Application of inulin in treating polycystic ovarian syndrome of high fat diet

Abstract

The invention discloses an application of inulin in preparation of a medicine for treating polycystic ovarian syndrome, especially the polycystic ovarian syndrome caused by high fat diet. Starting from the pathogenesis of the PCOS, research is carried out in the direction of'regulating host-intestinal dialogue by dietary fibers, promoting a tryptophan-quinolinic acid metabolic pathway to enhance an NAD + de novo synthesis pathway and improving PCOS oxidative stress and insulin resistance ', pathophysiology of the PCOS is researched from a brand new perspective, and the pathogenesis of a patient suffering from the PCOS is comprehensively researched in combination with metabonomics. And a new thought and a new target spot are provided for subsequent treatment.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of inulin in treating polycystic ovary syndrome in patients with a high-fat diet. Background Art

[0002] Polycystic Ovary Syndrome (PCOS) is a common reproductive endocrine disorder that causes female infertility. However, the pathogenesis of PCOS is unclear and the treatment is difficult. At present, drug treatment is the main treatment in clinic, and lifestyle intervention such as diet is also added. PCOS is a common reproductive dysfunction disease that causes female infertility in clinic. PCOS patients have ovarian dysfunction, poor development, and ovulation disorders. At the same time, they show corpus luteum function deficiency and abnormal progesterone secretion in the menstrual cycle, which in turn affects endometrial decidualization, endometrial receptivity and embryo implantation. In today's society, changes in diet and lifestyle, reduced intake of dietary fiber, and the popularization of high-fat diets have led to intestinal flora disorders and increased incidence of PCOS. Oxidative stress and insulin resistance are common complications of PCOS, so PCOS is also an endocrine disease, and the pathogenic genes of PCOS show pleiotropy, which are not only regulated by genes, but also affected by environmental factors. The diversity of clinical symptoms increases the difficulty of prevention, treatment and management of PCOS patients. Only by fully understanding its pathogenic mechanism can appropriate management and treatment measures be taken.

[0003] In recent years, studies have found that changes in dietary structure, that is, dietary fiber intake, can affect PCOS symptoms through intestinal flora. Studies in dietary therapy have shown that high dietary fiber can improve PCOS-related symptoms. Dietary fiber includes soluble and insoluble dietary fiber; it can regulate the intestinal microbiota and have an important impact on the metabolites of the microbiota, including short-chain fatty acids, tryptophan metabolites, and bile acid-related metabolites. It can effectively interact with the intestinal system host to regulate PCOS symptoms. Current studies have shown that intestinal flora disorders in PCOS may be related to pathways such as carbohydrates and short-chain fatty acids.

[0004] The intestinal flora of PCOS patients is unbalanced, which disrupts the stability and diversity of intestinal flora and forms pathological flora. The intestinal flora is also called the "hidden organ" because of its large variety and number and its significant impact on human health. A large number of literatures point out that PCOS patients have disordered intestinal flora. As we all know, obesity is a common symptom of PCOS patients, which can also promote intestinal flora disorder in PCOS patients. Clinical studies have shown that in the state of PCOS disease, it will lead to intestinal flora disorder in patients, resulting in ovarian dysfunction, insulin resistance, and reduced secretion of interleukin-22, which proves that intestinal flora disorder will directly affect the function of reproductive organs. The stability and diversity of intestinal flora in PCOS patients are disordered, forming pathological flora. Reasonable dietary intervention is of great significance for PCOS patients to control obesity and regulate insulin resistance, which can effectively improve sugar and lipid metabolism and promote the recovery of ovarian function. However, overeating and eating high-sugar and high-fat foods have been proven to aggravate the symptoms of PCOS. Studies have found that after dietary fiber is degraded by intestinal bacteria, a large amount of metabolites short-chain fatty acids (SCFAs) will be produced, which can improve obesity and metabolic syndrome.

[0005] Inulin is a reserve polysaccharide in plants, mainly derived from plants. Inulin molecules are composed of about 31 β-D-fructofuranose and 1 to 2 pyranose residues, and the fructose residues can be connected by β-2,1-bonds. The molecular formula of inulin is expressed as GFn, where G represents the terminal glucose unit, F represents the fructose molecule, and n represents the number of fructose units. Inulin is contained in the protoplasm of cells in a colloidal form. Unlike starch, it is easily soluble in hot water. Inulin is another form of energy storage in plants besides starch. It is an ideal functional food ingredient and a good raw material for the production of oligofructose, polyfructose, high fructose syrup, crystalline fructose and other products. At present, there are few studies on the application of inulin in PCOS. Summary of the invention

[0006] The purpose of the present invention is to solve the above problems and provide a method for treating polycystic ovary syndrome caused by a high-fat diet.

[0007] In order to achieve its purpose, the present invention adopts the following technical solution:

[0008] Application of inulin in preparing medicine for treating polycystic ovary syndrome.

[0009] The polycystic ovary syndrome is polycystic ovary syndrome in patients with a high-fat diet.

[0010] The inulin is passed through quinolinic acid-NAD + Metabolic pathways improve the pathogenesis of PCOS.

[0011] The inulin promotes the intestinal tryptophan-quinolinic acid metabolic pathway and enhances NAD + De novo synthesis.

[0012] The inulin increases tryptophan and decreases quinolinic acid.

[0013] The inulin improves colon flora.

[0014] The inulin improves insulin resistance.

[0015] The beneficial effects of the present invention are:

[0016] 1. At present, the research direction of PCOS is mainly focused on the pathological mechanism of PCOS reproductive system, while there are few studies on the intestinal flora combined with quinolinic acid-NAD+ metabolism. The present invention starts with the pathogenesis of PCOS, from "dietary fiber regulates host-intestinal dialogue, promotes tryptophan-quinolinic acid metabolic pathway to enhance NAD + The present invention studies the pathophysiology of PCOS from a new perspective, and combines metabolomics to comprehensively study the pathogenesis of PCOS patients, providing new ideas and targets for subsequent treatment.

[0017] 2. At present, in addition to drugs and surgery, life management is the primary treatment method, including controlling diet and exercise. In recent years, people's intake of high fat has increased and the intake of dietary fiber has decreased, which is an important factor leading to the onset of PCOS. The present invention starts with dietary fiber and explores how dietary fiber can improve the symptoms of PCOS. The present invention has found that inulin has a good application effect in the treatment of polycystic ovary syndrome with a high-fat diet, which provides a theoretical basis for the prevention and treatment of the disease and has good prospects for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results show that changes in vaginal smear cells can be used to monitor the estrous cycle of PCOS model mice.

[0019] Figure 2 The results of weight changes (A), serum hormone changes (B), estrous cycle changes (C), appearance changes (D), and HE staining of ovarian sections (E) of mice after modeling are shown.

[0020] Figure 3 Shown are the results of insulin and glucose tolerance tests 2 weeks after dietary intervention after modeling.

[0021] Figure 4 The results of serum hormone tests 2 weeks after dietary intervention after modeling are shown.

[0022] Figure 5 Shows the results of the analysis of microbial flora changes after dietary fiber intervention.

[0023] Figure 6Results of fecal central carbon analysis are shown.

[0024] Figure 7 Results of fecal MCF analysis are shown. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0027] Example 1

[0028] 1 Experimental methods

[0029] 1.1 Modeling and group rearing

[0030] All mouse operations were performed in accordance with the guidelines of the Animal Research Institute Committee. 66 female C56BL / 6 mice (21 days old) were purchased from the Experimental Animal Center of Chongqing Medical University.

[0031] Mice were housed in a pathogen-free environment at 22°C (room temperature), 50% relative humidity, and a light / dark (12h / 12h) cycle. The mouse polycystic ovary syndrome (PCOS) model was established by continuous subcutaneous injection of dihydrotestosterone (DHT) for 35 days. All mice were randomly divided into 2 groups: an oil group (n=16) and a DHT-induced group (n=50). Once a day, the oil group was subcutaneously injected with 0.1ml / 100 (g·d) sesame oil, and the DHT-induced group was subcutaneously injected with 6mg / 100 (g·d) DHT diluted in 0.1ml / 100 (g·d) sesame oil. After modeling, the mice were raised in groups with feed.

[0032] Feeds were purchased from Nantong Trofi Feed Technology Co., Ltd. and the rats were fed with four types of feeds in groups for 2 weeks:

[0033] Normal group: control feed (feed code LAD 3001G);

[0034] Inulin group: high inulin model feed (feed code LAD 3001G-X1);

[0035] High-fat group: 60% high-fat diet (feed code TP 23300-X1);

[0036] Inulin + high fat group: high-fat and high-inulin model diet (feed code TP 23300-X2).

[0037] The weight of each mouse was recorded every day. 22 days after the injection of DHT or sesame oil, vaginal smears were performed on all mice at 9:00 am every day to determine the cell type for 10 consecutive days to determine the estrous cycle of the mice. After the model was established, 8 mice in the oil group and 8 mice in the DHT-induced model group were euthanized (using intraperitoneal injection of chloral hydrate), blood was collected from the retro-orbital venous plexus, and bilateral ovaries were removed. The ovarian tissue was quickly cut into 1 mm 3 Small pieces were fixed with 10% formalin solution at 4°C for 24 hours, then embedded in paraffin for HE staining. The remaining ovaries were frozen at -80°C. After modeling, the remaining PCOS group mice were divided into 4 groups (n=8), and specific feed interventions were started respectively: normal group, inulin group, high-fat group, inulin + high-fat group, and the remaining oil group mice (n=8) were given normal group feed. During this feeding process, the mice were no longer given DHT or sesame oil, and the treatment was continued for 14 days. During this period, body weight and glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were measured. 1.2 Glucose tolerance test (IPGTT) and insulin tolerance test (ITT)

[0038] After the 10th day of feeding with a specific diet, the IPGTT test was performed after fasting but not water deprivation for 6 hours. A 2g / kg glucose solution was injected intraperitoneally, and tail vein blood samples were collected at 0, 30, 60, 90 and 120 minutes, and blood glucose levels were measured using a blood glucose meter. The next day, an ITT test was performed in the same fasting state, and 1IU / kg of diluted insulin solution was injected intraperitoneally, and tail vein blood was collected at the same time points to measure blood glucose concentration. The total area under the IPGTT and ITT glucose response curves (AUC) was then calculated to evaluate insulin resistance and glucose metabolism function.

[0039] 1.3 Analysis of estrous cycle

[0040] Vaginal smears were taken for 10 consecutive days, starting on day 22 after DHT or sesame oil treatment. The estrous cycle stage was examined optically by microscopic analysis of vaginal epithelial cell smears.

[0041] 1.4 Serum hormone measurement

[0042] Mice were anesthetized and blood was collected from the retro-orbital venous plexus. Serum components were separated by centrifugation and stored at -80°C for hormone detection. Enzyme-linked immunosorbent assay (ELISA) kits were used to measure serum testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) concentrations.

[0043] 1.5 Hematoxylin-eosin (H&E) and Mattson staining

[0044] The ovaries were removed immediately after blood sampling, cleaned and fixed in 10% formalin solution at 4°C for 24 h. The fixed ovary samples were dehydrated, embedded in paraffin and sliced ​​to 5 μm. These slices were stained with hematoxylin and eosin to examine the pathological structural changes of the ovaries. Ovarian collagen was stained with Mattson to show ovarian fibrosis in a high androgen environment.

[0045] 1.6 Metabolomics GC-MS of feces

[0046] Colonic fecal tissue samples (weight 20 mg ± 1 mg) were mixed with 400 μL of pre-cooled extraction solution containing 4 M NaOH, methanol and water in a ratio of 1:4:3, and 4 μL of D4-alanine internal standard at a concentration of 10 mM was added. The samples were homogenized at 30 Hz for 1 min using TissueLyser II (QIAGEN, Hilden, Germany). Subsequently, 300 μL of supernatant was separated and transferred to a clean glass tube for subsequent derivatization reaction. Briefly, 34 μL of pyridine and two 20 μL of MCF (methyl chloroformate) were added, followed by vortex mixing for 30 seconds. Then 400 μL of chloroform and 400 μL of 50 mM sodium bicarbonate solution were added and vortex mixed for 10 seconds to separate the derivatized metabolites from the lower chloroform phase before gas chromatography-mass spectrometry (GC-MS) analysis.

[0047] The derivatized samples were analyzed on an Intuvo 9000 gas chromatograph (GC) system equipped with a MSD5977B mass spectrometer (Agilent, CA, USA) with 70 eV electron emission. A 1 μL sample was separated and injected using a ZB-1701GC Intuvo capillary column (20 m × 0.18 mm × 0.18 μm, Agilent, CA, USA). The inlet was set to pulse splitless mode and maintained at 300 °C. Helium was used as the carrier gas with a constant flow rate of 1.0 mL / min. The chromatographic oven temperature was initially set to 45 °C for 1.5 min, then increased to 180 °C at a rate of 18 °C / min and maintained for 3.5 min, then increased to 220 °C at a rate of 80 °C / min and maintained for 3.5 min, then increased to 240 °C at a rate of 80 °C / min and maintained for 3.5 min, and finally increased to 280 °C at a rate of 160 °C / min and maintained for 4.5 min. The auxiliary temperature, mass spectrometer quadrupole and mass spectrometer source were set to 250 ° C, 230 ° C and 150 ° C, respectively. The detection mass range was set to 30 μm to 550 μm, the scanning speed was set to 3 scans per second, and the solvent delay was set to 5.5 minutes. AMDIS software and the MCF mass spectral library built by our laboratory (constructed using chemical standards) were used for metabolite deconvolution and identification. Compound identification was required to meet the MCF library spectral match of more than 95%, and the chromatographic retention index time matched the reference time of the alkane mixture within 50 intervals.

[0048] 1.7 Method for detecting central carbon in feces:

[0049] Weigh 10 mg of feces and combine with 500 μl of pre-cooled (4°C) 50°C methanol to precipitate protein. Then add 5ul of solution containing 20ng / mL of each internal standard (DL-tyrosine, DL-phenylalanine and 4-chloro-L-phenylalanine). Using TissueLyserII (Tissue Lyser II, Qiagen, Germany), the sample was homogenized at 30Hz for 1 minute and 3 clean steel beads were added. The sample was then vortexed for 60 seconds and incubated at -20°C for 1 hour. The extract was centrifuged at 16,000×g for 20 minutes and analyzed by LC-MS.

[0050] 2 Experimental results

[0051] 2.1 Construction of PCOS animal model

[0052] Figure 1 The results showed that the estrous cycle of PCOS model mice was altered by monitoring the changes in vaginal smear cells. The results showed that the estrus period was shortened, and the interphase and pro-estrus period were prolonged.

[0053] Figure 2 Mouse body weight ( Figure 2 A) serum hormones ( Figure 2 B) Four estrous cycles ( Figure 2 C) Appearance Figure 2 D), HE staining of ovarian sections ( Figure 2 E) Test results: The average weight of mice in the PCOS group was about 23g, and the average weight of mice in the control group was about 19g. The weight of mice in the PCOS group increased significantly; Changes in the proportion of hormone levels in the PCOS group: serum androgen, luteinizing hormone levels and luteinizing hormone / follicle-stimulating hormone ratio increased significantly; estrous cycle disorder in the PCOS group; anal-vaginal distance in the PCOS group was shortened; Ovarian sections of the PCOS group: characteristic polycystic ovary morphology, increased number of atretic follicles, and significantly reduced number of corpora lutea.

[0054] The above experimental results confirmed that the PCOS animal model was successfully established.

[0055] 2.2 Insulin and glucose tolerance tests

[0056] The results are as follows Figure 3 Shown: The results of insulin and glucose tolerance tests after 2 weeks of dietary intervention after modeling showed that inulin can improve insulin resistance in PCOS in a high-fat diet.

[0057] 2.3 Serum hormone testing after 2 weeks of dietary intervention after modeling

[0058] The results are as follows Figure 4 As shown: In the PCOS high-fat group, serum androgen T, luteinizing hormone level LH ​​and luteinizing hormone LH / follicle-stimulating hormone FSH ratio increased significantly, and inulin intervention can effectively improve the hormonal endocrine abnormalities of PCOS.

[0059] 2.4 Analysis of microbial flora after dietary fiber intervention: 16S rRNA gene sequencing

[0060] The results are as follows Figure 5 As shown:

[0061] α diversity ( Figure 5 A): No significant difference was found, although there was a certain upward trend

[0062] β diversity ( Figure 5 B): In the inulin intervention group, aggregation phenomenon occurred, indicating that inulin intervention can effectively improve the composition of the intestinal flora diversity in PCOS.

[0063] Species composition analysis / taxonomic composition analysis ( Figure 5 C): After inulin intervention, the bacterial flora showed differences, Bifidobacterium and Ileibacterium increased, and Firmicutes_C decreased significantly).

[0064] Potential risks of Firmicutes: 1. Associated with obesity: Studies have found that the ratio of Firmicutes to Bacteroidetes is related to body weight. A higher proportion of Firmicutes may be associated with obesity because these bacteria are better at extracting energy from food, leading to excessive energy intake. They may affect the body's metabolic rate and energy consumption and storage by regulating hormone levels and metabolic pathways. 2. Contains pathogenic bacteria: Although most bacteria in the Firmicutes are beneficial to the human body, some pathogenic bacteria also belong to this phylum. For example, Clostridium perfringens is a bacterium that causes gastrointestinal infections, and Staphylococcus aureus is a common cause of some serious infections. The presence of these pathogens may pose a threat to human health.

[0065] Bifidobacterium is a probiotic that has many benefits for human health. They are widely found in the human intestine and are one of the important members of the intestinal flora. The benefits of bifidobacteria include: Maintaining the balance of intestinal flora: Bifidobacteria can work together with other beneficial bacteria to inhibit the growth of harmful bacteria, thereby maintaining the balance of intestinal flora. This balance is essential for preventing intestinal diseases and promoting intestinal health. Promoting nutrient absorption: Bifidobacteria can break down complex carbohydrates in food and produce beneficial substances such as short-chain fatty acids, which help promote the absorption and utilization of nutrients in the intestine. Enhance immunity: Bifidobacteria can stimulate the development and function of the intestinal immune system, improve the body's immunity, and thus prevent the occurrence of infectious diseases. Improve intestinal function: Bifidobacteria can relieve intestinal problems such as constipation and diarrhea, improve intestinal peristalsis and secretion function, and make the intestine healthier.

[0066] Branch diagram of LEfSe analysis ( Figure 5 D) There are 23 different species among different phyla. The bar graph of LEfSe analysis shows that the difference colonies in the normal group are the most obvious.

[0067] In the high-fat PCOS_INU group (after adding inulin to high-fat diet PCOS mice): g_Schaedlerella, g_MD308, g_Butyricicoccus_A, f_Rikenellaceae were enriched ( Figure 5 E).

[0068] 2.6 Fecal central carbon analysis

[0069] The results are as follows Figure 6 Shown: NAD in cecal feces + The NAD + There was a significant increase, indicating that inulin does have an effect on NAD + The major de novo pathway for NAD synthesis involves tryptophan.

[0070] 2.7 Fecal MCF analysis

[0071] The results are as follows Figure 7 The results show that in the PCOS group, tryptophan decreased and quinolinic acid increased, indicating that tryptophan decreased in the PCOS group and quinolinic acid-NAD + Metabolism was inhibited. After intervention with inulin, tryptophan increased and quinolinic acid decreased. Especially in the high-fat PCOS group. The PCOS group showed that NAD + After the intervention of inulin dietary fiber, tryptophan increased and quinolinic acid decreased, which may promote the synthesis of quinolinic acid-NAD + Metabolism, improve energy metabolism in PCOS patients, improve insulin resistance, reduce oxidative stress, and improve the pathogenesis of PCOS.

[0072] 3 Analysis

[0073] NAD (nicotinamide adenine dinucleotide) is an indispensable redox coenzyme. NAD has three main biosynthetic pathways, including de novo synthesis from tryptophan, salvage synthesis from nicotinamide NAM, and Preiss-Handler synthesis from nicotinic acid. The de novo synthesis pathway is a supplement from tryptophan to the intestine, which can improve the intestinal enterocyte function.

[0074] Recent studies have found that supplementing with dietary fiber inulin promotes the utilization of nicotine by bacteria and increases the production of nicotinamide adenine dinucleotide (NAD + ) synthesis, revealing that dietary fiber affects the host and intestinal flora in the synthesis of NAD + The process maintains the homeostasis of the NAD+ pool. Tryptophan (Trp) is an essential amino acid that can only be obtained from food. The human body mainly relies on exogenous food intake, which plays an important role in normal growth and health. Trp is first absorbed in the small intestine after digestion in the gastrointestinal tract, and the remaining Trp reaches the colon; under the action of symbiotic bacteria, it is broken down into various metabolites. The kynurenine pathway is one of the three major metabolic pathways of tryptophan in the intestine. In intestinal epithelial cells, tryptophan enters the tryptophan degradation pathway (Kynurenine Pathway) through indoleamine 2,3-dioxygenase (IDO) 1, and generates kynurenine (Kyn) and its downstream products, such as quinolinic acid (QA), nicotinic acid, nicotinamide adenine dinucleotide and kynurenic acid (Kna). Studies have shown that intestinal flora plays an important role in activating IDO1. Quinolinic acid enters the Preiss-Handler pathway, starting with nicotinic acid, and is catalyzed by nicotinic acid phosphoribosyltransferase (NAPRT) enzymes to form NAD + .

[0075] The results of the present invention show that the intake of fiber dietary inulin by PCOS patients can improve the disorder of intestinal flora and thus regulate the NAD between the host intestines.+ Anabolism, improve intestinal flora, improve PCOS symptoms; the mechanism of action may be that dietary fiber regulates the host-intestinal flora "quinolinic acid-NAD + metabolism".

Claims

1. Application of inulin in the preparation of medicines for treating polycystic ovary syndrome.

2. The use according to claim 1, characterized in that: The polycystic ovary syndrome is polycystic ovary syndrome in patients with a high-fat diet.

3. The use according to claim 1 or 2, characterized in that: The inulin is passed through quinolinic acid-NAD + Metabolic pathways improve the pathogenesis of PCOS.

4. The use according to claim 3, characterized in that: The inulin promotes the intestinal tryptophan-quinolinic acid metabolic pathway and enhances NAD + De novo synthesis.

5. The use according to claim 4, characterized in that: The inulin increases tryptophan and decreases quinolinic acid.

6. The use according to claim 1 or 2, characterized in that: The inulin improves colon flora.

7. The use according to claim 1 or 2, characterized in that: The inulin improves insulin resistance.

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