Method for establishing intestinal dysfunction model by using zebra fish

Abstract

The invention relates to the field of zebra fish screening (evaluation) models, and discloses a method for establishing an intestinal dysfunction model by using zebra fish, which comprises the following steps: (1-1) selecting zebra fish greater than 5dpf and feeding the zebra fish in zebra fish culture water at the culture temperature of 26-30 DEG C; and (1-2) injecting different doses of escherichia coli into intestinal tracts of the zebrafish in the model experiment group, and constructing a zebrafish intestinal dysfunction model group. According to the method, escherichia coli cells are directly injected into the intestinal tract of the zebra fish by utilizing a microinjection technology for the first time, so that the sepsis-induced intestinal dysfunction model is established, the model is closer to the actual infection condition, and a more comprehensive and complex sepsis pathological process can be simulated. According to the method, the high-accuracy, scientific and reasonable zebra fish intestinal dysfunction model is successfully constructed by optimizing influence factors such as modeling concentration, evaluation indexes and multi-index verification selection. Compared with other methods, the model has the advantage of short modeling period, so that the detection cost is greatly reduced, and wide popularization and application are facilitated.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for establishing an intestinal dysfunction model using zebrafish. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection, which can rapidly develop into multiple organ failure and is one of the important causes of death in patients in the intensive care unit (ICU). In the development of sepsis, intestinal dysfunction is an important pathological link, manifested as intestinal barrier function destruction, intestinal microbial translocation, intestinal motility dysfunction, etc. Therefore, establishing a reliable intestinal dysfunction model is of great significance for studying the pathogenesis of sepsis and developing new treatment strategies.

[0003] At present, the commonly used sepsis models mainly include lipopolysaccharide (LPS)-induced models and Escherichia coli (E. coli) infection models. Among them, the LPS model is widely used because of its simple operation, but it mainly simulates one aspect of bacterial infection and cannot fully replicate the complexity of sepsis. For example, the LPS model is significantly different from clinical sepsis in terms of cytokine release patterns, immune system activation, etc., and cannot fully reflect the complexity of human sepsis. In contrast, the E. coli model is closer to the actual infection situation and can simulate a more comprehensive infection process, but its operation is relatively complicated and the cost is relatively high. Summary of the invention

[0004] The purpose of the present invention is to provide a method for establishing an intestinal dysfunction model using zebrafish.

[0005] The technical solution adopted by the present invention to solve the technical problem is: to provide a method for establishing an intestinal dysfunction model using zebrafish, comprising the following steps:

[0006] (1-1) Select zebrafish older than 5 dpf and raise them in zebrafish culture water at a culture temperature of 26-30°C;

[0007] (1-2) Different doses of Escherichia coli were injected into the intestines of zebrafish in the model experimental group to construct a zebrafish intestinal dysfunction model group.

[0008] Furthermore, it also includes:

[0009] (2-1) Setting up a normal control group, taking zebrafish of the same size as in step (1-1) and placing them in zebrafish culture water for culture as the normal control group;

[0010] (2-2) After 4-6 hours of treatment, the number of neutrophils in the normal control group and the model group was counted;

[0011] (2-3) After 40-56 h of treatment, the number of intestinal peristalsis of zebrafish in the normal control group and the model group was counted;

[0012] (2-4) Analyze the data and compare the differences between the normal control group and the model group; perform statistical analysis on the changes in the number of intestinal motility and the number of neutrophils. When the p-values ​​of the changes in the number of intestinal motility and the number of neutrophils between the normal control group and the model group are both less than 0.05, it indicates that the differences are statistically significant, indicating that the model was successfully constructed and a zebrafish intestinal dysfunction model was obtained.

[0013] Furthermore, the zebrafish selected in step (1-1) is a zebrafish of 5dpf to 7dpf.

[0014] Furthermore, the water quality conditions for zebrafish culture in step (1-1) are: 200 mg of instant sea salt is added to every 1 L of reverse osmosis water, the conductivity is 450-550 μS / cm; the pH value is 6.5-8.5; and the hardness is 50-100 mg / LCaCO3.

[0015] Furthermore, in step (1-1), the culture temperature is 28°C.

[0016] Furthermore, the different doses of Escherichia coli in step (1-2) include but are not limited to 10 2 CFU / tail, 10 3 CFU / tail, 10 4 CFU / tail, 10 5 CFU / tail and 10 6 CFU / tail.

[0017] The time for injecting E. coli in step (1-2) is selected between 10:00 and 11:30 of 5 dpf; the injection method is microinjection.

[0018] Furthermore, the zebrafish is selected from wild-type AB strain zebrafish or smooth muscle strain zebrafish.

[0019] Furthermore, the processing time in step (2-2) is 5 hours; and the processing time in step (2-3) is 48 hours.

[0020] As a model organism, zebrafish is widely used in the evaluation of intestinal models because of its similarities with humans in intestinal physiology and function. The following are several key points in the evaluation of intestinal models by zebrafish:

[0021] Gut microbiome research: Zebrafish and humans have certain similarities in the composition of their gut microbiota, making zebrafish an effective model for studying the interaction between the gut microbiome and host health. The zebrafish gut microbiome changes significantly during development, especially in the composition of the proteobacterial phylum.

[0022] Similarity of intestinal structure: The intestinal structure of zebrafish is highly homologous to that of mammals, including the proliferation and differentiation of intestinal epithelium, the formation of intestinal folds, and the distribution of intestinal microorganisms. These structural features make zebrafish a good model for studying intestinal function and disease.

[0023] Feasibility of genetic manipulation: Genetic manipulation of zebrafish is relatively easy, and specific functions of intestinal microorganisms can be studied through specific gene or microbiome manipulation, such as by introducing specific microorganisms into germ-free zebrafish larvae to explore the specific functions of intestinal microorganisms.

[0024] Highly conserved immune system: The zebrafish immune system is highly similar to that of humans, including two branches: specific immunity and nonspecific immunity. They have a variety of immune cell types, such as macrophages, neutrophils, and T cells, which play a role in immune responses, making zebrafish a powerful model for studying the human immune system.

[0025] The beneficial effects of the present invention are:

[0026] (1) The present invention is the first to use microinjection technology to directly inject Escherichia coli cells into the zebrafish intestine to establish a sepsis-induced intestinal dysfunction model, which is closer to the actual infection situation and can simulate a more comprehensive and complex sepsis pathological process.

[0027] (2) The present invention successfully constructed a highly accurate, scientific and reasonable zebrafish intestinal dysfunction model by optimizing the influencing factors such as modeling concentration, evaluation indexes and multi-index verification selection. Compared with other methods, this model has the advantage of a short modeling cycle, which greatly reduces the detection cost and facilitates wide promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the number of neutrophils in the zebrafish intestine after Escherichia coli induction of the present invention. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The technical solutions of the present invention are further described in detail below through specific embodiments.

[0030] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0031] Example:

[0032] 1. Materials and Methods

[0033] 1.1 Experimental Materials

[0034] Escherichia coli (E. coli): purchased from Guangdong Microbiological Culture Collection Center, stored at -20°C. Cultured at 30°C, 5% CO2, 150 rpm shaker, in LB medium.

[0035] Experimental animals: Wild-type AB strain zebrafish, raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to every 1L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / LCaCO3), bred and provided by our company's fish farming center, 5 days after fertilization (120 hpf), the experimental animal use license number is SYXK (Zhejiang) 2022-0004, and the breeding and management meets the requirements of the international AAALAC certification.

[0036] Wild-type AB zebrafish were bred by natural pair mating. Zebrafish aged 5 days post fertilization (120 hpf) were used in the sepsis-induced intestinal dysfunction model.

[0037] 1.2 Main instruments and reagents

[0038] Dissecting microscope (SZX7, OLYMPUS, Japan);

[0039] Microinjection instrument (IM300, Narishige, Japan);

[0040] Needle pulling instrument (PC-10, Narishige, Japan);

[0041] Motorized focusing and continuous zoom fluorescence microscope (AZ100, Nikon, Japan);

[0042] CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China);

[0043] Desktop large-capacity low-speed centrifuge (TD5A, Changsha Yingtai Instrument Co., Ltd., China);

[0044] Electronic turbidimeter (DensiCHEKTM Plus, bioMérieux, USA);

[0045] Constant temperature oscillator (HZ-9211K, Taicang Hualida Experimental Equipment Co., Ltd., China);

[0046] Vertical laminar flow clean bench (CA-1390-1, Shanghai Shangjing Purification Equipment Co., Ltd., China);

[0047] 6-well plate (Zhejiang Bellamy Biotechnology Co., Ltd., China).

[0048] 1.3 Reagents

[0049] Methylcellulose (Batch No. C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China);

[0050] LB medium (Batch No. 20220813, Qingdao Haibo Biotechnology Co., Ltd., China);

[0051] PBS (Cat. No. BL601A, Biosharp, China).

[0052] 2. Experimental Methods

[0053] 2.1 Zebrafish treatment

[0054] Wild-type AB strain zebrafish of 5 dpf (5 days after fertilization) were selected, and 10.0 nL of E. coli suspension containing different doses (see Table 1-2) was injected into the intestine of each zebrafish by microinjection. The injected zebrafish were randomly assigned to a 6-well plate, with 30 zebrafish placed in each well, and 3 mL of zebrafish culture water was added to serve as the model group. At the same time, a normal control group was set up. The zebrafish in the normal control group were not injected with E. coli, and the other conditions were consistent with the model group.

[0055] 2.2 Analysis of neutrophil count

[0056] After treatment at 28°C for 5 hours, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope to photograph the number of neutrophils. NIS-Elements D3.20 advanced image processing software was used to collect data and perform statistical analysis.

[0057] 2.3 Counting of intestinal peristalsis

[0058] After 48 hours of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope to record the intestinal peristalsis of the zebrafish. NIS-Elements D3.20 advanced image processing software was also used to collect video data, and the number of intestinal peristalsis was counted based on the video.

[0059] 2.4 Data Analysis

[0060] The data of neutrophil numbers and intestinal peristalsis times were expressed as mean ± SE and statistically analyzed using SPSS 26.0 software. If the p value was less than 0.05, the difference was considered statistically significant, indicating that the zebrafish sepsis-induced intestinal dysfunction model was successfully established.

[0061] 3. Results and Discussion

[0062] 3.1. Modeling and exploration

[0063] Under the experimental conditions, the highest dose of E. coli to induce zebrafish sepsis model was 10 4 CFU / tail. See Table 1-1 for details.

[0064]

[0065] Table 1-1. Results of the E. coli injection dose exploration experiment (n=30)

[0066] 3.2. Intestinal dysfunction model (phenotype)

[0067] Under the conditions of this experiment, E. coli can induce constipation in zebrafish. Figure 1 (Note: the green fluorescent particles in the figure are neutrophils) and Tables 1-1, 1-2, 1-3, and 1-4.

[0068]

[0069] Table 1-2. Experimental results of the number of intestinal peristalsis in zebrafish after induction with E. coli (n=10) As shown in Table 1-2, compared with the normal control group, **p<0.01, ***p<0.001.

[0070]

[0071] Table 1-3. Experimental results of the number of neutrophils in the zebrafish intestine after E. coli induction (n=10)

[0072] From 1-3, we can see that compared with the normal control group, ***p<0.001. 3 CFU / tail, 104 CFU / tail, the p values ​​were all less than 0.05, indicating that the zebrafish intestinal dysfunction model was successfully established.

[0073] Through statistical analysis of the above two main indicators, the number of neutrophils and the number of intestinal peristalsis, it is possible to comprehensively evaluate whether the zebrafish sepsis-induced intestinal dysfunction model is successfully constructed. This model can be used to further study the pathological mechanisms related to sepsis and drug screening in the process of new drug development.

[0074] 3.3. Intestinal dysfunction model (genetic verification)

[0075] 3.3.1 Sample preparation

[0076] Escherichia coli was transplanted into the intestine of 5dpf wild-type AB strain zebrafish by microinjection, with 10 4 CFU was used to establish a zebrafish sepsis model. The model zebrafish were randomly assigned to a 6-well plate, and 30 zebrafish were treated in each well (experimental group). The volume of each well was 3.00 mL, which was used as the model group. At the same time, a normal control group was set up. The zebrafish in the normal control group were not injected with E. coli, and the other conditions were consistent with the model group.

[0077] 3.3.2 Total RNA extraction and cDNA synthesis

[0078] After 2 days of treatment at 28°C, the total RNA of each group of zebrafish was extracted using an RNA rapid extraction kit, and the total RNA concentration and purity were measured using a UV-visible spectrophotometer. 2.00 μg of total RNA from zebrafish samples was taken and 20.0 μL of cDNA was synthesized according to the instructions of the cDNA first-strand synthesis kit.

[0079] 3.3.3q-PCR detection

[0080] The expression of β-actin, ahr1a, ahr2, cyp1a, kir2.2a and trpa1b genes was detected by q-PCR. β-actin was used as the internal reference for gene expression to calculate the relative RNA expression of ahr1a, ahr2, cyp1a, kir2.2a and trpa1b genes. The statistical results were expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0081]

[0082]

[0083] Table 1-4. Results of the evaluation experiment on the efficacy of samples in improving sepsis (genes) (n=3)

[0084] As shown in Tables 1-4, compared with the model group, *p<0.05, **p<0.01, ***p<0.001, indicating that the zebrafish intestinal dysfunction model was successfully constructed.

[0085]

[0086] Table 1-5. Results of the evaluation experiment on the efficacy of samples in improving sepsis (genes) (n=3)

[0087] As shown in Tables 1-5, compared with the model group, **p<0.01, ***p<0.001, indicating that the zebrafish intestinal dysfunction model was successfully constructed.

[0088] 3.3.4 Mechanisms of action of AHR signaling pathway, TRPA1 channel, and Kir2.2 and 2.1 potassium channels in sepsis-induced intestinal dysfunction in zebrafish

[0089] The AHR (aryl hydrocarbon receptor) signaling pathway plays an important role in the pathogenesis of sepsis. AHR is a ligand-activated transcription factor that is involved in regulating immune responses and maintaining intestinal barrier function. By regulating the AHR signaling pathway, the protective effect of intestinal epithelial cells can be enhanced and the inflammatory response can be reduced, thereby increasing the survival rate and improving the prognosis of patients with sepsis.

[0090] The TRPA1b (transient receptor potential ankyrin 1) channel is an ion channel expressed in a variety of cell types, which is involved in physiological processes such as sensing temperature changes and inflammatory responses. In terms of intestinal inflammation, TRPA1 may be activated by responding to inflammatory factors and oxidative stress products, thereby participating in the regulation of inflammatory responses. Studies have shown that inflammatory factors released after inflammation interact with TRPA1 to promote inflammatory responses, and TRPA1 also regulates the function of immune cells.

[0091] The mechanisms of action of AhR (aryl hydrocarbon receptor) in intestinal inflammation are interconnected. AhR is a transcription factor that can be activated by binding to its ligand, thereby regulating the expression of a series of downstream genes, including CYP1A1. CYP1A1 is a key enzyme in the AhR signaling pathway and is involved in the metabolism of a variety of compounds, including endogenous and exogenous substances. Studies have shown that CYP1A1 may play a role during sepsis by enhancing the inflammatory response and preventing the phagocytosis of bacteria in macrophages.

[0092] Kir2.2 is an inward rectifier potassium channel that plays an important role in regulating cell membrane potential. Although Kir2.2 is mainly expressed in cardiac tissue, studies have shown that Kir2.1 is a homologous channel of Kir2.2. Although these studies have focused on Kir2.1, due to the similarities between Kir2.2 and Kir2.1 in structure and function, it plays an important role in the activation of immune cells and inflammatory responses.

[0093] In inflammatory diseases, the activation of macrophages is closely related to the dynamic changes of intracellular metabolic pathways. In particular, Kir2.1 regulates the inflammatory response by maintaining the resting membrane potential of macrophages and affecting the cell's access to nutrients. Studies have found that the activity of Kir2.1 is essential for the inflammatory function of macrophages. The inactivation of Kir2.1 leads to cell membrane depolarization, which in turn affects the dynamics of cell membrane phospholipids, leading to the internalization of nutrient transport channels, reducing the external nutrient supply necessary during inflammation, and ultimately inhibiting inflammatory function.

[0094] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. A method for establishing an intestinal dysfunction model using zebrafish, characterized in that: The following steps are involved: (1-1) Select zebrafish older than 5 dpf and raise them in zebrafish culture water at a culture temperature of 26-30°C; (1-2) Different doses of Escherichia coli were injected into the intestines of zebrafish in the model experimental group to construct a zebrafish intestinal dysfunction model group.

2. The method for establishing an intestinal dysfunction model using zebrafish according to claim 1, characterized in that: Also includes: (2-1) Setting up a normal control group, taking zebrafish of the same size as in step (1-1) and placing them in zebrafish culture water for culture as the normal control group; (2-2) After 4-6 hours of treatment, the number of neutrophils in the normal control group and the model group was counted; (2-3) After 40-56 h of treatment, the number of intestinal peristalsis of zebrafish in the normal control group and the model group was counted; (2-4) Analyze the data and compare the differences between the normal control group and the model group; perform statistical analysis on the changes in the number of intestinal motility and the number of neutrophils. When the p-values ​​of the changes in the number of intestinal motility and the number of neutrophils between the normal control group and the model group are both less than 0.05, it indicates that the differences are statistically significant, indicating that the model was successfully constructed and a zebrafish intestinal dysfunction model was obtained.

3. The method for establishing an intestinal dysfunction model using zebrafish according to claim 1, characterized in that: The zebrafish selected in step (1-1) are 5dpf to 7dpf zebrafish.

4. The method for establishing an intestinal dysfunction model using zebrafish according to claim 1, characterized in that: The water quality conditions for zebrafish culture in step (1-1) are as follows: 200 mg of instant sea salt is added to every 1 L of reverse osmosis water, the conductivity is 450-550 μS / cm; the pH value is 6.5-8.5; and the hardness is 50-100 mg / LCaCO3.

5. The method for establishing an intestinal dysfunction model using zebrafish according to claim 1, characterized in that: In step (1-1), the culture temperature is 28°C.

6. The method for establishing an intestinal dysfunction model using zebrafish according to claim 1, characterized in that: Step (1-2) includes but is not limited to 10 2 CFU / tail, 10 3 CFU / tail, 10 4 CFU / tail, 10 5 CFU / tail and 10 6 CFU / tail. The time for injecting E. coli in step (1-2) is selected between 10:00 and 11:30 of 5 dpf; the injection method is microinjection.

7. The method for establishing an intestinal dysfunction model using zebrafish according to claim 1, characterized in that: The zebrafish was selected from wild-type AB strain zebrafish or smooth muscle strain zebrafish.

8. The method for establishing an intestinal dysfunction model using zebrafish according to claim 2, characterized in that: The treatment time in step (2-2) is 5 h; the treatment time in step (2-3) is 48 h.

Images