Metabolic disease caenorhabditis elegans model and construction method and application thereof

Abstract

The embodiment of the invention discloses a metabolic disease caenorhabditis elegans model as well as a construction method and application thereof, relates to the technical field of metabolic disease models, and aims to improve screening and research of metabolic disease active substances and product development. The construction method of the metabolic disease caenorhabditis elegans model comprises the following steps: placing eggs of wild nematodes or mutant nematodes on a nematode growth high-fat culture medium for culture, and placing the eggs in a constant-temperature incubator for culture to obtain the Multi-HFD model.

Description

Technical Field

[0001] This application relates to the technical field of metabolic disease models, and specifically relates to a Caenorhabditis elegans model for metabolic diseases and its construction method and application. Background Art

[0002] Metabolic syndrome is actually a lipid storage disorder that originates from ectopic lipid storage in obesity. Obesity is a symptom of excessive accumulation of neutral lipids such as triglyceride (TAG) in white adipocytes. Adipose tissue controls individual lipid homeostasis at the tissue level. However, at the cellular level, TAG is stored in lipid droplets (LDs). LDs, as unique intracellular organelles, consist of a neutral lipid core rich in TAG and sterol esters and a phospholipid monolayer decorated with LD proteins, and can dynamically regulate intracellular lipid and energy homeostasis. The main function of LDs is to dynamically regulate energy homeostasis through the cycle of biogenesis and consumption in response to energy surplus or demand. The disorder of this homeostasis often leads to many human metabolic disorders, including insulin resistance, type II diabetes, fatty liver, cardiovascular diseases, etc., which are common diseases threatening human health. The size, number of LDs, and the type and number of fatty acids in TAG are key factors in metabolic disorders, and their morphological research is of great significance for understanding obesity and ectopic lipid storage.

[0003] Currently, the animal models used to study metabolic diseases mainly include rats, mice, rhesus monkeys, miniature pigs, etc. The models are mainly established by methods such as diet induction, gene mutation, chemical / drug induction, etc. Although they can highly simulate human pathology and have the characteristics of high flexibility and high translational value, they have high costs, complex operations, long modeling cycles, and ethical restrictions, etc.

[0004] C. elegans is a soil-dwelling nematode about 1 mm long, completely transparent, non-toxic and harmless. Compared with traditional animal models, as a model for studying metabolic diseases, C. elegans has many advantages: it is small in size and completely transparent, which is convenient for observing its body structure, behavioral indicators and fluorescent protein labeling and localization under a microscope; it has a short life cycle and a short generation cycle, and is an ideal model for exploring transgenerational genetic effects; its genome sequencing is complete, and more than 60%-80% of its genes are homologous to human disease-related genes; it has powerful genetic tools and can conduct functional research on epigenetic problems; the whole map and invariant cell lineage ensure the consistency of cell analysis in experiments and eliminate the common tissue heterogeneity problems in rodent models; the genomic distribution and regulatory function of its epigenetic markers are highly conserved with the data of mammalian cell culture systems. Currently, C. elegans has been widely used in research fields such as transgenerational lipid metabolism, avoiding the inheritance of pathogenic behaviors, and intergenerational neuroprotective effects, and is a mature model organism for metabolic diseases. Summary of the Invention

[0005] The main purpose of this application is to provide a nematode Multi-HFD model and its construction method and application, aiming to improve the screening, research and product development of active substances for metabolic diseases.

[0006] To achieve the above object, the technical solution adopted in the embodiments of this application is as follows: A method for constructing a Caenorhabditis elegans model of metabolic diseases, placing the eggs of wild-type nematodes or mutant nematodes on a nematode growth high-fat medium for culture, and placing them in a constant temperature incubator for culture to obtain a Multi-HFD model.

[0007] As some alternative embodiments of this application, the nematode growth high-fat medium is prepared through the following steps:

[0008] Mix the cooked egg yolk with E. coli OP50 bacterial liquid in a ratio of 1:5 to prepare a high-fat diet bacterial liquid for standby; mix the high-fat diet bacterial liquid with absolute ethanol or sterilized pure water (final concentrations are both 0.2%) respectively for standby; mix the high-fat diet bacterial liquid with resveratrol (final concentrations are 50, 100, 200 μM) for standby; coat 300 μL of the above solution on an NGM feeding plate, air dry and seal with a film, and store at 4°C for standby.

[0009] As some alternative embodiments of this application, the OD600 of the E. coli OP50 bacterial liquid is 0.4 - 0.6.

[0010] As some alternative embodiments of this application, the culture temperature is 16°C - 20°C, the culture humidity is 40% - 60%, and the culture time is 2 - 4 days.

[0011] As some alternative embodiments of this application, the Multi-HFD model promotes the obesity phenotype by forming super-large lipid droplets; and synchronously reduces the physiological indexes related to lipid toxicity to promote the formation of super-large lipid droplets induced by fat synthesis.

[0012] As some alternative embodiments of this application, the physiological indexes related to lipid toxicity include head swing frequency, swallowing frequency and defecation frequency.

[0013] As some alternative embodiments of this application, the Multi-HFD model significantly increases the TAG level by upregulating the expression of the fat generation transcription factor SBP-1 and promoting the activity of the fatty acid synthase FASN-1, so as to form pathological characteristic super-large lipid droplets.

[0014] In some alternative embodiments of the present application, in the Multi-HFD model, the formation of pathological characteristic super-large lipid droplets is associated with genes daf-16, mdt-15, sbp-1, nhr-80, age-1, and the homologous genes of delta-9 desaturase (fat-5, fat-6, fat-7).

[0015] On the other hand, the embodiments of the present application also provide a Caenorhabditis elegans model for metabolic diseases, which is characterized by being constructed by the method described above.

[0016] On the other hand, the embodiments of the present application also provide an application of the Caenorhabditis elegans model for metabolic diseases as described above, which is used for screening drugs for improving metabolic diseases.

[0017] Compared with the prior art, based on the biological characteristics that the homology between Caenorhabditis elegans (C. elegans, simply referred to as nematode) and human genes reaches 60%-80%, this model induces the formation of super-large lipid droplets through continuous multi-generation high-fat diet treatment, and its characteristic manifestations are: the Multi-HFD model can promote the formation of super-large lipid droplets and synchronously trigger lipid toxicity-related physiological dysfunctions, including reduced head swing frequency, swallowing frequency, and defecation frequency. Morphological analysis shows that the body length of nematodes in the model group increases, the body width expands, and the body area increases significantly. Molecular mechanism research shows that this model leads to a significant increase in TAG levels by upregulating the expression of the fat generation transcription factor SBP-1 and promoting the activity of fatty acid synthase FASN-1, and finally forms pathological characteristic super-large lipid droplets. Further research shows that the formation of super-large lipid droplets depends on the occurrence of genes daf-16, mdt-15, sbp-1, nhr-80, age-1, and the homologous genes of delta-9 desaturase (fat-5, fat-6, fat-7). This model has the characteristics of stable phenotype, high repeatability, and high throughput advantages, providing an efficient experimental platform for studying the molecular mechanism of obesity-related metabolic syndrome and screening lipid metabolism regulating drugs. Description of the Drawings

[0018] Figure 1 Shows the formation of super-large lipid droplets in the Multi-HFD model of nematodes in different treatment groups (a, c) and the quantification of fat deposition (b);

[0019] Figure 2 Shows the changes in physiological indexes of the Multi-HFD model of nematodes in different treatment groups (a: swallowing frequency; b: head swing frequency; c: defecation frequency);

[0020] Figure 3Changes in the body shape of the Multi-HFD model of nematodes in different treatment groups (a: schematic diagram of body shape; b: body length; c: body width; d: body area);

[0021] Figure 4 Schematic diagrams of fluorescence intensity (a), TAG levels (b), SBP-1 (c), ATGL-1 (d), and FASN-1 (e) of the Multi-HFD model of nematodes in different treatment groups;

[0022] Figure 5 Schematic diagram (a) and result diagram (b) of the changes in the maximum lipid droplet diameter of the Multi-HFD model of nematode mutants CF1038, TJ1052, CE541, XA7702, and BX165 in different treatment groups;

[0023] Figure 6 Schematic diagram (a) and result diagram (b) of the changes in the maximum lipid droplet diameter of the Multi-HFD model of nematode mutants BX107, BX106, BX153, BX110, and BX156 in different treatment groups. Detailed implementation manners

[0024] It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0025] As described above, the Multi-HFD nematode model provided by the present application exhibits phenomena such as the formation of super-large lipid droplets in the body after modeling, impaired physiological functions, obesity, and increased fat synthesis. This model is similar to the phenomena of dietary polyunsaturated fatty acids promoting the formation of super-large lipid droplets and high-temperature-induced lipid droplet fusion to form super-large lipid droplets in biological processes, and this model has the characteristics of economy, effectiveness, and feasibility.

[0026] In addition, the present application also has the following advantages compared with the prior art:

[0027] The modeling method is simple: Utilizing the heredity of obesity, the model is established by continuous multi-generation high-fat diet treatment to promote the accumulation of fat in the body and form super-large lipid droplets. The growth cycle of nematodes is short, and subculture can be completed in 2 - 4 days. The operation is simple, and other adverse effects brought by chemical drugs are avoided.

[0028] High stability: Based on the standardized nematode synchronization culture and high-throughput processing flow (single batch processing capacity ≥ 2000 individuals), the Multi-HFD model can stably induce the formation of super-large lipid droplets in different treatment batches (n ≥ 15), different operators (n ≥ 3), and cross-year repeated experiments (2023 - 2025).

[0029] In the embodiments of the present application, the Multi-HFD nematode model forms extremely large lipid droplets in vivo, showing impaired physiological indices, including significantly reduced swallowing frequency, head swing frequency, and defecation frequency. Morphological analysis shows that the body length of nematodes in the model group increases, the body width expands, and the body area increases significantly. Molecular mechanism research shows that this model leads to a significant increase in TAG levels by upregulating the expression of sterol regulatory element-binding protein (SREBP1, SBP-1) and promoting the activity of fatty acid synthase 1 (FASN-1), ultimately forming pathologically characteristic extremely large lipid droplets, and then triggering lipid toxicity-related physiological dysfunction. Further research shows that the formation of extremely large lipid droplets depends on the key roles of genes daf-16, mdt-15, sbp-1, nhr-80, age-1, and the homologous genes of delta-9 desaturase (Stearoyl-CoA Desaturase, SCD) (fat-5, fat-6, fat-7) in lipid synthesis. After the above key lipid metabolism gene deletion mutants are treated with Multi-HFD, the phenotype of extremely large lipid droplets disappears.

[0030] Next, the technical solutions of the present application will be described in detail in conjunction with specific embodiments:

[0031] It should be noted that in the following embodiments, the structural formula of resveratrol is shown in formula (I),

[0032]

[0033] The final concentration of the action of resveratrol is preferably 50-200 μM, and further preferably 200 μM.

[0034] The biological model to which resveratrol is applied is preferably the Multi-HFD model, specifically manifested as continuously subjecting wild-type nematodes from the parent (P0) to the offspring (F1, F2) to a high-fat diet treatment, and then measuring various indices of the F2.

[0035] Example 1

[0036] (I) Experimental scheme

[0037] 1. Experimental materials

[0038] The eggs of wild-type nematodes or mutant nematodes are evenly divided into 4 groups and placed on 4 different nematode growth media (NGM) for culture, and placed in a constant temperature incubator (16-20 °C, humidity 40%-60%) for 2.5 d for subculture to obtain the offspring F2. The 4 media and the genotypes of the nematode mutants used are as follows:

[0039] Blank control group (OP50): Directly coat 300 μL of E. coli OP50 bacterial solution on NGM, air dry and then seal with a film, store at 4 °C for later use.

[0040] High-fat model group (CK): Mix cooked egg yolk with E. coli OP50 bacterial solution (the OD of the E. coli OP50 bacterial solution 600 is 0.4 - 0.6) in a ratio of 1:5 to prepare a high-fat diet bacterial solution for later use. Mix sterilized pure water with the high-fat diet bacterial solution, coat 300 μL of the working solution on NGM, air dry and then seal with a film, store at 4 °C for later use.

[0041] Solvent control group (ET): Mix an equal-concentration solvent (final concentration of 0.2% ethanol) with the high-fat diet bacterial solution, coat 300 μL of the working solution on NGM, air dry and then seal with a film, store at 4 °C for later use.

[0042] Resveratrol group (RES): Mix RES with the high-fat diet bacterial solution to fully form a working solution with a final concentration of 50 - 200 μM, coat 300 μL of the working solution on NGM, air dry and then seal with a film, store at 4 °C for later use; among them, the final concentration of the action of RES is preferably 200 μM.

[0043] Among them:

[0044] AG400: {fasn-1(av138[fasn-1::gfp])I.}

[0045] VS20: {hjIs67[atgl-1p::atgl-1::GFP+mec-7::RFP]}

[0046] TJ1052: {age-1(hx546)II.}

[0047] CF1038: {daf-16(mu86)I.}

[0048] BX165: {nhr-80(tm1011)III.}

[0049] CE541: {sbp-1(ep79)III.}

[0050] CE548: {epEx141[sbp-1::GFP::sbp-1+rol-6(su1006)]}

[0051] XA7702: {mdt-15(tm2182)III.}

[0052] BX107: {fat-5(tm420)V.}

[0053] BX106: {fat-6(tm331)IV.}

[0054] BX153: {fat-7(wa36)V.}

[0055] BX110: {fat-6(tm331)IV; fat-5(tm420)V.}

[0056] BX156: {fat-6(tm331)IV; fat-7(wa36)V.}

[0057] 2. Measurement methods

[0058] Measurement of the maximum lipid droplet diameter: After staining the lipid droplets of wild-type and mutant nematodes with or without intervention using Oil Red O staining method, take pictures under bright field at 40 times magnification of an upright fluorescence microscope, and measure the diameter of the largest lipid droplet among them using Image J software. At least 15 nematodes should be photographed in each experiment, and 3 biological replicates should be set for each treatment method.

[0059] Measurement of body size: Pick wild-type nematodes with / without intervention onto a glass slide containing 1% NaN 3 Anesthetize them, then place them under bright field at 4 times magnification of a fluorescence microscope for photographing, and then use Image J software for data analysis. Select the body length and the thickest part of the body of the nematodes to determine the body length, body width, and body area. At least 15 nematodes should be photographed in each experiment, and 3 biological replicates should be set for each treatment method.

[0060] Swallowing frequency: Observe and record the number of pharyngeal bulb movements of wild-type nematodes within 30 s under an optical stereomicroscope. At least 15 nematodes should be recorded in each group, and at least 3 biological replicates.

[0061] Head swing frequency: Record one swing of the nematode's head to the left or right as one time, and record the number of head swings within 30 s as the head swing frequency. At least 15 nematodes should be recorded in each group, and at least 3 biological replicates.

[0062] Defecation frequency: Observe and record the interval time between two defecation actions under an optical stereomicroscope. At least 15 nematodes should be recorded in each experiment, and 3 biological replicates should be set for each treatment method.

[0063] Measurement of TAG level: Collect about 1000 wild-type nematodes, wash them 2 - 3 times with M9 buffer to remove bacteria. For the homogenized samples, use triglyceride (TAG, Nanjing Jiancheng Bioengineering Institute, China) and a micro BCA TM Protein kit (Nanjing Jiancheng Bioengineering Institute, China), and detect according to the instructions of the manufacturer. Normalize the TAG content to the protein concentration. The samples should be detected at least three times with an enzyme-labeled instrument, and at least 3 biological replicates should be set for each treatment method.

[0064] Fluorescence intensity measurement: The mutants VS20, AG400, and CE548 were cultured for 2.5 days and then washed 2-3 times with M9 buffer to avoid interference of bacteria on the surface of nematodes with photography. Approximately 0.5 mL was left and 20 μL of 1% NaN 3 anesthetic was added. After they became rigid, the nematode-containing solution was taken onto a glass slide and photographed using the GFP mode on an upright fluorescence microscope. At least 15 nematodes were photographed in each experiment, and 3 biological replicates were set for each treatment method.

[0065] 3. Data processing

[0066] All experiments were repeated at least three times. The results were expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used for significance analysis using GraphPad Prism 8.0.1 version (GraphPad Software, Inc., San Diego, CA). Different lowercase letters (a, b, c) in the same column indicated significant differences (p < 0.05), which was statistically significant.

[0067] (2) Experimental results

[0068] 1. The Multi-HFD nematode model forms extremely large lipid droplets in vivo and promotes fat deposition

[0069] In this application, a high-fat exposure model (P0-F2 continuous high-fat diet intervention system) was innovatively established. This model simulates the human intergenerational metabolic memory effect through three generations of continuous high-fat feeding (Multi-HFD), provides a new experimental paradigm for studying the accumulation of chronic metabolic stress, and the following key results were observed: Oil Red O staining was performed on F2, and the quantitative analysis results showed ( Figure 1 b): After being fed with Multi-HFD, the fat deposition in nematodes increased (30.36% increase in the CK group, 30.50% increase in the ET group, p < 0.05), while the treatment with RES (p > 0.05) could not change the fat increase caused by Multi-HFD feeding ( Figure 1 b). This phenomenon suggests that continuous multi-generation high-fat exposure leads to a superimposed effect of metabolic stress in offspring, manifested as a state of lipid overload, and RES cannot reverse the overload damage. In addition, it was found that the diameter of lipid droplets in nematodes fed with Multi-HFD was significantly amplified, forming extremely large lipid droplets, suggesting that chronic lipotoxicity triggers a new mechanism of lipid droplet fusion.

[0070] To further explore this phenomenon, Image J was used to measure the diameter of the largest lipid droplet in nematodes. The results showed ( Figure 1 a, Figure 1c), After feeding with Multi-HFD, extremely large lipid droplets appeared in F2 (increased by 31.27 μm in the CK group and 28.22 μm in the ET group, p < 0.05). Although RES did not change the in vivo fat deposition (p > 0.05), it had a significant inhibitory effect on the diameter of extremely large lipid droplets in vivo, reducing it by 9.95 μm (p < 0.05).

[0071] Thus, this application reveals that: ① Continuous three-generation high-fat exposure can break through the metabolic compensation threshold and cause irreversible lipid toxicity accumulation (intergenerational superposition effect); ② The differential regulation of RES on the total lipid amount and lipid droplet morphology suggests that there is a regulatory pathway for lipid droplet fusion independent of total lipid metabolism. This discovery provides a new theoretical basis for developing intervention strategies for metabolic diseases targeting lipid droplet remodeling.

[0072] 2. The Multi-HFD nematode model shows impaired physiological functions

[0073] In nematodes fed with Multi-HFD, it was found that the diameter of their lipid droplets was significantly amplified, forming extremely large lipid droplets. The maximum lipid droplet diameter in the intestine of F2 nematodes increased by 28.22 μm - 31.27 μm compared with the OP50 control group (p < 0.05), suggesting that continuous multi-generation high-fat diet treatment may cause lipid toxicity in F2. Therefore, the physiological indexes of nematodes were further explored, and the swallowing frequency ( Figure 2 a), head swing frequency ( Figure 2 b), and defecation frequency ( Figure 2 c) of F2 were measured respectively. The results showed that after continuous high-fat diet feeding, the swallowing frequency of nematodes decreased by 33.94%, which may be related to the increased intestinal mechanical pressure caused by abnormal enlargement of lipid droplets; the head swing frequency decreased by 16.06%, reflecting that lipid overload may affect neurotransmitter conduction or energy supply for muscle contraction, and the defecation frequency decreased sharply by 75.55%, indicating that the lipid absorption-excretion balance was disrupted (p < 0.05). After treatment with RES, all physiological indexes were significantly improved. Among them, the swallowing frequency increased by 7.40%, indicating the improvement of intestinal mechanical function; the head swing frequency increased by 18.73%, suggesting the restoration of neuromuscular coordination; the defecation frequency increased by 23.08%, indicating the accelerated lipid turnover rate (p < 0.05), showing an improvement effect on the lipid toxicity of the Multi-HFD nematode model.

[0074] 3. The Multi-HFD nematode model is obese

[0075] To clarify the obese phenotype of the Multi-HFD nematode model, the body shape was further measured ( Figure 3 a), and its body length ( Figure 3 b), body width ( Figure 3 c), and body area ( Figure 3d), the results showed that after Multi-HFD treatment, the body length of C. elegans increased by 29.05%, the body width increased by 35.37%, and the body area increased by 120.90% (p<0.05), while the RES treatment group had no significant improvement on body length, body width, and body area (p>0.05), which was consistent with the quantitative results of Oil Red O staining. It indicated that the treatment of RES could not produce lipid-lowering effect on C. elegans treated with Multi-HFD.

[0076] This result suggested that Multi-HFD treatment might exacerbate lipid toxicity through the "size-metabolism positive feedback loop", that is, metabolic disorders were caused by body size expansion, and the metabolic disorders further led to the aggravation of overweight status, forming a vicious cycle, while the treatment of RES could not improve this phenotype.

[0077] 4. The Multi-HFD C. elegans model inhibits lipolysis and promotes lipogenesis

[0078] TAG is composed of a glycerol molecule and three fatty acid chains linked by ester bonds, and is the main energy storage form in animals (about 95% of the total lipids), mainly stored in lipid droplets of adipocytes. It is stored in lipid droplets of adipocytes in a non-hydrated form and is decomposed into glycerol and fatty acids for energy supply when energy is needed through the lipolysis mechanism. ATGL-1 is an enzyme that plays a key role in lipid metabolism, and its function has diverse regulatory mechanisms and biological effects in different species and physiological processes. Among them, the first step of hydrolysis of TAG (triacylglycerol) → DG (diacylglycerol) mediated by ATGL is the rate-limiting step. FASN-1 is the C. elegans homologous gene of fatty acid synthase, which is responsible for catalyzing the synthesis of long-chain fatty acids. The up-regulation of FASN-1 leads to an increase in TAG synthesis, while the up-regulation of ATGL-1 promotes TAG decomposition. The two form a dynamic balance under the regulation of the neuro-intestinal axis and insulin signaling pathway. SBP-1, as a member of the SREBP transcription factor family, directly regulates the expression of lipid generation-related genes such as FASN-1.

[0079] The results showed ( Figure 4 ), the content of TAG in the Multi-HFD C. elegans model increased sharply ( Figure 4 b), SBP-1 ( Figure 4 a, c) and the fluorescence intensity of FASN-1 ( Figure 4 a, e) increased significantly, but did not affect ATGL-1 ( Figure 4a, d) Fluorescence intensity indicates that after treatment with Multi-HFD, nematodes mainly increase their fat levels by promoting fat synthesis. Additionally, treatment with RES can significantly reduce the TAG level but cannot restore it to the normal level. It can inhibit fat synthesis by suppressing the fluorescence intensities of SBP-1 and FASN-1, and at the same time promote an increase in the fluorescence intensity of ATGL-1, promoting fat breakdown, thereby controlling fat content in a two-way manner by inhibiting fat synthesis and promoting fat breakdown to maintain homeostasis.

[0080] 5. The phenomenon of super-large lipid droplets disappears in the Multi-HFD nematode model after deletion of key genes in lipid metabolism

[0081] In nematodes, the IIS signaling pathway (Insulin / insulin-like growth factor signaling pathway) senses external nutritional signals through the receptor DAF-2 (homolog of insulin / IGF-1 receptor), activates the downstream phosphatidylinositol 3-kinase (AGE-1) and AKT kinase cascade reactions, and ultimately regulates the nuclear localization of the transcription factor DAF-16 / FOXO, regulating cellular glucose and lipid metabolism, growth, and differentiation. In the obese state, long-term high-fat diet or energy surplus can lead to abnormal activity of the IIS signaling pathway, manifested as insulin resistance, that is, the sensitivity of cells to insulin signals decreases, thereby triggering elevated blood sugar, increased fat synthesis, and ectopic lipid deposition.

[0082] As a member of the nuclear hormone receptor superfamily, NHR-80 may affect energy metabolism by regulating mitochondrial function. NHR-80 can maintain energy metabolism homeostasis by regulating the expression of genes related to fatty acid β-oxidation, and its abnormal function may lead to fat accumulation and the occurrence of metabolic syndrome.

[0083] SBP-1 (sterol regulatory element-binding protein SREBP homolog gene) directly regulates the expression of lipid generation-related genes such as FASN-1 to regulate lipid metabolism.

[0084] The transcriptional co-regulator MDT-15 is a subunit of the Mediator complex and can regulate various physiological aspects, including lipid metabolism, stress resistance, and lifespan. MDT-15 is a key regulator of the lipid synthesis pathway. By acting synergistically with SREBP, it activates the expression of fatty acid desaturases (such as FAT-2). Its abnormal expression may lead to adipose tissue inflammation and insulin resistance, thereby promoting the development of obesity-related metabolic syndrome.

[0085] After deletion of the genes daf-16, age-1, sbp-1, nhr-80, and mdt-15 respectively, the phenomenon of super-large lipid droplets caused by Multi-HFD treatment in wild-type nematodes disappears. Figure 5) It indicates its role in inducing lipid droplet formation and fusion, suggesting that the above genes play a key regulatory role in the process of lipid droplet formation and fusion. In addition, the nhr-80 deletion mutant exhibits unique phenotypic characteristics: although the formation of super-large lipid droplets is completely blocked, the Multi-HFD treatment can still induce a significant expansion of the lipid droplet diameter, indicating that the nhr-80 deletion can only partially reverse the abnormal lipid droplet phenotype, and there may be other pathways that can still promote lipid droplet formation and fusion after the failure of nhr-80 function, such as daf-16, age-1, sbp-1, mdt-15, etc. After RES treatment, the inhibitory effect on the expansion of the lipid droplet diameter disappears, indicating that RES depends on the above genes to play an inhibitory role in lipid droplet enlargement.

[0086] Based on the above results, it can be known that: daf-16 / age-1 is located upstream of the regulatory network and affects lipid synthesis by regulating sbp-1 expression; while nhr-80 and mdt-15 form a parallel pathway to regulate lipid droplet fusion respectively. It is worth noting that the residual lipid droplet enlargement effect in the nhr-80 defective mutant suggests the existence of a daf-16-independent regulatory pathway.

[0087] 6. The phenomenon of super-large lipid droplets disappears in the Multi-HFD nematode model after the deletion of SCD homologous genes

[0088] Delta-9 desaturase (also known as stearoyl-CoA desaturase, SCD) is a key enzyme that catalyzes the conversion of saturated fatty acid (SFA) to monounsaturated fatty acid (MUFA), and its products (such as oleic acid, palmitoleic acid) are important substrates for TAG synthesis. Studies have shown that an increase in SCD activity will lead to an increase in the proportion of MUFA in adipose tissue, promoting lipid droplet formation and fat deposition. A high-fat diet upregulates SCD expression by activating transcription factors (such as sbp-1 / SREBP, daf-16 / FOXO) and nuclear receptors (such as nhr-80), promoting fatty acid desaturation and fat accumulation.

[0089] In nematodes, the fat-5, fat-6, and fat-7 genes encode three different SCD homologs. Fat-5 mainly catalyzes the production of palmitoleic acid (C16:1n7) from palmitic acid (C16:0), while fat-6 and fat-7 mainly catalyze the production of oleic acid (C18:1n9) from stearic acid (C18:0), and fat-7 has a higher affinity for stearic acid. To further explore whether SCD is involved in the formation of super-large lipid droplets in the Multi-HFD nematode model, nematode mutants with corresponding gene deletions were used for Oil Red O staining and the measurement and analysis of the diameter of the largest lipid droplets in their bodies.

[0090] The results showed that (such as Figure 6 ), when fat-5 was individually absent or both fat-5 and fat-6 were absent, the absence of fat-5 inhibited the desaturation of palmitic acid (16:0), but still increased the lipid droplet diameter, indicating that the presence of fat-7 could retain part of the compensatory effect of stearic acid (18:0) desaturation to compensate for the function of SCD, while when fat-6 or fat-7 was individually absent or both fat-6 and fat-7 were absent, the formation of super-large lipid droplets was inhibited, further proving that the formation of super-large lipid droplets requires the participation of stearic acid desaturation. The function of RES completely depends on the integrity of the fat-6 / fat-7 double pathway. It is speculated that stearic acid desaturation (mediated by fat-7) is a necessary condition for the formation of super-large lipid droplets. There is a functional compensation mechanism in the SCD family, but fat-7 is irreplaceable; while RES exerts an intervention effect through the SCD-dependent pathway.

[0091] In summary, the Multi-HFD model can promote the formation of super-large lipid droplets and synchronously trigger lipid toxicity-related physiological dysfunctions, including reduced head swing frequency, swallowing frequency, and defecation frequency. Morphological analysis showed that the body length, body width, and body area of the nematodes in the model group increased significantly. Molecular mechanism studies showed that this model led to a significant increase in TAG levels by upregulating the expression of the adipogenesis transcription factor SBP-1 and promoting the activity of the fatty acid synthase FASN-1, ultimately forming pathological characteristic super-large lipid droplets. Further studies showed that the formation of super-large lipid droplets depends on the occurrence of genes such as daf-16, mdt-15, sbp-1, nhr-80, age-1, and the homologous gene of delta-9 desaturase. The resveratrol can significantly improve the formation of super-large lipid droplets and restore physiological functions, promote fat decomposition and inhibit fat generation to reduce fat accumulation, and regulate the lipid droplet diameter depending on genes such as daf-16, mdt-15, sbp-1, nhr-80, age-1, and the homologous gene of delta-9 desaturase.

[0092] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing a metabolic disease Caenorhabditis elegans model, characterized in that: The eggs of wild-type nematodes or mutant nematodes are placed on a nematode growth high-fat medium for culture, and then placed in a constant temperature incubator for culture to obtain a Multi-HFD model.

2. The method for constructing a metabolic disease Caenorhabditis elegans model according to claim 1, characterized in that: The nematode growth high-fat medium is prepared by the following steps: Mix the cooked egg yolk and E. coli OP50 bacterial solution in a ratio of 1:5 to prepare a high-fat diet bacterial solution for use; mix the high-fat diet bacterial solution with anhydrous ethanol or sterilized pure water for use; mix the high-fat diet bacterial solution with resveratrol for use; spread 300 μL of the above solution on an NGM feeding plate, seal the plate after drying, and store it at 4°C for use.

3. The method for constructing a metabolic disease Caenorhabditis elegans model according to claim 2, characterized in that: The OD600 of the E. coli OP50 bacterial solution is 0.4-0.

6.

4. The method for constructing a metabolic disease Caenorhabditis elegans model according to claim 1, characterized in that: The culture temperature is 16° C.-20° C., the culture humidity is 40%-60%, and the culture time is 2 days-4 days.

5. The method for constructing a metabolic disease Caenorhabditis elegans model according to claim 1, characterized in that: The Multi-HFD model promotes the obese phenotype by forming extra-large lipid droplets, and simultaneously reduces physiological indicators related to lipid toxicity to promote fat synthesis and induce the formation of extra-large lipid droplets.

6. The method for constructing a metabolic disease Caenorhabditis elegans model according to claim 5, characterized in that: The lipid toxicity-related physiological indicators include head shaking frequency, swallowing frequency and defecation frequency.

7. The method for constructing a metabolic disease Caenorhabditis elegans model according to claim 1, characterized in that: The Multi-HFD model upregulates the expression of the lipogenic transcription factor SBP-1 and promotes the activity of the fatty acid synthase FASN-1, resulting in a significant increase in TAG levels to form pathologically characteristic extra-large lipid droplets.

8. The method for constructing a metabolic disease Caenorhabditis elegans model according to claim 7, characterized in that: In the Multi-HFD model, the formation of pathologically characteristic extra-large lipid droplets is associated with genes daf-16, mdt-15, sbp-1, nhr-80, age-1 and delta-9 desaturase homologous genes.

9. A metabolic disease Caenorhabditis elegans model, characterized in that: The method is obtained by constructing according to any one of claims 1 to 7.

10. A use of the metabolic disease Caenorhabditis elegans model as claimed in claim 9, characterized in that: Used in screening drugs to improve metabolic diseases.

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