Construction method and application of caenorhabditis elegans hyperlipemia model

Abstract

The invention belongs to the technical field of biology, and relates to a construction method and application of a caenorhabditis elegans hyperlipemia model. The construction method of the caenorhabditis elegans hyperlipemia model comprises the following steps: S1, putting eggs obtained by synchronization into a culture medium without an escherichia coli culture solution, and culturing at constant temperature to obtain L1-stage caenorhabditis elegans; s2, transferring the L1-stage caenorhabditis elegans into a culture medium containing an escherichia coli culture solution and glucose, and culturing at constant temperature to obtain L4-stage caenorhabditis elegans; s3, transferring the L4-stage caenorhabditis elegans into a culture medium containing FUDR, an escherichia coli culture solution and glucose, and performing constant-temperature culture to obtain the adult-stage caenorhabditis elegans hyperlipemia model. The caenorhabditis elegans hyperlipemia model constructed by adopting an innovative staged culture strategy has the advantages of high stability, simplicity and convenience in operation, good repeatability and the like, and the lipid-lowering potential of active substances can be quickly screened and verified.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a construction method and application of a Caenorhabditis elegans hyperlipidemia model. Background Art

[0002] Hyperlipidemia refers to abnormally high levels of cholesterol and triglycerides in the blood, and is widely considered to be an important risk factor for cardiovascular disease. Its onset is usually accompanied by metabolic disorders, which are mainly affected by poor eating habits, genetic factors, and lack of exercise. In addition, hyperlipidemia often causes complications such as obesity, intestinal flora imbalance, and lipid metabolism disorders. Regulating blood lipid levels has become one of the key strategies for the treatment and prevention of hyperlipidemia, especially for intervention against peripheral fat accumulation and insulin resistance caused by long-term high-fat and high-sugar diets. This type of dietary pattern not only induces inflammatory responses, but also aggravates lipid metabolism imbalances, thereby promoting the occurrence of metabolic diseases. Therefore, reducing the incidence of hyperlipidemia has important social and health value.

[0003] Research on the lipid-lowering effects of food and medicine has developed rapidly in recent years. The lipid-lowering active ingredients of some food and medicine products have been identified, while for products whose active ingredients have not yet been identified, research usually focuses on the effects of their extracts. In these studies, efficacy evaluation is a key link, which not only provides a scientific basis for the development of active substances and drugs, but also lays the foundation for the screening of functional ingredients and in-depth analysis of the mechanism of action. As an alternative animal model, Caenorhabditis elegans provides a rapid and economical research advantage over traditional mammalian models, and is expected to accelerate technological breakthroughs in this field and promote the efficient use of research resources.

[0004] Caenorhabditis elegans is a model organism with a small body, short life cycle and simple structure. Its transparent body wall and highly organized characteristics facilitate precise positioning and imaging analysis using fluorescent dyes. The intestine of nematodes functions similarly to the liver of mammals in lipid synthesis and storage, and is its metabolic center and immune organ. Currently, the genome of Caenorhabditis elegans has been fully sequenced, and a total of 471 lipid metabolism-related genes have been identified, which have 60% homology with human genes. Therefore, Caenorhabditis elegans has become one of the classic models for studying lipid metabolism regulation and related diseases (such as obesity, Alzheimer's disease and hyperlipidemia).

[0005] However, in studies based on Caenorhabditis elegans, there are significant differences in the evaluation of the lipid-lowering effects of active substances in different experimental designs. Traditional methods usually analyze nematodes directly after culturing from the L1 stage to the L4 stage. The results may be biased towards developmental characteristics and it is difficult to fully reflect the physiological state of the adult stage and the long-term effects of active substances. At the same time, the short culture cycle may ignore the cumulative effects of lipid metabolism disorders and oxidative stress, affecting the accuracy of the results. In addition, processing samples from the L4 stage is likely to miss the early intervention window, making it difficult to observe the preventive role of active substances in lipid metabolism regulation, and may miss its dynamic effects on nematode reproduction, metabolic regulation and aging-related indicators.

[0006] Therefore, an improved model construction method is urgently needed to more comprehensively evaluate the lipid-lowering effects of active substances and reveal their potential mechanisms. Summary of the invention

[0007] The present invention innovatively adopts a phased culture strategy to expose Caenorhabditis elegans to a high-concentration glucose environment to simulate the hyperlipidemia state caused by excessive lipid accumulation; subsequently, by detecting the lipid droplet storage level, triglyceride and free fatty acid content, and expression changes of lipid metabolism-related genes in the nematodes treated with dietary active substances or drugs, the lipid-lowering efficacy of the active substances or drugs is accurately evaluated; the Caenorhabditis elegans hyperlipidemia model constructed by the present invention has the advantages of high stability, simple operation, good repeatability, etc., and can quickly screen and verify the lipid-lowering potential of active substances.

[0008] To achieve this technical purpose, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for constructing a hyperlipidemia model of Caenorhabditis elegans, the method comprising the following steps:

[0010] S1. The synchronized eggs are placed in a culture medium without E. coli culture medium, and cultured at a constant temperature for 9-12 hours to obtain L1 stage Caenorhabditis elegans;

[0011] S2. transferring the L1 stage Caenorhabditis elegans to a culture medium containing Escherichia coli culture medium and glucose, and culturing at a constant temperature for 24 hours until the nematodes develop to the L4 stage, thereby obtaining L4 stage Caenorhabditis elegans;

[0012] S3. The L4 stage Caenorhabditis elegans were transferred to a culture medium containing FUDR, Escherichia coli culture medium and glucose, and cultured at a constant temperature for 70-73 hours to obtain an adult Caenorhabditis elegans hyperlipidemia model.

[0013] Preferably, the glucose in step S2 and step S3 is D-anhydrous glucose, and the concentration of the D-anhydrous glucose is 40 mM.

[0014] Preferably, the E. coli culture medium in step S2 and step S3 is OP50 culture medium, and the OD of the OP50 culture medium is 600 It is 0.4-0.6.

[0015] In a second aspect, the present invention provides a method for constructing a hyperlipidemia model of Caenorhabditis elegans, the method comprising the following steps:

[0016] S1. The synchronized eggs were placed in an empty culture medium without OP50 Escherichia coli culture medium, and incubated in a constant temperature incubator at 20°C for 9-12 hours to obtain L1 stage Caenorhabditis elegans;

[0017] S2. transferring the L1 stage Caenorhabditis elegans to a culture medium containing Escherichia coli culture medium and glucose, and incubating in a constant temperature incubator at 20° C. for 24 hours until the nematodes develop to the L4 stage, thereby obtaining L4 stage Caenorhabditis elegans;

[0018] S3. The L4 stage Caenorhabditis elegans were transferred to a culture medium containing FUDR, Escherichia coli culture medium and glucose, and cultured at a constant temperature for 72 hours to obtain an adult Caenorhabditis elegans hyperlipidemia model.

[0019] In a third aspect, a hyperlipidemia model of Caenorhabditis elegans is provided, wherein the hyperlipidemia model of Caenorhabditis elegans is constructed by the construction method described in the present invention.

[0020] In a fourth aspect, there is provided a use of the Caenorhabditis elegans hyperlipidemia model of the present invention for evaluating active substances in treating or preventing hyperlipidemia caused by fat accumulation.

[0021] Preferably, the active substance is a fish maw heparin mucopolysaccharide extract, and the concentration of the active substance is 0.25-1 mg / mL.

[0022] More preferably, the active substance is fish bladder heparin mucopolysaccharide.

[0023] In the present invention, the structure of the fish bladder heparin mucopolysaccharide is:

[0024] [→4GlcUAβ1→3GalNAc(4S)β1→]

[0025] In the present invention, the fish bladder heparin mucopolysaccharide is a chondroitin sulfate A analog, and the preparation method thereof refers to Example 1 in patent CN112107590B.

[0026] Preferably, the active substance is administered in the form of a liquid.

[0027] Preferably, the active substance can alleviate lipid accumulation induced by high glucose concentration, including a significant decrease in lipid droplet density in Oil Red O staining, and a significant decrease in triglyceride and free fatty acid content.

[0028] Preferably, the active substance can upregulate the relative expression level of genes related to lipid metabolism.

[0029] Preferably, the lipid metabolism related genes include nhr-49 gene, fat-5 gene, fat-6 gene and fat-7 gene.

[0030] In a fifth aspect, there is provided a use of the Caenorhabditis elegans hyperlipidemia model of the present invention in screening candidate substances with lipid-lowering effects.

[0031] In a sixth aspect, there is provided a use of the Caenorhabditis elegans hyperlipidemia model of the present invention for evaluating drugs in treating or preventing hyperlipidemia caused by fat accumulation.

[0032] The existing technology uses trehalose to culture L4 stage Caenorhabditis elegans, and also observes lipid droplet accumulation by fat staining in Caenorhabditis elegans. However, this culture method may have the following problems: 1) Only the L4 stage is treated, which cannot simulate the complete pathological process of lipid accumulation from the early stage of Caenorhabditis elegans to the adult stage, reducing the model's ability to evaluate the long-term effects of active substances; 2) The lipid droplet accumulation is mainly observed by fat staining in Caenorhabditis elegans, and the pathological state and potential mechanism of the Caenorhabditis elegans obesity model are not fully evaluated, affecting the applicability of the model for active substance screening; 3) Trehalose is a non-reducing sugar that needs to be decomposed into glucose by trehalase before it can be metabolized and utilized; the metabolic rate of trehalose is slow and is easily affected by enzyme activity and environmental conditions (such as temperature and bacterial liquid composition) in Caenorhabditis elegans, resulting in large fluctuations in the degree of fat accumulation, affecting the stability and repeatability of the model; 4) The existing technology mostly uses 24-well plate liquid culture. First, a shaker is required for culture, which is cumbersome to operate and increases the possibility of experimental errors. Secondly, C. elegans is in a suspended state, and its contact with exogenous substances (such as trehalose and active substances) is uneven, which can easily lead to unstable induction effects.

[0033] In contrast, the advantages of the present invention are: through synchronized culture, the complete development cycle from L1 stage to adult stage is covered, ensuring the consistency of the physiological state of nematodes. A staged culture strategy is adopted to ensure the gradual induction of lipid accumulation and the observation of dynamic changes in physiological state. The stability and dynamic response ability of the model can be used for multi-dimensional index evaluation, screening and verification of the lipid-lowering effect of active substances, and support further research on its mechanism. Glucose is the core substance of cellular energy metabolism. It is mainly metabolized through glycolysis and tricarboxylic acid cycle in Caenorhabditis elegans to produce ATP for energy supply. The metabolic pathway of glucose is stable and efficient, and its sensitivity to the external environment (such as temperature and bacterial liquid composition) is low. Therefore, directly using glucose for modeling is conducive to the repeatability of the model and the comparability of the results. In addition, the present invention adopts solid culture medium treatment, which can make the distribution of exogenous substances uniform and easy to standardize, better simulate the feeding behavior and metabolic state of Caenorhabditis elegans closer to natural physiological conditions, and has the characteristics of simple operation and low cost.

[0034] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0035] (1) The present invention proposes a method for constructing a hyperlipidemia model of C. elegans based on staged treatment, which provides a stable and efficient experimental platform for evaluating the lipid-lowering effects of active substances or drugs. Compared with the traditional method of only culturing C. elegans to the L4 stage, the present invention can comprehensively reflect its physiological and biochemical state by covering the complete developmental cycle of C. elegans from the L1 stage to the adult stage. The method of the present invention optimizes the construction strategy of the hyperlipidemia model, taking into account both early induction and long-term observation, which helps to accurately evaluate the preventive and sustained effects of active substances or drugs on lipid metabolism disorders, and lays a solid scientific foundation for in-depth exploration of the lipid metabolism regulation mechanism.

[0036] (2) Through the Oil Red O staining experiment, the hyperlipidemia model of Caenorhabditis elegans constructed by the present invention showed significant fat accumulation characteristics, and the fat content increased by 62.25% compared with the normal group (blank group). Further biochemical tests showed that the triglyceride and free fatty acid contents in the model group increased by 163.97% and 42.71%, respectively. Molecular level analysis showed that the expression of genes related to lipid metabolism changed significantly. The above results show that this method successfully induced lipid metabolism disorders in Caenorhabditis elegans, simulated the pathological state of hyperlipidemia, further verified the successful construction of the hyperlipidemia model, and provided a reliable model tool for lipid metabolism research and disease mechanism exploration.

[0037] (3) The lipid-lowering activity of the fish bladder heparin mucopolysaccharide extract was evaluated using the constructed Caenorhabditis elegans hyperlipidemia model. The experimental results showed that the extract could significantly reduce the lipid droplet level of the model group nematodes, while reducing the accumulation of triglycerides and free fatty acids. Gene expression analysis showed that the fish bladder heparin mucopolysaccharide extract could significantly upregulate the expression of the fatty acid β-oxidation regulatory gene nhr-49 and promote the expression of the Δ9 fatty acid desaturase genes fat-5, fat-6 and fat-7. It can be seen that the extract improves lipid metabolism disorders through multi-pathway synergistic effects and exerts a significant lipid-lowering effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solution of the present invention, the drawings involved in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the description 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.

[0039] Figure 1 The Oil Red O staining results of the hyperlipidemia models of Caenorhabditis elegans in different experimental groups; A is the Oil Red O staining images of the hyperlipidemia models of Caenorhabditis elegans in different experimental groups; B is the quantification of the Oil Red O staining fat fluorescence intensity (lipid droplet area ratio) of the hyperlipidemia models of Caenorhabditis elegans in different experimental groups.

[0040] Figure 2 A is the measurement result of lipid accumulation in the hyperlipidemia model of Caenorhabditis elegans in different experimental groups; A is the measurement result of triglyceride content in the hyperlipidemia model of Caenorhabditis elegans in different experimental groups; B is the measurement result of free fatty acid content in the hyperlipidemia model of Caenorhabditis elegans in different experimental groups.

[0041] Figure 3 A is the determination result of the relative expression level of lipid metabolism genes in the hyperlipidemia models of Caenorhabditis elegans in different experimental groups; A is the determination result of the relative expression level of nhr-49 gene in the hyperlipidemia models of Caenorhabditis elegans in different experimental groups; B is the determination result of the relative expression level of fat-5 gene in the hyperlipidemia models of Caenorhabditis elegans in different experimental groups; C is the determination result of the relative expression level of fat-6 gene in the hyperlipidemia models of Caenorhabditis elegans in different experimental groups; D is the determination result of the relative expression level of fat-7 gene in the hyperlipidemia models of Caenorhabditis elegans in different experimental groups. DETAILED DESCRIPTION

[0042] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0043] Example 1

[0044] Method for constructing a hyperlipidemia model in Caenorhabditis elegans

[0045] This embodiment provides a method for constructing a hyperlipidemia model of Caenorhabditis elegans, comprising the following steps:

[0046] In this example, wild-type C. elegans N2 was used as the research object, and the nematodes were synchronized using a lysis solution (2M NaOH, 10% NaHOCl, and ddH2O), and the obtained eggs were placed on an empty culture medium without OP50 E. coli culture medium, and incubated in a 20°C constant temperature incubator for 9-12 hours to obtain L1 stage C. elegans. The L1 stage nematodes were transferred to a culture medium containing E. coli culture medium and glucose, and continued to be cultured in a 20°C constant temperature incubator for 24 hours until the nematodes developed to the L4 stage. The L4 stage nematodes were transferred to a culture medium containing 200 μM FUDR, E. coli culture medium, and glucose, and continued to be cultured in a 20°C constant temperature incubator for 72 hours, and finally a hyperlipidemia model of C. elegans in the adult stage was constructed.

[0047] Example 2

[0048] Culture conditions of fish swim bladder treated with heparin-like mucopolysaccharide

[0049] The fish swim heparin mucopolysaccharide lyophilized powder was dissolved in sterile water and sterilized using a bacterial filter with a diameter of 0.22 μm. The fish swim heparin mucopolysaccharide solution was mixed with OP50 Escherichia coli culture medium (culture medium), and three treatment concentrations were set after dilution: low-dose group (0.25 mg / mL), medium-dose group (0.50 mg / mL) and high-dose group (1.00 mg / mL). The blank group nematodes were cultured on standard growth agar plates; the model group was cultured on agar plates containing 40 mM D-anhydrous glucose. The synchronized L1 stage nematodes were transferred to agar culture plates containing OP50 Escherichia coli culture and fish swim heparin mucopolysaccharide, and placed in a constant temperature incubator at 20°C for further culture.

[0050] Example 3

[0051] Effects of fish swim bladder heparin-like mucopolysaccharides on lipid droplet levels in the hyperlipidemia model of Caenorhabditis elegans

[0052] After washing the nematodes 3 times with PBST buffer, 600 μL of 60% isopropanol was added, and the mixture was rotated at room temperature for 3 minutes. After removing the supernatant, 600 μL of 60% ORO staining solution was added and rotated at room temperature for 2 hours. The nematodes were washed 3 times, and a small amount of sample was taken and placed on a slide for imaging. The experiment was repeated at least three times, and the number of nematodes in each group was not less than 30. The images were quantitatively analyzed using ImageJ software, and the data were analyzed by one-way ANOVA using SPSS software to compare the significance between the groups. Different letters indicate statistically significant differences between the groups (P<0.05).

[0053] Oil red O staining is the most intuitive indicator for observing the fat storage site and lipid accumulation degree of Caenorhabditis elegans, so it is used to evaluate the effect of fish swim bladder heparin mucopolysaccharide on the improvement of lipid droplet levels. Figure 1 This is a 40-fold magnification of Oil Red O staining of Caenorhabditis elegans. It can be seen that the lipid droplet staining intensity of the model group nematodes increased significantly, and the fat content increased by 62.25% compared with the blank group. The treatment of fish bladder heparin mucopolysaccharide significantly reduced the staining area, and the high-dose group reduced the lipid level by 35.42%, indicating that its intervention effect was significant.

[0054] Example 4

[0055] Effects of fish swim bladder heparin-like mucopolysaccharides on lipid accumulation in the hyperlipidemia model of Caenorhabditis elegans

[0056] The wild-type N2 treated with the experiment was collected to determine the triglyceride content and free fatty acid content in Caenorhabditis elegans. The determination method was based on the instructions of the detection kit (triglyceride: Nanjing Jiancheng Bioengineering Institute; free fatty acid: Beijing Box Biotechnology Co., Ltd.). The experiment was repeated at least three times, and the number of experimental nematodes collected in each group was about 10,000. The experimental data were presented as "mean ± standard deviation". SPSS software was used to perform one-way ANOVA on the data to compare the significance between the groups. Different letters indicate statistically significant differences between the groups (P<0.05).

[0057] Among them, the triglyceride content in Caenorhabditis elegans is as follows Figure 2 As shown in A, the free fatty acid content in Caenorhabditis elegans is Figure 2 As shown in B. Figure 2 A in the figure shows that the triglyceride content in the model group was significantly higher than that in the blank group, with a content of 1.293±0.114mmol / g prot, an increase of 163.97%. Treatment with different concentrations of fish maw heparin mucopolysaccharide significantly reduced the triglyceride content, and the triglyceride content in the high-dose group of fish maw heparin mucopolysaccharide treatment was reduced by 56.76%. Figure 2As shown in Figure B, the free fatty acid content in the model group was significantly higher than that in the control group (0.0842±0.0061 vs 0.0590±0.0078umol / g prot), while the fish bladder heparin mucopolysaccharide treatment significantly reduced the free fatty acid content. In particular, the high-dose group of fish bladder heparin mucopolysaccharide treatment reduced the free fatty acid content by 26.64%, indicating that fish bladder heparin mucopolysaccharide has an improving effect on fat deposition.

[0058] Example 5

[0059] Determination of the relative expression of lipid metabolism genes in the hyperlipidemia model of Caenorhabditis elegans constructed by fish swim bladder heparin mucopolysaccharide

[0060] RNA was extracted from the Caenorhabditis elegans sample using an RNA extraction kit, and then purified by ethanol precipitation. cDNA was obtained by reverse transcription, and then the premix was added according to the instructions of the RNA fluorescence quantitative kit. The relative expression levels of nhr-49 gene, fat-5 gene, fat-6 gene and fat-7 gene were determined by RT-qPCR. actin-1 was selected as the internal reference gene, and 2 -△△Ct Calculation method. The nhr-49 gene is a key gene target of the nuclear hormone signaling pathway in Caenorhabditis elegans. It has similar sequence homology to mammalian peroxisome proliferator-activated receptors (PPARs) and can activate fatty acid β-oxidation. The fat-5, fat-6, and fat-7 genes are encoded by Δ9 fatty acid desaturase, which is the rate-limiting enzyme in fatty acid synthesis and can promote the transport of fatty acids into the mitochondrial matrix. The relative expression levels of the nhr-49 gene, fat-5 gene, fat-6 gene, and fat-7 gene are shown in Figure 2. Figure 3 As shown, the experiment was repeated at least three times, and the number of nematodes collected in each group was about 10,000. The experimental data were presented as "mean ± standard deviation", and the data were analyzed by one-way ANOVA using SPSS software to compare the significance among the groups. Different letters indicate statistically significant differences among the groups (P<0.05).

[0061] Depend on Figure 3 As shown in A, compared with the blank group, the model group significantly downregulated the expression level of the nhr-49 gene, while the treatment with different concentrations of fish bladder heparin mucopolysaccharide showed a dose-dependent upregulation trend of nhr-49 expression, which was increased by 2.85 times, 3.23 times and 3.84 times respectively compared with the model group. Figure 3 B in Figure 3 C in Figure 3D in the figure shows that compared with the blank group, the model group significantly inhibited the expression of fat-6 and fat-7 genes. Under the intervention of different concentrations of fish bladder heparin mucopolysaccharide, the gene expression of △9 fatty acid desaturase (fat-5, fat-6 and fat-7) was upregulated to varying degrees. The expression of fat-6 gene was upregulated to 1.06 times and 1.27 times of that in the model group in the low-dose group and the medium-dose group; the expression of fat-7 in the low-dose group, the medium-dose group and the high-dose group was 1.86 times, 1.66 times and 1.81 times of that in the model group, respectively. This shows that fish bladder heparin mucopolysaccharide extract significantly promotes the expression of fatty acid synthesis genes and △9 fatty acid desaturase genes, and can effectively play the role of improving lipid metabolism in the body.

[0062] The above experimental results show that the method for constructing a hyperlipidemia model of C. elegans based on staged treatment provides a stable and efficient experimental platform for evaluating the lipid-lowering effect of active substances or drugs. Compared with the traditional method of only culturing nematodes to the L4 stage, the present invention can fully reflect the physiological and biochemical state of nematodes by covering the complete developmental cycle of nematodes from the L1 stage to the adult stage. The method of the present invention optimizes the construction strategy of the hyperlipidemia model, taking into account both early induction and long-term observation, which helps to accurately evaluate the preventive and sustained effects of active substances or drugs on lipid metabolism disorders, and lays a solid scientific foundation for in-depth exploration of the lipid metabolism regulation mechanism.

[0063] Through the Oil Red O staining experiment, the hyperlipidemia model of Caenorhabditis elegans constructed by the present invention showed significant fat accumulation characteristics, and the fat content increased by 62.25% compared with the normal group (blank group). Further biochemical tests showed that the triglyceride and free fatty acid contents in the model group increased by 163.97% and 42.71%, respectively. Molecular level analysis showed that the expression of genes related to lipid metabolism changed significantly. The results showed that this method successfully induced lipid metabolism disorders in Caenorhabditis elegans, simulated the pathological state of hyperlipidemia, further verified the successful construction of the hyperlipidemia model, and provided a reliable model tool for lipid metabolism research and disease mechanism exploration.

[0064] The present invention uses a constructed Caenorhabditis elegans hyperlipidemia model to evaluate the lipid-lowering activity of the fish bladder heparin mucopolysaccharide extract. The experimental results show that the extract can significantly reduce the lipid droplet level of the nematodes in the model group, while reducing the accumulation of triglycerides and free fatty acids. Gene expression analysis shows that the fish bladder heparin mucopolysaccharide extract can significantly upregulate the expression of the fatty acid β-oxidation regulatory gene nhr-49 and promote the expression of Δ9 fatty acid desaturase genes fat-5, fat-6 and fat-7. The results show that the extract improves lipid metabolism disorders through multi-pathway synergistic effects and exerts a significant lipid-lowering effect.

[0065] The above-described embodiments are part of the embodiments of the present invention, but not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but rather represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A method for constructing a hyperlipidemia model of Caenorhabditis elegans, characterized in that: The construction method comprises the following steps: S1. The synchronized eggs are placed in a culture medium without E. coli culture medium, and cultured at a constant temperature for 9-12 hours to obtain L1 stage Caenorhabditis elegans; S2. transferring the L1 stage Caenorhabditis elegans to a culture medium containing Escherichia coli culture medium and glucose, and culturing at a constant temperature for 24 hours until the nematodes develop to the L4 stage, thereby obtaining L4 stage Caenorhabditis elegans; S3. The L4 stage Caenorhabditis elegans were transferred to a culture medium containing FUDR, Escherichia coli culture medium and glucose, and cultured at a constant temperature for 70-73 hours to obtain an adult Caenorhabditis elegans hyperlipidemia model.

2. The construction method according to claim 1, characterized in that: The glucose in step S2 and step S3 is D-anhydrous glucose, and the concentration of the D-anhydrous glucose is 40 mM.

3. The construction method according to claim 1, characterized in that: The E. coli culture medium in step S2 and step S3 is OP50 culture medium, and the OD of the OP50 culture medium is 600 It is 0.4-0.

6.

4. A Caenorhabditis elegans hyperlipidemia model, characterized in that: The Caenorhabditis elegans hyperlipidemia model is constructed by the construction method described in any one of claims 1 to 4.

5. Use of the Caenorhabditis elegans hyperlipidemia model according to claim 4 for evaluating active substances in treating or preventing hyperlipidemia caused by fat accumulation.

6. The use according to claim 5, characterized in that: The active substance is a fish bladder heparin mucopolysaccharide extract, and the concentration of the active substance is 0.25-1 mg / mL.

7. The use according to claim 5, characterized in that: The active substance can alleviate lipid accumulation induced by high concentration of glucose.

8. The use according to claim 5, characterized in that: The active substance can upregulate the relative expression of genes related to lipid metabolism.

9. The use according to claim 8, characterized in that: The lipid metabolism related genes include nhr-49 gene, fat-5 gene, fat-6 gene and fat-7 gene.

10. Use of the Caenorhabditis elegans hyperlipidemia model according to claim 4 in screening candidate substances with lipid-lowering effects.

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