Application of intestinal probiotics and metabolites thereof in treating autism spectrum disorder

Abstract

The invention relates to the technical field of biological medicines, in particular to application of intestinal probiotics and metabolites thereof in treating autism spectrum disorders. According to the application disclosed by the invention, six intestinal bacteria which are in causal negative correlation with ASD are identified for the first time from the perspective of genetics, and the effects of ruminococcus gavreuii of the ruminococcus and the effects of Satasella wadsworensis of the ruminococcus on the autism spectrum disorder are verified. The invention also discloses a kit for detecting the autism spectrum disorder of the Autism spectrum disorder and a kit for detecting the autism spectrum disorder of the Autism spectrum disorder of the Autism spectrum disorder of the Autism spectrum disorder of the Autism spectrum disorder of the Autism spectrum disorder of the Autism spectrum disorder of the Autism spectrum disorder of the Autism spectrum disorder of the Autism spectrum disorder. Experiments prove that the traditional Chinese medicine composition can significantly improve social defects of model mice and has no side effects on normal mice. The invention provides new targets and candidate substances for developing probiotic preparations, metabolite supplements or functional foods for preventing, relieving or treating ASD, and has important clinical application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of a kind of intestinal probiotics and its metabolites in treating autism spectrum disorder. BACKGROUND

[0002] Autism spectrum disorder, also known as autism or autism spectrum disorder, is a pervasive developmental disorder affecting social interaction, communication ability and behavior pattern. At present, the etiology of autism is still unclear, and the pathogenesis still needs to be explored, and genetic factors are closely related.

[0003] Now, there is still no specific drug for treating autism, and the treatment means is very limited. In recent years, it has been found that intestinal microbiota (GM) plays an important role in the pathogenesis of ASD through the “gut-brain axis”, and the prior art (patent application CN119064592A) mainly uses intestinal flora metabolites as diagnostic markers. However, how to transform these findings into effective treatment means is still a great challenge. SUMMARY

[0004] The purpose of the present application is to overcome the deficiencies in the prior art and provide an ASD treatment scheme based on intestinal probiotics and its metabolites.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides application of intestinal probiotics and its metabolites in preparing a drug or preparation for preventing and / or treating autism spectrum disorder, wherein the intestinal probiotics include at least one of the following genera: Ruminiclostridium5, Ruminococcaceae UCG-005, Ruminococcus1, Sutterella, Dorea and Turicibacter.

[0006] The present application provides a group of probiotics and metabolites thereof with potential for treating ASD, by Mendelian randomization method, for the first time combining the evidence of the causal effect of strains on ASD from the perspective of genetics, the probiotics comprising at least one of the following genera: Ruminiclostridium 5 and Ruminococcus 1 (especially strains capable of increasing the levels of metabolites Delta-CEHC and docosadienoic acid), Ruminococcaceae UCG-005 (especially strains capable of regulating the level of serotonin), Sutterella (especially strains capable of regulating the level of N-acetyl-L-glutamine), and reveals the intermediary mechanism of its action by regulating specific plasma metabolites (serotonin, N-acetyl-L-glutamine, Delta-CEHC, docosadienoic acid). Based on the above causal evidence, the present application provides a brand-new ASD treatment strategy directly targeting the gut microbiome and its metabolic pathways.

[0007] Preferably, the intestinal probiotics comprise Ruminococcus 1 and / or Sutterella.

[0008] Preferably, the Ruminococcus 1 comprises Ruminococcus gauvreauii with the accession number DSM 19829, and the Sutterella comprises Sutterella wadsworthensis with the accession number CCUG 69352.

[0009] Preferably, the autism spectrum disorder comprises 16p11.2 microduplication (DP) type autism, in particular 16p11.2 dp / + (DP) type autism.

[0010] Preferably, the treatment refers to improvement of the core symptom of ASD, i.e. social impairment.

[0011] In the second aspect, the present application provides a use of a group of intestinal probiotics and metabolites thereof in the preparation of a product for preventing and / or treating autism spectrum disorder, the intestinal probiotics comprising at least one of the following genera: Ruminiclostridium 5, Ruminococcaceae UCG-005, Ruminococcus 1, Sutterella, Dorea, and Turicibacter.

[0012] Preferably, the intestinal probiotic bacteria comprise Ruminococcus 1 and / or Sutterella.

[0013] Preferably, the Ruminococcus 1 comprises Ruminococcus gauvreauii with the accession number of DSM 19829, and the Sutterella comprises Sutterella wadsworthensis with the accession number of CCUG 69352.

[0014] Preferably, the autism spectrum disorder comprises 16p11.2 microduplication (DP) type autism, in particular 16p11.2 dp / + (DP) type autism.

[0015] The present application further provides Ruminococcus gauvreauii and Sutterella wadsworthensis screened from the Mendelian randomization method, and it is proved through DP autism model mouse experiments that the above-mentioned bacteria liquid with an effective dose can significantly improve the social defects of model mice, and has no side effects on normal mice. The present application provides a new target and candidate substance for developing probiotic preparations, metabolite supplements or functional foods for preventing, alleviating or treating ASD, and has important clinical application prospects.

[0016] Preferably, the product comprises food and dietary supplements.

[0017] In a third aspect, the present application provides a medicine or microbial preparation for preventing and / or treating autism spectrum disorder, comprising intestinal probiotic bacteria and / or metabolites thereof, and a pharmaceutically acceptable carrier; the probiotic bacteria comprise Ruminococcus 1 and / or Sutterella; the effective amount of viable bacteria of the intestinal probiotic bacteria in the medicine or microbial preparation is not less than 1×10 8 CFU / mL.

[0018] Preferably, the Ruminococcus 1 comprises Ruminococcus gauvreauii with the accession number of DSM 19829, and the Sutterella comprises Sutterella wadsworthensis with the accession number of CCUG 69352.

[0019] The present application has the following beneficial effects: The present application screens the genera with significant genetic negative causal correlation with ASD from the intestinal microorganism whole genome association study (GWAS) summary data through bidirectional Mendelian randomization analysis. The present application further verifies the effect of Ruminococcus gauvreauii and Sutterella wadsworthensis screened out from bidirectional Mendelian randomization analysis on autism spectrum disorder. And through the DP autism model mouse experiment, it is confirmed that the effective dose of the above-mentioned bacterial liquid can significantly improve the social deficiency of the model mouse, and has no side effects on normal mice. The present application not only provides a new source for preparing a drug for treating social deficiency of DP mice, but also discovers the medicinal value of intestinal probiotics and their metabolites. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 1 is a scatter plot of the causal effect of intestinal microorganisms on autism spectrum disorder (ASD).

[0021] Figure 2 Figure 2 is a causal relationship diagram of two-step Mendelian randomization analysis of intestinal microorganisms, plasma metabolites and autism spectrum disorder.

[0022] Figure 3 Figure 3 is a three-chamber social experiment showing that an effective amount of bacterial liquid can improve the social deficiency of DP mice; A is a three-chamber social principle diagram, B is a contact time statistical diagram of Ruminococcus gauvreauii bacteria on the social ability of DP mice, and C is a contact time statistical diagram of Sutterella wadsworthensis bacteria on the social ability of DP mice. DETAILED DESCRIPTION

[0023] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific examples.

[0024] The experimental methods used in the present application are conventional methods unless otherwise specified, and the materials, reagents, etc. used are conventional commercially available products and can be obtained from commercial channels unless otherwise specified.

[0025] The strains used in the experiments of the present application are purchased from Ningbo Mingzhou Biotechnology Co., Ltd., wherein Ruminococcus gauvreauii has a preservation number of DSM 19829 and is preserved in the German DSM Microorganism and Cell Culture Collection Center, and Sutterella wadsworthensis has a preservation number of CCUG 69352 and is preserved in the University of Gothenburg Culture Collection Center in Sweden. The above two strains can be purchased from commercial channels.

[0026] Example 1: Causal relationship analysis between gut microbiota, plasma metabolites and autism spectrum disorder This example aims to elucidate the association between gut microbiota and autism spectrum disorder (ASD) from a causal perspective by Mendelian randomization, a genetic method, and reveal the key mediating role of plasma metabolites.

[0027] 1. Data sources and preprocessing: This study integrated the summary data from three large-scale genome-wide association studies (GWAS). The gut microbiota data came from the MiBioGen consortium, covering 211 microbial taxa of 18,340 individuals; the plasma metabolite data came from the Canadian Longitudinal Study on Aging (CLSA), including 1,458 metabolites and their ratios of 8,299 individuals; the ASD data came from the Danish Psychiatric Central Research Register (DPCRR), including 18,382 cases and 27,969 controls.

[0028] Instrumental variable selection: From the GWAS data of exposure (microbiota or metabolites), single nucleotide polymorphisms (SNPs) significantly associated with exposure were selected as instrumental variables (IVs). The selection criteria were: P < 1 x 10 -5 and removed linkage disequilibrium within 10,000 kb with a threshold of r² < 0.001. The F-statistics of all selected SNPs were greater than 10, effectively avoiding weak instrumental variable bias.

[0029] 2. Identification of causal effects of gut microbiota on ASD: Inverse-variance weighted method (IVW) was used as the main analysis method to systematically evaluate the causal effects of 119 genus-level gut microbiota on ASD.

[0030] The scatter plot of the causal effects of gut microbiota on autism spectrum disorder (ASD) is shown in Figure 1 The analysis identified 6 gut bacterial genera with significant genetic associations with ASD, namely Ruminiclostridium5, Ruminococcus1, Ruminococcaceae UCG-005, Sutterella, Dorea, and Turicibacter. This finding suggests that these 6 bacterial communities are not only statistically associated with ASD, but are more likely to play a causal role in the disease development process. From a genetic perspective, natural genetic variation in these bacterial colonies is considered one of the reasons for the difference in individual risk of developing ASD.

[0031] 3. Mediating role analysis of plasma metabolites: To explore the pathway of "gut microbes→ plasma metabolites→ ASD", the inventors performed a two-step Mendelian randomization analysis. The causal effect of gut microbes on plasma metabolites was assessed, followed by the causal effect of these gut-microbe-affected plasma metabolites on ASD.

[0032] The schematic diagram and results of the two-step Mendelian randomization analysis are shown in FIG. 2. The analysis revealed a complete causal chain. The results showed that the genetic susceptibility of ASD did not act directly, but exerted its influence by changing specific gut microbiota (and their downstream plasma metabolites), thereby affecting the disease progression of ASD from a genetic perspective. This confirmed that plasma metabolites are the key functional mediators of gut microbes affecting the risk of ASD. Figure 2

[0033] Through rigorous genetic causal inference, the inventors not only confirmed that specific gut microbes are risk factors for ASD, but more importantly, first mapped the causal pathway of "microbes-metabolites-disease". This finding transformed the abstract gut-brain axis association into a specific, interventionable molecular target, providing a direct and solid genetic theoretical cornerstone for the subsequent development of diagnostic tools and therapeutic strategies based on microbes and their metabolites (such as supplementing probiotics or metabolites).

[0034] Example 2: Strains screened based on causal effect 1. Criteria for strain screening The probiotic strains involved in the present application are strictly based on the results of two-way Mendelian randomization analysis. As shown in FIG. 2, the inventors generated a scatter plot of the causal effect between gut microbiota and ASD by variance weighting method. From these genera with significant causal effects, the inventors screened out the genera with protective negative causal association with ASD as the target, including Ruminococcus and Sutterella. Specifically, the inventors used Ruminococcus gauvreauii and Sutterella wadsworthensis for subsequent experiments. Figure 1

[0035] 2. Culture of strains ​​In an anaerobic workstation (85% N2, 10% CO2, 5% H2), the strain freeze-dried powder was serially diluted with anaerobic pure water, and then coated on a pre-reduction selective medium (modified GAM agar medium) for the target genus. Incubate at 37°C under anaerobic conditions for 48-96 hours. According to the colony morphology, color and size, the first generation of colonies is inoculated on the selective medium for the above target genus (Ruminococcus gauvreauii, PYG liquid medium; Sutterella wadsworthensis, chocolate agar plate) with an inoculation loop for multiple purification until a pure culture is obtained.

[0036] Example 3: Verification of the effect of the target strain on the improvement of the social behavior of DP autism model mice This example aims to verify the improvement effect of Sutterella wadsworthensis (Sutterella wadsworthensis) and Ruminococcus gauvreauii (Ruminococcus gauvreauii) cultured in Example 2 on the core symptom of autism, social deficiency, through in vivo animal experiments.

[0037] 1. Experimental animals and model construction The present application 16p11.2 dp / + (DP) autism model mice were purchased from the United States Jax Lab laboratory, StockNumber.013129 Strain Name: B6129SDp(7Slx1b-Sept1)5Aam, and then bred, and 6-8 week old male DP and wild type (WT) male mice produced by cohabitation were used for experiments.

[0038] The DP model mice were randomly divided into a control group, a Sutterella wadsworthensis treatment group, a Ruminococcus gauvreauii treatment group, and a wild type (WT) mouse group as a normal control group. Each group had at least 8 mice.

[0039] 2. Preparation of bacterial solution and gavage scheme The Sutterella wadsworthensis and Ruminococcus gauvreauii strains were inoculated into their suitable culture media for large-scale culture, respectively, and the bacterial bodies in the logarithmic growth phase were collected, resuspended with sterile PBS after centrifugation, and the concentration of the bacterial solution was adjusted to 1×10 9 CFU / mL. The mice in the treatment groups were given the corresponding bacterial solution at a fixed time every day, and the gavage volume was 100 μL per mouse (i.e., the daily dose was about 1×10 8(CFU / animal). The model group and the normal group were given an equal volume of sterile saline by gavage daily. The intervention period was 2 consecutive weeks.

[0040] 3. Behavioral Evaluation – Three-Room Social Experiment After a two-week intervention, a three-room social experiment device was used (see schematic diagram). Figure 3 A) Assess the social behavior of mice, with the main analytical indicators being the direct contact and sniffing time between the experimental mice and the "stimulated mouse" and "empty cage" areas.

[0041] 4. Experimental Results like Figure 3 As shown in Figure B, compared with the wild-type control group, the DP model control group mice had significantly reduced contact time with the stimulated mice, exhibiting obvious social deficits. In contrast, the DP mice treated with Ruminococcus gauvreauii bacterial solution via gavage showed a significant increase in contact time with the stimulated mice, approaching wild-type levels.

[0042] like Figure 3 As shown in Figure C, similarly, treatment with *Sutterella wadsworthensis* bacterial solution significantly increased the contact time between DP model mice and the stimulated mice, effectively improving their social deficit behaviors. Throughout the experiment, neither the DP mice treated with either bacterial solution nor healthy wild-type mice showed significant adverse reactions such as weight loss, disheveled fur, or reduced activity, indicating that the bacterial solution at this treatment dose had no significant side effects on normal mice.

[0043] This embodiment demonstrates through rigorous animal behavioral experiments that using 1×10 8 Gavage administration of Ruminococcus gauvreauii or Sutterella wadsworthensis bacterial suspensions selected by Mendelian randomization analysis at a dose of CFU / day for two consecutive weeks safely and effectively improved social deficits in DP autism model mice. This provides direct preclinical pharmacodynamic evidence for the formulation of these strains into drugs for the treatment of ASD.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of a group of intestinal probiotic bacteria and their metabolites for the preparation of a medicament or a preparation for the prevention and / or treatment of autism spectrum disorders, characterized in that, The intestinal probiotic bacteria comprise at least one of the following genera: Ruminiclostridium5, Ruminococcaceae UCG-005, Ruminococcus1, Sutterella, Dorea, Turicibacter.

2. Use according to claim 1, wherein The intestinal probiotic bacteria comprise Ruminococcus1 and / or Sutterella.

3. Use according to claim 2, wherein the compound is ###0002### The Ruminococcus1 comprises Ruminococcus gauvreauii having the accession number DSM 19829; the Sutterella comprises Sutterella wadsworthensis having the accession number CCUG 69352.

4. The use according to claim 1, wherein The autism spectrum disorder comprises 16p11.2 microduplication (DP) autism.

5. Use of a gut probiotic and metabolites thereof for the preparation of a product for the prevention and / or treatment of autism spectrum disorders, characterized in that, The intestinal probiotic bacteria comprise at least one of the following genera: Ruminiclostridium5, Ruminococcaceae UCG-005, Ruminococcus1, Sutterella, Dorea, Turicibacter.

6. The use according to claim 5, wherein the compound is ###0002### The Ruminococcus1 comprises Ruminococcus gauvreauii having the accession number DSM 19829; the Sutterella comprises Sutterella wadsworthensis having the accession number CCUG 69352.

7. The use according to claim 5, wherein the compound is ###0002### The autism spectrum disorder comprises 16p11.2 microduplication (DP) autism.

8. The use according to claim 5, wherein the compound is ###0002### The product comprises a food, a dietary supplement.

9. A medicament or microbial preparation for preventing and / or treating autism spectrum disorder, characterized by, Intestinal probiotic bacteria and / or metabolites thereof, and a pharmaceutically acceptable carrier; the probiotic bacteria comprising Ruminococcus 1 and / or Sutterella; the live effective amount of intestinal probiotic bacteria in the drug or microbial preparation being not less than 1 x 10 8 CFU / mL.

10. The medicament or microbial preparation for preventing and / or treating autism spectrum disorder according to claim 9, wherein The Ruminococcus1 comprises Ruminococcus gauvreauii having the accession number DSM 19829; the Sutterella comprises Sutterella wadsworthensis having the accession number CCUG 69352. The autism spectrum disorder comprises 16p11.2 microduplication (DP) autism.

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