A method for constructing a 5-hydroxymethylfurfural-induced oxidative stress model
By adding 5-hydroxymethylfurfural to the Drosophila culture medium, an oxidative stress model was constructed, which solved the problem of the lack of suitable animal models in the existing technology and realized a simple, economical, and high-throughput toxicity evaluation and screening of 5-hydroxymethylfurfural.
Patent Information
- Application Number
- CN202410595682.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-14
- Publication Date
- 2026-01-06
- Estimated Expiration
- 2044-05-14
AI Technical Summary
There is a lack of suitable animal models for studying the toxicity of 5-hydroxymethylfurfural. In vivo experiments are costly and raise ethical concerns, while in vitro experiments are complex. The Drosophila melanogaster model has not been widely used for the toxicity evaluation of 5-hydroxymethylfurfural.
An oxidative stress model was constructed by adding 5-hydroxymethylfurfural to the Drosophila culture medium. The activities of ROS, SOD, CAT enzymes and MDA in the Drosophila developmental cycle and hemolymph were measured to establish a high-throughput screening platform.
A fruit fly model for evaluating the side effects of byproducts of food toxicity was successfully constructed, assessing oxidative damage and performing high-throughput screening, providing a simple and economical method for toxicity evaluation.
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Figure CN118318798B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of animal model evaluation technology, and specifically relates to a method for constructing a 5-hydroxymethylfurfural-induced oxidative stress model. Background Technology
[0002] 5-Hydroxymethylfurfural (5-HMF) is a common food processing contaminant, primarily formed through Maillard reactions and caramelization. Notably, 5-HMF is present in levels several orders of magnitude higher in most foods than in other associated contaminants such as acrylamide and advanced glycation end products (AGEs), and is widely found in a variety of foods, including honey, coffee, dried fruit, fruit juice, breakfast cereals, bread, milk powder, and alcoholic beverages.
[0003] Current research on the associated hazardous substance 5-hydroxymethylfurfural (5-HMF) largely relies on in vivo or in vitro cell experiments. In vivo experiments face limitations due to the lengthy and costly mammalian modeling process, as well as ethical concerns. In vitro experiments only assess toxicity at the single-cell level, while animal models are far more complex than cellular models. Therefore, suitable animal models for investigating the side effects of 5-HMF are lacking.
[0004] Despite increasing attention being paid to the harmful effects of byproducts such as 5-hydroxymethylfurfural (5-HMF), there are currently almost no reports on the use of the Drosophila melanogaster model for toxicity evaluation of 5-HMF. Drosophila melanogaster possesses numerous advantages, including ease of rearing, short lifespan, high reproductive capacity, simple chromosomes, and numerous easily observable mutant phenotypes, making it one of the most classic and important model organisms in scientific research. Furthermore, the antioxidant defense and metabolic systems in Drosophila melanogaster are conserved with those of mammals. Summary of the Invention
[0005] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a method for constructing a 5-hydroxymethylfurfural-induced oxidative stress model.
[0006] The objective of this invention is achieved through the following technical solution:
[0007] A method for constructing a 5-hydroxymethylfurfural-induced oxidative stress model includes the following steps:
[0008] S1. Fruit flies were cultured and divided into a control group and an experimental group. The control group was cultured in a conventional culture medium without exposure to 5-hydroxymethylfurfural. The experimental group was cultured in a culture medium containing 5-hydroxymethylfurfural and exposed to 5-hydroxymethylfurfural. After anesthetizing the fruit flies with CO2, male and female fruit flies that emerged within 8 hours were collected. Each tube of the control group and the experimental group contained 20 male and 20 female fruit flies. After laying eggs for 12 hours, the adult fruit flies were removed. After waiting for 4-5 days for the eggs to develop into third-instar larvae, the fruit fly animal model was obtained.
[0009] S2, Determination of the developmental cycle of fruit flies;
[0010] S3. Quantitative analysis of ROS in Drosophila hemolymph;
[0011] S4. Determine the activity of SOD and CAT enzymes and the content of MDA.
[0012] The purity of 5-hydroxymethylfurfural in step S1 is ≥99%, and the concentration used (i.e., the final concentration of 5-hydroxymethylfurfural in the culture medium) is 5mM-50mM.
[0013] The fruit fly development cycle determination in step S2 specifically involves placing 50 pairs of adult male and female fruit flies in a normal culture medium to lay eggs for 1 hour. After the adults are removed, fertilized eggs of uniform shape and size are selected under a stereomicroscope and cultured in an experimental culture medium. The observations are conducted at fixed times and locations every day, and the development time from egg to prepupa and from egg to adult is calculated.
[0014] The quantitative analysis of ROS in Drosophila hemolymph described in step S3 is performed as follows: Weigh third-instar Drosophila larvae, wash with PBS, blot dry with filter paper, place in a 1.5 mL centrifuge tube, add PBS buffer at a mass-to-volume ratio of 1 g: 9 mL, homogenize thoroughly with a bead mill, centrifuge the homogenate at 7500 rpm and 4℃ for 10 min, after centrifugation, add 190 μL of homogenate supernatant diluted 5-10 times and 10 μL of O12 fluorescent probe to a black 96-well fluorescent plate; incubate at 37℃ in the dark for 30 min, place in a continuous wavelength multifunctional microplate detection platform, and perform quantitative analysis using an excitation wavelength of 488 nm and an emission wavelength of 526 nm.
[0015] The determination of SOD, CAT enzyme activity and MDA content in step S4 is specifically performed as follows: Weigh a certain amount of third-instar fruit fly larvae, wash them with PBS, blot dry with filter paper, place them in a 1.5 mL centrifuge tube, add PBS buffer at a mass-to-volume ratio of 1 g: 9 mL, homogenize thoroughly with a bead mill, centrifuge the homogenate at 7500 rpm and 4℃ for 10 min, collect the supernatant, and determine the SOD, CAT enzyme activity and MDA content. Each indicator is standardized using the absorbance of the sample protein, and the protein concentration of each group of samples is determined according to the BCA protein quantification kit method.
[0016] The conventional culture medium is corn flour medium, and the formula of corn flour medium is: 9.6 g corn flour, 9.6 g sucrose, 3.2 g yeast powder, 0.8 g agar powder, 0.2 g methylparaben, 0.8 mL propionic acid, 2 mL anhydrous ethanol, and 100 mL distilled water; the culture environment is: constant temperature incubator, temperature is 25±1℃, relative humidity is 50%~60%, and the photoperiod is 12L:12D.
[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0018] This invention constructs an animal model for studying the side effects of byproducts of food by feeding fruit flies food containing 5-hydroxymethylfurfural (5-HMF). Normal food is used as a control. The antioxidant stress capacity of the fruit flies in vivo and in the intestines is measured after feeding them food containing 5-HMF. This invention can be used to establish and evaluate animal models of oxidative damage caused by byproducts of food by fruit flies, and to establish a high-throughput screening platform for bioactive substances that can mitigate oxidative damage caused by byproducts. Attached Figure Description
[0019] Figure 1 The effects of 5-HMF exposure on the developmental cycle of Drosophila.
[0020] Figure 2 The effect of 5-HMF exposure on ROS levels in Drosophila.
[0021] Figure 3 The effects of 5-HMF exposure on the activity of antioxidant enzymes and MDA content in Drosophila. Detailed Implementation
[0022] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Example 1
[0023] S1. Experimental fruit flies were cultured and divided into two groups: a control group, which was cultured in a conventional culture medium without exposure to 5-hydroxymethylfurfural; and an experimental group, which was cultured in a culture medium containing 5-hydroxymethylfurfural and exposed to 5-hydroxymethylfurfural. After anesthetizing the fruit flies with CO2, male and female fruit flies that emerged within 8 hours were collected. Each tube of the control group and the experimental group contained 20 male and 20 female fruit flies. After 12 hours of egg laying, the adult fruit flies were removed. After waiting 4-5 days for the eggs to develop into third-instar larvae, the fruit fly animal model was obtained.
[0024] S2, Determination of the developmental cycle of fruit flies: 50 pairs of male and female fruit flies aged 3 days were placed in a normal culture medium to lay eggs. After laying eggs for 1 hour, the adult flies were removed and fertilized eggs of uniform morphology and size were selected under a stereomicroscope and cultured in an experimental culture medium. The observation was carried out at fixed times and locations every day and the results were recorded. The development time from egg to prepupa and from egg to adult was calculated respectively.
[0025] S3, Quantitative analysis of ROS in Drosophila hemolymph: A certain amount of third-instar Drosophila larvae were weighed, washed with PBS, and dried with filter paper. They were placed in a 1.5 mL centrifuge tube, and PBS buffer was added at a mass-to-volume ratio of 1 g: 9 mL. The mixture was homogenized thoroughly using a bead mill. The homogenate was centrifuged at 7500 rpm and 4℃ for 10 min. After centrifugation, 190 μL of the homogenate supernatant diluted 5-10 times and 10 μL of O12 fluorescent probe were added to a black 96-well fluorescent plate. The plate was incubated at 37℃ in the dark for 30 min, and then placed in a continuous wavelength multi-functional microplate detection platform for quantitative analysis using an excitation wavelength of 488 nm and an emission wavelength of 526 nm.
[0026] S4, determination of SOD, CAT enzyme activity and MDA content: A certain amount of third-instar fruit fly larvae were weighed, washed with PBS, and dried with filter paper. They were placed in a 1.5 mL centrifuge tube, and PBS buffer was added at a mass-to-volume ratio of 1 g: 9 mL. The mixture was homogenized thoroughly using a bead mill. The homogenate was centrifuged at 7500 rpm and 4℃ for 10 min, and the supernatant was collected. The SOD, CAT enzyme activity and MDA content were determined. Each indicator was standardized using the absorbance of the sample protein. The protein concentration of each group of samples was determined according to the BCA protein quantification kit method.
[0027] The experimental data were subjected to the following statistical analysis:
[0028] Data were plotted and analyzed using GraphPad Prism 9 and SPSS 16.0 software. All experimental results are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used to assess differences between groups. Different letters indicate statistical differences between groups.
[0029] Figure 1The effects of 5-HMF exposure on the developmental cycle of Drosophila were presented, with data expressed as mean ± standard deviation, N = 10. The figures show that at low concentrations of 5 mM and 12.5 mM, pupation and emergence times were not affected. However, at high concentrations of 25 mM and 50 mM, compared to the control group, pupation time was prolonged by 1.72 days and 2.21 days, respectively, and emergence time was prolonged by 1.37 days and 1.91 days, respectively, indicating that development time is delayed with increasing ingestion concentration.
[0030] Figure 2 The effect of 5-HMF exposure on ROS levels in Drosophila was investigated. Data are expressed as mean ± standard deviation, N = 3. The figure shows that exposure to 5 mM, 12.5 mM, 25 mM, and 50 mM 5-HMF significantly increased ROS concentrations in third-instar Drosophila larvae (p < 0.05), increasing by 34.94%, 27.41%, 23.33%, and 40.44% respectively compared to the control group. This indicates that 5-HMF exposure can increase the content of oxygen free radicals in the body.
[0031] Figure 3 The effects of 5-HMF exposure on the activity of antioxidant enzymes and MDA content in Drosophila were investigated. Data are expressed as mean ± standard deviation, N = 3. With increasing ingestion concentration, the MDA content in third-instar larvae increased (p < 0.05), increasing by 21.49% and 31.39% compared to the control group at concentrations of 25 mM and 50 mM, respectively. This indicates that when 5-HMF ingestion reaches a certain concentration, oxygen free radicals accumulate in large quantities, leading to lipid peroxidation and a significant increase in MDA content. Superoxide dismutase (SOD) is the first line of defense against free radicals. SOD enzyme activity was found to gradually decrease with increasing 5-HMF ingestion concentration, decreasing by 12.61%, 11.33%, and 17.40% at concentrations of 12.5 mM, 25 mM, and 50 mM, respectively (p < 0.05). In catalase, increasing 5-HMF uptake promoted CAT enzyme secretion (p<0.05). At concentrations of 12.5 mM, 25 mM, and 50 mM, enzyme activity increased by 4.87%, 21.19%, and 21.55%, respectively. This further indicates that 5-hydroxymethylfurfural induces oxidative stress in third-instar larvae of Drosophila, leading to redox imbalance.
[0032] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A method for constructing a 5-hydroxymethylfurfural-induced oxidative stress model, characterized by The method comprises the following steps: S1, culture fruit flies, and divide them into a control group and an experimental group; the control group is cultured in a conventional culture medium without 5-hydroxymethyl furfural exposure; the experimental group is cultured in a culture medium containing 5-hydroxymethyl furfural, and the fruit flies are exposed to 5-hydroxymethyl furfural; after the fruit flies are anesthetized with CO2, the male and female fruit flies that have emerged within 8 hours are collected, and the control group and the experimental group are each loaded with 20 male and female fruit flies per tube; after the fruit flies lay eggs for 12 hours, the adult fruit flies are poured out, and after the eggs develop into third instar larvae for 4-5 days, the fruit fly animal model is obtained; the purity of the 5-hydroxymethyl furfural is greater than or equal to 99%, and the concentration used is 25 mM-50 mM; S2, fruit fly development cycle determination; S3, quantitative analysis of ROS in fruit fly hemolymph; S4, determination of SOD, CAT enzyme activity and MDA content.
2. The construction method of claim 1, wherein: The fruit fly development cycle determination in step S2 is specifically as follows: 50 pairs of male and female fruit fly adults are placed in a normal culture medium to lay eggs for 1 hour, then the adults are poured out, and fertilized eggs of uniform size are picked out under a body microscope and cultured in an experimental culture medium; the development time from egg to prepupa and from egg to adult is calculated by observing and recording at a fixed time and point every day.
3. The method of construction of claim 1, wherein: The quantitative analysis of ROS in fruit fly hemolymph in step S3 is specifically as follows: fruit fly third instar larvae are weighed, washed with PBS, and then the water is absorbed with filter paper; the fruit fly third instar larvae are placed in a 1.5 mL centrifuge tube, and PBS buffer is added at a mass-volume ratio of 1 g:9 mL; the homogenate is fully homogenized by using a bead mill, and then centrifuged at 7500 rpm and 4°C for 10 min; after centrifugation, 190 μL of the homogenate supernatant diluted by 5-10 times and 10 μL of O12 fluorescent probe are added to a fluorescent special black 96-well plate; the plate is incubated at 37°C for 30 min in the dark, and then placed in a continuous wavelength multifunctional microplate detection platform; quantitative analysis is performed by using an excitation wavelength of 488 nm and an emission wavelength of 526 nm.
4. The method of construction of claim 1, wherein: The determination of SOD, CAT enzyme activity and MDA content in step S4 is specifically as follows: a certain amount of fruit fly third instar larvae is weighed, washed with PBS, and then the water is absorbed with filter paper; the fruit fly third instar larvae are placed in a 1.5 mL centrifuge tube, and PBS buffer is added at a mass-volume ratio of 1 g:9 mL; the homogenate is fully homogenized by using a bead mill, and then centrifuged at 7500 rpm and 4°C for 10 min; the supernatant is taken to determine the SOD, CAT enzyme activity and MDA content; the absorbance of the sample protein is used for standardization treatment of each index, and the protein concentration of each group of samples is determined according to the determination method of the BCA protein quantitative kit.
5. The construction method according to any one of claims 1 to 4, characterized in that: The conventional culture medium is a corn powder culture medium, and the formula of the corn powder culture medium is as follows: corn powder 9.6 g, sucrose 9.6 g, yeast powder 3.2 g, agar powder 0.8 g, nipagin methyl ester 0.2 g, propionic acid 0.8 mL, anhydrous ethanol 2 mL, and distilled water 100 mL; the culture environment is as follows: a constant temperature incubator, a temperature of 25±1°C, a relative humidity of 50%-60%, and a light cycle of 12L:12D.