Application of resveratrol in preparation of product for preventing and / or treating unexplained recurrent abortion

Abstract

The invention discloses application of resveratrol in preparation of a product for preventing and / or treating unexplained recurrent abortion, and belongs to the technical field of biological medicine. A small dose of resveratrol is subjected to intragastric administration treatment on a mouse with unknown recurrent abortion, so that the resveratrol not only reduces the embryo absorption rate, but also improves the inflammatory response of the decidua tissue of the mouse, improves the Sirt1 level of the decidua tissue of the mouse, and prevents the generation and development of the sterile inflammation of the maternal-fetal interface of the mouse with unknown recurrent abortion; further, the pregnancy rate of the mice with unexplained recurrent abortion is effectively protected and treated. The invention provides a new way and method for treating recurrent abortion caused by unknown reasons.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of resveratrol in preparing a product for preventing and / or treating unexplained recurrent abortion. Background Art

[0002] Recurrent spontaneous abortion (RSA) is a common complication in women of childbearing age, which refers to the occurrence of two or more clinical pregnancy failures, with an incidence rate of up to 1-5%. The etiology of RSA is diverse, including chromosomal abnormalities, age, antiphospholipid syndrome, uterine malformations, thrombosis, hormone or metabolic disorders, infection, autoimmunity, sperm quality and lifestyle issues. In 50-75% of RSA patients, the cause cannot be determined, which is called unexplained recurrent spontaneous abortion (URSA). Since the etiology and pathogenesis of URSA have not been fully clarified, there is currently no unified diagnostic standard internationally for the diagnosis of the disease. At the same time, the treatment method for URSA is also unclear, and there is a lack of targeted and effective treatment plans. At present, targeted preventive treatment is mainly based on the cause. For women at high risk, commonly used intervention measures include active lymphocyte immunotherapy, intravenous immunoglobulin, glucocorticoids, cyclosporine A, G-CSF, progesterone, TNF-α inhibitors and anticoagulant therapy. However, the routine use of these treatments is not recommended in existing guidelines or expert consensus on miscarriage. Therefore, research on URSA is still very important. In-depth understanding of the causes and pathogenic mechanisms of URSA and the development of new targeted treatments are of great significance for reducing the incidence of URSA and promoting healthy pregnancy.

[0003] In recent years, a large number of studies have shown that the occurrence of unexplained recurrent spontaneous abortion is closely related to the imbalance of maternal and fetal immune tolerance. Compared with normal pregnancy, URSA patients show destruction of the maternal-fetal interface, irregular cell arrangement, higher nuclear rupture rate, and increased infiltrating cells in the chorionic villi and decidua.

[0004] Resveratrol (3,4,5-trihydroxy-trans-stilbene, RES) is a common non-flavonoid polyphenol compound, widely distributed in various plants such as the root of Polygonum cuspidatum, peanuts, and grape skins. It can be separated and purified by supercritical fluid extraction, Soxhlet extraction, enzymatic hydrolysis, etc. It has been found to have multiple pharmacological effects such as anti-inflammatory, antioxidant, anti-aging, and anti-tumor. It has been confirmed to be a natural compound that can effectively activate NAD+-dependent deacetylases - Sirtuins family proteins. SIRT1 is the most studied mammalian sirtuin at present. It is a NAD+ (nicotinamide adenine dinucleotide)-dependent histone deacetylase that participates in the regulation of metabolism, aging, cell survival, autophagy, etc. SIRT1 can activate a variety of proteins and transcription factors (such as HMGB1, PGC-1α, NF-κB, NLRP-3, etc.) through deacetylation, promote mitochondrial biosynthesis, and participate in many physiological and pathological processes, such as oxidative stress and cell apoptosis. SIRT1 plays an important regulatory role in inflammatory response. Resveratrol has been shown to significantly improve inflammatory-related diseases. It not only significantly reduces the levels of pro-inflammatory markers IL-6 and TNF-α in the liver of mice with non-alcoholic fatty liver disease induced by a high-fat diet, but also inhibits NLRP3 activation-induced cell pyroptosis, playing a positive protective role in liver function. In addition, resveratrol can also protect the myocardium of endotoxemia mice by mediating the miR-149 / HMGB1 axis and regulating the ferroptosis pathway. It is precisely because of these characteristics that resveratrol has become a valuable therapeutic tool for the treatment of various diseases related to Sirt1.

[0005] There are also reports on the role of resveratrol in pregnancy-related diseases. Wang et al.'s study showed that supplementing 40 mg / kg of resveratrol may promote breast cell proliferation and antioxidant capacity through mitochondrial autophagy and regulating the intestinal microbiota of pregnant mice. In addition, resveratrol can improve uterine mitochondrial function in mice on a long-term high-fat diet, thereby improving obesity-related adverse pregnancy outcomes. However, there are currently no reports on the clinical use of resveratrol to treat unexplained recurrent spontaneous abortion. Summary of the invention

[0006] The present invention provides an application of resveratrol in preparing a product for preventing and / or treating unexplained recurrent abortion. The chemical formula of resveratrol is C 14 H 12 O3, CAS registration number is 501-36-0.

[0007] Preferably, the product is a medicine.

[0008] More preferably, resveratrol is the only active ingredient in the medicament.

[0009] More preferably, the medicine further contains medically acceptable excipients.

[0010] More preferably, the dosage form of the drug is an injection, solution, emulsion, suspension, suppository, ointment, cream, spray, drops, powder, granules, granules, capsule, pill, tablet, patch or sustained-release preparation.

[0011] The present invention also provides an application of a composition in preparing a product for preventing and / or treating unexplained recurrent spontaneous abortion, wherein the composition contains resveratrol.

[0012] Preferably, the product is a medicine.

[0013] More preferably, the medicine further contains medically acceptable excipients.

[0014] More preferably, the dosage form of the drug is an injection, solution, emulsion, suspension, suppository, ointment, cream, spray, drops, powder, granules, granules, capsule, pill, tablet, patch or sustained-release preparation.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention relates to the use of resveratrol in the preparation of a drug for preventing and treating unexplained recurrent abortion. The present invention treats unexplained recurrent abortion mice with a small dose of resveratrol by intragastric administration, which not only reduces the embryo absorption rate, but also improves the inflammatory response of the mouse decidual tissue, increases the Sirt1 level of the mouse decidual tissue, prevents the occurrence and development of aseptic inflammation at the maternal-fetal interface of the unexplained recurrent abortion mice, and further effectively protects and treats the pregnancy rate of the unexplained recurrent abortion mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The intervention group in Example 1 was given a small dose of resveratrol by gavage before and after cage combination, and the control group and URSA group were given the same dose of solvent by gavage. The mice were dissected and tissues were obtained on the 14th day of pregnancy. (A) The left and right uterine horns, fetuses and placentas of normal pregnant mice, aborted mice and mice treated with different doses (25, 50, 100 mg / kg / d) of drugs. (B) Statistical chart of the number of embryos in each group.

[0018] Figure 2 The decidual tissue pathology of normal pregnant mice, aborted mice and mice treated with the drug at a dose of 50 mg / kg / d in Example 1. (A) H&E staining results of decidual tissue of normal pregnant mice, aborted mice and mice treated with resveratrol. (B) Quantitative evaluation of cell infiltration in the decidual tissue of the three groups of mice by counting, scale bar is 50 μm, *** The difference between the two groups was significant (P < 0.001).** It means there is a significant difference between the groups (P<0.01).

[0019] Figure 3 The expression of Sirt1 protein in the decidual tissue of normal pregnant mice, aborted mice and mice treated with drugs at a dose of 50 mg / kg / d in Example 1. (A) Immunohistochemical detection of the expression and distribution of Sirt1 protein in the decidual tissue of normal pregnant mice, aborted mice and mice treated with resveratrol. (B) ImageJ was used to quantitatively evaluate the average optical density value of Sirt1 protein expression in the decidual tissue of the three groups of mice, and the scale bar is 50 μm. * It means there is a significant difference between the groups (P<0.05). DETAILED DESCRIPTION

[0020] The preparation method of the resveratrol liquid preparation in the following examples is to dissolve resveratrol powder (MedChemExpress) in a solvent of PEG300 and normal saline in a ratio of 1:1.

[0021] Example 1

[0022] The experiment of the present invention of low-dose resveratrol protecting unexplained recurrent spontaneous abortion based on Sirt1 molecule is as follows:

[0023] (1) The implementation of the technology of low-dose resveratrol targeting Sirt1 molecule to protect unexplained recurrent miscarriage is as follows:

[0024] Female CBA / J mice, male DBA / 2J mice, and male BALB / c mice aged 6-8 weeks were purchased from Beijing Huafukang Biotechnology Co., Ltd. All mice were adaptively raised in a standard environment with a constant temperature of 21-23°C and a humidity of 50-60% for 1 week and were given autoclaved water and food.

[0025] After one week of adaptive feeding, all female CBA / J mice were randomly divided into three groups, namely the control group (Control group, CBA / J♀×BALB / c♂), the model group (URSA group, CBA / J♀×DBA / 2J♂) and the treatment group (URSA+RES group, CBA / J♀×DBA / 2J♂), with 8 mice in each group, and then the right ear of each female mouse was marked with an ear tag for another week of adaptive feeding. (Modeling group: CBA / J female mice are prone to miscarriage when mating with male DBA / 2J male mice, and the reason is unknown, so it is often used as a URSA mouse model. Bleeding can be observed at the vaginal opening of female mice during pregnancy, and when the mice were killed on the 14th day of pregnancy, reabsorption of the fetus can be observed, which is a manifestation of miscarriage). Subsequently, the female mice in the treatment group were given resveratrol by gavage at a dose of 25, 50, and 100 mg / kg / d at 18:00 in the evening for prevention; the mice in the control group and the modeling group were given the same dose of solvent by gavage at the same time, and all drinking water was replaced every two days. After 7 consecutive days of gavage, at 18:00 in the evening, every two female CBA / J mice in the control group were caged with one male BALB / c mouse, every two female CBA / J mice in the modeling group were caged with one male DBA / 2J mouse, and every two female CBA / J mice in the treatment group were caged with one male DBA / 2J mouse. All female mice were checked and separated before 8:00 in the morning of the next day, and female mice with vaginal plugs were recorded as gestational day 0 (GD0). Subsequently, the food intake, activity, vaginal bleeding, and body weight of mice in each group were observed and recorded every day. All female mice stopped gavage on the 7th day (GD7) after plugs were seen. All mice were euthanized on the 14th day of pregnancy (GD14), and the eyeballs were removed to obtain venous blood, which was placed at room temperature for two hours and then centrifuged at 3000rpm for 10 minutes at 4 degrees. The supernatant was aspirated and placed in a -80℃ refrigerator for later use. At the same time, the mouse uterus, embryos, placenta and decidua were separated, and all tissues were fixed with 4% paraformaldehyde and stored at -80℃. The total number of fetuses and the number of live fetuses were counted at the same time. The absorbed fetuses were small and black with necrosis and bleeding. The embryo absorption rate was calculated as the number of absorbed embryos / total number of embryos × 100%.

[0026] (2) The protective effect of resveratrol on mice with unexplained recurrent spontaneous abortion was tested as follows:

[0027] Absorption tire detection:

[0028] When the mice were raised to the 14th day of pregnancy, they were killed by cervical dislocation, their eyes were quickly removed to collect blood, and the abdominal cavity was opened to remove the uterus and take pictures. Then the fetus, placenta, resorbed fetus and decidua were separated, and the number of surviving embryos and resorbed fetuses was counted.

[0029] H&E staining:

[0030] After the mouse decidual tissue was fixed with 4% paraformaldehyde for 24-48 hours, all samples were dehydrated with 70%, 80%, 90%, and 100% alcohol gradients, and then embedded in paraffin after immersion in alcohol and xylene mixture and xylene, and the wax blocks were cut into 4μm thick paraffin sections. The paraffin sections were immersed in xylene for 20 minutes, repeated once, and then immersed in 100%, 90%, 80%, and 70% alcohol for 2 minutes, washed twice with distilled water, stained with hematoxylin for 3 minutes, and washed with running water for 5 minutes after the color was observed under a microscope. Then, eosin staining was performed for 2 minutes, and the staining effect was observed under a microscope after gradient alcohol dehydration, and then xylene was permeabilized for 30 minutes, and then all sections were observed with a resin sealant and a fully automatic high-resolution panoramic imaging analysis system.

[0031] Detection of Sirt1 protein distribution in decidual tissue:

[0032] The previously prepared mouse decidua paraffin sections were baked in an oven at 60°C for 2 hours, then dewaxed with xylene and gradient alcohol, and then the sections were permeabilized with 2% Triton solution (Langjieke Technology Co., Ltd., China, membrane permeabilizer, which plays a role in nuclear perforation) for 20 minutes, and then repaired with sodium citrate antigen repair solution. After blocking endogenous peroxidase (Zhongshan Jinqiao, China) for 10 minutes, the sections were incubated with Sirt1 (1:200 dilution) at 4°C overnight, and then all samples were incubated with reaction enhancement solution (Zhongshan Jinqiao, China) for 20 minutes, washed, incubated with enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer (Zhongshan Jinqiao, China) for 20 minutes, and stained with DAB color development kit (diaminobenzidine, Zhongshan Jinqiao, China). After the sections were sealed with resin, positive cells were observed under the fully automatic high-resolution panoramic imaging analysis system. DAB is a common substrate for horseradish peroxidase. Under the catalysis of horseradish peroxidase, DAB will produce brown precipitate.

[0033] At present, the experiments related to the present invention are completed in the Key Laboratory of Gamete and Reproductive Tract Abnormalities of the National Health Commission. The experimental research and results are as follows:

[0034] There were 8 mice in the control group, 8 pregnant mice, 66 live embryos, 4 absorbed fetuses, and an embryo absorption rate of 5.7%.

[0035] There were 8 mice in the modeling group, 8 pregnant mice, 48 surviving embryos, 20 absorbed fetuses, and an embryo absorption rate of 29.4%.

[0036] There were 8 mice in the treatment group with a resveratrol dose of 25 mg / kg / d, 8 pregnant mice, 53 surviving embryos, 10 absorbed fetuses, and an embryo absorption rate of 15.9%.

[0037] There were 8 mice in the treatment group with a resveratrol dosage of 50 mg / kg / d, 8 pregnant mice, 60 surviving embryos, 5 absorbed fetuses, and an embryo absorption rate of 7.7%.

[0038] There were 8 mice in the treatment group with a resveratrol dose of 100 mg / kg / d, 8 of which were pregnant mice. The number of surviving embryos was 55, the number of absorbed fetuses was 14, and the embryo absorption rate was 20.3%.

[0039] like Figure 1 As shown: the uterus and placenta / fetus of the control group, modeling group and drug intervention groups at doses of 25, 50, and 100 mg / kg / d. Among them, the embryos of the mice in the control group were basically the same size and ruddy in color. In the modeling group, some embryos and placentas were smaller, or even no embryos were formed. In the three treatment groups, there were fewer cases of fetal absorption. Compared with the control group, the embryo absorption rate of the modeling group was 29.4%. Compared with the modeling group, after resveratrol treatment, the embryo absorption rates of the three treatment groups decreased significantly, which were 15.9%, 7.7%, and 20.3%, respectively. Among them, the embryo absorption rate of the resveratrol treatment group with a dose of 50 mg / kg / d decreased most significantly, so this group of mice was selected as the treatment group in subsequent experiments.

[0040] like Figure 2 As shown: There are more immune cells infiltrating at the maternal-fetal interface of URSA mice. Among them, the number of decidual infiltrating cells in the model group mice was significantly more than that in the control group. In the decidual tissue, the URSA group had dense cells, irregular arrangement, and diffuse cell nuclei compared with the control group. However, the treatment group after resveratrol intervention was significantly improved.

[0041] like Figure 3 As shown: The expression of Sirt1 protein in the decidual tissue of the three groups of mice was different. Among them, the expression of Sirt1 in the decidual tissue of the modeling group mice was significantly reduced compared with the control group (brown area), while the expression of Sirt1 increased after resveratrol treatment.

[0042] The above results show that in the mouse model of unexplained recurrent spontaneous abortion, the decidual tissue cells at the maternal-fetal interface of the mice are disordered, with a large number of immune cells infiltrating, and the expression of Sirt1 protein is reduced. After treatment with resveratrol (RES), the immune microenvironment at the maternal-fetal interface and the expression of Sirt1 protein can be improved, which effectively proves that Sirt1 can be used as a target for the prevention, protection and treatment of resveratrol in URSA disease, and can provide a theoretical basis for the prevention and treatment of clinical URSA patients.

[0043] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of resveratrol in the preparation of products for preventing and / or treating unexplained recurrent spontaneous abortion.

2. The use according to claim 1, characterized in that: The product described is a drug.

3. The use according to claim 2, characterized in that: Resveratrol is the only active ingredient in the drug.

4. The use according to claim 2 or 3, characterized in that: The medicine also contains medically acceptable auxiliary materials.

5. The use according to claim 4, characterized in that: The dosage form of the drug is injection, solution, emulsion, suspension, suppository, ointment, cream, spray, drop, powder, granule, granule, capsule, pill, tablet, patch or sustained-release preparation.

6. Use of a composition in the preparation of a product for preventing and / or treating unexplained recurrent spontaneous abortion, characterized in that: The composition contains resveratrol.

7. The use according to claim 6, characterized in that: The product described is a drug.

8. The use according to claim 7, characterized in that: The medicine also contains medically acceptable auxiliary materials.

9. The use according to claim 8, characterized in that: The dosage form of the drug is injection, solution, emulsion, suspension, suppository, ointment, cream, spray, drop, powder, granule, granule, capsule, pill, tablet, patch or sustained-release preparation.

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