consideration for the safety of offspring. Therefore, while improving their own internal environment, their role in preventing offspring reproductive and metabolic disorders and blocking intergenerational / transgenerational genetic effects at an early stage is unknown and has little potential.

[0005] The caloric restriction (CR) diet improves the body's energy balance and overall metabolic flexibility by reducing the total amount of food intake, prolonging the fasting time, and increasing the fasting-eating rhythm. Both clinical and animal experiments have found that caloric restriction can reduce body weight and body fat, reduce blood lipids and liver lipids, and improve glucose tolerance and insulin sensitivity. Caloric restriction can significantly improve the quality of eggs in obese women. However, there are currently no reports on improving female reproductive metabolic disorders and offspring health caused by high androgen exposure by adjusting dietary regimens. Summary of the invention

[0006] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide an application of a calorie-restricted dietary regimen in the preparation of a product for improving female reproductive metabolic disorders and hereditary abnormalities caused by hyperandrogen exposure. The present invention has found that adjusting the dietary regimen can improve female reproductive metabolic disorders and offspring health caused by hyperandrogen exposure.

[0007] In order to achieve the above object, the technical solution of the present invention is: The first aspect of the present invention provides a use of a calorie-restricted diet regimen in the preparation of a product for improving female reproductive metabolic abnormalities caused by high androgen exposure; The calorie-restricted diet is a food with a total energy reduction of 10-40% based on the standard diet.

[0008] The second aspect of the present invention provides a use of a calorie-restricted diet regimen in the preparation of a product for blocking the inheritance of abnormal female reproductive metabolism caused by high androgen exposure; The calorie-restricted diet is a food with a total energy reduction of 10-40% based on the standard diet.

[0009] In some embodiments of the present invention, blocking the inheritance of abnormal female reproductive metabolism caused by high androgen exposure includes reversing the gene of abnormal DNA methylation level of eggs caused by high androgen exposure.

[0010] Preferably, the gene is selected from Cacna1s, Atp1a2, Gna11, Kcnmb4, Gck, Camk2b, Atp1b2, Gip, Adcy3, Ryr2, Adcy2, Cacna1d, Adcy4, Adcy8, Adcy6, Glp1r, Atf6b, Camk2a, Plcb3, Rapgef4, Creb3l1, Gnas, Kcnmb3, Creb3l4, Stx1a, Adcyap1r1, Cacna1c, Kcnn4, Atp1a3, Trpm4, Kcnj11, Abcc8, Kcn At least one of u1, Kcnn1, Prkaca, Adcy7, Fxyd2, Cck, Creb3, Pik3cd, Tbc1d1, Ulk1, Cacna1f, Acacb, Gpr119, Camkk2, Foxo3, Pparg, Eef2, Lipe, Srebf1, Akt2, Fasn, Akt1s1, Ppp2r5c, Cpt1c, Akt1, Gys1, Pfkp, Ppp2r2d, Pik3r1, Ccnd1, Cpt1b, Insr, Tsc1, Ppp2cb, Crtc2 and Pik3r2.

[0011] It should be noted that the standard diet plan is a reasonable healthy diet plan, and its total intake is the recommended intake or the average intake of the normal population.

[0012] In some embodiments of the present invention, the calorie restricted dietary regimen is implemented for ≥ 4 weeks.

[0013] In some embodiments of the present invention, the calorie-restricted diet is a food with a total energy reduction of 10-30% based on the original diet.

[0014] In some embodiments of the present invention, the calorie-restricted diet is a food with a total energy reduction of 20% based on the original diet.

[0015] In some embodiments of the present invention, the reproductive metabolic abnormalities include metabolic disorders and reproductive disorders; The metabolic disorders include disorders of basic development and metabolic indicators, sugar metabolism indicators and lipid metabolism indicators.

[0016] The reproductive disorders include, but are not limited to, infrequent ovulation and prolonged menstrual cycles, polycystic ovarian morphological changes, increased circulating free testosterone levels, increased anorectal distance, increased AMH, or clinical manifestations of hyperandrogenism such as hirsutism and acne.

[0017] In some embodiments of the present invention, the basic development and metabolic indicators include but are not limited to body weight, BMI, body fat percentage, food intake, metabolic rate, respiratory entropy and heat production.

[0018] In some embodiments of the present invention, the carbohydrate metabolism index includes but is not limited to fasting blood glucose, fasting insulin, glucose tolerance test, insulin tolerance and insulin secretion test.

[0019] In some embodiments of the present invention, the lipid metabolism indicators include but are not limited to biochemical indicators such as peripheral blood triglycerides, cholesterol, low-density lipoprotein, high-density lipoprotein, and liver tissue lipid accumulation and adipocyte morphology.

[0020] In some embodiments of the present invention, in the original diet plan, the intake ratio of protein, fat and carbohydrates is 20-30% protein, 10-15% fat and 60-70% carbohydrates.

[0021] In some embodiments of the present invention, the intake ratio of protein, fat and carbohydrates is 20-25% protein, 10-15% fat and 60-65% carbohydrates. In some embodiments of the present invention, the original dietary regimen also contains vitamins, amino acids and minerals.

[0022] In some embodiments of the present invention, the vitamins include vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B6, vitamin B2, niacin, pantothenic acid, biotin, choline and folic acid.

[0023] In some embodiments of the invention, the amino acids include methionine, cystine, lysine, tryptophan, arginine, leucine, isoleucine, threonine, valine, histidine, phenylalanine and tyrosine.

[0024] In some embodiments of the invention, the minerals include sodium, magnesium, potassium, copper, iron, manganese, zinc, selenium and iodine.

[0025] The third aspect of the present invention also provides the use of a calorie-restricted diet regimen in preparing a mouse model for improving female reproductive metabolic abnormalities caused by high androgen exposure.

[0026] In some embodiments of the present invention, the calorie-restricted diet is a diet with a total energy reduction of 10-40% based on the normal diet of female mice with abnormal reproductive metabolism caused by high androgen exposure. The calorie-restricted diet is implemented for ≥ 4 weeks.

[0027] The third aspect of the present invention also provides the use of a calorie-restricted diet regimen in preparing a model for recovery of female reproductive metabolic abnormalities in mice offspring caused by high androgen exposure.

[0028] In some embodiments of the present invention, the application is to feed female mice with abnormal reproductive metabolism caused by high androgen exposure with a calorie-restricted diet to obtain intervention mouse offspring.

[0029] In some embodiments of the present invention, the calorie-restricted diet is a diet with a total energy reduction of 10-40% based on the normal diet of female mice with abnormal reproductive metabolism caused by high androgen exposure. The calorie-restricted diet is implemented for ≥ 4 weeks.

[0030] The beneficial effects of the present invention are: This invention uses IVF-ET for the first time to clarify the key role of eggs in the inheritance of reproductive and metabolic disorders mediated by hyperandrogen exposure. In addition, a large number of experiments have determined that a 20% calorie restriction diet can improve the abnormal reproductive metabolic phenotypes of the current generation and offspring exposed to hyperandrogenism. And further, through single-cell whole-genome methylation sequencing of eggs after calorie restriction and bioinformatics analysis screening, the key effector genes that may play a role in this diet are explored, which is expected to become an early intervention target for the inheritance of abnormal reproductive metabolism and abnormal phenotypes of offspring caused by hyperandrogen exposure in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 This is a schematic diagram of the research process of the role of dietary adjustment in blocking reproductive metabolic disorders and inheritance caused by high androgen exposure in Example 1 of the present invention; Figure 2 It is the identification of the metabolic and reproductive phenotypes of parent-offspring mice under the prenatal androgen exposure (PNA) genetic system in Example 1 of the present invention; wherein A is a schematic diagram of the breeding process of PNA exposure and parent-offspring mice, B is the change in body weight, CD is the change in visceral and subcutaneous body fat rate, E is a typical visceral adipose tissue morphology, F is a typical liver pathological morphology, G is the result of the glucose tolerance test, H is the result of the insulin tolerance test, I is the change in the insulin release index, J is the anorectal distance test, K is a typical vulvar hairy morphology, L is the change in vaginal opening time, and M is the statistics and typical changes of estrous cycle changes; Figure 3 The figures are the changes in reproductive metabolic indicators of PNA mice after intervention with different calorie restriction diets of the present invention; wherein A is the change in body weight after intervention with different diets, BC is the change in body fat percentage, and D is the change in ovarian weight; Figure 4 It is the detailed changes of metabolic indicators of PNA mice after the present invention implements 20% calorie restriction diet adjustment; wherein A is a flowchart of the implementation of different grouping diet strategies, B is the weight change, CD is the change of visceral and subcutaneous body fat rate, E is a typical visceral fat tissue morphology diagram, F is a typical liver pathological morphology diagram, G is the liver tissue triglyceride level test result, H is the liver tissue cholesterol level test result, I is the glucose tolerance test result, J is the insulin tolerance test result, K is the change of insulin release index, L is the metabolic cage oxygen consumption change test result, M is the metabolic cage energy consumption change test result, N is the metabolic cage respiratory exchange rate change test result, O is the metabolic cage activity change test result; Figure 5 It is the identification of offspring and metabolic reproductive phenotypes under the genetic intervention system of the present invention; wherein A is a schematic diagram of the offspring breeding process after the mother's dietary intervention of the present invention, B is the change of offspring weight, CD is the change of offspring visceral and subcutaneous body fat rate, E is the test result of offspring glucose tolerance test, F is the test result of offspring insulin tolerance test, G is the change of offspring insulin release index, H is the test result of offspring metabolic cage energy consumption change, I is the test result of offspring metabolic cage respiratory exchange rate change, J is offspring anal-genital distance test, K is the change of offspring vaginal opening time, and L is the statistical situation of offspring estrous cycle change; Figure 6 It is a graph of the differential methylation analysis results of the mice eggs exposed to high androgen after adjustment of the 20% calorie restriction diet of the present invention; wherein A is the change in the overall DNA methylation level of the four groups, B is the change in the DNA methylation level of each regulatory element in the four groups, C is the recognition of the promoter-differentially methylated region, D is the enrichment of the promoter-differentially methylated gene pathway, and E is the STRING protein interaction network diagram of the key pathway of promoter differential methylation enrichment. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0034] The technical solution of the present invention is as follows: First, a mouse model of prenatal androgen exposure was established, and its offspring were bred through in vitro fertilization and embryo transfer (IVF-ET). Through metabolic and reproductive phenotype testing, it was clarified how reproductive metabolic disorders are inherited in this genetic system and the role of the egg in mediating the inheritance of abnormal phenotypes.

[0035] Secondly, a scientifically feasible calorie restriction diet plan was established. By implementing different degrees of calorie restriction diets on PNA mice and evaluating their effectiveness in improving the reproductive metabolic phenotype, the best dietary strategy was selected.

[0036] Thirdly, we obtained the offspring of intervention mice and clarified the health benefits of the offspring of mice following a calorie restricted diet and the key role of eggs in improving the metabolic and reproductive disorders of the offspring after maternal calorie restriction.

[0037] Fourthly, we obtained the eggs of mice after the intervention, and screened the effect pathways and potential targets of calorie restriction that may block the inheritance of reproductive and metabolic abnormalities caused by kaohsiung exposure through WGBS sequencing and bioinformatics analysis.

[0038] The present invention also provides The following combination Figure 1-6 The present invention is further described in detail, wherein: Figure 1 Schematic diagram of the experimental design of the present invention.

[0039] Example 1 1. Establish a PNA mouse model and breed its offspring through IVF-ET, detect the metabolic and reproductive phenotypes of the mice, and clarify the genetic effects of the animal model and the role of eggs in the inheritance of metabolic and reproductive disorders in offspring 1.1 Establishment of a mouse model of prenatal androgen exposure Pregnancy time of 10-12 week old wild-type C57BL / 6J female mice was precisely calculated and caged. From embryonic day 16.5 to 18.5 (E16.5 to E18.5), 250 μg dihydrotestosterone (DHT, dissolved in 100 μL corn oil) or the same amount of corn oil in the control group was subcutaneously injected into the interscapular region every day. After the mice were born (F1), the number of pups and the sex ratio of the two groups of mothers were kept the same to ensure balanced feeding; the mice were weaned at 21 days of age and then 5 mice were fed in a cage. All animals were kept in a specific pathogen free (SPF) environment with a 12-hour light-12-hour dark cycle, constant temperature and humidity.

[0040] 1.2 Offspring breeding strategy The F1 generation female mice were subjected to normal ovulation induction, i.e., each F1 mouse was injected with 5 IU pregnant mare serum gonadotrophin (PMSG), and 48 hours later, 5 IU human chorionic gonadotropin (HCG) was injected. 16 hours later, mature MII eggs were taken out from the ampulla of the mouse oviduct. The taken out MII oocytes were placed in fertilization fluid, and sperm that had been successfully capacitated in advance were added. They were cultured in a CO2 incubator for a period of time. After fertilization, the excess sperm was washed off, and the cells were cultured to the 2-cell stage. The 2-cell embryos were transplanted into the oviduct of the pseudo-pregnant mice. The transplanted female mice were kept in a cage with 2 mice, and the mice were born about 19 days later. The feeding conditions of the F2 generation mice were the same as those of the F1. The animal model construction and breeding strategies are as follows: Figure 2 As shown in A.

[0041] 1.3 Metabolic phenotyping After 21 days of lactation, mice began to eat independently. Their body weight was monitored once a week until the mice were sampled. Sugar metabolism experiments were performed at 15 to 18 weeks of age, including glucose tolerance test (GTT), insulin tolerance test (ITT), and insulin releasing test (IRT). The CLAMS small animal metabolic cage test was then performed to monitor the mouse's food intake, metabolic rate, respiratory entropy, heat production and other indicators. Finally, samples were collected at 18-20 weeks of age after fasting overnight, and the tissues were accurately weighed. The lipid accumulation in the liver was detected by Oil Red O staining, and the fat morphology was observed by HE staining after paraffin embedding and sectioning. The Prilep kit was used to detect the levels of triglycerides and cholesterol in serum, and the lipid accumulation in the liver was observed by Oil Red O staining, and the fat morphology was observed by HE staining after paraffin embedding and sectioning.

[0042] The detection methods of metabolism-related indicators are as follows: a. Sugar metabolism experiment In GTT, mice were fasted for 16 hours overnight, and 20% glucose balanced salt solution was intraperitoneally injected at 2 g glucose per kg of fasting body weight of mice; in ITT, mice were fasted for 6 hours, and 0.75 IU insulin was intraperitoneally injected at 0 min, 15 min, 30 min, 60 min, and 120 min of fasting body weight of mice. Blood glucose of mice was measured by tail tip blood at 0 min, 15 min, 30 min, 60 min, and 120 min, and subsequent analysis was performed. In the same way as GTT, blood was collected from the tail vein at 0 and 15 minutes after glucose injection, and serum insulin levels were monitored by ELISA.

[0043] b.CLAMS small animal metabolic cage test Before conducting the metabolic cage experiment, dual-energy X-rays were used to detect the lean mass and fat mass of mice for subsequent experimental data correction. After the test, the mice rested for 2 days and were placed in a CLAMS metabolic cage with a temperature set at 24°C. The other indices were normal, except for the calorie-restricted feeding of mice at 15:00 every day, and the system remained stable at other times.

[0044] c. Oil red O staining of liver to detect lipid deposition Liver frozen sections were adhered at room temperature for more than 30 minutes; after rewarming, the tissue was fixed with 4% PFA in a humidified box for about 15-30 minutes; washed with distilled water for 1-2 minutes × 2 times to remove the embedding agent; rinsed with 60% isopropanol for 2 minutes to facilitate oil red staining; stained with oil red working solution for 10 minutes; generally added once, and then added again after 5 minutes, and the slides must not be dry; 60% isopropanol was added with a gun tip to separate the slides; washed with distilled water; stained with hematoxylin for 1 minute, washed with tap water to reverse blue; washed with distilled water for the last time; glycerol gelatin was used to seal the slides. Take out the slides from the distilled water, gently shake off the water drops on them, dip the glycerol gelatin with a large gun tip and drop it on the tissue, and quickly add a cover glass. Dry overnight in a fume hood.

[0045] d. Dehydrated fat and pancreatic tissue, embedded in paraffin, and then stained with HE After the adipose tissue was fixed with PFA overnight, the fixative was washed off with PBS and placed in a dehydrator for tissue dehydration. The dehydrated tissue was embedded in paraffin and sliced ​​at a thickness of 5 µm. The sliced ​​tissue was placed on a slide and dried. The slices were routinely stained with HE using a Leica automatic stainer. After staining, the slices were sealed with neutral gum, dried in a fume hood, and photographed to collect images.

[0046] like Figure 2 As shown, the fat mass of mice exposed to high androgen and the offspring developed from eggs exposed to androgen alone increased significantly ( Figure 2 C, 2D), the overall weight gain of the offspring mice was particularly obvious ( Figure 2 B), accompanied by a significant increase in the volume of fat cells in two generations of mice ( Figure 2 E), increased lipid accumulation in the liver ( Figure 2 F). The offspring may even have decreased glucose tolerance and insulin sensitivity ( Figure 2 G, 2H), decreased insulin production index ( Figure 2 These results suggest that direct exposure to high androgen levels can lead to metabolic dysfunction in mice and that their eggs can independently mediate the inheritance of metabolic damage phenotypes.

[0047] 1.4 Reproductive phenotype detection Anogenital distance (AGD) was measured with a vernier caliper on the day of weaning, and each mouse was measured three times to reduce errors. All mice were then monitored daily until vaginal opening appeared, and the age of vaginal opening (age in days) was recorded. Vaginal smears were performed on adult mice aged 12-16 weeks for more than 14 consecutive days to detect the estrous cycle. Two experimenters independently evaluated the cytological results of the vaginal smears, and then statistical analysis was performed on the changes in the estrous cycle.

[0048] like Figure 2 As shown, PNA mice and their offspring showed different degrees of AGD extension ( Figure 2 J) Vulva hairiness ( Figure 2 K) and other high androgen manifestations, the vaginal opening of the offspring mice appeared earlier ( Figure 2 L), estrous cycle disorder ( Figure 2 These results suggest that direct exposure to high androgen levels can lead to reproductive phenotype disorders in mice and that their eggs can independently mediate the inheritance of reproductive dysfunction.

[0049] 2. Establish a calorie-restricted diet strategy and identify the best diet plan through reproductive metabolic index testing 2.1 Implementation of calorie-restricted diet Calorie restriction should be performed after mice reach adulthood. Calorie restriction before adulthood will affect the normal development of mice. After 12-week-old F1 female mice are placed in a single cage, their daily food intake is weighed for one week and the food intake is calculated. After the start of calorie restriction, the mice are still placed in a single cage, and their daily food intake is accurately weighed and restricted to 20%, 30%, and 40% (see Table 1 for feed formula). They are fed at 15:00 every day, and no feed is added at other times, and they drink water normally. After about a week of adaptation according to different degrees of calorie restriction, the feeding method remains consistent, and the standard intervention time is no less than 4 weeks.

[0050] Note: After calorie restriction is initiated by limiting the daily food intake by 20%, 30%, and 40%, the daily food intake of mice will be reduced by 20%, 30%, and 40% based on their original food intake.

[0051] Table 1-1 Feed formula for different degrees of calorie restriction

[0052] Table 1-2 Feed formula for different degrees of calorie restriction

[0053] Table 1-3 Feed formulas for different degrees of calorie restriction

[0054] like Figure 3 As shown in the figure, after implementing the above-mentioned different degrees of calorie restriction diet, the metabolic damage of PNA mice was significantly improved, as shown by a significant decrease in body weight and visceral fat ( Figure 3 A, 3B), and it decreases more dramatically as the degree of calorie restriction increases. Subcutaneous fat is the "good fat" in the body and has metabolic protection compared to visceral fat. A 40% drastic calorie restriction will cause a significant reduction in subcutaneous fat ( Figure 3 C), there may be some risks to the overall metabolism, so it will not be considered in the future. The local effects of calorie restriction on the ovaries were observed, and 30% calorie restriction caused a sharp decrease in ovarian weight ( Figure 3 D), which has a significant impact on its own function. Relatively speaking, the effect of 20% calorie restriction is more moderate, which can reduce body weight and visceral fat weight, and has little effect on subcutaneous fat and ovaries. Therefore, a 20% calorie restriction diet was subsequently used to adjust the diet of PNA mice.

[0055] 2.2 Detection of metabolic parameters in mice after calorie restriction diet After implementing the calorie-restricted diet for at least 4 weeks, the mice were tested for more detailed metabolic indicators ( Figure 4 A) to clarify its improvement effect. These indicators include basic development and metabolic indicators (weight, body fat percentage, food intake, metabolic rate, respiratory entropy, heat production, etc.), sugar metabolism indicators (GTT, ITT, IRT, etc.), lipid metabolism indicators (lipid accumulation in the liver, adipocyte morphology, etc.). The detection method is the same as described above (1.3 Metabolic phenotype detection).

[0056] Test results such as Figure 4 As shown, 20% caloric restriction can reduce the body weight of PNA mice ( Figure 4 B) and visceral fat content ( Figure 4 C, without affecting its subcutaneous fat ( Figure 4 D), fat cell volume decreases ( Figure 4 E), decreased lipid accumulation in the liver ( Figure 4 F), triglyceride and total cholesterol levels decreased ( Figure 4 G, 4H). In terms of glucose metabolism, calorie restriction can significantly improve glucose tolerance and insulin sensitivity in mice and stimulate insulin secretion ( Figure 4 IK). In terms of energy metabolism, calorie restriction reduces the oxygen consumption and nocturnal heat production of mice ( Figure 4 L, 4M), respiratory exchange rate and activity showed a significant fasting-feeding rhythm ( Figure 4 N,4O), among which the respiratory exchange rate decreased in the fasting state, indicating increased lipid utilization. The above results show that 20% calorie restriction significantly improves the glucose and lipid metabolism function of PNA mice and promotes the formation of fasting-fasting rhythm.

[0057] Obtain offspring of mice adjusted to a calorie-restricted diet and clarify the impact of this strategy on the reproductive metabolic health of offspring exposed to high androgen levels 3.1 Obtain offspring of PNA mice adjusted to a 20% calorie-restricted diet by IVF-ET like Figure 5 As shown in A, in order to focus on the phenotypic changes in offspring caused by changes in the eggs, the PNA mice (F1) with adjusted diets were used to obtain offspring (F2) through in vitro fertilization and embryo transfer. The surrogate mothers were all wild-type female mice fed normally. After birth, the F2 mice were given a normal diet after weaning, and the environmental conditions were the same as above.

[0058] 3.2 Detection of PNA-like metabolism and reproductive indicators in offspring The detection methods of related indicators are the same as above (1.4 Reproductive phenotype detection). After the PNA mice were given a 20% calorie restriction diet, the weight of the offspring mice decreased ( Figure 5 B), less fat ( Figure 5 C, 5D), impaired glucose tolerance, insulin resistance, and decreased insulin secretion capacity, etc., were improved ( Figure 5 EG, and was comparable to the control group. In addition, reproductive function damage in offspring mice, such as prolonged AGD, early vaginal opening, and estrous cycle disorders, was also restored after calorie restriction in mothers exposed to high androgen levels ( Figure 5 JL).

[0059] Methylation sequencing of PNA mouse eggs after calorie restriction was performed to screen methylation-related targets in eggs where calorie restriction exerts its effects 4.1 Construction and sequencing of DNA methylation library F1 mice in the calorie restriction group and the control group were stimulated to ovulate and oocytes were collected. Specifically, 48 hours after the mice were injected with PMSG, the freshly collected ovaries were dissected in M2 medium (M7167, Sigma), and then oocytes with good morphology, uniform size and appropriate developmental stage were carefully selected under a microscope for subsequent experiments. The library was constructed using the universal bisulfite method. 16-20 oocytes from 3 mice were mixed in each sample, lysed and then bisulfite converted. The methylation library was obtained after PCR amplification and paired-end deep sequencing was performed on the Illumina HiSeqX-ten sequencer. All clusters that passed the screening were converted into FASTQ files using the standard Illumina pipeline.

[0060] 4.2 Post-sequencing analysis All bisulfite sequencing reads were initially trimmed to remove adapters and low-quality bases using Trimmomatic (v 0.33). They were subsequently aligned to the mouse reference genome (mm10) in paired-end mode using Bismark (v 0.16.3). Subsequently, reads were ordered and PCR duplicates removed using Samtools and Picard.

[0061] The overall methylation level was calculated from the average DNA methylation level of all covered CpG sites, and the methylation level of different genomic elements was determined by calculating the average methylation level of all covered CpG sites in the corresponding genomic region (refer to UCSC genome browser annotation). Differentially methylated regions were identified by sliding windows across the entire genome. The entire mouse genome was divided into 1000 bp windows and slid forward in steps of 500 bp. Those windows that contained at least 5 covered CpG sites in all mouse egg samples were retained for subsequent comparative analysis. Fisher's exact test was performed on each window using the methylKit (version 1.14) software package, and SLIM was used to correct for multiple testing. Windows with a methylation difference of more than 0.2 (q value < 0.05) were annotated as inter-group DMRs, and promoter DMRs were defined as DMRs with at least 1 bp overlap in the promoter region of a gene (2 kb upstream and downstream of the transcription start site of the gene).

[0062] Subsequent gene function analysis included Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using the R software package clusterProfiler55 4.12.0, Internet network analysis of enriched KEGG pathways through the Metascape website (http: / / metascape.org / ), visualization with Cytoscape 3.8.2, and STRING functional protein association network analysis and visualization using the STRING website (https: / / string-db.org / ), in order to further explore the effects of different diets on the methylation level of PNA mouse eggs.

[0063] like Figure 6 As shown in the figure, the methylation status was observed as a whole, and it was found that the global methylation ( Figure 6 A) or each regulatory element ( Figure 6 B), all showed abnormally increased methylation, which was improved after calorie restriction. Further observation of the differential methylation status of the gene promoter regions in each group and pathway enrichment analysis revealed that the genes associated with abnormally increased promoter hypermethylation in the eggs of mice exposed to intrauterine high androgen and improved by calorie restriction were significantly enriched in calcium channel and insulin secretion-related signaling pathways ( Figure 6 CE), this finding corresponds to the reduced insulin secretion capacity of PNA mouse offspring and its improvement after maternal calorie restriction. The abnormally reduced promoter hypomethylation regions in the eggs of mice exposed to intrauterine high androgen and improved by calorie restriction were significantly enriched in the AMPK signaling pathway ( Figure 6 CE), among which the hypomethylation of the promoter of key transcription factors such as Srebf1 may correspond to the enhanced lipid synthesis capacity and is related to the increased lipid accumulation in the offspring of PNA mice, and the increased methylation of Srebf1 after the intervention of maternal calorie restriction also corresponds to the significant reduction in lipid accumulation after its improvement. The genes whose abnormal DNA methylation levels in eggs caused by high androgen exposure can be reversed by the calorie restriction diet are as follows (Table 2), which may be the key effector genes for the diet to block the inheritance of abnormal phenotypes in high androgen offspring, and are expected to become early intervention and early warning targets for clinical application in the future.

[0064] Table 2 Genes with abnormal DNA methylation levels that can be reversed by CR intervention in 68 oocytes exposed to high androgen levels

[0065] In summary, in response to the unknown role of eggs in mediating the inheritance of abnormal phenotypes caused by high androgen exposure, the present invention uses a mouse model of prenatal androgen exposure (PNA), innovatively utilizes in vitro fertilization-embryo transfer (IVF-ET) and uses systematic phenotypic experiments to confirm that eggs exposed to intrauterine high androgen exposure can independently mediate the inheritance of reproductive and metabolic disorders in offspring.

[0066] In view of the fact that current clinical strategies are unable to comprehensively improve the complex symptoms of women with hyperandrogenism, the metabolic and reproductive abnormalities of offspring have not received attention, and there is a lack of effective means to block the genetic problems of reproductive metabolic abnormalities, the present invention innovatively proposes a dietary intervention strategy to reduce total calorie intake while ensuring the intake of essential nutrients, aiming to explore its potential benefits for the metabolic and reproductive health of parents and offspring exposed to hyperandrogenism.

[0067] In response to the lack of targeted intervention for genetic diseases caused by hyperandrogen exposure, this paper combines single-cell whole genome bisulfite sequencing (WGBS) of hyperandrogen-exposed oocytes after calorie restriction in vivo for the first time to screen methylation effect targets and finds that insulin secretion and AMPK signaling pathways are the key to mediating abnormal genetic diseases induced by hyperandrogenism and responding to calorie restriction intervention, further locking in Adcy3 , Gnas , Srebf1Promoter methylation changes of genes such as these may be key effector targets, and in the future they are expected to provide valuable scientific basis for the early and effective prevention and control of egg-borne diseases caused by Kaohsiung exposure and clinical early warning intervention.

[0068] Based on the content of the present invention, targeted improvement of the metabolic sensitivity and flexibility of animal models and clinical patients with high androgen exposure, and pre-pregnancy adjustment measures for weight management such as metformin, GLP1 analogs, AMPK pathway activators such as Aldometanib and other drug intervention programs, or other dietary adjustment programs, including but not limited to time-restricted eating, low-carbohydrate diet, low-fat diet and other dietary intervention strategies, the above intervention measures alone or in combination with a calorie-restricted diet, their effects on female reproductive metabolic disorders caused by high androgen exposure and blocking genetic effects may have consistent effects with the present invention, and should all be included in the scope of protection of the present invention.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of calorie-restricted diet in the preparation of products for improving female reproductive metabolic abnormalities caused by high androgen exposure; The calorie-restricted diet is a food with a total energy reduction of 10-40% based on the standard diet.

2. The use of calorie-restricted diet regimens in the preparation of products that block the inheritance of abnormal female reproductive metabolism caused by high androgen exposure; The calorie-restricted diet is a food with a total energy reduction of 10-40% based on the standard diet; Preferably, the blocking of the inheritance of abnormal female reproductive metabolism caused by high androgen exposure includes reversing the gene of abnormal DNA methylation level of eggs caused by high androgen exposure; Further preferably, the gene is selected from Cacna1s, Atp1a2, Gna11, Kcnmb4, Gck, Camk2b, Atp1b2, Gip, Adcy3, Ryr2, Adcy2, Cacna1d, Adcy4, Adcy8, Adcy6, Glp1r, Atf6b, Camk2a, Plcb3, Rapgef4, Creb3l1, Gnas, Kcnmb3, Creb3l4, Stx1a, Adcyap1r1, Cacna1c, Kcnn4, Atp1a3, Trpm4, Kcnj11, Abcc8, K At least one of cnu1, Kcnn1, Prkaca, Adcy7, Fxyd2, Cck, Creb3, Pik3cd, Tbc1d1, Ulk1, Cacna1f, Acacb, Gpr119, Camkk2, Foxo3, Pparg, Eef2, Lipe, Srebf1, Akt2, Fasn, Akt1s1, Ppp2r5c, Cpt1c, Akt1, Gys1, Pfkp, Ppp2r2d, Pik3r1, Ccnd1, Cpt1b, Insr, Tsc1, Ppp2cb, Crtc2 and Pik3r2.

3. The use according to claim 1 or 2, characterized in that: The calorie-restricted diet regimen is implemented for ≥ 4 weeks.

4. The use according to claim 1 or 2, characterized in that: The calorie-restricted diet is a diet in which the total energy is reduced by 10-30% on the basis of the standard diet; Preferably, the calorie-restricted diet is a food with a total energy reduction of 20% based on the original diet.

5. The use according to claim 1 or 2, characterized in that: The reproductive metabolic abnormalities include metabolic disorders and reproductive disorders; The metabolic disorders include disorders of basic development and metabolic indicators, sugar metabolism indicators and lipid metabolism indicators; The reproductive disorders include infrequent ovulation and prolonged menstrual cycle, polycystic ovarian morphological changes, increased circulating free testosterone levels, increased anorectal distance, increased AMH or hirsutism and acne.

6. The use according to claim 5, characterized in that The basic development and metabolic indicators include body weight, BMI, body fat percentage, food intake, metabolic rate, respiratory entropy and heat production; The sugar metabolism indexes include fasting blood sugar, fasting insulin, glucose tolerance test, insulin tolerance and insulin secretion test; The lipid metabolism indicators include peripheral blood triglycerides, cholesterol, low-density lipoprotein, high-density lipoprotein, liver tissue lipid accumulation, and adipocyte morphology.

7. The use according to claim 1 or 2, characterized in that: In the standard diet plan, the intake ratio of protein, fat and carbohydrates is 20-30% protein, 10-15% fat and 60-70% carbohydrates.

8. The use according to claim 7, characterized in that The intake ratio of protein, fat and carbohydrates is 20~25% protein, 10~15% fat and 60~65% carbohydrates.

9. The use according to claim 7, characterized in that The standard dietary regimen also contains vitamins, amino acids and minerals.

10. The use according to claim 9, characterized in that The vitamins include vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B6, vitamin B2, niacin, pantothenic acid, biotin, choline and folic acid; The amino acids include methionine, cystine, lysine, tryptophan, arginine, leucine, isoleucine, threonine, valine, histidine, phenylalanine and tyrosine; Such minerals include sodium, magnesium, potassium, copper, iron, manganese, zinc, selenium and iodine.

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