阅读说明：本技术 海藻提取物gv971在制备治疗多囊卵巢综合征药物中的应用 (Application of seaweed extract GV971 in preparation of medicine for treating polycystic ovarian syndrome ) 是由 刘海鸥 徐丛剑 张菲菲 柳妍 李智 蔡青青 王阳 于 2021-11-16 设计创作，主要内容包括：本发明涉及海藻提取物GV971在制备治疗多囊卵巢综合征药物中的应用。海藻提取物GV971是酸性寡糖糖链类(AOSC)化合物,本发明实验表明GV971能够治疗多囊卵巢综合征、调节激素水平、改善生殖异常表型或调节肠道菌群。GV971可以改善来曲唑小鼠的代谢及生殖异常表型,并且这种改善作用是通过调节肠道菌群的失调,进而改善了卵巢局部炎症激活状态而实现的。海藻提取物GV971来源天然,安全可靠,无明显毒副作用,适合临床推广使用。(The invention relates to application of seaweed extract GV971 in preparing a medicine for treating polycystic ovarian syndrome. The alga extract GV971 is an Acid Oligosaccharide Sugar Chain (AOSC) compound, and the experiment of the invention shows that the GV971 can treat polycystic ovarian syndrome, regulate the hormone level, improve the abnormal reproductive phenotype or regulate the intestinal flora. GV971 can improve the metabolic and reproductive abnormality phenotype of letrozole mice, and the improvement is realized by regulating the dysregulation of intestinal flora and further improving the activation state of local ovarian inflammation. The alga extract GV971 has natural source, safety, reliability, no obvious toxic or side effect and is suitable for clinical popularization and use.)

1. Application of Sargassum extract in preparing medicine for treating polycystic ovarian syndrome is provided.

2. Application of Sargassum extract in preparing medicine for regulating hormone level is provided.

3. Application of seaweed extract in preparing medicine for improving reproductive abnormality phenotype is provided.

4. Application of seaweed extract in preparing medicine for regulating intestinal flora is provided.

5. The use of claim 2, wherein the hormone is insulin, androgen or luteinizing hormone.

6. The use according to claim 4, wherein the intestinal flora is intestinal flora α or intestinal flora β.

7. The use as claimed in any one of claims 1 to 4, wherein the seaweed extract is GV 971.

8. A pharmaceutical composition for treating polycystic ovarian syndrome is characterized in that the pharmaceutical composition takes seaweed extract GV971 as an active ingredient and further comprises a pharmaceutically acceptable carrier.

9. A pharmaceutical composition for regulating hormone level, which is characterized in that the pharmaceutical composition takes seaweed extract GV971 as an active ingredient and further comprises a pharmaceutically acceptable carrier.

10. A pharmaceutical composition for improving the reproductive abnormality phenotype, which comprises a seaweed extract GV971 as an active ingredient and further comprises a pharmaceutically acceptable carrier.

11. The pharmaceutical composition for regulating the intestinal flora is characterized in that the pharmaceutical composition takes seaweed extract GV971 as an active ingredient and further comprises a pharmaceutically acceptable carrier.

12. The pharmaceutical composition of any one of claims 8 to 11, wherein the pharmaceutical composition is in the form of an external or internal dosage form.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is in the form of a patch, a paste, an ointment, a gel, a film coating agent, a cataplasm, a spray, a capsule, a granule, a tablet, a pill, an oral liquid or an injection.

14. The seaweed extract GV971 is used as a target for screening drugs for treating polycystic ovarian syndrome, drugs for regulating hormone level, drugs for improving reproductive abnormality phenotype or drugs for regulating intestinal flora.

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a seaweed extract GV971 in preparation of a medicine for treating polycystic ovarian syndrome.

Background

Polycystic ovarian syndrome (PCOS) is a common disorder of fertility in women caused by a complex endocrine and metabolic abnormality, characterized by chronic anovulation (ovulatory dysfunction or loss) and hyperandrogenism (excess production of male hormones in women), with the predominant clinical manifestations of irregular menstrual cycle, infertility, hirsutism and/or acne, being the most common endocrine disorders in women.

Seaweed extract GV971 is an Acidic Oligosaccharide Sugar Chain (AOSC) compound. AOSCs derived from Laminaria japonica (Echlonia Kurome Okam), a family of Alariaceae, were able to antagonize A β -induced apoptosis in mouse hippocampal neurons. A series of AOSC derivatives are synthesized, and the GV971 is found to be directly combined with A beta to prevent SH-SY5Y cell apoptosis induced by the A beta. GV971 is well absorbed orally and passes the blood-brain barrier rapidly; can also improve mouse cognitive disorder induced by scopolamine and Abeta. Clinical phase III experiments show that GV971 has good safety, obviously improves cognitive impairment of AD patients, and is expected to become a novel anti-AD clinical medicine.

Literature panhanbo, linmianming, goergei, trewei, research progress of marine natural products against alzheimer's disease [ J ]. chinese marine drugs, 2019,38 (02): 73-82, GV971 is disclosed as being capable of ameliorating cognitive dysfunction caused by Alzheimer's disease. The literature Jiang Lung, Sun Quanching, Li Li, Lu Fuping, Liu Fufeng. molecular instruments inter the inhibition Effect of GV971Components Derived from Marine acids oligonucleotides against the formation transformation of A β 42Monomers [ J ]. ACS chemical metabolism, 2021: it is disclosed that GV971 may ameliorate cognitive function decline caused by Alzheimer's disease by remodeling gut flora or by antiviral effects on brain HHV-1. However, the application of the seaweed extract GV971 in the preparation of the medicine for treating polycystic ovarian syndrome is not reported at present. GV971 has the following structural formula:

disclosure of Invention

The invention aims to provide the application of seaweed extract GV971 in preparing a medicine for treating polycystic ovarian syndrome aiming at the defects in the prior art.

In order to achieve the purpose, the invention adopts the technical scheme that:

in a first aspect, the application of the seaweed extract in preparing the medicine for treating polycystic ovarian syndrome is provided.

In another aspect of the present invention, there is provided the use of an extract of seaweed for the manufacture of a medicament for regulating hormone levels.

In another aspect of the invention, there is provided the use of an extract of seaweed in the manufacture of a medicament for improving a reproductive abnormality phenotype.

In another aspect of the invention, there is provided the use of a seaweed extract in the manufacture of a medicament for modulating intestinal flora.

Preferably, the hormone is insulin, androgen or luteinizing hormone.

Preferably, the intestinal flora is intestinal flora alpha or intestinal flora beta.

The seaweed extract as described in any of the above is GV 971.

In another aspect of the invention, a pharmaceutical composition for treating polycystic ovarian syndrome is provided, wherein the pharmaceutical composition takes seaweed extract GV971 as an active ingredient, and further comprises a pharmaceutically acceptable carrier.

In another aspect of the present invention, a pharmaceutical composition for regulating hormone levels is provided, wherein the pharmaceutical composition comprises the alga extract GV971 as an active ingredient, and further comprises a pharmaceutically acceptable carrier.

In another aspect of the present invention, a pharmaceutical composition for improving the phenotype of abnormal reproduction is provided, wherein the pharmaceutical composition comprises the alga extract GV971 as an active ingredient, and further comprises a pharmaceutically acceptable carrier.

In another aspect of the present invention, a pharmaceutical composition for regulating intestinal flora is provided, wherein the pharmaceutical composition comprises seaweed extract GV971 as an active ingredient, and further comprises a pharmaceutically acceptable carrier.

The dosage form of any one of the pharmaceutical compositions is an external dosage form or an internal dosage form.

Preferably, the dosage form of the pharmaceutical composition is a patch, a paste, an ointment, a gel, a coating agent, a cataplasm, a spray, a capsule, a granule, a tablet, a pill, an oral liquid or an injection.

In another aspect of the invention, the application of the seaweed extract GV971 as a target point in screening drugs for treating polycystic ovarian syndrome, drugs for regulating hormone level, drugs for improving reproductive abnormality phenotype or drugs for regulating intestinal flora is provided.

The invention has the advantages that:

1. the seaweed extract GV971 can effectively improve metabolic abnormalities such as weight gain, ovary weight gain, body fat gain, glucose tolerance damage and the like of mice with letrozole-induced polycystic ovary syndrome; improve the disturbance of the estrus cycle of the mice, reduce the androgen level of the mice, improve the anovulation of the mice and increase the number of pregnant embryos of the mice.

2. The seaweed extract GV971 improves ovarian polycystic change of letrozole mice, increases corpus luteum quantity, reduces cystic follicle, and improves ovarian morphological abnormality and ovulation abnormality of letrozole mice.

3. The alga extract GV971 can regulate the imbalance of intestinal flora to down-regulate the expression of Cyp17a1, the proinflammatory factors IL-17rb and IL-34, the ovarian functional genes Col6a5, Vcam1, Fads1 and the like, and down-regulate the serum proinflammatory factor IL-18, thereby improving the local inflammation activation state of ovaries and effectively treating the metabolic and reproductive abnormality phenotype of letrozole mice.

Drawings

Figure 1 experimental endpoint body weight statistics for four groups of mice.

Figure 2 experimental endpoint ovarian weight statistics for four groups of mice.

Figure 3 experimental endpoint fat pad weight statistical plots for four groups of mice.

FIG. 4 is a statistical chart of insulin resistance experiments in four groups of mice.

Figure 5 is a statistical plot of the glucose tolerance experiments for four groups of mice.

Figure 6 typical estrus cycle for placebo controlled mice.

Figure 7 typical estrus cycle of letrozole group mice.

Figure 8. typical estrus cycle for placebo gavage GV971 mice.

Figure 9 typical estrus cycle of letrozole gavage GV971 mice.

Figure 10 statistical plot of serum testosterone levels of four groups of mice.

FIG. 11 is a statistical chart of serum dehydrotestosterone levels of four groups of mice.

Figure 12 is a statistical plot of serum luteinizing hormone levels for four groups of mice.

Fig. 13 is a statistical chart of fertility experiments for four groups of mice.

FIG. 14 is a graph of typical ovarian HE staining in placebo mice.

FIG. 15 is a graph of typical ovarian HE staining of letrozole mice.

FIG. 16 is a graph of typical ovarian HE staining of placebo mouse GV971 gavage.

FIG. 17 is a graph of typical ovarian HE staining of letrozole mouse GV971 gavage.

Figure 18 ovarian corpus luteum count statistical plot for four groups of mice.

Figure 19 ovarian vesicle count statistics for four groups of mice.

Figure 20 ovarian transcriptome sequencing cluster analysis heatmap of four groups of mice.

FIG. 21 is a graph of the clustering pattern of ovarian transcriptome sequencing PCA for four groups of mice.

Figure 22, ovarian transcriptome sequencing differential gene volcano plot of group C and L mice.

Figure 23 ovarian transcriptome sequencing differential gene volcano plots of L and LG group mice.

Figure 24 functional enrichment plot of ovarian transcriptome sequencing differential gene KEGG for group C and L mice.

Figure 25 functional enrichment plot of ovarian transcriptome sequencing differential gene KEGG for L and LG group mice.

FIG. 26 is a statistical chart of the expression levels of IL-18 in ovarian tissues of four groups of mice.

FIG. 27 is a statistical plot of serum IL-18 levels in four groups of mice.

FIG. 28 is a statistical chart of the intestinal flora alpha diversity of four groups of mice.

FIG. 29 is a graph of the intestinal flora beta diversity PCA clustering pattern of four groups of mice.

FIG. 30 is a LEfSe multi-level species difference discriminant analysis chart of intestinal flora of four groups of mice.

FIG. 31 is a graph showing correlation analysis between the serum IL-18 level of mice and the genus Difference.

Detailed Description

The following detailed description of the present invention will be made with reference to the accompanying drawings.

Example 1 animal experiments with algal extract GV971 for treatment of polycystic ovarian syndrome

1 materials and methods

1.1 laboratory mice

Female C57BL/6 mice at 3 weeks of age were purchased from Shanghai Jiesi laboratory animals, Inc., SPF grade. The male mice used for fertility tests were 10 weeks old and normal in fertility and purchased from shanghai jequirity laboratory animals ltd, SPF grade.

1.2 mice feeding

All mice are raised in 3-5 animals/cage in an endocrine focus laboratory in Shanghai city, the raising temperature is maintained at 21-25 ℃, the illumination time is 12L:12D of artificial circadian rhythm, and drinking water is freely taken. The animals used in the experiment were approved by animal ethics committee of the secondary obstetrical and gynecological hospital of repeated denier.

1.3 establishment of PCOS mouse model

Letrozole animal model, C57BL/6 female mice 21 days old were embedded with letrozole sustained release tablets (50ng/d) 3mm in diameter in the neck for 21 days, and the control group was embedded with placebo for 21 days.

1.4 gavage experiment of GV971

Starting at the same time of molding, GV971 (national standard H20190031) is filled with 100mg/kg of body weight, prepared with 0.9 wt% of normal saline, and the same amount of normal saline is filled into the stomach of the control group once a day.

The experiment yielded four groups of mice: placebo control group (C), letrozole group (L), placebo mouse gavage GV971 group (G), letrozole mouse gavage GV971 group (LG).

1.5 detection of estrus cycle in mice

From the 7 th day of the molding, 9 am of vaginal smear were taken every day until the molding was completed. Dipping: a small amount of sterile physiological saline is sucked by a sterilized 10ul gun head, gently inserted into the vagina of a mouse, and blown and sucked for a plurality of times. Smearing: the smear is made by placing physiological saline with vaginal contents on a glass slide, and then the smear is naturally dried by spreading in air (until the slide becomes white). Fixing: the slide is fixed in 95% ethanol for 30-60 min. Dyeing: dyeing by Giemsa dyeing method. And (4) observing results: the smear is placed under a microscope and histological changes of the vaginal smear are observed to determine the different phases of the estrous cycle changes.

Estrus precursor (P): microscopically, it can be seen that the vaginal smear contains most of the nucleated epithelial cells, some are single, some are in the form of sheet, and some are accompanied by a small amount of white blood cells.

Estrus (E): the smear contains many enucleated keratinized squamous cells, large and flat, irregular edges, and no or few leukocytes and epithelial cells in the visual field.

Postestrus (M): the smear has a decrease in keratinized epithelium, and many leukocytes and nucleated epithelium are present.

Estrus interval (D): the mouse vaginal mucosa is thin and almost all leukocytes are in the vaginal smear.

1.6 sugar tolerance test

(1) After the mice were replaced in clean cages for 12h of fasting (normal drinking water was guaranteed during the fasting period), the weight of each mouse was weighed and the mice were individually labeled to allow rapid identification of the mice tested during the experiment.

(2) The tail end of the mouse tail is cut off by 1-2mm by scissors, after the tail is lightly pressed to enrich the blood into one drop, the blood sugar level of the mouse is measured by a blood sugar tester and is recorded as fasting blood sugar.

(3) A2 g/kg dose of glucose solution was injected intraperitoneally into each mouse.

(4) Measuring fasting blood glucose value, and measuring blood glucose value of mouse by tail vein blood sampling 15min, 30min, 60min, 90min, and 120min after glucose injection.

1.7 insulin resistance assay

(1) After replacing the mice with clean cages for 4h (normal drinking water during fasting), each mouse was weighed

And the mice are individually labeled so that the mice tested can be quickly identified during the experiment.

(2) The tail end of the mouse tail is cut off by 1-2mm by scissors, after the tail is lightly pressed to enrich the blood into one drop, the blood sugar level of the mouse is measured by a blood sugar tester and is recorded as fasting blood sugar.

(3) Insulin was injected intraperitoneally at a dose of 1.5IU/kg per mouse.

(4) Measuring fasting blood glucose value, and measuring blood glucose value of mouse by tail vein blood sampling 15min, 30min, 60min, 90min, and 120min after glucose injection.

1.8 fertility detection

(1) Purchasing 10-week-old male C57BL/6 mice, and caging the mice with sexually mature female mice without experimental use at a ratio of 1:1, wherein the female mice are separated from male and female mice after being tied;

(2) after the female mouse is determined to be pregnant, the male mouse which is combined with the female mouse is proved to have normal sexual ability and can be used, and if the female mouse in the same cage can not be pregnant, the male mouse is replaced;

(3) selecting a sufficient number of male mice with normal fertility, and combining the male mice with female mice to be detected in a ratio of 1: 1;

(4) after the suppository is seen, the female mouse is dissected 10 days later, and the number of embryos in the uterus is counted.

1.9 detection of IL-18 in serum hormones, serum and ovarian tissues

Centrifuging 3000g of the obtained blood for 10min, subpackaging the upper layer serum, freezing at-80 ℃, taking out the ovarian tissue, grinding and homogenizing in PBS containing protease inhibitor, subpackaging, freezing at-80 ℃, and measuring the content of each hormone by enzyme-linked immunosorbent assay according to the instruction of a testosterone quantitative determination kit, an LH quantitative determination kit, an DHEAS quantitative determination kit and an IL-18 quantitative determination kit.

1.10 ovarian morphometry

Fixing ovaries in 4% paraformaldehyde for 24h, performing conventional gradient dehydration, transparency, wax immersion, embedding, slicing and the like, preparing paraffin sections, and baking at 37 ℃ and then storing at normal temperature for later use. The xylene wash was performed 2 times for 5min each time. Gradient hydrating 100%, 95%, 90%, 80%, 70% alcohol for 5min and 2 times, and washing with distilled water twice after finishing. After 15min of hematoxylin counterstaining, washing for 2 times with distilled water for 5 min; soaking in 1% hydrochloric acid ethanol for 30s, and washing with distilled water for 5min for 2 times; dyeing with 1% eosin solution for 5min, washing with distilled water for 5min for 2 times; dehydrating with 70%, 80%, 90%, 95%, and 100% alcohol gradient for 2 times (5 min per gradient); washing with xylene for 5min for 2 times; and (5) sealing the neutral gum. After completion of the preparation, the ovarian morphology was observed under a light microscope to evaluate the morphological structural changes of the ovarian tissues of the mice in each group.

1.11 Total RNA extraction and inflammatory factor polymerase-chain reaction of ovarian tissue

(1) Washing mouse ovary tissue with PBS 3 times, adding 1ml Trizol for homogenizing; mixing Trizol and tissue, standing for 5-10min, transferring into 1.5ml EP tube, and adding Trizol to make total volume reach 1 ml.

(2) 200. mu.l of chloroform was added thereto in 1/5 volumes of the added Trizol, and the mixture was mixed by inversion and stirred vigorously for 30 seconds to give a milky emulsion. The mixture was kept on ice for 5min, and the aqueous phase and the organic phase were observed to separate slowly.

(3) Centrifuge at 12,000rpm for 10min at 4 ℃ and aspirate the upper aqueous phase into a new 1.5ml EP tube (without touching the DNA layer).

(4) Mixing with isopropanol of equal volume, mixing, and standing on ice for 10 min.

(5) Centrifugation was carried out at 12,000rpm for 10min at 4 ℃ and the supernatant was discarded, leaving RNA precipitated at the bottom of the tube.

(6) Adding 75% ethanol 1ml (prepared with DEPC water), washing the precipitate, gently shaking the centrifuge tube to suspend the precipitate, centrifuging at 4 deg.C and 12,000rpm for 5min, discarding the supernatant, and minimizing the residual liquid. Repeat for 1 time.

(7) Air drying at room temperature for 5-10 min. Add 20ul sterile DEPC water to dissolve the RNA sample.

(8) 1ul of RNA was taken for determination of RNA concentration. OD values quantitate RNA concentration.

(9) Reverse transcription into cDNA, and polymerase-chain reaction with each inflammation factor primer.

1.12 ovarian tissue transcriptome sequencing experiments

(1) Extracting total RNA:

total RNA was extracted from tissue samples, the concentration and purity of the extracted RNA was determined using a Nanodrop2000, RNA integrity was determined by agarose gel electrophoresis, and RIN was determined by Agilent 2100. The total amount of RNA required by single library construction is more than or equal to 1ug, the concentration is more than or equal to 35 ng/mu L, OD260/280 is more than or equal to 1.8, and OD260/230 is more than or equal to 1.0.

(2) Oligo dT enriched mRNA:

eukaryotic mRNA has a structure with a ploy A tail at the 3' end, and mRNA can be separated from total RNA by performing A-T base pairing with ploy A by using magnetic beads with oligo (dT) for analyzing transcriptome information.

(3) Fragmentation of mRNA:

the Illumina Novaseq 6000 platform is used for sequencing short sequence fragments, and mRNA obtained by enrichment is a complete RNA sequence with the average length of several kb, so random interruption is needed. The fragmentation buffer is added to randomly fragment mRNA, and small fragments of about 300bp are separated by magnetic bead screening.

(4) Reverse synthesis of cDNA:

under the action of reverse transcriptase, hexa-base random primers (random hexamers) are added, mRNA is used as a template to carry out inversion synthesis on one-strand cDNA, and then two-strand synthesis is carried out to form a stable double-stranded structure.

(5) Connecting the adaptor:

the double-stranded cDNA structure is sticky-ended, and is filled with End Repair Mix to be a flat End, and then added with an "A" base at the 3' End for connection with a Y-shaped adaptor.

(6) And (3) performing on-machine sequencing on an Illumina platform:

enriching a library, and amplifying 15 cycles by PCR;

② recovering target strips by 2 percent agarose gel;

thirdly, quantifying TBS380(PicoGreen), and mixing and loading the TBS380 and the PicoGreen according to the data proportion;

performing bridge PCR amplification on the cBot to generate clusters;

illumina platform sequencing (PE library, read length 2X 150 bp).

1.13 sequencing and analysis of the microbial diversity of the mouse intestinal tract

(1) Fresh mouse feces were collected in 1.5ml EP tubes, frozen at-80 ℃ after rapid freezing with liquid nitrogen.

(2) Sequencing microbial diversity: according toThe soil DNA Kit (Omega Bio-Tek, Norcross, GA) was used for total DNA extraction of microbial community, and the DNA concentration and purity were measured by using NanoDrop2000 NanoDrop UV-vis; DNA extraction quality was determined by 1% agarose gel electrophoresis and PCR amplification of the 16SrRN gene V3-V4 region was performed using 338F (5'-ACTCCTACGGGAGGCAGCAG-3') (SEQ ID NO:1) and 806R (5 '-GGACTACHVGGGTWTCTAAT-3') (SEQ ID NO:2) (GeneAmp 9700, ABI, Waltham, Mass.). Mixing PCR products of the same sample, recovering the PCR products by using 2% agarose Gel, purifying the recovered products by using AxyPrep DNA Gel Extraction Kit, detecting by using 2% agarose Gel electrophoresis, and using Quantus^TMThe recovered product was detected and quantified by a Fluorometer (Promega, USA). The library was constructed using the NEXTFLEX Rapid DNA-Seq Kit. Sequencing was performed using the mises pe300 platform from Illumina.

(3) Sequence splicing and annotation: the quality control is carried out on the original sequencing sequence by Fastp software, and the splicing is carried out by Flash software. The Usearch software performed OTU clustering on the sequences according to 97% similarity and eliminated chimeras. And (3) comparing each sequence with a Silva database (SSU132) by using an RDP classifier, setting the comparison threshold value to be 70%, and obtaining a species classification annotation result.

(4) And (3) data analysis: alpha diversity analysis, species composition analysis, species comparison analysis, species difference analysis, and defining characteristic upregulated genera.

(5) Generating a co-occurrence network using weighted gene co-expression network analysis (WGCNA) based on OTU arcsin-square root transition count data, inputting data of more than 15 samples including OTU root transformation technique data, body weight, serum T, LH, FSH, E2, P, and DHT values, and whether it is a PCOS animal model (expressed as 0 or 1); selecting and constructing a directed network, and selecting a proper power value, namely the constructed network conforms to the scale-free network characteristics; displaying each module by a hierarchical clustering tree; pearson correlation between the modules and the metabolic trait was calculated using module eigen otu defined as the first principal component of the module, and a correlation map between the modules was plotted. The significance of the correlation is determined by the asymptotic p-value. Visualization of the network is realized by using a gplot function in a snaR package, and a disease-related microorganism species biorarker is found.

2 results of the experiment

2.1 basic index

Fig. 1 to 5 illustrate that letrozole induces the mice to have metabolic abnormalities such as weight gain, ovarian weight gain, parasgonadal fat pad increase, impaired glucose tolerance and insulin resistance, and after glucose administration or insulin injection, the blood glucose level of the mice in the L group is significantly increased and the blood glucose is reduced more slowly than that of the control group. The above metabolic abnormality phenotype was improved by gavage treatment with GV 971. (note: fig. 1-3, compared to group L,^★P＜0.05，^★★P＜0.01，^★★★p is less than 0.001; fig. 4 and 5, group C compared to group L,^★P＜0.05，^★★P＜0.01，^★★★p is less than 0.001, and compared with LG group,^#P＜0.05)。

2.2 estrus cycle

Figures 6 to 9 represent the estrus cycle of four groups of mice. Typical estrus cycles for mice in the placebo group (fig. 6), those in the letrozole group (fig. 7), those in the placebo gavage GV971 group (fig. 8), and those in the letrozole group (fig. 9). Wherein P is the prophase of estrus, E is the estrus, M is the anaphase of estrus, and D is the estrus interval). The estrus cycle is an important index for evaluating the sexual cycle of the mice, the estrus of normal mice is 4-5 days, the prophase of estrus (P), the estrus (E), the anaphase of estrus (M) and the estrus interval (D) repeatedly appear, and the disorder of the estrus cycle is an important characteristic of PCOS mice.

In figure 7, it is shown that letrozole mice are chronically in estrus, whereas the estrus cycle of the GV971 gavaged letrozole mice shows repeated appearance of each estrus period, and there is no significant difference between GV971 gavaged placebo and control groups.

2.3 sex hormone levels

Figures 10 to 12 show the serum sex hormone levels of four groups of mice. Serum testosterone levels of letrozole mice were significantly elevated (figure 10), dehydrotestosterone levels and luteinizing hormone levels (figure 11, figure 12), while elevated androgen levels were also an important pathological manifestation and diagnostic criteria for PCOS. However, the letrozole mice subjected to GV971 intervention had significantly reduced testosterone levels and significantly reduced LH levels compared to letrozole mice. (note: in comparison with the L groups,^★P＜0.05，^★★P＜0.01，^★★★P＜0.001)

2.4 fertility

Fig. 13 represents fertility experiments for four groups of mice. Compared with the control group, the number of the pregnant embryos of the mice in the letrozole group is obviously reduced, the number of the pregnant embryos of the letrozole mice is increased by the intervention of GV971, the fertility is improved, and the GV971 improves the ovarian ovulation abnormity of the letrozole mice. (note: in comparison with the L groups,^★P＜0.05，^★★P＜0.01)

2.5 ovarian morphology, number of follicles and number of corpus luteum

Fig. 14-17 show ovarian morphology in control, letrozole, placebo gavage GV971, and letrozole gavage GV971 mice. FIG. 14 shows the size of the control groupTypical ovarian HE staining in mice, fig. 15 typical ovarian HE staining in mice in letrozole group, fig. 16 typical ovarian HE staining in mice in placebo gavage GV971 group, fig. 17 typical ovarian HE staining in mice in letrozole gavage GV971, fig. 18 number of four groups of mouse ovarian luteal bodies, fig. 19 number of four groups of mouse alveolar follicles (where, x denotes corpus luteum, # denotes cystic follicle, compare with L group,^★P＜0.05，^★★P＜0.01)。

fig. 14 to 17 show that the luteal phase count of letrozole mice is obviously reduced compared with the control group, and the number of vesicular follicles is obviously increased, which indicates that letrozole induces the mice to have ovarian polycystic-like morphological change, while after GV971 drying prognosis, the number of luteal phases is increased, and the number of vesicular follicles is obviously reduced, which indicates that GV971 improves the ovarian morphological abnormality and ovulation abnormality of PCOS model mice.

2.6 ovarian transcriptome sequencing analysis

Figure 20 presents a heatmap of a clustering model of ovarian transcriptome sequencing of four groups of mice. It can be seen that the LG group mice and the L group mice are clustered by themselves, and the clustering patterns of the C group and the G group are similar (the first three columns on the left are samples LG1, LG3 and LG6, respectively, the expression of related genes of the three columns is obviously up-regulated and presents different clustering patterns from the other three groups; the three groups of L13, L12 and L7 present the same clustering patterns; and the clustering patterns of C18, G22, C15 and C16 are closer).

FIG. 21 presents a clustered PCA plot of ovarian transcriptome sequencing for four groups of mice. It can be seen that the LG group mice self-formed a clustering pattern.

Figures 22 and 23 represent the differential gene volcano plots between C and L, L and LG. It can be seen that the genes of group L which are up-regulated compared with group C include proinflammatory factors such as IL-17rb, IL-33, IL-13ra2, etc., functional genes related to ovarian angiogenesis and tissue proliferation such as Col6a5, Col4a3, Vcam1, Fads1, etc., and genes related to androgen synthesis promotion by aromatase such as Hsd17b3, Cyp27a1, Cyp17a1, etc. Indicating that the ovary of the mice in letrozole group is locally in an immune-activated, androgen synthesis-activated state (FIG. 22). The expression of LG group Cyp17a1 subjected to the dry prognosis of GV971 is down-regulated, and the expressions of proinflammatory factors IL-17rb and IL-34, ovarian functional genes Col6a5, Vcam1, Fads1 and the like are also down-regulated, so that the intervention of GV971 improves the local immune activation state of ovaries of PCOS-like mice and the expression of ovarian tissue structure hyperplasia (figure 23).

By the KEGG enrichment analysis of the C and L, L and LG differential genes, fig. 24 and 25, the inflammation and immune related pathway activation, as well as metabolic pathway activation, were shown for group L versus group C (fig. 25); whereas intervention of GV971 resulted in improved activation of local ovarian inflammation-associated pathways in group L, notably with down-regulation of the intestinal immune pathways (fig. 26).

FIGS. 26 and 27 show that intervention of GV971 down-regulates the expression of local IL-18 in the ovary in group L (FIG. 26), and that serum IL-18 levels are significantly down-regulated (FIG. 27). (note: in comparison with the L groups,^★P＜0.05，^★★P＜0.01，^★★★P＜0.001)

2.7 intestinal flora

Fig. 28 to 31 show the intestinal flora changes in four groups of mice. It can be seen that the intestinal flora α diversity of the letrozole mice was significantly reduced compared to the control group, whereas the reduction of intestinal flora α diversity was well-established as a change in PCOS, whereas intervention of GV971 restored α diversity (figure 28: compared to group L,^★p is less than 0.05). The clustering pattern of the beta-diversity of the intestinal flora of letrozole mice was different from that of the LG group, while the clustering pattern of the C group was approximately the same as that of the G group, indicating that the intervention of GV971 altered the beta-diversity of the intestinal tract of PCOS-like mice (fig. 29). By analyzing the genus difference at the level of enterobacteria in four groups of mice, it can be seen that the abundance of norak _ f _ Murebacteriaceae in the mice with trozole was increased compared to the control group, while the intervention of GV971 down-regulated the abundance of this genus (FIG. 30: difference between groups: group)^★P is less than 0.05). And in the Spearman correlation analysis of mouse serum IL-18 with the differential genus, it was found that the IL-18 level was positively correlated with the aforementioned abundance level of norak _ f _ muribacteriaceae (fig. 31:^★P＜0.05，^★★p < 0.01, the darker the cell color, the stronger the correlation).

The results show that GV971 can improve the abnormal metabolism and reproduction phenotype of letrozole mice, and the improvement is realized by regulating the dysregulation of intestinal flora and further improving the local inflammation activation state of ovaries.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> affiliated obstetrical and gynecological hospital of double-denier university

Application of <120> seaweed extract GV971 in preparation of medicine for treating polycystic ovarian syndrome

<130> /

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<400> 1

actcctacgg gaggcagcag 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

ggactachvg ggtwtctaat 20