Branched chain fatty acid and application thereof in inhibiting and killing caenorhabditis elegans

Abstract

The invention belongs to the technical field of biotoxicity testing, and discloses branched chain fatty acid and application thereof in inhibiting and killing caenorhabditis elegans. Model organism caenorhabditis elegans are adopted as a tested organism, 3-methylvaleric acid is adopted as an exogenous medicine, an experimental group and a control group with different concentration gradients are set, the death rates of the caenorhabditis elegans in 24 hours, 48 hours and 72 hours are calculated, and the death rate of the caenorhabditis elegans in the experimental group is calculated. And an LC50 value is calculated. Apparent structure analysis and transcriptome test analysis are carried out by means of an electron microscope, fluorescent staining and the like, and the toxic action mechanism of exogenous drugs on nematodes is explored. The research systematically reveals the toxicity mechanism of 3-methylvaleric acid for the first time, and provides a theoretical basis for the future research and development of 3-methylvaleric acid.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological toxicity testing, and in particular relates to a branched-chain fatty acid and an application thereof in inhibiting and killing Caenorhabditis elegans. Background Art

[0002] Short-chain fatty acids (SCFAs) are saturated fatty acids with fewer than six carbon atoms. Due to their high volatility, they are also called volatile fatty acids and are metabolites of intestinal microbial fermentation. SCFA research spans multiple disciplines, including microbiology, metabolism, immunology, neuroscience, and clinical medicine.

[0003] (1) Flavors and fragrances Short-chain fatty acids are used in the food additive and flavor and fragrance industries because of their distinctive odor. The "Hygienic Standards for the Use of Food Additives" stipulates usage standards. Different short-chain fatty acids naturally produce different aromas. For example, 4-methylvaleric acid, 3-methylvaleric acid, and 2-methylvaleric acid have different aroma intensities due to the different locations of their branches. Liu Yuping and others measured the relative strength of their aromas and found that the order was 4-methylvaleric acid > 3-methylvaleric acid > 2-methylvaleric acid. Experimental results show that the position of the methyl group has a certain influence on the properties of branched-chain fatty acids. When the concentration of certain branched-chain fatty acids and phenolic substances is sufficient, they can contribute unique flavors. The butyric acid flavor of the cow's milk cheese Romano cheese is improved by 2-methylbutyric acid and 2-ethylbutyric acid, providing a sweet and fruity taste.

[0004] (2) Biomedicine Most current studies have shown that in the medical field, short-chain fatty acids play a role in intestinal health and bacterial balance. Butyrate is the main energy source for colonic epithelial cells, which can maintain the integrity of the intestinal barrier, prevent the leakage of pathogens and toxins, and enhance the intestinal barrier function. Butyrate reduces intestinal inflammation by inhibiting the NF-κB pathway and has a significant effect on diseases such as inflammatory bowel disease and irritable bowel syndrome. Butyrate can also inhibit the growth of pathogens such as Escherichia coli by lowering the intestinal pH value and promote the proliferation of probiotics such as lactic acid bacteria.

[0005] Short-chain fatty acids are also closely linked to the development and improvement of a variety of diseases. They affect the central nervous system through immune pathways and are associated with neuropsychiatric disorders such as anxiety, depression, and autism. Animal studies have shown that short-chain fatty acids may delay the development of Alzheimer's and Parkinson's diseases. Short-chain fatty acids suppress tumors by enhancing immune function through, for example, activating cytotoxic T cells. They may also reduce damage to the intestinal mucosa caused by chemotherapy drugs and improve patient tolerance. Acetic acid and propionic acid reduce the risk of atherosclerosis by regulating cholesterol metabolism and blood pressure. Butyric acid has a potential inhibitory effect on colon cancer by inducing cancer cell apoptosis and inhibiting proliferation.

[0006] (3) Research significance Short-chain fatty acids, as key substances in the interaction between intestinal microorganisms and the host, are core molecules connecting diet, microbiota and health. Research on their functions not only deepens the understanding of the "microbiota-host symbiosis" mechanism, but also provides new targets for the treatment of metabolic diseases, immune disorders, neurodegenerative diseases and cancer. They have important scientific value and clinical application potential. However, the research challenge lies in the complexity of the mechanism and individual differences. The composition of the microbiota, the genetic background of the host and the dietary structure will affect the effect of short-chain fatty acids. The effects of short-chain fatty acids in different tissues may be opposite. These all require more detailed research, so the study of short-chain fatty acids remains a large and profound research field.

[0007] Currently, most research focuses on traditional short-chain fatty acids, primarily examining their antioxidant activity or their beneficial effects on disease. However, the toxicity of monomethylated branched-chain fatty acids is relatively rare. 3-Methylvaleric acid is a high-grade flavoring and fragrance, often used for fragrance and flavoring. It plays a vital role in the electronic liquid crystal industry, pharmaceutical synthesis, and flavoring and tobacco flavoring industries. However, the effects of 3-methylvaleric acid on the human body have yet to be systematically studied.

[0008] Through the above analysis, the problems and defects of the existing technology are as follows: (1) Currently, most studies are on traditional short-chain fatty acids, and most of the studies are on their antioxidant activity or their positive effects on diseases, while there are fewer studies on the toxicity of monomethylated branched-chain fatty acids.

[0009] (2) 3-Methylvaleric acid is a high-grade flavoring and fragrance, often used for flavoring and seasoning. It plays an important role in the electronic liquid crystal industry, pharmaceutical synthesis, edible flavoring, and tobacco flavoring industries. Currently, there has been no systematic research on the effects of 3-methylvaleric acid on the human body. Summary of the Invention

[0010] In response to the problems existing in the prior art, the present invention provides a branched-chain fatty acid and its application in inhibiting and killing Caenorhabditis elegans.

[0011] The present invention is achieved by providing a branched-chain fatty acid and its application in inhibiting and killing Caenorhabditis elegans, comprising: Step 1: Use the model organism Caenorhabditis elegans as the test organism and 3-methylvaleric acid as the exogenous drug. Set up experimental and control groups with different concentration gradients, calculate the mortality of nematodes at 24 h, 48 h, and 72 h, and calculate the LC 50 value; Step 2: Perform epigenetic structure analysis and transcriptome analysis with the help of electron microscopy and fluorescent staining to explore the toxic mechanism of exogenous drugs on nematodes.

[0012] Furthermore, the apparent structure analysis is performed by electron microscopy, fluorescent staining, etc.: (1) Fluorescence staining analysis of nematode cell necrosis; (2) Fluorescence staining analysis of lipofuscin accumulation in nematodes; (3) Determination and analysis of ROS in nematodes.

[0013] Furthermore, the nematode cell necrosis fluorescence staining analysis: The experimental group treated with 3-methylvaleric acid showed more red fluorescence than the control group, indicating that the cell membrane of the nematodes treated with 3-methylvaleric acid ruptured, allowing the dye to enter the nematodes' bodies and exhibit obvious red fluorescence under red excitation light; this shows that the drug ruptures the nematode cell membrane and enters the nematodes' bodies, triggering a series of changes in physiological and biochemical indicators, thereby shortening the nematode's lifespan; fluorescence intensity statistics also show that the experimental group has a more significant fluorescence effect than the control group.

[0014] Furthermore, the nematode lipofuscin accumulation was analyzed by fluorescence staining: Lipofuscin in nematodes is autofluorescent. Under green excitation light, the experimental group treated with 3-methylvaleric acid showed more green fluorescence than the control group, indicating that 3-methylvaleric acid caused a large accumulation of lipofuscin in the nematodes, accelerating the aging and death of the nematodes; fluorescence intensity statistics also showed that the experimental group had a more significant fluorescence effect than the control group.

[0015] Furthermore, the ROS in the nematode body was measured and analyzed: The experimental group treated with 3-methylvaleric acid showed more green fluorescence compared to the control group, indicating that 3-methylvaleric acid can stimulate oxidative stress in nematodes and accelerate aging or death of nematodes; fluorescence intensity statistics show that the experimental group has a more significant fluorescence effect than the control group; this is consistent with the results of GO enrichment analysis, which suggests that 3-methylvaleric acid can affect the nematode's defense mechanism, weaken the nematode's antioxidant defense mechanism, induce oxidative stress in nematodes, and accumulate a large amount of reactive oxygen free radicals in the body, thereby accelerating aging or death of nematodes.

[0016] Another object of the present invention is to provide a branched fatty acid toxicity effect system on Caenorhabditis elegans, comprising: Step 1: Use the model organism Caenorhabditis elegans as the test organism and 3-methylvaleric acid as the exogenous drug. Set up experimental and control groups with different concentration gradients, calculate the mortality of nematodes at 24 h, 48 h, and 72 h, and calculate the LC 50 value; Step 2: Perform epigenetic structure analysis and transcriptome analysis with the help of electron microscopy and fluorescent staining to explore the toxic mechanism of exogenous drugs on nematodes.

[0017] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows: The present invention uses the model organism Caenorhabditis elegans as the test organism, uses 3-methylvaleric acid as the exogenous drug, sets up experimental groups and control groups with different concentration gradients, calculates the corrected mortality of the nematodes at 24 h, 48 h, and 72 h, and calculates the LC 50 The researchers used electron microscopy and fluorescence staining to analyze the structure of the nematode, measure physiological and biochemical parameters, and then conduct transcriptome analysis to explore the toxic mechanisms of exogenous drugs in nematodes. This study systematically reveals the toxic mechanism of 3-methylvaleric acid for the first time, providing a theoretical basis for future research and development of 3-methylvaleric acid.

[0018] 3-Methylvaleric acid, a fresh, fruity top note, is used in apple and strawberry flavors, as well as cheese flavors. As an additive, evaluating its dosage and toxicity is crucial. This study investigates the toxicity mechanism of this additive, demonstrating the potential commercial value of its future usage.

[0019] The toxicity of 3-methylvaleric acid was systematically studied for the first time. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a flow chart of the branched-chain fatty acids and their application in inhibiting and killing Caenorhabditis elegans provided by an embodiment of the present invention.

[0021] Figure 2 This is a structural block diagram of the system for the toxic effects of branched fatty acids on Caenorhabditis elegans provided by an embodiment of the present invention.

[0022] Figure 3 This is a SEM image of the apparent structure of Caenorhabditis elegans provided in an embodiment of the present invention.

[0023] Figure 4 This is a fluorescent staining image of Caenorhabditis elegans cell necrosis provided by an embodiment of the present invention.

[0024] Figure 5 This is a fluorescent staining image of lipofuscin accumulation in Caenorhabditis elegans provided by an embodiment of the present invention.

[0025] Figure 6 This is a ROS fluorescence staining image of Caenorhabditis elegans provided by an embodiment of the present invention.

[0026] Figure 7 GO annotation histograms provided by an embodiment of the present invention: (a) Z1-CK; (b) Z2-CK; (3) Z3-CK; (4) Z4-CK.

[0027] Figure 8 This is a gene clustering dendrogram provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0029] like Figure 1 As shown, the embodiment of the present invention provides a branched-chain fatty acid and its application in inhibiting and killing Caenorhabditis elegans, comprising the following steps: S101, using the model organism Caenorhabditis elegans as the test organism, 3-methylvaleric acid as the exogenous drug, setting up experimental groups and control groups with different concentration gradients, calculating the mortality of the nematodes at 24 h, 48 h, and 72 h, and calculating the LC 50 value; S102, using electron microscopy and fluorescent staining to conduct epigenetic structural analysis and transcriptome testing and analysis, to explore the toxic mechanism of exogenous drugs on nematodes.

[0030] The embodiment of the present invention provides an apparent structural analysis using electron microscopy, fluorescent staining, etc.: (1) Fluorescence staining analysis of nematode cell necrosis; (2) Fluorescence staining analysis of lipofuscin accumulation in nematodes; (3) Determination and analysis of ROS in nematodes.

[0031] Fluorescence staining analysis of nematode cell necrosis provided in the embodiments of the present invention: The experimental group treated with 3-methylvaleric acid showed more red fluorescence than the control group, indicating that the cell membrane of the nematodes treated with 3-methylvaleric acid ruptured, allowing the dye to enter the nematodes' bodies and exhibit obvious red fluorescence under red excitation light; this shows that the drug ruptures the nematode cell membrane and enters the nematodes' bodies, triggering a series of changes in physiological and biochemical indicators, thereby shortening the nematode's lifespan; fluorescence intensity statistics also show that the experimental group has a more significant fluorescence effect than the control group.

[0032] Fluorescence staining analysis of lipofuscin accumulation in nematodes provided in the embodiments of the present invention: Lipofuscin in nematodes is autofluorescent. Under green excitation light, the experimental group treated with 3-methylvaleric acid showed more green fluorescence than the control group, indicating that 3-methylvaleric acid caused a large accumulation of lipofuscin in the nematodes, accelerating the aging and death of the nematodes; fluorescence intensity statistics also showed that the experimental group had a more significant fluorescence effect than the control group.

[0033] The present invention provides an analysis of ROS in nematodes: The experimental group treated with 3-methylvaleric acid showed more green fluorescence compared to the control group, indicating that 3-methylvaleric acid can stimulate oxidative stress in nematodes and accelerate aging or death of nematodes; fluorescence intensity statistics show that the experimental group has a more significant fluorescence effect than the control group; this is consistent with the results of GO enrichment analysis, which suggests that 3-methylvaleric acid can affect the nematode's defense mechanism, weaken the nematode's antioxidant defense mechanism, induce oxidative stress in nematodes, and accumulate a large amount of reactive oxygen free radicals in the body, thereby accelerating aging or death of nematodes.

[0034] like Figure 2 As shown, the embodiment of the present invention provides a branched fatty acid toxic effect system on Caenorhabditis elegans, including: The calculation module is used to use the model organism Caenorhabditis elegans as the test organism, 3-methylvaleric acid as the exogenous drug, set up experimental groups and control groups with different concentration gradients, calculate the mortality rate of the nematode at 24 h, 48 h, and 72 h, and calculate the LC 50 value; The analysis module is used to perform epigenetic structure analysis with the help of electron microscopy and fluorescent staining, conduct transcriptome testing and analysis, and explore the toxic mechanism of exogenous drugs on nematodes.

[0035] The present invention is specifically implemented: 1. Toxicity test According to the experimental results (Table 1), the corrected mortality of nematodes by 3-methylvaleric acid was 98.99% at 24 h, 100.00% at 48 h, and 100.00% at 72 h. Its LC 50 =196.33 μg / mL. Table 1 Nematicidal activity results of 3-methylvaleric acid Notes: “-”: indicates corrected mortality ≤ 10%, no nematicidal activity; “+”: indicates that the corrected mortality rate is 10% to 30% and the nematocidal activity is weak; “ + + ”: indicates a corrected mortality rate of 30% to 50% and moderate nematicidal activity; “ + + + ”: indicates that the corrected mortality rate is 50% to 80% and the nematode killing activity is relatively strong; “ + + + + ”: indicates that the corrected mortality rate is greater than 80% and the nematode killing activity is strong; “ / ”: Indicates that it cannot be seen clearly under a microscope.

[0036] Effects of 2-methylvaleric acid on the surface structure of nematodes The nematodes in the control group showed a naturally curved shape, with a plump body, a smooth surface, a normal epidermis of the head and body, and a clear lateral line on the side of the body ( Figure 3 a, c). In contrast, the experimental group showed a stiff body, unclear transverse stripes on the head, and obvious depressions, wrinkles, and tears in the lateral lines and epidermis of various parts of the body, with an uneven body surface ( Figure 3 b, d). This indicates that 3-methylvaleric acid severely damages the epidermal structure of C. elegans, visually demonstrating the physical damage to the nematode epidermis.

[0037] 3. Effects of 3-methylvaleric acid on nematode cell necrosis, ROS, and lipofuscin (1) Fluorescence staining analysis of nematode cell necrosis The experimental group treated with 3-methylvaleric acid showed more red fluorescence than the control group ( Figure 4 (a, b) shows that the cell membrane of nematodes treated with 3-methylvaleric acid ruptures, allowing the dye to enter the nematode body, resulting in obvious red fluorescence under red excitation light. This indicates that the drug ruptures the cell membrane of the nematode and enters the nematode body, causing changes in a series of physiological and biochemical indicators, thereby shortening the nematode lifespan. Fluorescence intensity statistics ( Figure 4 c) also showed that the experimental group had a more significant fluorescence effect than the control group.

[0038] (2) Fluorescence staining analysis of nematode lipofuscin accumulation Lipofuscin in nematodes has autofluorescence. Under green excitation light, the experimental group treated with 3-methylvaleric acid showed more green fluorescence than the control group ( Figure 5 a, b) This indicates that 3-methylvaleric acid causes a large accumulation of lipofuscin in the nematode, accelerating its aging and death. Fluorescence intensity statistics Figure 5 c, also showed that the experimental group had a more significant fluorescence effect than the control group.

[0039] (3) Analysis of ROS in C. elegans The experimental group treated with 3-methylvaleric acid showed more green fluorescence than the control group ( Figure 6a, b) indicate that 3-methylvaleric acid can stimulate oxidative stress in nematodes, accelerating aging or death. Fluorescence intensity statistics Figure 6 c shows that the experimental group had a more significant fluorescence effect than the control group. This is consistent with the results of GO enrichment analysis, which suggests that 3-methylvaleric acid can affect the nematode's defense mechanism, weakening its antioxidant defense mechanism, inducing oxidative stress in the nematode and causing a large accumulation of reactive oxygen free radicals in the body, thereby accelerating aging or even death.

[0040] 4. Transcriptome testing (1) Enrichment analysis The GO database functional annotation was used to perform enrichment analysis of differentially expressed genes. Figure 7 The following is a GO functional annotation map of the control group and different treatment groups (Z2, Z3, and Z4). As can be seen, the treated and control groups were primarily enriched in biological processes and cellular components. Z1-CK was primarily enriched in macromolecule metabolism (GO:0043170), defense response (GO:0006952), cellular process regulation (GO:0050794), membrane-bound organelles (GO:0043227), and intracellular organelles (GO:0043229). Z2-CK was primarily enriched in macromolecule metabolism (GO:0043170), positive regulation of organisms (GO:0048518), plasma membrane-bound cellular projection assembly (GO:0120025), membrane-bound organelles (GO:0043227), intracellular organelles (GO:0043229), and membraneless organelles (GO:0043228). Z3-CK was significantly enriched in macromolecule metabolism (GO:0043170), defense response (GO:0006952), cellular process regulation (GO:0050794), membrane-bound organelles (GO:0043227), and intracellular organelles (GO:0043229). Z4-CK was significantly enriched in macromolecule metabolism (GO:0043170), transport (GO:0006810), cellular process regulation (GO:0050794), membrane-bound organelles (GO:0043227), and intracellular organelles (GO:0043229). GO enrichment analysis suggests that 3-methylvaleric acid can affect nematode defense mechanisms, potentially damaging the cell wall and membrane, leading to damage of the cuticle and a direct and significant effect on epidermal morphology, consistent with SEM observations.

[0041] (2) Genetic analysis of WGCNA A weighted correlation network (WGCNA) analysis was constructed. Eleven modules were identified, including black, magenta, cyan, blue, purple, pink, brown, yellow, green, red, and gray, containing 155, 65, 2627, 1301, 58, 142, 1083, 1056, 713, 239, and 298 genes, respectively. A network analysis diagram with a weight threshold greater than 0.15 was constructed using WGCNA. This network analysis identified 24 core genes in the black module, including WBGene00000748, WBGene00000748, and WBGene00000707, which are N-terminal domain proteins of nematode cuticle collagen, and WBGene0000021236, which is involved in regulating the nematode longevity pathway.

[0042] Figure 8 Gene clustering dendrogram. Each leaf corresponds to a gene. A total of 11 merged modules were identified using a threshold of 0.15 within the framework of the weighted gene co-expression network (based on a threshold of 0.15). The transcriptome test results showed that during the toxicity of 3-methylvaleric acid to nematodes, differential genes were upregulated and downregulated at the molecular level. Through differential gene expression, it was found that 3-methylvaleric acid had a very significant effect on the oxidative stress (defense response), neuropeptide pathway, cell apoptosis and energy metabolism of nematodes. As the concentration increased, the enrichment pathways of differential genes were different. Multiple pathways worked synergistically to induce strong oxidative stress in nematodes, destroying the nematode epidermal structure (especially the cuticle). The drug solution penetrated into the nematode, causing the nematode lysosome to rupture and inducing cell apoptosis and necrosis.

[0043] The specific application fields or related products of the present invention.

[0044] Applications of 3-Methylvaleric Acid: 1. 3-Methylvaleric acid, an organic synthesis intermediate, is often used as an intermediate in the synthesis of other organic compounds. It can be used to produce ester, amide, and acyl chloride derivatives, which have important applications in medicine, pesticides, and chemicals.

[0045] 2. Flavors and Fragrances: 3-Methylvaleric acid has a unique herbal aroma with a slightly grassy flavor and is a food flavoring permitted by national regulations. It can be used in the production of soft drinks, cold drinks, jellies, puddings, and other foods, and can also be used in the blending of flavors and fragrances.

[0046] This study uses Caenorhabditis elegans as a model organism to investigate the toxicity mechanism of 3-methylvaleric acid. Over 40% of C. elegans' genes are homologous to humans, making it useful for studying human physiology and disease. As a short-chain fatty acid, 3-methylvaleric acid's unique chemical structure offers potential for functional diversity. The study of its toxicity mechanism aims to explore its potential as a food additive. Furthermore, its nematicidal activity is highly effective, suggesting potential applications in agriculture for the development of effective nematicides for disease control.

[0047] Toxicity tests using 3-methylvaleric acid at different concentration gradients demonstrated its strong nematicidal activity. Scanning electron microscopy (SEM) surface structural testing revealed severe surface structural damage, including physical damage to the nematode epidermis. Fluorescence testing revealed that the lysosomal membranes of nematodes treated with 3-methylvaleric acid underwent extensive rupture, releasing a large amount of hydrolytic enzymes within the lysosomes, disrupting the normal intracellular structure and causing cell necrosis. Acridine orange dye is permeable to cell membranes, and the 3-methylvaleric acid-treated group exhibited more green fluorescence compared to the control group, indicating that the cell membranes of nematodes treated with 3-methylvaleric acid ruptured, allowing the dye to enter the nematodes. This results in distinct green fluorescence under green excitation light. Entry of 3-methylvaleric acid into the nematodes may disrupt the structure of certain normal proteins or organelles, leading to massive apoptosis in nematode cells. Lipofuscin and ROS measurements in the nematodes showed that the 3-methylvaleric acid-treated group exhibited greater green fluorescence compared to the control group, indicating that 3-methylvaleric acid causes a significant accumulation of lipofuscin in the nematodes and stimulates oxidative stress, accelerating aging or death. Transcriptome analysis suggests that 3-methylvaleric acid affects the nematode's defense mechanisms, potentially damaging the cell wall and membrane, leading to damage to the nematode's cuticle and a direct and significant impact on epidermal morphology, consistent with SEM observations.

[0048] In summary, 3-methylvaleric acid treatment significantly altered the gene expression profile of C. elegans, with differentially expressed genes primarily enriched in defense responses (GO:0006952). Lysosomal rupture triggers the accumulation of lipofuscin and ROS. Lipofuscin is present in lysosomes, and lysosomal rupture leads to the loss or inactivation of numerous hydrolytic enzymes, resulting in an increase in lipofuscin. Transcriptome data also showed significant differences in the peroxidase metabolic pathway caused by 3-methylvaleric acid, suggesting that 3-methylvaleric acid may induce oxidative stress by inhibiting the antioxidant system, resulting in a decrease in ROS scavenging capacity.

[0049] The present invention uses multiple indicators such as nematode epigenetic structure, nematode cell apoptosis, cell necrosis, lysosomes, ROS, and nematode transcriptome to measure the nematocidal activity of drugs, analyzing the toxic action mechanism from multiple dimensions, which has great research significance in toxicology.

[0050] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. An application of a branched-chain fatty acid in inhibiting and killing Caenorhabditis elegans, characterized in that: The application includes subjecting Caenorhabditis elegans to the action of 3-methylvaleric acid, setting a plurality of treatment groups with different concentrations, recording the death data of each group after treatment for 24 hours, 48 ​​hours and 72 hours, and calculating the half lethal concentration LC50.

2. The use according to claim 1, characterized in that The method further comprises observing the morphological structure of the treated Caenorhabditis elegans, wherein the observation is performed by performing sample imaging analysis using a scanning electron microscope or a transmission electron microscope.

3. The use according to claim 1, characterized in that The method also includes fluorescent staining detection of cell necrosis in nematodes, wherein the dye can penetrate into the cell membrane rupture site and emit a visible light signal under the excitation of a specific wavelength.

4. The use according to claim 1, wherein It also includes the detection of fluorescence accumulation of lipofuscin in nematodes, and the use of an automated imaging system to acquire images and perform intensity statistics on the stained samples.

5. The use according to claim 1, characterized in that The method also includes quantitative determination of reactive oxygen species in nematodes, wherein the determination uses a ROS fluorescent probe to perform color reaction analysis on the treated sample.

6. The use according to claim 1, wherein It also includes high-throughput transcriptome analysis of samples from the treatment group and the control group, and the analysis includes differential gene screening and enrichment pathway annotation.

7. The use according to any one of claims 1 to 6, characterized in that The 3-methylvaleric acid is a high-purity chemical reagent and is dispersed in a liquid culture medium at a constant temperature for use in nematode exposure experiments.

8. A test system for the toxic effects of branched-chain fatty acids on Caenorhabditis elegans, characterized in that: The system includes a concentration setting module, a treatment and cultivation module, a mortality rate collection module and a data calculation module, and is used to measure the toxicity response of nematode samples under different treatment conditions.

9. The test system according to claim 8, wherein: It also includes a microscopic detection module and an image processing module for realizing the quantitative analysis of the morphology of individual nematodes and the staining status.

10. The test system according to claim 8 or 9, characterized in that: It also includes a sample RNA extraction module and a transcriptome sequencing module, which are used to obtain the sample's full gene expression profile data and perform bioinformatics analysis.

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