Construction method and application of liver injury animal model
The liver injury model was constructed by gavage of dibutyl phthalate of different concentrations in mice, which solved the shortcomings of dibutyl phthalate toxicology research in the liver field, and achieved in-depth research on the mechanism of liver damage and drug development.
Patent Information
- Application Number
- CN202411585741.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-08
- Publication Date
- 2025-08-29
AI Technical Summary
The toxicological mechanism of dibutyl phthalate in the prior art has not been fully discovered, especially in the liver field, and there is little research, and there is a lack of effective animal models of liver damage.
Animal model of liver injury was constructed by gavage of different concentrations of dibutyl phthalate (10-250 mg/kg b.w./day) to 5-6 weeks of age male mice, combined with liver pathological changes, serum markers and liver tissue oxidative stress markers.
It provides an animal model of liver injury with simple operation and strong controllability, which can study the toxic mechanism of dibutyl phthalate on the liver and is used to develop anti-hepatic injury drugs and functional foods.
Smart Images
Figure CN120549032A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of toxicology, and specifically relates to a method for constructing and applying a liver injury animal model. Background Art
[0002] Dibutyl phthalate (DBP) is a widely used low-molecular-weight phthalate ester. It is a plasticizer commonly found in plastic products and can also be detected in some skincare products, such as perfumes, lotions, and cosmetics. Due to its widespread use, human exposure to DBP has gradually increased. The biological damage caused by DBP is multifaceted. Studies have shown that exposure can cause adverse reproductive effects and affect the normal development of rat pups. DBP exposure has also been linked to developmental disorders and increased mortality in zebrafish, possibly due to damage to mitochondrial DNA. Exposure to DBP also significantly inhibits acetylcholinesterase activity, leading to neurological disturbances in the fish's brain. Furthermore, DBP has been shown to increase the risk of anxiety and impair memory, demonstrating its potential neurotoxicity. Acute DBP exposure can also significantly damage the small intestine. This demonstrates the significant biological toxicity of DBP.
[0003] Currently, research on dibutyl phthalate has become a hot topic. However, its toxicological mechanisms have not yet been fully explored. Most toxicological studies focus on the neurological field, with few reports on the liver field. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for constructing an animal model of liver injury and its application. The construction method is simple and highly controllable, and provides a reliable research basis for studying the liver injury mechanism of dibutyl phthalate.
[0005] The invention provides a method for constructing an animal model of liver damage. The method comprises the following steps: administering dibutyl phthalate to the animal by gavage; the gavage dose of the dibutyl phthalate is 10 to 250 mg / kg bw / day.
[0006] As a preferred embodiment, the solvent of dibutyl phthalate is edible oil.
[0007] As a preferred embodiment, the animals include: male mice aged 5 to 6 weeks.
[0008] As a preferred embodiment, the duration of the gavage is 25 to 30 days.
[0009] As a preferred embodiment, the method further comprises: subjecting the animal to adaptive culture for 5 to 8 days before gavage.
[0010] As a preferred embodiment, the effect evaluation factors of the liver injury animal model include at least one of the following:
[0011] (1) Pathological changes of the liver;
[0012] (2) changes in serum marker levels;
[0013] (3) changes in the levels of oxidative stress markers in liver tissue;
[0014] (4) The degree of liver damage.
[0015] The present invention provides a liver injury animal model obtained by the above-mentioned construction method.
[0016] The present invention provides the use of the above-mentioned construction method or the above-mentioned liver injury animal model in evaluating drugs for resisting liver injury caused by dibutyl phthalate.
[0017] The present invention provides the use of the above-mentioned construction method or the above-mentioned liver injury animal model in the preparation of anti-liver injury drugs.
[0018] The present invention provides the use of the above-mentioned construction method or the above-mentioned liver injury animal model in the preparation of functional foods.
[0019] Beneficial Effects: The present invention provides a method for constructing an animal model of liver injury, comprising the steps of: administering dibutyl phthalate orally to an animal; the oral dose of dibutyl phthalate is 10 to 250 mg / kg b.w. / day. The method is simple to operate, and experiments using different concentrations of dibutyl phthalate revealed that all concentrations caused liver injury, with the degree of injury increasing with increasing dibutyl phthalate concentration. Therefore, the degree of injury in the model can be controlled by oral dose, facilitating research into the mechanism of liver injury caused by dibutyl phthalate and related fields, and possessing promising application value.
[0020] The present invention provides an animal model of liver injury obtained by the above-mentioned construction method, which can meet the demand for mouse models in the study of the mechanism of phthalate liver toxicity.
[0021] The present invention provides methods for constructing animal models of liver injury or applications of such animal models. The liver injury mice constructed in the present invention can be used as a model for liver injury to study the genetic pathways, proteins, and metabolites that change during liver injury. This can be used to investigate the mechanisms of liver injury caused by dibutyl phthalate (DBP), and to develop new functional foods to repair DBP-damaged livers. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1The following are the results of pathological sections of mouse liver after oral administration of different doses of dibutyl phthalate;
[0023] Figure 2 The figure shows the results of the activity determination of aspartate aminotransferase and alanine aminotransferase in the serum of mice after oral administration of different doses of dibutyl phthalate;
[0024] Figure 3 The figure shows the results of the activity determination of glutathione peroxidase, superoxide dismutase and myeloperoxidase in the liver of mice after oral administration of different doses of dibutyl phthalate. DETAILED DESCRIPTION
[0025] The invention provides a method for constructing a liver injury animal model. The method comprises the following steps: administering dibutyl phthalate to the animal by gavage; the gavage dose of the dibutyl phthalate is 10 to 250 mg / kg b.w. / day.
[0026] In an embodiment of the present invention, animals are gavaged with dibutyl phthalate at a dose of 10 to 250 mg / kg bw / day, such as 10 mg / kg bw / day, 50 mg / kg bw / day, 100 mg / kg bw / day, 150 mg / kg bw / day, 200 mg / kg bw / day, or 250 mg / kg bw / day. In a specific embodiment, the dibutyl phthalate has a purity greater than 99%.
[0027] In an embodiment of the present invention, the solvent for dibutyl phthalate is an edible oil, such as corn oil or soybean oil, specifically corn oil, which is safe, chemically stable, and inexpensive. In a specific embodiment, dibutyl phthalate dissolved in corn oil is used as an intervention agent and continuously gavaged into animals to establish an animal model of liver injury.
[0028] In the present embodiment, the animals include: male mice aged 5 to 6 weeks; mice in this age range are of suitable weight for manipulation and are in their prime, with minimal other physiological factors. Female mice are more susceptible to hormonal cycles, so male mice are used. In a specific embodiment, the male mice are C57BL / 6J and are 5 weeks old.
[0029] In the embodiment of the present invention, the animals are cultured for 5 to 8 days before oral administration. In a specific embodiment, male mice are cultured for 7 days under the conditions of sufficient food and sterile drinking water to ensure that the living environment of the mice is hygienic and comfortable.
[0030] In an embodiment of the present invention, the duration of the gavage is 25 to 30 days, specifically 28 days. In a specific embodiment, the mice are gavaged continuously for 28 days, only once a day, at 10:30 a.m. every day. 12 hours after the 28th gavage, the mice are stopped from being fed feed, but the mice are not stopped from being fed water.
[0031] In an embodiment of the present invention, the effect evaluation factors of the liver injury animal model include at least one of the following:
[0032] (1) Pathological changes of the liver; the pathological changes include: damage and / or inflammation;
[0033] (2) Changes in serum marker levels; the serum markers include: alanine aminotransferase and / or aspartate aminotransferase;
[0034] (3) Changes in the levels of liver tissue oxidative stress markers; the liver tissue oxidative stress markers include: one or more of glutathione peroxidase, superoxide dismutase, and myeloperoxidase;
[0035] (4) The degree of liver damage.
[0036] The present invention provides a liver injury animal model obtained by the above-mentioned construction method.
[0037] The present invention provides the use of the aforementioned construction method or animal model of liver injury for evaluating drugs that protect against dibutyl phthalate-induced liver injury. The animal model of liver injury constructed in the present invention can be used to study the hepatotoxic mechanisms of dibutyl phthalate, specifically, to investigate the relevant gene pathways, proteins, and metabolites that change during liver injury.
[0038] The present invention provides the use of the above-mentioned construction method or the above-mentioned liver injury animal model in the preparation of anti-liver injury drugs.
[0039] The present invention provides the use of the above-mentioned construction method or the above-mentioned liver injury animal model in the preparation of functional foods. The liver injury animal model constructed by the present invention can be used to develop new functional foods that have the effect of repairing liver damage caused by dibutyl phthalate.
[0040] To further illustrate the present invention, the construction method and application of a liver injury animal model provided by the present invention are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.
[0041] Example 1
[0042] Method for establishing mouse liver injury model:
[0043] (1) Five-week-old SPF-grade C57BL / 6J male mice were housed in sterile cages and provided with ample fresh sterile drinking water and food. A 12-hour light and dark cycle was maintained daily, and the cage bedding, drinking water, and food were changed every three days. Prior to the dibutyl phthalate gavage experiment, the mice were acclimated to the diet for one week.
[0044] (2) Dissolve dibutyl phthalate (purity greater than 99%) in corn oil (reagent grade) to prepare 20 mL of solutions at concentrations of 0 mg / mL, 1 mg / mL, 5 mg / mL, and 25 mg / mL, respectively. Vortex thoroughly for 60 seconds and store in a -20°C refrigerator. Prepare fresh solutions every 5 days.
[0045] (3) On day 8 (i.e., after completion of adaptive training), the mice in step (1) were randomly divided into four groups, namely, a control group (CON), a low-dose group (LOW), a medium-dose group (MIDDLE), and a high-dose group (HIGH), and were gavaged with the dibutyl phthalate corn oil solution prepared in step (2). The gavage dose of dibutyl phthalate in the control group was 0 mg / kg b.w. / day, the low-dose group was 10 mg / kg b.w. / day, the medium-dose group was 50 mg / kg b.w. / day, and the high-dose group was 250 mg / kg b.w. / day.
[0046] (4) Starting from the eighth day of adaptive culture, the mice were weighed and recorded daily at 10:30 a.m., according to the gavage dose in step (3). The mice were then gavaged according to their weight. The weight, food intake, and water intake of the C57BL / 6J mice were measured daily during the gavage period.
[0047] (5) The operation in step (4) was performed by gavage at the same time every day for 28 consecutive days.
[0048] (6) After the last gavage in step (5), mice were fasted from food but not water starting at 10:30 p.m. (i.e., 12 hours after the last gavage). At 10:30 a.m. the next day, the mice were weighed, anesthetized with isoflurane, blood was collected from the eyeballs, centrifuged at 3000 rpm for 10 minutes, the supernatant was collected, the color was checked for normality, and the serum was stored in a -80°C refrigerator. The mice were then immediately killed by spinal dislocation, and the liver and other tissues were obtained by dissection. The tissue samples were thoroughly washed with phosphate buffer solution, quenched with liquid nitrogen for 30 seconds, weighed on a balance, and stored in a -80°C refrigerator.
[0049] Example 2
[0050] The pathological conditions of mice with liver injury were analyzed:
[0051] (1) The mouse liver obtained in step (6) of Example 1 was placed in a paraformaldehyde fixative, stained with hematoxylin-eosin, and sliced. The hematoxylin-eosin stained slices of the liver were observed under a microscope and photographed. The results were as follows: Figure 1 shown.
[0052] from Figure 1 It can be seen that the liver suffered obvious damage and inflammation after oral administration of dibutyl phthalate.
[0053] (2) The serum obtained in step (6) of Example 1 was taken out of the refrigerator, thawed on ice, and the activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were detected. The results are shown in Table 1 and Figure 2 Alanine aminotransferase and aspartate aminotransferase activity assay kits were purchased from Nanjing Jiancheng Bioengineering Institute.
[0054] GraphPad Prism 8.0 software was used for bar graphing and statistical analysis of the data. The results are presented as mean ± standard deviation. Comparisons between the two groups were analyzed using the t-test. * indicates a t-test P value less than 0.05, ** indicates a t-test P value less than 0.01, and *** indicates a t-test P value less than 0.001.
[0055] Table 1 Serum AST and ALT activities in mice after oral administration at different doses
[0056] Group AST activity (U / L) ALT activity (U / L) Control group (CON) 44.7±15.3 27.7±11.7 Low dose group (LOW) 60.6±13.6 38.2±17.7 Medium dose group (MIDDLE) 87.1±14.5 45.5±11.1 High dose group (HIGH) 91.6±11.7 52.5±10.5
[0057] From Table 1 and Figure 2 As can be seen in the results, the activities of serum markers aspartate aminotransferase and alanine aminotransferase were significantly increased. The aspartate aminotransferase activities in the control, low-dose, medium-dose, and high-dose groups were 44.7±15.3U / L, 60.6±13.6U / L, 87.1±14.5U / L, and 91.6±11.7U / L, respectively. The alanine aminotransferase activities in the control, low-dose, medium-dose, and high-dose groups were 27.7±11.7U / L, 38.2±17.7U / L, 45.5±11.1U / L, and 52.5±10.5U / L, respectively.
[0058] (3) The liver tissue obtained in step (6) of Example 1 was taken out of the refrigerator, 0.1 g was weighed on a balance, 900 μL of physiological saline was added, and the mixture was frozen and ground at 12000 rpm for 8 minutes. 500 μL of the ground 10% liver homogenate was taken to detect myeloperoxidase (MPO) activity. The remaining homogenate was centrifuged at 4°C and 10000 rpm for 10 minutes, 100 μL of the supernatant was taken, and 3.9 mL of pure water was added to dilute it to prepare a liver homogenate supernatant dilution solution. The total superoxide dismutase activity (SOD) and glutathione peroxidase (GSH-Px) activity were detected. The results are shown in Table 2 and Figure 3 Glutathione peroxidase, superoxide dismutase, and myeloperoxidase activity assay kits were purchased from Nanjing Jiancheng Bioengineering Institute.
[0059] Table 2 GSH-Px, SOD and MPO activities in the liver of mice after oral administration at different doses
[0060] Group GSH-Px activity (U / mgprot) SOD activity (U / mgprot) MPO activity (U / g) Control group (CON) 306.4±36.8 166.4±13.1 0.12±0.06 Low dose group (LOW) 253.0±12.8 140.2±18.5 0.15±0.09 Medium dose group (MIDDLE) 213.1±30.9 114.2±23.0 0.27±0.07 High dose group (HIGH) 198.8±50.3 105.0±7.0 0.24±0.06
[0061] From Table 2 and Figure 3 As can be seen, glutathione peroxidase activity decreased, superoxide dismutase activity decreased, and myeloperoxidase activity increased, indicating significant oxidative stress in the liver. The glutathione peroxidase activity in the control, low-dose, medium-dose, and high-dose groups was 306.4±36.8U / mg prot, 253.0±12.8U / mg prot, 213.1±30.9U / mg prot, and 198.8±50.3U / mg prot, respectively. The superoxide dismutase activity in the control, low-dose, medium-dose, and high-dose groups was 166.4±13.1U / mg prot, 140.2±18.5U / mg prot, 114.2±23.0U / mg prot, and 105.0±7.0U / mg prot, respectively. Among them, the myeloperoxidase activities of the control group, low-dose group, medium-dose group and high-dose group were 0.12±0.06U / g, 0.15±0.09U / g, 0.27±0.07U / g and 0.24±0.06U / g, respectively.
[0062] It can be seen that the construction method provided by the present invention can be used to construct a model liver injury mouse with a high modeling success rate. The degree of damage in the model can be regulated by gavage dose. It can be used to study the related gene pathways, proteins and metabolites that change during liver injury, and to develop new functional foods to repair the liver damaged by dibutyl phthalate, and has good application value.
[0063] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.
Claims
1. A method for constructing an animal model of liver injury, characterized in that: The construction method comprises the following steps: administering dibutyl phthalate to animals by gavage; the gavage dosage of the dibutyl phthalate is 10 to 250 mg / kg bw / day.
2. The construction method according to claim 1, characterized in that The solvent of the dibutyl phthalate is edible oil.
3. The construction method according to claim 1, characterized in that The animals include: male mice aged 5 to 6 weeks.
4. The construction method according to claim 1, characterized in that The duration of the gavage is 25 to 30 days.
5. The construction method according to claim 1, characterized in that The method also includes: culturing the animals for 5 to 8 days before gavage.
6. The construction method according to claim 1, characterized in that The effect evaluation factors of the liver injury animal model include at least one of the following: (1) Pathological changes of the liver; (2) changes in serum marker levels; (3) changes in the levels of oxidative stress markers in liver tissue; (4) The degree of liver damage.
7. An animal model of liver injury obtained by the construction method according to any one of claims 1 to 6.
8. Use of the construction method according to any one of claims 1 to 6 or the liver injury animal model according to claim 7 in evaluating drugs that inhibit liver injury caused by dibutyl phthalate.
9. Use of the construction method according to any one of claims 1 to 6 or the liver injury animal model according to claim 7 in the preparation of anti-liver injury drugs.
10. Use of the construction method according to any one of claims 1 to 6 or the liver injury animal model according to claim 7 in the preparation of functional foods.