Use of indole-3-acetamide and a strain producing the same for preparing a medicine for preventing or treating polycystic ovary syndrome

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of indole-3-acetamide and a producing strain thereof in preparation of a medicine for preventing or treating polycystic ovary syndrome. The present inventors first find that indole-3-acetamide can significantly improve estrous cycle disorder, insulin resistance, blood hormone disorder and polycystic-like changes of ovaries of a polycystic ovary syndrome model mouse, and does not cause obvious reproductive toxicity. The present inventors also find that a gas Collins bacteria produced by the indole-3-acetamide producing strain or an engineering bacteria based on an indole-3-acetamide synthetase can also play the same metabolic improvement role, and can be used for preparation of the medicine for preventing or treating polycystic ovary syndrome.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically, the invention relates to the application of indole-3-acetamide and its producing bacteria in the preparation of drugs for the prevention or treatment of polycystic ovary syndrome. Background Technology

[0002] Declining fertility rates have become a serious and undeniable public health problem worldwide. Among the many complex causes of female infertility, reproductive endocrine disorders consistently rank first, and polycystic ovary syndrome (PCOS) is one of the most common core causes of anovulatory infertility in clinical practice, accounting for more than 50% of all anovulatory infertility cases. PCOS is a chronic endocrine and metabolic disorder characterized by hyperandrogenemia, infrequent or complete anovulation, and polycystic ovarian morphology. Patients often also experience varying degrees of insulin resistance, which further exacerbates the accumulation of hyperandrogenemia, creating a vicious cycle of mutual influence. In addition to the core reproductive system abnormalities, this disease can lead to a series of long-term complications, including central obesity, a significantly increased risk of type 2 diabetes, dyslipidemia, and increased potential cardiovascular disease risks. Some patients may also experience long-term menstrual irregularities, hirsutism, acne, anxiety, and depression. The overall clinical presentation is complex and highly heterogeneous, with significant differences in symptom combinations and severity among different patients.

[0003] However, to date, the underlying pathogenesis of PCOS remains incompletely understood, and a unified core pathogenic target has not been identified. Most clinical interventions still rely on symptomatic treatment for different symptoms, such as using short-acting contraceptives to regulate menstrual cycles, using ovulation-inducing drugs to help follicle development, and using metformin to improve insulin resistance. A radical treatment targeting the root cause of the disease has not yet been developed. Due to the highly heterogeneous clinical manifestations of PCOS, the core issues vary greatly among patients. Standardized and individualized treatment has long been a key and challenging issue for obstetrics and gynecology, reproductive medicine, and endocrinology. Existing clinical interventions still have limitations, including single-target effects, significant long-term side effects, and poor response in some patients. Therefore, there is an urgent need in this field for novel therapeutic active ingredients that can precisely target the core pathological aspects of PCOS while simultaneously regulating reproductive endocrine function and improving metabolic status, providing safer and more effective intervention options for clinical practice. Summary of the Invention

[0004] Indole-3-acetamide (IAM or indoleacetamide for short) is produced by *Colinella aerogenes* (…). Collinsella aerofaciensTryptophan metabolites produced by specific gut microbiota, such as α, are widely present in the human population, but their physiological functions, especially their effects on reproductive endocrine and metabolic diseases, have not been reported. The inventors' research results show that indole-3-acetamide can significantly improve abnormal glucose metabolism and reduce insulin resistance in in vivo animal models and in vitro cell models: (1) improve abnormal glucose metabolism and reduce insulin resistance; (2) correct sex hormone disorders: reduce hyperandrogenemia (significantly decrease serum testosterone T level), downregulate luteinizing hormone (LH) level, and restore the typical hormonal imbalance state of PCOS; (3) restore ovulation function: reduce the number of cystic follicles in the ovary, increase the number of corpora lutea, reverse the anovulatory state of PCOS model mice, and restore the estrous cycle. In addition, after colonization by Collins aerogenes, which can produce indole-3-acetamide, and engineered bacteria based on indole-3-acetamide synthase, they can also exert the above-mentioned metabolic and reproductive improvement effects in treating PCOS, and it has been confirmed that this effect depends on the indole-3-acetamide produced by bacterial metabolism. Based on this, the present invention was completed.

[0005] Specifically, the present invention is achieved through the following technical solutions: This invention provides the use of indole-3-acetamide or its producing bacteria in the preparation of a medicament for the prevention or treatment of polycystic ovary syndrome in subjects with this need.

[0006] Alternatively, in the above applications, the indole-3-acetamide producing bacteria are Collins bacteria or engineered bacteria based on indole-3-acetamide synthase.

[0007] Preferably, the Collins spp. is Collins gas-producing bacteria (… Collinsella aerofaciens ).

[0008] Alternatively, in the above applications, the subject is a mammal.

[0009] Preferably, the mammal is a human.

[0010] Alternatively, in the above applications, the drug may also contain a pharmaceutically acceptable carrier, diluent, or excipient.

[0011] Alternatively, in the above applications, the drug is in oral dosage form.

[0012] Preferably, the oral dosage form is selected from one or more of the following: granules, capsules, tablets, powders, oral liquids, suspensions, or emulsions.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. The inventors have for the first time revealed the therapeutic effect of indole-3-acetamide on PCOS, covering three core pathological links: glucose metabolism, sex hormones and ovulation, providing a novel strategy for the prevention and treatment of PCOS.

[0014] 2. The inventors have for the first time revealed the therapeutic effect of the indole-3-acetamide-producing bacterium Collins on PCOS, which can achieve endogenous and continuous production of indole-3-acetamide, avoiding the potential safety issues of directly supplementing chemical substances, and verifying its ameliorative effect on PCOS.

[0015] 3. The inventors have for the first time disclosed the therapeutic effect of constructing engineered bacteria based on indole-3-acetamide synthase on PCOS, which can achieve endogenous and continuous production of indole-3-acetamide, avoiding the potential safety issues of directly supplementing chemical substances, and verifying the improvement effect on PCOS. Attached Figure Description

[0016] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 Effects of indole-3-acetamide on the estrous cycle in DHEA mice. Images are shown as mean ± SEM. P: proestrus; E: estrus; M / D: metestrus / diestrus. One-way ANOVA was used to assess the significance of each group. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0017] Figure 2 The effect of indole-3-acetamide on glucose tolerance test (GTT) results in DHEA mice. Images are shown as mean ± SEM. To more clearly illustrate differences between groups, the area under the curve was calculated, and one-way ANOVA was used to assess the significance of each group. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0018] Figure 3 Effect of indole-3-acetamide on insulin tolerance test (ITT) results in DHEA mice. Images are shown as mean ± SEM. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0019] Figure 4 Effects of indole-3-acetamide on serum sex hormones in DHEA mice. Images are shown as mean ± SEM. One-way ANOVA was used to assess the significance of each group. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0020] Figure 5 Effects of indole-3-acetamide on polycystic ovarian changes in DHEA mice. Images are shown as mean ± SEM. One-way ANOVA was used to assess the significance of each group. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0021] Figure 6 The content of indole-3-acetamide synthesized by *Colinella aerogenes* in DHEA mice. Images are shown as mean ± SEM. Independent samples t-test was used. This indicates that P < 0.01 compared to the model group.

[0022] Figure 7 Effects of *Colinus* aerogenes on the estrous cycle in DHEA mice. Images are shown as mean ± SEM. P: proestrus; E: estrus; M / D: metestrus / diestrus. One-way ANOVA was used to assess the significance of each group. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0023] Figure 8 The effect of *Collinusella oxyphylla* on the glucose tolerance test (GTT) results in DHEA mice. Images are shown as mean ± SEM. To more clearly illustrate differences between groups, the area under the curve was calculated, and one-way ANOVA was used to assess the significance of each group. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0024] Figure 9 Effect of *Colinella aerogenes* on insulin tolerance test (ITT) results in DHEA mice. Images are shown as mean ± SEM. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0025] Figure 10 Effects of *Colinbus aerogenes* on serum sex hormones in DHEA mice. Images are shown as mean ± SEM. One-way ANOVA was used to assess the significance of each group. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0026] Figure 11 Effects of *Collinusella oxyphylla* on polycystic ovarian changes in DHEA mice. Images are shown as mean ± SEM. One-way ANOVA was used to assess the significance of each group. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0027] Figure 12 The content of indole-3-acetamide produced by engineered bacteria based on indole-3-acetamide synthase. Images are shown as mean ± SEM. Independent samples t-test was used. Indicates association with Escherichia coli E. coli The group comparison showed P < 0.01.

[0028] Figure 13 The effect of engineered bacteria based on indole-3-acetamide synthase on the estrous cycle in DHEA mice. Images are shown as mean ± SEM. P: proestrus; E: estrus; M / D: metestrus / diestrus. One-way ANOVA was used to assess the significance of each group. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0029] Figure 14 The effect of engineered bacteria based on indole-3-acetamide synthase on the glucose tolerance test (GTT) results in DHEA mice. Images are shown as mean ± SEM. To more clearly illustrate differences between groups, the area under the curve was calculated, and one-way ANOVA was used to assess the significance of each group. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0030] Figure 15 The effect of engineered bacteria based on indole-3-acetamide synthase on insulin tolerance test (ITT) results in DHEA mice. Images are shown as mean ± SEM. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0031] Figure 16Effects of engineered bacteria based on indole-3-acetamide synthase on serum sex hormones in DHEA mice. Images are shown as mean ± SEM. One-way ANOVA was used to assess the significance of each group. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group.

[0032] Figure 17 The effect of engineered bacteria based on indole-3-acetamide synthase on polycystic ovarian changes in DHEA mice. Images are shown as mean ± SEM. One-way ANOVA was used to assess the significance of each group. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0034] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0035] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0036] Example 1: Comparison of the ameliorative effects of indole-3-acetamide on dehydroepiandrosterone (DHEA)-induced PCOS mouse models Materials: Blood glucose test strips (Roche), blood glucose meter (Roche), dehydroepiandrosterone (DHEA) (Sigma), sex hormone assay kit. The dosage administered via gavage was 10 mg / kg body weight, three times per week.

[0037] Establishment of the mouse model: C57 mice were purchased from Jicui Yaokang. The temperature was 20-24℃, the humidity was 50-60%, the light was 12 hours (8:00-20:00), the soundproof environment was maintained, and the mice were allowed free access to food and water. The experiment was conducted after the mice had adapted to the environment for one week.

[0038] Methods: Eighteen female C57 mice, aged 3-4 weeks and weighing 10-12g, were randomly divided into: (1) Normal control group: daily subcutaneous injection of sesame oil and oral administration of PBS three times a week for 3 weeks; (2) Experimental control group: daily subcutaneous injection of DHEA (6mg / 100g) and oral administration of PBS three times a week for 3 weeks; (3) Indole-3-acetamide treatment group (i.e., experimental group): daily subcutaneous injection of DHEA and oral administration of indole-3-acetamide (10 mg / kg) three times a week for 3 weeks. Estrogen cycles were measured daily 10 days after DHEA injection. The estrous cycles of mice in the experimental control group were significantly disordered, proving that the model was successfully established. Oral glucose tolerance and insulin tolerance were measured before the last administration. After the last administration, blood was collected, centrifuged at 3000 rpm at 4℃, and serum sex hormone levels were measured. Ovarian sections were examined for pathological changes.

[0039] Results: Compared with the model control group, indole-3-acetamide treatment significantly improved insulin resistance-related indicators during the estrous cycle in mice, and improved ovarian status and sex hormone levels. (See attached figures) Figures 1-5 .

[0040] Example 2: Comparison of the ameliorative effects of *Colinella aerogenes* on dehydroepiandrosterone (DHEA)-induced PCOS mouse models Materials: Blood glucose test strips (Roche), blood glucose meter (Roche), dehydroepiandrosterone (DHEA) (Sigma), sex hormone assay kit. Bacterial culture administered via gavage at a rate of 2 x 10. 8 CFU was administered to each mouse once a week.

[0041] Establishment of the mouse model: C57 mice were purchased from Jicui Yaokang. The temperature was 20-24℃, the humidity was 50-60%, the light was 12 hours (8:00-20:00), the soundproof environment was maintained, and the mice were allowed free access to food and water. The experiment was conducted after the mice had adapted to the environment for one week.

[0042] Methods: Eighteen female C57 mice, aged 3-4 weeks and weighing 10-12g, were randomly divided into: (1) Normal control group: daily subcutaneous injection of sesame oil and weekly gavage with PBS for 3 weeks; (2) Experimental control group: daily subcutaneous injection of DHEA (6mg / 100g) and weekly gavage with PBS for 3 weeks; (3) Collins gas-producing bacteria group (i.e., experimental group): daily subcutaneous injection of DHEA and weekly gavage with Collins gas-producing bacteria (2 x 10g) 8 CFU was administered for 3 weeks. Estrogenic cycles were monitored daily 10 days after DHEA injection. The estrous cycles of mice in the experimental control group were significantly disrupted, proving the successful establishment of the model. Oral glucose tolerance and insulin tolerance were measured before the last administration. After the last administration, blood was collected, centrifuged at 3000 rpm at 4℃, and serum sex hormone levels were measured. Ovarian sections were examined for pathological changes.

[0043] Results: Compared with the experimental control group, treatment with *Colinella aerogenes* significantly improved estrous cycles and insulin resistance-related indicators in mice by increasing indole-3-acetamide levels, and also improved ovarian status and sex hormone levels in mice. (See attached results). Figures 6-11 .

[0044] Example 3: Engineered bacteria constructed based on indole-3-acetamide synthase (IAM-E. coli) Comparison of the ameliorative effects of dehydroepiandrosterone (DHEA) on PCOS mouse models Materials: Blood glucose test strips (Roche), blood glucose meter (Roche), dehydroepiandrosterone (DHEA) (Sigma), sex hormone assay kit. Bacterial culture was administered via gavage at a dose of 2 x 10⁻⁶. 8 CFU was administered to each mouse once a week.

[0045] Engineered bacteria constructed based on indole-3-acetamide synthase (IAM-E. coli) Construction: The corresponding IAM synthase gene was inserted into the genome of *E. coli* Nissle 1917 (attb site of the *EcN-GFP* genome) using CRISPR-Cas9 technology. The pTargetF plasmid was removed by culturing the strain alone at 30°C with 0.5 mM IPTG and 50 μg / mL kanamycin for over 14 hours. Finally, the strain was cultured overnight in LB medium at 42°C to remove the pCas plasmid. A strain expressing IAM was successfully constructed. (IAM-E. coli) .

[0046] Establishment of the mouse model: C57 mice were purchased from Jicui Yaokang. The temperature was 20-24℃, the humidity was 50-60%, the light was 12 hours (8:00-20:00), the soundproof environment was maintained, and the mice were allowed free access to food and water. The experiment was conducted after the mice had adapted to the environment for one week.

[0047] Methods: Eighteen female C57 mice, aged 3-4 weeks and weighing 10-12g, were randomly divided into: (1) Normal control group: daily subcutaneous injection of sesame oil and weekly gavage with PBS for 3 weeks; (2) Experimental control group: daily subcutaneous injection of DHEA (6mg / 100g) and weekly gavage with PBS for 3 weeks; (3) Engineered bacteria IAM-E. coli Group (i.e., experimental group): DHEA was administered subcutaneously daily and engineered bacteria was administered via gavage once a week. IAM-E. coli (2 x 10) 8 CFU was administered for 3 weeks. Estrogenic cycles were monitored daily 10 days after DHEA injection. The estrous cycles of mice in the experimental control group were significantly disrupted, proving the successful establishment of the model. Oral glucose tolerance and insulin tolerance were measured before the last administration. After the last administration, blood was collected, centrifuged at 3000 rpm at 4℃, and serum sex hormone levels were measured. Ovarian sections were examined for pathological changes.

[0048] Results: Compared with the experimental control group, treatment with engineered bacteria significantly improved estrous cycles and insulin resistance-related indicators in mice by increasing indole-3-acetamide content, and also improved ovarian status and sex hormone levels in mice. (See attached figures.) Figures 12-17 .

[0049] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The use of indole-3-acetamide or its producing bacteria in the preparation of a medicine for the prevention or treatment of polycystic ovary syndrome in subjects who require it.

2. The application according to claim 1, characterized in that: The bacteria that produce indole-3-acetamide are either Collins bacteria or engineered bacteria based on indole-3-acetamide synthase.

3. The application according to claim 2, characterized in that: The Collins species mentioned is *Colins*, which produces gas (… Collinsella aerofaciens ).

4. The application according to claim 1, characterized in that: The subjects were mammals.

5. The application according to claim 4, characterized in that: The mammal in question is a human.

6. The application according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable carriers, diluents, or excipients.

7. The application according to claim 6, characterized in that: The drug is an oral dosage form.

8. The application according to claim 7, characterized in that: The oral dosage form is selected from one or more of the following: granules, capsules, tablets, powders, oral liquids, suspensions, or emulsions.

Images