Construction method and application of caenorhabditis elegans inflammation model

Abstract

The invention discloses a construction method and application of a caenorhabditis elegans inflammation model, belongs to the technical field of model biology, and provides a construction method of a caenorhabditis elegans inflammation model, which comprises the following steps: putting L1-stage caenorhabditis elegans into a lipopolysaccharide solution, and carrying out induced culture to obtain induced cultured caenorhabditis elegans; placing the caenorhabditis elegans in a chromosome system, performing staining culture and centrifugation to obtain dyed caenorhabditis elegans, and placing the dyed caenorhabditis elegans on a culture medium for recovery to obtain the caenorhabditis The surface of the caenorhabditis elegans is provided with a transparent cuticle anatomical structure, the intestinal barrier damage condition is easy to observe after the intestinal tract is dyed by a dye, and the cell proliferation, tissue development and tissue integrity of the caenorhabditis elegans are easy to carry out fluorescence microscope and morphological analysis. A caenorhabditis elegans inflammation model is constructed through lipopolysaccharide induction, and a new experimental animal model is provided for screening anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention belongs to the field of model biological technology, and in particular relates to a construction method and application of a Caenorhabditis elegans inflammation model. Background Art

[0002] Caenorhabditis elegans ( Caenorhabditis elegans ), a tiny nematode with a body length of only about 1mm, was the first multicellular eukaryotic organism whose genome was completely sequenced in the late 1990s. C. elegans has the characteristics of clear genetic background, simple individual structure, high reproduction rate, short life cycle, and easy culture conditions, which make it a major genetic model organism. It is reported that 60% to 80% of human genes have homologous genes in the C. elegans genome. As a self-fertilized hermaphrodite, C. elegans has practical advantages in experimental biology and genetic screening. It can verify the accuracy of experiments in large quantities, and the cost of breeding and preservation is low. It survives for about 21 days at 20°C, which can greatly shorten the test cycle. Therefore, C. elegans is an excellent model for studying aging-related diseases and drug toxicity.

[0003] Inflammation is an immune response produced by the body. When the body is infected or exposed to endotoxins such as lipopolysaccharide (LPS), the balance of the body will be disturbed. Acute inflammation occurs when tissues are injured or infected, but in the case of continuous stimulation, acute inflammation will develop into chronic inflammation, leading to a series of diseases.

[0004] When Caenorhabditis elegans is infected by pathogens and inflammation is induced, the oxidative balance in the body is disrupted, resulting in impaired intestinal barrier function. Along with the oxidative imbalance, a large amount of ROS is produced, which further damages the intestinal barrier function and aggravates the inflammatory response. The immune system of Caenorhabditis elegans is based on several signaling cascades, including a Toll-like receptor, three mitogen-activated protein kinases (MAPKs), a transforming growth factor-β (TGF-β), insulin-like receptors (ILRs), and programmed cell death (PCD) pathways. Infection of Caenorhabditis elegans with pathogenic bacteria, fungi, and viruses triggers pathogen-specific gene expression programs in many tissues in the body. The expression of upstream signaling pathways has promoted the discovery and characterization of multiple evolutionarily conserved signaling pathways. Therefore, pathways mediated by p38 MAPK, GATA transcription factors, and oxidative stress responses play an important role in different infection situations in Caenorhabditis elegans. A large number of studies have found that after Caenorhabditis elegans ingests pathogenic bacteria and triggers inflammation, it relies on its simplified intestinal epithelium to fight infection and further triggers the transcription of genes encoding antimicrobial peptides and enzymes. The expression of these genes increases the host's survival rate during infection. The p38 and ERK protein kinase genes of the MAPK pathway in Caenorhabditis elegans are essential for the regulation of immune and inflammatory signals in the body and are highly homologous to human homologs. In addition, studies have found that Caenorhabditis elegans mainly achieves inflammation regulation by activating PMK-1 / P38 MAPKS and upregulating GATA's transcription factor ELT-2.

[0005] At present, there is no patent for using LPS to induce the construction of a Caenorhabditis elegans inflammation model. There is insufficient data to support the selection of concentrations for Caenorhabditis elegans inflammation modeling, and there are no systematic reports on using Caenorhabditis elegans as an animal model for studying inflammation. Summary of the invention

[0006] In order to solve the above technical problems, the present invention proposes a method for constructing and applying a Caenorhabditis elegans inflammation model, and constructs a Caenorhabditis elegans inflammation model by inducing different concentrations of lipopolysaccharide (LPS), thereby providing a new experimental animal model for screening anti-inflammatory drugs.

[0007] To achieve the above-mentioned purpose, the present invention provides a method for constructing a Caenorhabditis elegans inflammation model, comprising the following steps: placing L1 stage Caenorhabditis elegans in a lipopolysaccharide solution with a concentration of 0.125-1 μg / mL, inducing culture, and obtaining induced cultured Caenorhabditis elegans; placing the induced cultured Caenorhabditis elegans in a staining system, staining culture, centrifuging, and obtaining stained Caenorhabditis elegans; placing the stained Caenorhabditis elegans on a culture medium for recovery, and obtaining a Caenorhabditis elegans inflammation model.

[0008] Preferably, the induction culture temperature is 18-22° C., and the induction culture time is 20-28 h.

[0009] Preferably, the staining system is prepared by mixing Escherichia coli solution and brilliant blue solution in a volume ratio of 1:1.

[0010] Further preferably, the Escherichia coli bacterial solution is Escherichia coli OP50 bacterial solution, and the mass concentration of the brilliant blue solution is 10%.

[0011] Preferably, the temperature of the dyeing culture is 18-22° C., and the time of the dyeing culture is 2-4 hours.

[0012] Preferably, the centrifugal temperature is 18-22° C., the centrifugal speed is 2000-2400 rpm, and the centrifugal time is 1-3 min.

[0013] Preferably, the dyeing degree of the stained Caenorhabditis elegans is full body cavity dyeing, and the full body cavity dyeing means that the dye permeates from the intestine into the body cavity, the dye spreads throughout the body cavity, and the dye cannot be discharged through the recovery process.

[0014] Preferably, the culture medium is NGM culture medium, the recovery temperature is 18-22° C., and the recovery time is 40-50 min.

[0015] Preferably, the method further comprises verifying the construction of the Caenorhabditis elegans inflammation model, and measuring the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity of the Caenorhabditis elegans inflammation model.

[0016] The present invention also provides application of the Caenorhabditis elegans inflammation model constructed by the construction method in screening ingredients with efficacy in treating digestive system inflammation.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention utilizes the anatomical structure of the transparent stratum corneum on the surface of Caenorhabditis elegans, the characteristics of the intestinal barrier damage that can be easily observed after the intestine is stained with dyes, and the advantages of being convenient for fluorescence microscopy and morphological analysis of cell proliferation, tissue development and tissue integrity, and constructs a Caenorhabditis elegans inflammation model by LPS induction, providing a new experimental animal model for screening anti-inflammatory drugs. By intuitively judging the intestinal permeability of inflammatory Caenorhabditis elegans and measuring the pro-inflammatory cytokine TNF-α, the optimal LPS concentration for constructing the inflammation model is determined. Fluorescence microscopy is then used to measure the fluorescence intensity of reactive oxygen species (ROS), glutathione (GSH) and lipofuscin in Caenorhabditis elegans, as well as the malondialdehyde content (MDA) and superoxide dismutase (SOD) activity in Caenorhabditis elegans, verifying the effect of LPS induction on oxidative stress indicators in inflammatory Caenorhabditis elegans.

[0018] LPS is mainly transferred to the intestinal system of C. elegans through the pharynx, which not only increases intestinal permeability, but also is accompanied by excessive production of ROS and excessive consumption of GSH, thus leading to redox imbalance in C. elegans and exacerbating inflammatory response. After induction with LPS at a concentration of 0.5 μg / mL, it was found that the fluorescence intensity of ROS and lipofuscin was significantly enhanced compared with the normal group, which can effectively reduce the level of GSH and promote the production of ROS and lipofuscin in C. elegans, effectively causing redox imbalance in C. elegans. At the same time, the content of MDA, a biomarker of oxidative stress, was significantly increased in LPS-induced C. elegans. SOD is the main antioxidant enzyme in C. elegans. After LPS induction, the SOD activity in C. elegans was lower than that in the normal group of C. elegans, indicating that LPS caused the accumulation of MDA in C. elegans and reduced the activity of SOD, thereby causing oxidative stress damage and inflammatory damage in C. elegans. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The staining degree judgment standard of intestinal permeability damage in Caenorhabditis elegans, where A means no staining in the intestine, B means staining inside the intestine, C means staining outside the intestine, and D means staining in the whole body cavity; Figure 2 The figure is the effect of different concentrations of lipopolysaccharide on the TNF-α content in Caenorhabditis elegans, in which a, b, c, d and e indicate significant differences; Figure 3 It is a statistical diagram of the percentage of damaged intestinal permeability after different concentrations of lipopolysaccharide were induced in Caenorhabditis elegans; Figure 4 The effects of different concentrations of lipopolysaccharide on reactive oxygen species, glutathione and lipofuscin in Caenorhabditis elegans are shown in Figure 2. Figure 5 The figures show the effects of different concentrations of lipopolysaccharide on the levels of malondialdehyde and superoxide dismutase in Caenorhabditis elegans, where A shows the effect on the malondialdehyde content in Caenorhabditis elegans, and B shows the effect on the superoxide dismutase content in Caenorhabditis elegans. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the contents of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] Source of materials used in the present invention: Escherichia coli Escherichia coli OP50 was obtained from Sichuan University of Science and Chemical Engineering; LB medium was purchased from Sinopharm Chemical Reagent Co., Ltd.; animal cell protein lysis buffer kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.; enzyme-linked immunosorbent assay for TNF-α was purchased from Beijing Institute of Optical Analysis; malondialdehyde kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.; and superoxide dismutase kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0027] Example 1. Cultivation and Synchronization of Caenorhabditis elegans 1.1 Escherichia coli E. coli Preparation of OP50 bacterial culture Escherichia coli Escherichia coliPreparation of OP50: First, E. coli After OP50 was spread, cultured and streaked, a single colony was picked and inoculated into LB liquid culture medium and cultured at 37°C for 16 h. E. coli OP50 was placed in a 65°C water bath for 30 min, and the cells were collected by centrifugation at 5000 rpm for 3 min, resuspended in sterile water and stored in a 4°C refrigerator for later use.

[0028] 1.2 Preparation of main reagents Preparation of NGM medium: (1) Weigh 3g NaCl, 17g agar and 2.5g peptone, add 975mL ultrapure water and mix well, then sterilize at 121℃ for 20min. Place the cooled culture medium in a clean bench, add 25mL of 1mol / L potassium phosphate buffer, 1mL of 1mol / L MgSO4 solution, 1mL of 1mol / L CaCl2 solution, and 1mL of 5mg / mL cholesterol solution, and shake gently to mix. Pour the mixed culture medium into the culture dish quickly, and the volume of the liquid should account for about 2 / 3 of the total volume of the culture dish.

[0029] (2) After the agar in the culture dish solidifies, place the dish upside down in the clean bench for 24 hours.

[0030] (3) Bacteria coating: absorb 150mL E. coli Drop OP50 bacterial solution to the center of the culture medium and spread it evenly with an applicator. During the bacterial spreading process, be careful not to let the triangular rod touch the inner wall of the culture dish, and keep the bacterial circle about 1 cm away from the inner wall of the culture dish. Dry it in a clean bench and irradiate it with ultraviolet light for 60 minutes. After the bacterial solution is completely attached, turn the culture dish upside down and store it in a 4°C refrigerator for later use.

[0031] Preparation of potassium phosphate buffer solution (1 mol / L): weigh 54.1 g KH2PO4 and 17.8 g K2HPO4, add ultrapure water, mix well, and make up to 500 mL. Autoclave at 121°C for 20 min.

[0032] Preparation of M9 buffer: weigh 2.5g NaCl, 3g Na2HPO4, 1.5g KH2PO4, add ultrapure water to 500mL, and sterilize by high pressure at 121℃ for 20min. After the solution cools, add 1mL MgSO4 solution (1mol / L) in the clean bench, shake and mix well for later use.

[0033] Preparation of K solution: weigh 1.5g NaCl and 1.18g KCl, add ultrapure water, mix well and make up to 500mL, sterilize at 121℃ for 20min and set aside.

[0034] Preparation of C. elegans lysis solution: weigh 0.5g sodium hypochlorite, add 5mL sodium hydroxide solution (1M) and mix well, then add 5mL sterile water and mix well. This lysis solution should be prepared and used immediately, because sodium hypochlorite is easily decomposed by light.

[0035] 1.3 Subculture of Caenorhabditis elegans Agar block transfer method: Sterilize the scalpel by burning it with an alcohol lamp. After cooling, cut a small square (1 cm × 1 cm) of culture medium containing Caenorhabditis elegans and place it upside down on a plate coated with E. coli On new NGM medium plates with OP50, Caenorhabditis elegans will autonomously crawl onto the new plates to obtain food.

[0036] 1.4 Synchronized culture of Caenorhabditis elegans Rinse the plate with 1 mL of M9 buffer and collect the L4 stage Caenorhabditis elegans into a 1.5 mL sterile EP tube. Centrifuge the solution containing Caenorhabditis elegans at 2200 rpm for 2 min at 4 ° C, discard the supernatant to obtain the Caenorhabditis elegans precipitate, and wash the worm twice with M9 buffer under the same conditions as above. Resuspend the Caenorhabditis elegans precipitate in 1 mL of lysis buffer, gently shake it up and down several times, and then break the worm body by alternating vortex shaking for 10s and 15s twice, for a total of 5 minutes. Subsequently, add M9 buffer to terminate the reaction (change to a large centrifuge tube), centrifuge at 2200 rpm and 20 ° C for 2 min, and remove the supernatant. Add M9 buffer again and wash repeatedly 5 times until there is no smell of hypochlorous acid. Use a pipette to transfer the washed Caenorhabditis elegans eggs to the sterile solid NGM culture medium and place it in a constant temperature incubator at 20 ° C for 16 hours to obtain Caenorhabditis elegans at the same growth and development level L1 stage for subsequent related experiments.

[0037] 2. Lipopolysaccharide induces intestinal inflammation in Caenorhabditis elegans Lipopolysaccharide (LPS) is the most common drug for inflammation modeling. It can cause inflammatory response by binding to host cell surface receptors and activating inflammatory cytokine gene expression. LPS was used to induce the intestinal inflammation model of Caenorhabditis elegans, and the LPS concentrations were set to 0μg / mL, 0.03125μg / mL, 0.125μg / mL, 0.5μg / mL, 1μg / mL, and 1.5μg / mL. Dilute the 3mg / mL LPS stock solution with K solution to the above concentrations, and pipette 200μL into each 48-well plate. Pick 20 synchronized Caenorhabditis elegans from each well plate, and repeat each concentration gradient experiment 3 times. Finally, seal the surface of the well plate with a breathable sealing film and induce culture in a constant temperature and humidity incubator at 20℃ for 24h.

[0038] 3. Determination of Inflammatory Factors in Caenorhabditis elegans Protein was extracted according to the animal cell protein lysis buffer kit. 1 mL of mixed protein lysis buffer was added to the Caenorhabditis elegans for homogenization and ultrasonic treatment on ice, and then centrifuged at 12000×g for 5 min at 4°C. The content of tumor necrosis factor (TNF-α) was determined by enzyme-linked immunosorbent assay.

[0039] LPS is a Gram-negative bacteria (G - ) is the main component of the cell wall and is often used to construct animal intestinal inflammation models. Figure 2 As shown in the figure, with the increase of LPS concentration, the content of inflammatory factor TNF-α in C. elegans was significantly upregulated. When the LPS concentration was 0μg / mL, 0.03125μg / mL, 0.125μg / mL, 0.5μg / mL, 1μg / mL and 1.5μg / mL, the corresponding content of inflammatory factor TNF-α in C. elegans was 285.64±9.43pg / mL, 346.43±2.62pg / mL, 428.09±8.92pg / mL, 433.97±6.95pg / mL, 490.08±11.01pg / mL and 625.72±2.92pg / mL, respectively. The significant increase in the level of inflammatory factor TNF-α proves that the inflammatory response is intensifying, which shows that LPS can effectively induce inflammation in C. elegans.

[0040] 4. Determination of Intestinal Permeability in Caenorhabditis elegans After inducing C. elegans with different concentrations of LPS for 24 hours, the induced cultured C. elegans were transferred to a 200 μL staining system consisting of 100 μL of E. coli OP50 solution and 100 μL of brilliant blue solution (10%, w / v). C. elegans were stained and cultured for 3 hours at 20°C. There were 20 C. elegans in each group, and the experiment was repeated three times. After the staining was completed, the cells were centrifuged at 2200 rpm and 20°C for 2 minutes. After the dye on the surface of the worm body was cleaned, the stained C. elegans were placed on NGM culture medium to recover for 45 minutes. Since C. elegans is transparent, the distribution of the dye in the intestinal cavity and body cavity can be clearly observed. If the intestine of C. elegans is not damaged, the dye that enters the body with food will be excreted during the recovery process or only a small amount will remain in the intestine. If the intestine of C. elegans is damaged, the brilliant blue dye will leak out of the intestine into the body cavity. The recovery process can expel the excess dye from the body, but the dye that has entered the body cavity will still remain in the body cavity. According to the distribution location and degree of staining of the brilliant blue dye in the intestine, it can be divided into intestinal no staining, intestinal staining, intestinal staining and body cavity full staining (such as Figure 1 Middle A, Figure 1 Middle B, Figure 1 Middle C and Figure 1The percentage of C. elegans with intestinal permeability damage was calculated as follows: the number of C. elegans with no intestinal infection, intestinal infection, extraintestinal infection, and full body cavity infection was counted, and the proportion of the total number of C. elegans in the experiment was counted.

[0041] Unstained intestine: When the intestine of Caenorhabditis elegans is not damaged, the brilliant blue dye enters the body with food and is excreted during the recovery process, and no brilliant blue dye is observed in the intestine.

[0042] Intestinal dyeing: When the intestine of C. elegans is not damaged, the brilliant blue dye enters the body with food and is not excreted or not completely excreted during the recovery process, and brilliant blue dye is observed in the intestine.

[0043] Extraintestinal dyeing: When the intestine of C. elegans is damaged, the brilliant blue dye penetrates from the intestine into the outer layer of the intestine, the brilliant blue dye is observed outside the intestine, and it cannot be excreted through the recovery process.

[0044] Full body cavity staining: When the intestine of Caenorhabditis elegans is damaged, the brilliant blue dye seeps from the intestine into the body cavity. At this time, the brilliant blue dye spreads throughout the body cavity and cannot be excreted through the recovery process.

[0045] LPS can cause oxidative stress in C. elegans and damage its intestinal permeability. Different concentrations of LPS were used to induce C. elegans, and the degree of damage to the intestinal permeability of C. elegans was used to evaluate whether the inflammation model was successfully established. Figure 3As shown in the figure, after 24 hours of induction with different concentrations of LPS, the low-dose group of LPS had no effect on the intestinal barrier function of Caenorhabditis elegans, and there was almost no intestinal extra-intestinal staining or total body cavity staining in Caenorhabditis elegans. The proportion of damaged intestinal permeability in Caenorhabditis elegans increased with the increase of LPS concentration. At 0.125μg / mL LPS, the intestine of Caenorhabditis elegans began to be damaged, resulting in intestinal extra-intestinal staining, indicating that the intestinal barrier was damaged and caused the leakage of brilliant blue dye. And at 0.5μg / mL LPS, total body cavity staining began to appear, and the brilliant blue dye diffused throughout the body of Caenorhabditis elegans, and with the increase of LPS concentration, the proportion of total staining in Caenorhabditis elegans also gradually increased. The higher the proportion of extra-intestinal staining and total body cavity staining, the higher the concentration of LPS, and the stronger the ability of Caenorhabditis elegans to damage the intestine. The results of intestinal damage in six groups of LPS concentrations are as follows: 0μg / mL LPS group (0%), 0.03125μg / mL LPS group (0%), 0.125μg / mL LPS group (9.77%), 0.5μg / mL LPS group (32.91%), 1μg / mL LPS group (45.45%), 1.5μg / mL LPS group (68.18%). The proportion of intestinal damage in C. elegans at a concentration of 0.5μg / mL LPS is 32.91%, proving that C. elegans has an inflammatory effect but little damage at this concentration of LPS. Therefore, the present invention intends to use LPS at a concentration of 0.5μg / mL to construct a C. elegans intestinal inflammation model.

[0046] 5. Determination of Reactive Oxygen Species, Glutathione and Lipofuscin in Caenorhabditis elegans After 24 hours of induction with different concentrations of LPS, the induced cultured Caenorhabditis elegans were transferred to 200 μL of fluorescent probe dye. The fluorescent probe dye for reactive oxygen species (ROS) was prepared by K solution with a concentration of 20 μM 2,7-dichlorofluorescein diacetate (DCFH-DA) solution, which was cultured at 20°C in the dark for 3 hours. The fluorescent probe dye for glutathione (GSH) was a 40 μM 2,3-naphthalenedicarbaldehyde (NDA) solution, which was cultured at 20°C in the dark for 2.5 hours. The Caenorhabditis elegans in the well plate were then quickly washed three times with M9 buffer to fully wash the dye. The Caenorhabditis elegans were then anesthetized with 5 mM levamisole, placed on a 2% agarose pad, and photos were quickly collected through the FITC channel of a fluorescence microscope. Lipofuscin can spontaneously fluoresce, so there is no need to perform any staining on C. elegans. The excitation wavelength of the fluorescence microscope is adjusted to 510-560nm, and the emission wavelength is adjusted to 590-650nm. A single C. elegans is observed and photographed to determine lipofuscin.

[0047] The redox balance in C. elegans mainly depends on the balance between ROS and GSH. LPS is mainly transferred to the intestinal system of C. elegans through the pharynx, which not only increases the permeability of the intestine, but is also accompanied by excessive production of ROS and excessive consumption of GSH, thus leading to redox imbalance in C. elegans and aggravating the inflammatory response. The NDA probe can quickly diffuse into the body of C. elegans and specifically bind to GSH under physiological conditions, reflecting the GSH content through green fluorescence intensity, while ROS can cause DCFH-DA cleavage, resulting in increased fluorescence intensity. Figure 4 As shown, after induction with 0.5 μg / mL LPS, the average fluorescence intensity of the control group (0 μg / mL LPS) was 1.18±0.28, and the average fluorescence intensity of the induced group (0.5 μg / mL LPS) was 0.67±0.09. At the same time, the fluorescence intensity of ROS (3.59±0.17) and lipofuscin (9.62±0.55) in the 0.5 μg / mL LPS group was significantly enhanced compared with that in the control group (1.08±0.06) and lipofuscin (2.56±0.17). This indicates that 0.5 μg / mL LPS-induced inflammation in C. elegans can effectively reduce GSH levels and promote the production of ROS and lipofuscin in C. elegans, effectively causing an imbalance in the redox reaction in C. elegans.

[0048] 6. Determination of Malondialdehyde and Superoxide Dismutase Content in Caenorhabditis elegans After inducing with different concentrations of LPS for 24 hours, the induced Caenorhabditis elegans were collected, washed and centrifuged with M9 solution, and 1 mL of extract at 4°C was added. The extract was homogenized on ice for 10 minutes using a homogenizer. After the extract was collected, the freeze-thaw cycle was repeated 3 times. The malondialdehyde (MDA) and superoxide dismutase (SOD) kit instructions were followed for determination. The results were quantified as total protein concentration and measured using an ultra-micro visible spectrophotometer.

[0049] To further verify whether LPS induction can effectively build inflammation in C. elegans and cause oxidative stress damage, the present invention measured the MDA content and SOD activity of C. elegans. As a biomarker of oxidative stress, the MDA content in C. elegans increased significantly after LPS induction (e.g. Figure 5 SOD is the main antioxidant enzyme in C. elegans. After LPS induction, the activity of SOD in C. elegans was significantly reduced (as shown in Figure 2A). Figure 5 This proves that LPS induces the accumulation of MDA in C. elegans, significantly increases the MDA content, and reduces the activity of SOD, thereby causing oxidative stress damage to the body and aggravating inflammatory damage in C. elegans.

[0050] The methods for constructing C. elegans inflammation models in the prior art are divided into LPS induction and pathogenic bacteria infection (Pseudomonas aeruginosa, Salmonella typhimurium, etc.) infection methods. Studies on the existing LPS-induced C. elegans inflammation model show that the LPS concentration selection range is between 0.4μg / mL and 100μg / mL, and the death of C. elegans is mainly judged by the movement reaction and swallowing action when touched, and there is still a lack of effective experimental data support; while the pathogenic bacteria construct the C. elegans inflammation model based on the bacterial toxins produced by the pathogenic bacteria to act on the body to cause inflammation, and the strength of the infection is uncertain. The present invention further provides a method for intuitively judging the intestinal damage of C. elegans and evaluating whether the C. elegans inflammation model is successful based on the obtained experimental support data, and the indicator support system provides a more efficient and convenient experimental animal model for screening anti-inflammatory drugs.

[0051] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a Caenorhabditis elegans inflammation model, characterized in that: The following steps are involved: L1 stage Caenorhabditis elegans are placed in a lipopolysaccharide solution with a concentration of 0.125-1 μg / mL, induced and cultured to obtain induced and cultured Caenorhabditis elegans; the induced and cultured Caenorhabditis elegans are placed in a staining system, stained and cultured, and centrifuged to obtain stained Caenorhabditis elegans; the stained Caenorhabditis elegans are placed on a culture medium to recover, and a Caenorhabditis elegans inflammation model is obtained.

2. The construction method according to claim 1, characterized in that: The induction culture temperature is 18-22° C., and the induction culture time is 20-28 h.

3. The construction method according to claim 1, characterized in that: The staining system is prepared by mixing Escherichia coli solution and brilliant blue solution in a volume ratio of 1:

1.

4. The construction method according to claim 3, characterized in that: The Escherichia coli bacterial solution is Escherichia coli OP50 bacterial solution, and the mass concentration of the brilliant blue solution is 10%.

5. The construction method according to claim 1, characterized in that: The temperature of the dyeing culture is 18-22° C., and the time of the dyeing culture is 2-4 hours.

6. The construction method according to claim 1, characterized in that: The centrifugal temperature is 18-22° C., the centrifugal speed is 2000-2400 rpm, and the centrifugal time is 1-3 min.

7. The construction method according to claim 1, characterized in that: The dyeing degree of the stained Caenorhabditis elegans is full body cavity dyeing, and the full body cavity dyeing means that the dye permeates from the intestine into the body cavity, the dye spreads throughout the body cavity, and the dye cannot be discharged through the recovery process.

8. The construction method according to claim 1, characterized in that: The culture medium is NGM culture medium, the recovery temperature is 18-22° C., and the recovery time is 40-50 min.

9. The construction method according to claim 1, characterized in that: The method also includes verifying the construction of a Caenorhabditis elegans inflammation model and measuring the malondialdehyde content and superoxide dismutase activity of the Caenorhabditis elegans inflammation model.

10. Use of the Caenorhabditis elegans inflammation model constructed by the construction method according to any one of claims 1 to 9 in screening ingredients with efficacy in treating digestive system inflammation.

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