Application of indoleacetic acid in diagnosis and treatment of gestational diabetes mellitus

Abstract

The invention discloses application of indoleacetic acid in diagnosis and treatment of gestational diabetes mellitus. Clinical queue research finds that the concentration of serum indoleacetic acid of pregnant women in a gestational diabetes mellitus group is remarkably lower than that of pregnant women in a healthy control group, and high diagnosis efficiency is achieved. Animal experiments further prove that in a high fat diet induced mouse model with gestational diabetes mellitus, the exogenous indoleacetic acid is supplemented, so that the fasting blood glucose of a female mouse with gestational diabetes mellitus can be reduced, abnormal glucose tolerance and insulin resistance can be improved, and the indoleacetic acid is free of embryotoxicity, safe and reliable. The application provides a new thought and means for early diagnosis and effective treatment of gestational diabetes mellitus, and has a wide application prospect.

Description

Technical Field

[0001] This application belongs to the field of biomedicine, specifically relating to the application of indoleacetic acid in the diagnosis and treatment of gestational diabetes. Background Technology

[0002] Gestational diabetes mellitus (GDM) is a common complication of pregnancy, posing a serious threat to the short- and long-term health of both mother and child. Currently, the diagnosis of GDM mainly relies on the oral glucose tolerance test (OGTT) in mid-pregnancy. However, this method has limitations, including a late detection window, cumbersome procedures, and the inability to provide early warning and intervention. In terms of prevention and treatment, existing methods mainly focus on lifestyle interventions and, when necessary, insulin therapy. However, adherence to lifestyle interventions varies, and insulin therapy carries risks of hypoglycemia and inconvenience. Therefore, there is an urgent clinical need to identify patients with gestational diabetes mellitus early and to develop safe and effective new prevention or treatment strategies. Summary of the Invention

[0003] To overcome the shortcomings of the prior art, this application provides the application of indoleacetic acid in the diagnosis and treatment of gestational diabetes mellitus.

[0004] To achieve the above objectives, this application adopts the following technical solution:

[0005] The first aspect of this application provides the use of a reagent for detecting indoleacetic acid levels in a sample in the preparation of products for diagnosing gestational diabetes.

[0006] Furthermore, the reagent can be used to detect the level of indoleacetic acid in a sample by any of the following methods: chromatography, mass spectrometry, spectroscopy, chromatography-mass spectrometry, enzyme-linked immunosorbent assay (ELISA), radiochemical analysis, nuclear magnetic resonance spectroscopy, light scattering analysis, and turbidimetry.

[0007] Furthermore, the reagent also includes a detectable marker.

[0008] Furthermore, the samples include blood, urine, and saliva.

[0009] Furthermore, the product includes reagent kits, chips, and test strips.

[0010] A second aspect of this application provides a product for diagnosing gestational diabetes mellitus, the product comprising a reagent for detecting the level of indoleacetic acid in a sample.

[0011] Furthermore, the reagent can be used to detect the level of indoleacetic acid in a sample by any of the following methods: chromatography, mass spectrometry, spectroscopy, chromatography-mass spectrometry, enzyme-linked immunosorbent assay (ELISA), radiochemical analysis, nuclear magnetic resonance spectroscopy, light scattering analysis, and turbidimetry.

[0012] Furthermore, the product includes reagent kits, chips, and test strips.

[0013] The third aspect of this application provides the use of indoleacetic acid in the preparation of medicaments for the prevention / treatment of gestational diabetes.

[0014] Furthermore, the medication also includes other medications for treating gestational diabetes.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0016] Furthermore, the pharmaceutically acceptable excipients include one or more of the following: fillers, binders, diluents, sweeteners, flavorings, preservatives, disintegrants, lubricants, flow aids, and antioxidants.

[0017] A fourth aspect of this application provides a medicament for treating gestational diabetes mellitus, said medicament comprising indoleacetic acid.

[0018] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0019] Furthermore, the dosage forms of the drug include gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0020] The fifth aspect of this application provides a method for treating gestational diabetes mellitus, the method comprising administering indoleacetic acid.

[0021] The advantages and beneficial effects of this application are as follows:

[0022] This application, through a clinical cohort study, found that serum indoleacetic acid (IAA) concentrations in pregnant women with gestational diabetes mellitus (GDM) were significantly lower than those in healthy control groups, demonstrating high diagnostic efficacy. Further animal experiments showed that in a high-fat diet-induced GDM mouse model, exogenous IAA supplementation reduced fasting blood glucose, improved glucose tolerance and insulin resistance in pregnant mice with GDM, and was safe and reliable with no embryotoxicity. This application provides new ideas and methods for the early diagnosis and effective treatment of GDM, and has broad application prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0024] Figure 1 This is a concentration distribution diagram of indoleacetic acid between the gestational diabetes mellitus group and the healthy control group;

[0025] Figure 2 This is the ROC plot of indoleacetic acid in the diagnosis of gestational diabetes mellitus in the group and the healthy control group;

[0026] Figure 3 This is a graph showing the IPGTT results;

[0027] Figure 4 This is a graph showing the results of IPITT;

[0028] Figure 5 This is the FBG result image;

[0029] Figure 6 This is a pregnancy safety assessment chart.

[0030] In the figure, * / # / $ P<0.05, ** / ## / $$ P<0.01, *** / ### / $$$ P<0.001. Detailed Implementation

[0031] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] This application provides the use of a reagent for detecting indoleacetic acid levels in a sample in the preparation of products for diagnosing gestational diabetes.

[0033] In some embodiments, the level or content of indoleacetic acid may be one or more of the following: the absolute or relative amount or concentration of indoleacetic acid; the presence or absence of indoleacetic acid; the range of the amount or concentration of indoleacetic acid; the minimum and / or maximum amount or concentration of indoleacetic acid; the average amount or concentration of indoleacetic acid; and / or the median or concentration of indoleacetic acid.

[0034] In some implementations, indoleacetic acid (IAA) may be used alone or in combination in diagnostic tests to assess a subject's gestational diabetes status. Gestational diabetes status includes the presence or absence of gestational diabetes. Monitoring the course of gestational diabetes, such as disease progression, may also be included. Based on the subject's gestational diabetes status, additional procedures may be indicated, including, for example, additional diagnostic tests or treatment procedures.

[0035] In some embodiments, a sample refers to a biological sample obtained or derived from a source of purpose as described in this application. In some embodiments, the source of purpose includes an organism, such as an animal or a human. In some embodiments, the biological sample includes biological tissue or fluid. In some embodiments, the biological sample may be or includes bone marrow; blood; blood cells; ascites; tissue or fine-needle biopsy samples; body fluids containing cells; free-floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid; peritoneal fluid; pleural fluid; feces; lymph; skin swabs; oral swabs; nasal swabs; washes or lavages, such as catheter lavages or bronchoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; other body fluids, secretions and / or excretions; and / or cells therein, etc. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the sample is a “primary sample” obtained directly from the source of purpose by any suitable means. For example, in some embodiments, primary biological samples are obtained by methods selected from: biopsy (e.g., fine-needle aspiration or tissue biopsy), surgical tissue, collection of bodily fluids (e.g., blood, lymph, feces, etc.). In some embodiments, as will be apparent from the context, the term "sample" refers to a preparation obtained through processing (e.g., by removing one or more components of the primary sample and / or by adding one or more reagents to the primary sample). For example, semi-permeable membrane filtration is used. As a preferred embodiment, the sample is blood.

[0036] The reagent also includes a detectable marker.

[0037] In some embodiments, a detectable label refers to a composition capable of generating a detectable signal indicating the presence of a metabolite in a sample being measured. Suitable labels include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent components, magnetic particles, and bioluminescent components. Therefore, a label is any composition detectable by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrochemical, optical, chemical detection devices, or any other suitable device. In some embodiments, a label can be visually detected without the aid of a device. The term "label" is used to refer to any chemical group or portion having a detectable physical property, or any compound capable of causing a chemical group or portion to exhibit a detectable physical property, such as an enzyme that catalyzes the conversion of a substrate into a detectable product. Labels also encompass compounds that inhibit the expression of a particular physical property. A label can also be a compound that is a member of a binding pair, the other member of which has a detectable physical property.

[0038] Among them, radioactive isotopes include but are not limited to 3 H, 14 C 35 S, 125 I, 131 I.

[0039] Enzymes include, but are not limited to, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and acetylcholinesterase.

[0040] Fluorescent molecules include, but are not limited to, FITC, rhodamine, and lanthanide phosphors.

[0041] The products include reagent kits, chips, and test strips.

[0042] In some embodiments, the kit components may be packaged in an aqueous medium or in a lyophilized form. Suitable containers in the kit typically include at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container for holding one component, and preferably, for appropriate aliquoting. When more than one component is present in the kit, the kit will also typically include a second, third, or other additional container for separately holding the additional components. However, different combinations of components may be contained in a single vial. The kit of this application will also typically include a container for containing the reactants, sealed for commercial sale. Such a container may include injection-molded or blow-molded plastic containers for holding the desired vials.

[0043] The solid support of the kit can be plastic, silicon wafer, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate or slide.

[0044] This application provides the use of indoleacetic acid in the preparation of medicaments for the prevention / treatment of gestational diabetes.

[0045] The medication also includes other medications for treating gestational diabetes.

[0046] In some embodiments, the other drugs for treating gestational diabetes or for improving metabolism include, but are not limited to, insulin drugs (such as insulin), oral hypoglycemic agents (such as metformin, glibenclamide), etc.

[0047] The drug also includes pharmaceutically acceptable excipients.

[0048] In some embodiments, pharmaceutically acceptable excipients refer to those compounds, materials, compositions, and / or dosage forms that, to the extent of reasonable medical judgment, are suitable for use in contact with tissues of humans and animals without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio. Pharmaceutically acceptable excipients include fillers, binders, diluents, sweeteners, flavorings, preservatives, disintegrants, lubricants, flow aids, antioxidants, or mixtures thereof.

[0049] In some embodiments, suitable fillers include, but are not limited to, lactose, microcrystalline cellulose, starch, pregelatinized starch, calcium phosphate, calcium sulfate, calcium carbonate, mannitol, sorbitol, xylitol, sucrose, maltose, fructose, dextrose, maltodextrin, etc.

[0050] In some embodiments, suitable binders include, but are not limited to, starch, natural sugars, corn sweeteners, natural and synthetic gums, cellulose derivatives, gelatin, povidone, polyethylene glycol, waxes, sodium alginate, alcohols, water, etc.

[0051] In some embodiments, suitable diluents include, but are not limited to, calcium carbonate, dicalcium phosphate, calcium phosphate, calcium sulfate, microcrystalline cellulose, microcrystalline silicified cellulose, powdered cellulose, glucose binder, dextrose, fructose, lactitol, anhydrous lactose, lactose monohydrate, lactose dihydrate, lactose trihydrate, mannitol, sorbitol, starch, pregelatinized starch, sucrose, talc, xylitol, maltodextrin, maltitol, etc.

[0052] In some embodiments, suitable sweeteners include, but are not limited to, alitane, acesulfame potassium, aspartame, D-tryptophan, dextran, erythritol, fructose, galactose, glycerol, glycyrrhizin, glucose, isomaltitol, xylitol, xylose, lactitol, lactose, levulose, maltitol, maltodextrin, maltol, maltose, mannitol, corn syrup, neohesperidin dihydrochalcone, neotame, saccharin, siclamate, sorbitol, sucralose, sucrose, tagatose, sematrandextrin, trehalose, etc.

[0053] In some embodiments, suitable flavoring agents include, but are not limited to, natural flavoring oils, anethole, acetic acid, ascorbic acid, phosphoric acid, fumaric acid, lactic acid, lemon, linalool, malic acid, menthol, eucalyptol, orange, citric acid, cinnamone, tartaric acid, thymol, vanilla flavor, strawberry, etc.

[0054] In some embodiments, suitable preservatives include, but are not limited to, parabens, phenol, chlorocresol, alkyl parabens, benzoic acid and its salts, boric acid and its salts, citric acid and its salts, sorbic acid and its salts, neutral preservatives, mercury preservatives, quaternary compounds, etc.

[0055] In some embodiments, suitable disintegrants include, but are not limited to, sodium starch glycolate, croscarmellose, croscarmellose carboxymethyl cellulose, starch, microcrystalline cellulose, and mixtures thereof.

[0056] In some embodiments, suitable lubricants include, but are not limited to, long-chain fatty acids and their hydrates or solvates, such as magnesium stearate and stearic acid, talc, glyceryl ester waxes and mixtures thereof.

[0057] In some embodiments, suitable flow aids include, but are not limited to, colloidal silica.

[0058] In some embodiments, suitable antioxidants include, but are not limited to, tocopherol, ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylated hydroxyanisole, edetate, their hydrated or solvated forms, and mixtures thereof.

[0059] In some embodiments, the dosage form of the drug includes gastrointestinal dosage forms and non-gastrointestinal dosage forms. Exemplarily, the gastrointestinal dosage forms include, but are not limited to: tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release formulations, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets; the non-gastrointestinal dosage forms include, but are not limited to: injectable dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and transdermal dosage forms.

[0060] In some embodiments, the injectable dosage forms include, but are not limited to, various injectables such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections; the respiratory dosage forms include, but are not limited to, sprays, aerosols, and powder inhalers; the cavity dosage forms include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, and pills, for use in the rectum, vagina, urethra, nasal cavity, and ear canal; the mucosal dosage forms include, but are not limited to, eye drops, nasal drops, ointments, mouthwashes, sublingual tablets, adhesive tablets, and films; and the skin dosage forms include, but are not limited to, topical solutions, lotions, liniments, ointments, plasters, pastes, and patches.

[0061] In some embodiments, the appropriate dosage of the drug provided in this application can be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, gender, condition, diet, administration time, route of administration, excretion rate and responsiveness. Skilled physicians can usually easily determine the prescription and the desired dosage that is effective for treatment, as long as it can produce the expected therapeutic and / or preventive effect on the disease or related symptoms, such dosage is within the protection scope of this application.

[0062] In some embodiments, the subject of the drug is an animal, preferably a mammal (human and non-human animals), including but not limited to: humans, non-human primates (especially higher primates, such as macaques, cynomolgus monkeys, stump-tailed macaques, rhesus monkeys, shrews, golden snub-nosed monkeys, and tree shrews), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, any livestock or pets, etc.; in a preferred embodiment, the subject is a human.

[0063] In some implementations, prevention or control includes: (1) suppressing the onset of disease in subjects or patients who may be at risk of disease and / or susceptible to disease but have not yet experienced or shown any pathology or symptoms of disease, and / or (2) slowing the onset of any or all pathology or symptoms of disease in subjects or patients who may be at risk of disease and / or susceptible to disease but have not yet experienced or shown any pathology or symptoms of disease.

[0064] In some implementations, the treatment includes: (1) suppressing the disease (e.g., preventing further development of the pathology and / or symptoms) in a subject or patient experiencing or exhibiting the pathology or symptoms of the disease; (2) alleviating the disease (e.g., reversing the pathology and / or symptoms) in a subject or patient experiencing or exhibiting the pathology or symptoms of the disease; and / or (3) producing any measurable reduction of the disease or its symptoms in a subject or patient experiencing or exhibiting the pathology or symptoms of the disease.

[0065] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0066] Example 1

[0067] 1. Experimental materials

[0068] The specific experimental materials are shown in Table 1.

[0069] Table 1 Experimental materials for Example 1

[0070]

[0071] 2. Experimental Methods

[0072] (1) This nested case-control study is based on the prospective cohort study conducted by Peking Union Medical College Hospital—the Maternal Endocrine Metabolic Health and Outcomes Study (MEMO Study, NCT05952739). 300 pregnant women in their 13th month of pregnancy were recruited between July 2023 and October 2025. +7 Pregnant women with singleton pregnancies were included. Women meeting any of the following criteria were excluded: ① severe pregnancy complications; ② pre-existing major heart, liver, kidney, hematologic, or autoimmune diseases; ③ other diseases that may affect gut microbiota or metabolomics, including inflammatory bowel disease, irritable bowel syndrome, and celiac disease; ④ previous gastrointestinal or biliary tract surgery (including bariatric surgery and cholecystectomy); ⑤ a history of smoking, alcohol abuse, or drug use. Based on the 2020 American Diabetes Association guidelines, a 75-gram oral glucose tolerance test (OGTT) was performed between 24 and 28 weeks of gestation. The original cohort included 50 women diagnosed with gestational diabetes who provided adequate blood samples before the mid-trimester OGTT. The 50 gestational diabetes cases were individually matched 1:1 with 50 healthy controls based on age, parity, pre-pregnancy body mass index (BMI), and gestational age at mid-trimester blood collection.

[0073] (2) After a 12-hour overnight fast, blood samples were collected from pregnant women between 20 and 28 weeks of gestation (before the OGTT) and centrifuged at 1500×g for 10 minutes at 4°C. The resulting serum was transferred to microcentrifuge tubes and stored at -80°C until the day of the targeted tryptophan metabolomics analysis.

[0074] (3) After thawing the sample stored at -80℃ on ice, vortex for 10 seconds and transfer 50 μL of the sample to a centrifuge tube. Add 250 μL of methanol solution (containing 10 pL of internal standard working solution with a concentration of 250 ng / mL) to the centrifuge tube, vortex for 3 min to mix, and then let it stand at -20℃ for 30 min. Centrifuge at 12000 rpm for 10 min at 4℃. Collect 150 μL of supernatant, centrifuge again, and transfer 100 μL of supernatant for liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis.

[0075] (4) Chromatography-mass spectrometry acquisition conditions

[0076] The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) (ExionLC). TM AD (https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS) (QTRAP® 6500+, https: / / sciex.com.cn / ).

[0077] Chromatographic conditions:

[0078] 1) Chromatographic column: Waters ACQUITY UPLC HSS T3 C18 column (1.8μm, 100mm×2.1mm id);

[0079] 2) Mobile phase: Phase A, ultrapure water (containing 0.1% formic acid); Phase B, acetonitrile (containing 0.1% formic acid);

[0080] 3) Flow rate: 0.35 mL / min; column temperature: 40℃; injection volume: 2 μL;

[0081] 4) Mobile phase gradient: 0 min A / B is 90:10 (V / V), 1 min A / B is 90:10 (V / V), 6 min A / B is 5:95 (V / V), 7 min A / B is 5:95 (V / V), 7.1 min A / B is 90:10 (V / V), 10 min A / B is 90:10 (V / V).

[0082] Mass spectrometry conditions:

[0083] The electrospray ionization (ESI) source operates at a temperature of 550°C, with a mass spectrometry voltage of 5500V in positive ion mode and -4500V in negative ion mode, using a curtain gas (CUR) of 35 psi. In the QTRAP® 6500+, each ion pair is scanned and detected based on optimized declustering potential (DP) and collision energy (CE).

[0084] (5) Qualitative and quantitative analysis

[0085] A Metal Vare Database (MWDB) was constructed based on standards for qualitative analysis of mass spectrometry data. Quantitative analysis was performed using multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometry. After obtaining mass spectrometry data from different samples, the chromatographic peaks of all target analytes were integrated, and quantitative analysis was performed using a standard curve.

[0086] 1) Data Processing: Mass spectrometry data were acquired using Analyst 1.6.3 software. MultiQuant 3.0.3 software was used to integrate and correct the chromatographic peaks in the samples, referring to the retention times and peak shapes of the standards, to ensure the accuracy of qualitative and quantitative analysis.

[0087] 2) Plotting standard curves: Prepare standard solutions with different concentrations of 0.01 ng / mL, 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, and 5000 ng / mL, and obtain the chromatographic peak intensity data of the corresponding quantitative signals for each concentration of standard; plot the standard curves for different substances with the ratio of external standard to internal standard concentration as the x-axis and the ratio of external standard to internal standard peak area as the y-axis.

[0088] 3) Calculate the metabolite concentration: Substitute the integral peak surface of all detected samples into the linear equation of the standard curve for calculation, and then further substitute it into the following formula to calculate the final concentration data of the metabolite in the actual sample.

[0089] The concentration of metabolites in a liquid sample (ng / mL) = c*V1 / 1000 / V2

[0090] Meaning of each letter in the formula:

[0091] c: The concentration value (ng / mL) obtained by substituting the integrated peak area in the sample into the standard curve.

[0092] V1: Total volume of extract (μL);

[0093] V2: Sample volume transferred (mL).

[0094] (6) Statistical analysis

[0095] Data analysis was performed using IBM SPSS Statistics 27 software. The Mann-Whitney test was used to compare differences between the two groups, with a significance level set at P < 0.05. Receiver operating characteristic (ROC) curves were plotted using GraphPad Prism 9.5.1 software, with sensitivity (true positive rate) on the Y-axis and 1-specificity (false positive rate) on the X-axis.

[0096] 3. Experimental Results

[0097] Figure 1 Box plots show the concentration distribution of the blood metabolic marker indoleacetic acid (IAA) between the gestational diabetes mellitus (GDM) group and the healthy control group (Con). The serum IAA concentration in the GDM group was significantly lower than that in the healthy control group (P<0.001).

[0098] Figure 2 The ROC plot shows the diagnostic efficacy of indoleacetic acid (IAA) as a blood metabolic marker in gestational diabetes mellitus (GDM) in the group and the healthy control group. The area under the ROC curve (AUC) was 0.703 (95% confidence interval: 0.602, 0.804, P<0.001).

[0099] Example 2

[0100] 1. Experimental materials

[0101] The specific experimental materials are shown in Table 2.

[0102] Table 2 Experimental Materials for Example 2

[0103]

[0104] 2. Experimental Methods

[0105] (1) Four-week-old female C57BL / 6J mice and nine-week-old male C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK-2021-0006) and housed in a specific pathogen-free (SPF) standard facility with an independent ventilation cage system of Beijing Medcona Biotechnology Co., Ltd. (SYXK-2023-0062). They had free access to food and water, and the temperature was 22°C~24°C, the humidity was 40%~60%, and the standard 12h circulating light was provided.

[0106] (2) Before the experiment, the mice were fed a normal diet. After one week of acclimatization, the baseline weight of the female C57BL / 6J mice was measured. After matching their weights, they were randomly divided into three groups: normal diet group (Con), gestational diabetes mellitus group (GDM), and gestational diabetes mellitus + indoleacetic acid group (GDM+IAA). The normal diet group was fed a normal diet and received PBS solution by gavage daily. The gestational diabetes mellitus group was fed a high-fat diet and received PBS solution by gavage daily. The gestational diabetes mellitus + indoleacetic acid group was fed a high-fat diet and received 20 mg / (kg*d) of indoleacetic acid solution (dissolved in PBS) by gavage daily.

[0107] (3) Four weeks after intervention, female mice underwent an intraperitoneal glucose tolerance test (IPGTT). After changing the bedding at 8:00 PM, the mice were fasted for 12 hours (8:00 PM - 8:00 AM) but allowed free access to water, and their weight was measured. The experiment began at 8:00 AM, and tail vein blood was collected from the mice for blood glucose measurement using a glucometer. After measuring fasting blood glucose (BG0), the mice were intraperitoneally injected with 20% glucose solution (2 g / kg body weight), and blood glucose was measured at 15 minutes (BG15), 30 minutes (BG30), 60 minutes (BG60), 90 minutes (BG90), and 120 minutes (BG120) after the glucose load. The area under the curve (AUC) of the glucose tolerance test is calculated based on the blood glucose value using the formula: (BG0 + 2BG15 + BG30) × 0.125 + (BG30 + 2BG60 + 2BG90 + BG120) × 0.25.

[0108] (4) After 5 weeks of intervention, female mice underwent an intraperitoneal insulin tolerance test (IPITT). After changing the bedding at 8:00 AM, mice were fasted for 4 hours (8:00 AM - 12:00 PM) but allowed free access to water, and their weight was measured. The experiment began at 12:00 PM, and tail vein blood was collected from the mice for blood glucose measurement. After BG0 was measured, mice were intraperitoneally injected with insulin (Humulin R, 0.75 U / kg body weight), and blood glucose was measured at 15 minutes (BG15), 30 minutes (BG30), 60 minutes (BG60), 90 minutes (BG90), and 120 minutes (BG120) after the insulin load. The area under the insulin tolerance test curve (AUC) was calculated based on the blood glucose values, using the same formula as above.

[0109] (5) After acclimatizing to a normal diet for one week, 9-week-old male mice were paired with 10-week-old female mice at a ratio of 1:2. Mating was restricted for 5 days. The female mice were checked for vaginal plugs at 8:00 am every day. If a vaginal plug was observed, it was recorded as day 0.5 of pregnancy.

[0110] (6) Pregnant mice were housed in individual cages, and pre-pregnancy intervention continued. Fasting blood glucose (FBG) was measured in pregnant mice during the second trimester (12.5 days of gestation). After changing the clean bedding at 8:00 a.m., the mice were fasted for 6 hours but allowed free water (8:00 a.m. - 2:00 p.m.), and fasting blood glucose was measured in the tail vein.

[0111] (7) After fasting for 10 hours (22:00pm-8:00am the next day) on day 18.5 of pregnancy, pregnant mice were anesthetized by intraperitoneal injection of barbiturates (100~200mg / kg) and the embryos in the uterus were counted and weighed.

[0112] 3. Experimental Results

[0113] IPGTT results are as follows Figure 3 As shown, compared with the control group, the blood glucose levels and blood glucose AUC (P<0.001) of pregnant mice in the gestational diabetes mellitus group were significantly increased at 30 minutes (P<0.001), 60 minutes (P<0.001), 90 minutes (P<0.001), and 120 minutes (P<0.001) after glucose loading. Compared with the gestational diabetes mellitus group, indoleacetic acid supplementation significantly reduced blood glucose levels and blood glucose AUC (P<0.001) at 15 minutes (P<0.05), 30 minutes (P<0.05), 60 minutes (P<0.001), 90 minutes (P<0.001), and 120 minutes (P<0.05) after glucose loading in pregnant mice with gestational diabetes mellitus.

[0114] IPITT results are as follows Figure 4As shown, compared with the control group, the blood glucose levels and blood glucose AUC (P<0.001) of pregnant mice in the gestational diabetes mellitus group were significantly increased at 0 minutes (P<0.001), 15 minutes (P<0.001), 30 minutes (P<0.001), 60 minutes (P<0.001), 90 minutes (P<0.001), and 120 minutes (P<0.001) after insulin loading. Compared with the gestational diabetes mellitus group, indoleacetic acid supplementation significantly reduced blood glucose levels and blood glucose AUC (P<0.01) at 15 minutes (P<0.05), 30 minutes (P<0.01), and 60 minutes (P<0.05) after insulin loading in pregnant mice with gestational diabetes mellitus.

[0115] FBG results as follows Figure 5 As shown, compared with the control group, the fasting blood glucose level at 12.5 days of gestation in maternal mice with gestational diabetes mellitus was significantly increased (P<0.001). Compared with the gestational diabetes mellitus group, indoleacetic acid supplementation significantly reduced the fasting blood glucose level at 12.5 days of gestation in maternal mice with gestational diabetes mellitus (P<0.001).

[0116] Pregnancy safety assessment results such as Figure 6 As shown, there were no significant differences in the number and weight of embryos at 18.5 days of gestation among the control group, the gestational diabetes mellitus group, and the gestational diabetes mellitus + indoleacetic acid group (P>0.05). This demonstrates that indoleacetic acid supplementation can reduce fasting blood glucose, improve glucose tolerance and insulin resistance in gestational diabetes mellitus mice, and is safe and reliable without embryotoxicity.

[0117] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of reagents for detecting indoleacetic acid levels in samples in the preparation of products for diagnosing gestational diabetes.

2. The application according to claim 1, characterized in that, The reagent is used to detect the level of indoleacetic acid in a sample by any of the following methods: chromatography, mass spectrometry, spectroscopy, chromatography-mass spectrometry, enzyme-linked immunosorbent assay, radiochemical analysis, nuclear magnetic resonance spectroscopy, light scattering analysis, and turbidimetry. Preferably, the reagent further includes a detectable marker.

3. The application according to claim 2, characterized in that, The samples included blood, urine, and saliva.

4. The application according to claim 1, characterized in that, The products include reagent kits, chips, and test strips.

5. A product for diagnosing gestational diabetes, characterized in that, The product includes a reagent for detecting the level of indoleacetic acid in a sample; Preferably, the reagent is used to detect the level of indoleacetic acid in the sample by any of the following methods: chromatography, mass spectrometry, spectroscopy, chromatography-mass spectrometry, enzyme-linked immunosorbent assay, radiochemical analysis, nuclear magnetic resonance spectroscopy, light scattering analysis, and turbidimetry; Preferably, the product includes a reagent kit, a chip, and a test strip.

6. Application of indoleacetic acid in the preparation of drugs for the prevention / treatment of gestational diabetes mellitus.

7. The application according to claim 6, characterized in that, The medication also includes other medications for treating gestational diabetes.

8. The application according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include one or more of the following: fillers, binders, diluents, sweeteners, flavorings, preservatives, disintegrants, lubricants, flow aids, and antioxidants.

9. A medicine for treating gestational diabetes mellitus, characterized in that, The drug includes indoleacetic acid; Preferably, the drug further includes pharmaceutically acceptable excipients.

10. The medicament according to claim 9, characterized in that, The dosage forms of the drug include those administered via the gastrointestinal tract and those administered outside the gastrointestinal tract.

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