Construction method and application of Eriocheir sinensis intestinal brain axis research model

Abstract

The invention discloses a construction method and application of an Eriocheir sinensis gut-brain axis research model, according to the method, metronidazole, neomycin and vancomycin are added to commercial feed according to the mass ratio of 10: 10: 5 g / kg to prepare compound antibiotic feed, and the Eriocheir sinensis is continuously fed for 96 h. According to the method, a standardized research model capable of synchronously and controllably presenting the causal relation of'intestinal flora disturbance-nervous system response 'is constructed in crustacean for the first time. The model is short in construction period, high in stability and high in repeatability, and a key research tool is provided for deeply analyzing the enteric-cerebral axis interaction mechanism of the eriocheir sinensis and even crustacean, screening neural active substances and evaluating neurotoxicity of environmental stress.

Description

TECHNICAL FIELD

[0001] The present application relates to a construction method and application of a Chinese mitten crab gut-brain axis research model, and belongs to the technical field of intersection of aquatic animal physiology, microbiology and neurobiology. BACKGROUND

[0002] Chinese mitten crab is an important freshwater crustacean, and its intestinal microbiota plays a key role in digestion, growth and immune regulation. The central nervous system of Chinese mitten crab is mainly composed of brain ganglion, thoracic ganglion and intestinal ganglion, which undertakes core functions such as behavior regulation, sensory conduction and stress response. Recent studies have shown that intestinal microbiota can remotely affect the function and behavior of the central nervous system through the "gut-brain axis" two-way communication system. However, compared with mammals, the study of crustacean gut-brain axis is still in its infancy, and there is a lack of a standardized research model that can synchronously and controllably disturb intestinal flora and quantitatively detect the response of the nervous system.

[0003] In the prior art, research on crustacean intestinal flora is mostly focused on composition analysis or phenotypic effects on growth and immunity, but there is no systematic method for specifically disturbing intestinal flora and synchronously observing the molecular and functional responses of the nervous system. This limits the in-depth understanding of the interaction mechanism between microbial flora and the nervous system of crustaceans, and hinders the development of new breeding strategies based on this principle (such as neuroprotective probiotics). Therefore, developing a standardized gut-brain axis research model is an important technical gap that needs to be broken through in this field. SUMMARY

[0004] The purpose of the present application is to provide a Chinese mitten crab gut-brain axis research model with short construction period, stable intervention effect, and multi-level verification of microbiome and nervous system, as well as a construction method and application thereof.

[0005] The construction method of the Chinese mitten crab gut-brain axis research model according to the present application comprises the following steps: feeding Chinese mitten crabs with feed mixed with mixed antibiotics, and obtaining a Chinese mitten crab intestinal flora deletion model after 48-96 hours of feeding; after the treatment is completed, the model is verified whether it is successfully constructed through intestinal microbiota intervention effect verification and nervous system response effect verification; the mixed antibiotics are composed of metronidazole, neomycin and vancomycin.

[0006] Further, the addition amount of metronidazole, neomycin and vancomycin is 10-18: 10-18: 5-9 g / kg of feed.

[0007] Further, the feeding frequency is twice a day, and the feeding amount is 4%-5% of the body weight of the crab.

[0008] Further, the Eriocheir sinensis is temporarily raised in a constant-temperature circulating water culture system for one week before being fed with the mixed antibiotic-spiked feed, and the water temperature is 24±1℃.

[0009] Further, the water temperature is kept constant and the dissolved oxygen is sufficient during the feeding.

[0010] Further, the preparation step of the mixed antibiotic-spiked feed comprises: mixing metronidazole, neomycin and vancomycin, dissolving them in a solvent, and uniformly spraying on the surface of the feed and air-drying in a cool room.

[0011] Further, the intervention effect verification of the intestinal microbiota comprises plate counting method, scanning electron microscope observation and 16S rRNA gene sequencing to verify the biomass and / or community structure of intestinal microorganisms; the response effect verification of the nervous system comprises targeted metabolomics analysis of thoracic ganglion, and detection of the expression level of neurotransmitter receptor-related genes in brain ganglion and thoracic ganglion; when there is a significant difference between the treated Eriocheir sinensis and the conventional Eriocheir sinensis, it proves that the model is successfully constructed.

[0012] Further, the neurotransmitter receptor-related genes comprise GABA receptor genes GABA 1AR and GABA 2BR ; dopamine receptor genes DA 1AR and DA 2R ; serotonin receptor genes 5-HT 1BR and 5-HT 2BR ; glutamate receptors NMDA 1AR , NMDA 2AR and NMDA 2BR .

[0013] Further, the imbalance verification of the intestinal microbiota further comprises: collecting feces or intestinal contents, calculating CFU by plate culture method, and taking the bacterial flora clearance rate > 90% as the judgment standard for effective clearance of the microbiota.

[0014] Further, the 16S rRNA gene sequencing evaluates the change of the microbial community structure by principal component analysis or species composition abundance change.

[0015] Further, the targeted metabolomics analysis detects neural metabolites by ultra-high performance liquid chromatography-tandem mass spectrometry, and adopts principal component analysis to judge the separation of the metabolic profiles between groups; the neural metabolites include but are not limited to at least one of gamma-aminobutyric acid, dopamine and 5-hydroxytryptamine.

[0016] The Eriocheir sinensis enterobrain axis research model constructed by the above-mentioned method of the application is applied to screening substances for adjusting the neural function of crustaceans or enhancing the neural stress tolerance of crustaceans, and the substances include probiotics, prebiotics or feed additives.

[0017] The research model of the gut-brain axis of the Chinese mitten crab constructed by the above-mentioned method of the present invention is applied to assess the toxic effects of environmental stress on the nervous system of crustaceans through the microbiome pathway.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. High construction efficiency: Using a specific ratio of compound antibiotics, stable and efficient induction of gut microbiota imbalance can be achieved within 96 hours. The construction cycle is short, which can quickly provide an experimental carrier for the study of gut-brain axis mechanisms.

[0019] 2. Comprehensive evaluation system: A multi-level data and validation system has been established, encompassing microbial quantity, community structure, neural metabolites, and receptor gene expression, forming a two-way validation system of "microbiome-neuron" to ensure the stability and reliability of model construction and a complete chain of evidence.

[0020] 3. Highly innovative and practical: This research model has constructed a microbial intervention system in crustaceans that can stably output nervous system response signals, providing a new tool for studying the relationship between the microbiome and the nervous system.

[0021] 4. Standardization and reproducibility: It provides clear antibiotic ratios, processing times, sampling points, and validation indicators, and the operation process is standardized, making it easy to repeat and apply in different laboratories.

[0022] 5. Broad application prospects: The constructed model can not only be used for basic mechanism research (such as the analysis of gut-brain axis signaling pathways), but also be directly applied to probiotic / meta-screening, functional feed additive evaluation, and high-throughput assessment of the neurotoxicity of environmental pollutants or stress factors, thus possessing both scientific research and industrial application value. Attached Figure Description

[0023] Figure 1 Line graph showing the change in bacterial clearance rate in feces and intestinal contents of group AN over time (Note: AN is the antibiotic group). Figure 2 Scanning electron micrographs of intestinal tissues from the CN and AN groups (Note: CN is the control group, and AN is the antibiotic group). Figure 3 The diagram shows the PCA analysis based on 16S rRNA sequencing (left) and the phylum-level species composition analysis (right). Figure 4 PCA analysis plot (left) and differential metabolite statistical analysis plot (right) for thoracic ganglion-targeted metabolomics; Figure 5 Bar chart showing the expression levels of neurotransmitter receptor-related genes in the brain and thoracic ganglia of the CN and AN groups (Note: CN is the control group, AN is the antibiotic group; * indicates significant difference).P <0.05, ** indicates P <0.01, *** indicates P <0.001); Figure 6 Scanning electron micrographs of the intestines of the heat stress groups HS and AH, and differential microbial community structures of the CN, AN, HS and AH groups (Note: CN is the control group, AN is the antibiotic group, HS is the heat stress group, and AH is the antibiotic + heat stress group). Figure 7 Bar chart showing the expression levels of neurotransmitter receptor-related genes in brain and thoracic ganglia in the CN, AN, HS, and AH groups (Note: CN is the control group, AN is the antibiotic group, HS is the heat stress group, and AH is the antibiotic + heat stress group; * indicates significant difference). P <0.05, ** indicates P <0.01, *** indicates P <0.001). Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0025] Example 1: Construction of a research model of the gut-brain axis of the Chinese mitten crab 1. Laboratory Animals: Healthy, vigorous Chinese mitten crabs with intact appendages and an average weight of 18±2g were selected and temporarily housed in a laboratory breeding system for one week to allow them to acclimatize to the laboratory environment. The water temperature was kept constant at 24±1℃, and sufficient dissolved oxygen was ensured. Normal commercial feed (New Hope Liuhe Co., Ltd.) was provided twice daily, morning and evening, with the total daily feed amount being approximately 4%-5% of the animals' total body weight.

[0026] 2. Preparation of compound antibiotic feed: Metronidazole (10 g / kg), neomycin (10 g / kg), and vancomycin (5 g / kg) were added to normal commercial feed at the following mass ratio. The three antibiotics were accurately weighed according to the required total feed volume, mixed, dissolved in sterile distilled water, and evenly sprayed onto the feed surface. The mixture was then air-dried in a cool, shaded place at room temperature and stored for later use. The control group feed was treated with an equal volume of sterile water.

[0027] 3. Experimental Grouping and Rearing Management: Crabs were fasted for 24 hours before the formal experiment. 120 crabs were randomly selected and divided into two groups, with three replicates per group and 20 crabs per replicate. Control Group (CN): Fed normal commercial feed. Antibiotic Group (AN): Fed feed containing a specific compound antibiotic. Crabs were fed twice daily at fixed times (9:00 AM and 5:00 PM) for 96 consecutive hours. During this period, the crabs' feeding and activity levels were closely monitored to ensure effective intake of the antibiotic-containing feed.

[0028] 4. Sample collection: (1) After the start of the experiment, at 0h, 12h, 24h, 48h, 72h and 96h, 5 crabs were randomly selected from each group. 0.1g of feces and 0.1g of intestinal contents (feces removed) were mixed with 9 mL of sterile PBS by vortexing. The CFU was calculated by plate counting method, and the change in intestinal microbiota clearance rate was then calculated. (2) 96h after feeding, the intestinal contents, brain ganglia and thoracic ganglia of the remaining 30 experimental crabs were collected using aseptic techniques. They were quickly frozen in liquid nitrogen and then transferred to a -80℃ ultra-low temperature freezer for storage. The intestinal contents were analyzed by scanning electron microscopy and 16S rRNA gene sequencing. The thoracic ganglia were used for subsequent targeted metabolomics analysis. The remaining brain ganglia and thoracic ganglia were used for expression analysis of neurotransmitter-related genes. Relative quantification analysis by real-time quantitative PCR was performed using the ChamQ SYBR qPCR Master Mix kit (ROX-free) (Nanjing Novizan Biotechnology Co., Ltd., China) on a LightCycler96 system (Roche, Switzerland). All PCR primer sequences are detailed in Table 1.

[0029] Table 1 Primer sequence information

[0030] 5. Experimental results: (1) The line graph of gut microbiota clearance rate ( Figure 1 It can be seen that as the antibiotic treatment time is extended, the clearance rate gradually increases. After 12 hours of treatment, the clearance rate of fecal flora stabilizes at over 90%, and after 48 hours of treatment, the clearance rate of intestinal flora stabilizes at over 90%. (2) Figure 2 As shown, a large number of intestinal microorganisms were tightly adhered in the intestinal tissue of the CN group, while only sporadic microorganisms or microbial fragments were visible in the intestinal tissue of the AN group after 96 hours of treatment. (3) 16S rRNA sequencing analysis of intestinal microbiota ( Figure 3 The results showed that the CN and AN groups formed two independent and distant clusters, and the composition of gut microbiota at the phylum level was significantly different, proving that antibiotics caused the gut microbiota to shift from a state of "high diversity and balanced composition" to an imbalanced state of "low diversity and single bacteria dominance". (4) Targeted metabolomics detection of thoracic ganglia ( Figure 4PCA analysis showed that the neural metabolites of the CN group and the AN group were significantly separated. The differential metabolite statistical diagram showed that the overall changes of various nerve-related metabolites in the thoracic ganglia of the CN group and the AN group were significantly different. Further analysis revealed that compared with the CN group, the AN group had significantly upregulated levels of various key neural metabolites (such as 5-hydroxytryptophan (5-HT), dopamine (DA) and histamine (HA)). (5) The expression levels of key neural receptor genes in the brain ganglia and thoracic ganglia were detected by qRT-PCR technology. GABA 1AR , GABA 2BR , DA 1AR , DA 2R , 5-HT 1BR , 5-HT 2BR , NMDA 1AR , NMDA 2AR and NMDA 2BR The expression levels of both groups were significantly higher than those of the CN group ( P <0.05). The above results collectively demonstrate that a research model of the gut-brain axis of the Chinese mitten crab, characterized by gut microbiota depletion and specific neurological molecular responses, was successfully constructed by feeding with a compound antibiotic for 96 hours.

[0031] Example 2: Application of the research model in heat stress-induced neurotoxicity 1. Experimental Design: To investigate the role of gut microbiota in regulating host neural stress response, a heat stress experiment was conducted using the model described in this invention. Based on Example 1, after feeding normal commercial feed and antibiotic feed for 96 hours respectively, the subjects were subjected to continuous heat stress for 96 hours. The heat stress temperature was set at 32°C, which is higher than their optimum growth temperature and can cause physiological stress. Feeding was stopped during the heat stress period. The experiment was divided into four groups: control group (CN): fed normal commercial feed at an optimal temperature of 24°C for 96 hours; antibiotic group (AN): fed feed containing a specific compound antibiotic for 96 hours at an optimal temperature of 24°C; heat stress group (HS): fed normal commercial feed for 96 hours followed by 96 hours of heat stress at 32°C; antibiotic + heat stress group (AH): fed feed containing a specific compound antibiotic for 96 hours followed by 96 hours of heat stress at 32°C.

[0032] 2. Sample collection: After the treatment was completed according to the settings, intestinal, brain ganglia and thoracic ganglia were collected from each group for subsequent analysis.

[0033] 3. Experimental Results: Scanning electron microscopy of the intestines showed a significant reduction in gut microbiota in the AN and AH groups compared to the CN group. Furthermore, the gut microbiota biomass in the AH group was significantly lower than that in the HS group. PCA analysis revealed clear separation between the CN and AN group samples, indicating that antibiotic treatment significantly altered the gut microbiota structure. Simultaneously, the HS and AH groups also showed a separation trend, suggesting that gut microbiota deficiency altered the body's response pattern to heat stress. Figure 6 Furthermore, in the cerebral and thoracic ganglia, the mRNA expression levels of GABA receptors, dopamine receptors, serotonin receptors, and glutamate receptors were mostly significantly higher in AN and HS than in CN. P <0.05%, the AH group showed a sharp decrease, significantly lower than the HS group ( P <0.05), and there was a significant interaction between antibiotics and heat stress ( P <0.05). These results indicate that an intact gut microbiota is an important exogenous regulator for maintaining neural homeostasis. Under heat stress alone, the body can compensate by upregulating the expression of relevant receptors; however, when the microbiota is eliminated, this key regulatory pathway is blocked, leading to the inability to properly induce the expression of neurotransmitter-related receptor genes, thus causing nerve damage. The significant interaction between antibiotics and heat stress demonstrates that the state of the microbiota and heat stress synergistically regulate neural function, and that the microbiota is a key factor determining stress response patterns. This proves the practical value of this model in assessing the neurotoxicity of environmental stresses through the microbial pathway.

Claims

1. A method for constructing a model of the gut-brain axis of Eriocheir sinensis, characterized in that, The method comprises the following steps: The Eriocheir sinensis is fed with the feed mixed with the mixed antibiotics, and the intestinal flora deficiency model of the Eriocheir sinensis is obtained after 48-96 hours; after the treatment, the model is verified whether it is successfully constructed through the intestinal flora intervention effect verification and the nervous system response effect verification; the mixed antibiotics are composed of metronidazole, neomycin and vancomycin.

2. The method for constructing the ENS model of Eriocheir sinensis according to claim 1, wherein, The adding amount of the metronidazole, the neomycin and the vancomycin is 10-18:10-18:5-9 g / kg of the feed.

3. The method of claim 1, wherein the method further comprises the step of: The feeding frequency is twice a day, and the feeding amount is 4%-5% of the weight of the crabs. ​ 4. The method of claim 1, wherein the method further comprises the step of: 4-1) constructing a Chinese mitten crab enteric-brain axis research model by using the Chinese mitten crab enteric-brain axis research model of claim 1. The Eriocheir sinensis is temporarily raised in a constant-temperature circulating water culture system for one week before being fed with the feed mixed with the mixed antibiotics, and the water temperature is 24±1℃.

5. The method of claim 1, wherein the method further comprises the step of: 5-1) constructing a Chinese mitten crab enteric-brain axis research model by using the Chinese mitten crab enteric-brain axis research model of claim 1. The water temperature is kept constant and the dissolved oxygen is sufficient during the feeding.

6. The method for constructing the intestinal-brain axis research model of the Chinese mitten crab according to claim 1, characterized in that, The preparation steps of the feed mixed with the mixed antibiotics comprise the following steps: the metronidazole, the neomycin and the vancomycin are mixed and dissolved in a solvent, and are uniformly sprayed on the surface of the feed and are air-dried in a cool room.

7. The method for constructing the intestinal-brain axis research model of the Chinese mitten crab according to claim 1, characterized in that, The intestinal flora intervention effect verification comprises the plate counting method, the scanning electron microscope observation and the 16S rRNA gene sequencing verification of the intestinal microbial biomass and / or community structure; the nervous system response effect verification comprises the targeted metabolomics analysis of the thoracic ganglion, the detection of the expression level of the neurotransmitter receptor related gene in the brain ganglion and the thoracic ganglion; when there is a significant difference between the treated Eriocheir sinensis and the conventional Eriocheir sinensis, it is proved that the model is successfully constructed.

8. The method of claim 7, wherein the method further comprises the step of: 8-1) constructing a Chinese mitten crab enteric-brain axis research model by using the Chinese mitten crab enteric-brain axis research model of claim 1. The neurotransmitter receptor-related genes include GABA receptor genes GABA 1AR and GABA 2BR ; dopamine receptor genes DA1AR and DA2R ; serotonin receptor genes 5-HT1BR and 5-HT2BR ; glutamate receptors NMDA 1AR , NMDA 2AR and NMDA 2BR .

9. The use of the model of the gut-brain axis of Eriocheir sinensis constructed by the method of claims 1-8 in screening substances for regulating the nervous function of crustaceans or enhancing the stress tolerance of the nervous system, characterized in that, The substances comprise probiotics, prebiotics or feed additives.

10. The application of the Eriocheir sinensis intestinal-brain axis research model constructed by the method of claims 1-8 in the evaluation of the toxic effects of environmental stress on the nervous system of crustaceans through the microbiota pathway.

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