Method for constructing liver fibrosis animal model and application thereof
By adding adenine to animal feed, a liver fibrosis model was constructed, overcoming the limitations of existing methods and achieving a simple, rapid, and low-cost liver fibrosis model suitable for drug screening and disease research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENZHEN TRADITIONAL CHINESE MEDICINE HOSPITAL
- Filing Date
- 2026-03-05
- Publication Date
- 2026-04-17
AI Technical Summary
Existing methods for constructing animal models of liver fibrosis suffer from problems such as complex operation, high cost, low success rate, high toxicity, or limited applicability, making it difficult to effectively simulate the multi-factor intertwined process of human liver fibrosis.
Adenine was used as a feed ingredient to induce liver fibrosis in experimental animals. Mice, rats, or guinea pigs were fed a diet containing 0.2% to 0.8% adenine to stably establish a liver fibrosis model.
It has achieved the construction of a liver fibrosis model that is simple to operate, has a high success rate, and is low in cost. It can rapidly induce the expression of TGF-β1 and α-SMA, simulate the complex process of human liver fibrosis, and is suitable for drug screening and disease research.
Smart Images
Figure CN121867336A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology and relates to a method for constructing an animal model of liver fibrosis and its application. Background Technology
[0002] Liver fibrosis refers to the excessive repair response of the liver in response to various chronic injuries (such as viral infections, alcohol, metabolic abnormalities, etc.), resulting in an abnormal increase and deposition of extracellular matrix (mainly collagen) in the liver. This is a key intermediate step in the progression of liver disease to cirrhosis.
[0003] Animal models of liver fibrosis are important tools for studying the pathogenesis of liver fibrosis, drug screening, and the development of treatment strategies. Currently, there are a variety of mature modeling methods, which can be mainly divided into the following categories according to different induction methods: (1) Chemical induction, including induction with carbon tetrachloride and thioacetamide. Carbon tetrachloride generates free radicals under the action of hepatocyte pigment P450, causing lipid peroxidation, hepatocyte necrosis, activation of hepatic stellate cells (HSCs), and promotion of collagen deposition. Thioacetamide is metabolized in the liver into an active intermediate, causing DNA damage, oxidative stress, and inflammation, thereby inducing fibrosis; (2) Alcohol induction, long-term ethanol intake leads to oxidative stress, acetaldehyde accumulation, intestinal flora imbalance, and endotoxemia, activating Kupffer cells and HSCs; (3) Bile duct ligation, surgical ligation of the common bile duct, causing cholestasis, bile duct epithelial damage, inflammatory infiltration, and periportal fibrosis; (4) Genetic engineering, such as constructing transgenic / gene knockout mice, such as TGF-β overexpression, Smad3 knockout, and leptin receptor-deficient mice.
[0004] However, existing methods all have certain limitations. While chemical induction is commonly used and can induce fibrosis relatively quickly, it faces core problems: high toxicity leading to high animal mortality, individual differences affecting the uniformity of results, severe early fibrosis potentially interfering with the analysis of fibrosis-specific mechanisms, and carbon tetrachloride itself carries carcinogenic risks. Bile duct ligation can rapidly induce typical cholestatic fibrosis, but its applicability is limited: the procedure is difficult in mice, with high failure rates and high mortality rates from complications (ascites, infection), and it can only simulate the fibrosis pattern of this specific etiology, failing to reflect damage pathways caused by viruses or alcohol. Alcohol induction, while directly addressing the etiology, has an excessively long induction period, generally weak fibrosis, and requires expensive specialized feed. Although genetic engineering methods can precisely study specific gene pathways, their construction and maintenance costs are extremely high, single-gene alterations often cannot fully simulate the complex pathogenesis of multiple factors in humans, and phenotypes may be influenced by animal strain background.
[0005] In conclusion, it is of great significance to develop a simple, short-cycle, and low-cost method for constructing animal models of liver fibrosis. Summary of the Invention
[0006] In view of the shortcomings of existing technologies and practical needs, this invention provides a method for constructing an animal model of liver fibrosis and its application, with the aim of developing a simple and highly successful method for constructing a liver fibrosis model.
[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of adenine in the construction of animal models of liver fibrosis.
[0008] This invention discovers that adenine can be effectively used to construct liver fibrosis models. After administration to animals, it can stably induce liver fibrosis, providing a new approach for constructing liver fibrosis models.
[0009] Preferably, the characteristics of the animal model of liver fibrosis include elevated expression levels of TGF-β1 and α-SMA.
[0010] Secondly, the present invention provides a method for constructing an animal model of liver fibrosis, the method comprising: An animal model of liver fibrosis was established by feeding experimental animals with adenine-containing feed.
[0011] The present invention further designs a method for constructing an animal model of liver fibrosis, which is simple to operate and can stably construct the model simply by feeding, without the need for complicated operations such as gavage.
[0012] Preferably, the experimental animal includes any one of mice, rats, or guinea pigs.
[0013] Preferably, the rats include any one of SD rats, Wistar rats, or LEW rats.
[0014] Preferably, the mass percentage of adenine in the feed is 0.2% to 0.8%, for example, it can be 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.75% or 0.8%, preferably 0.7% to 0.8%, and more preferably 0.75%.
[0015] Preferably, the feed comprises general animal feed.
[0016] Preferably, the adenine-containing feed is provided in a way that allows the rats to eat freely with sufficient feed.
[0017] Thirdly, the present invention provides the application of the method for constructing an animal model of liver fibrosis as described in the first aspect in screening drugs for treating, alleviating or preventing liver fibrosis.
[0018] Fourthly, the present invention provides a liver fibrosis model, which is obtained by the method for constructing an animal model of liver fibrosis described in the first aspect.
[0019] In this invention, adenine can be used to induce liver fibrosis, and corresponding organ or cell models can be obtained.
[0020] Fifthly, the present invention provides a method for screening drugs for treating, alleviating, or preventing liver fibrosis, the method comprising: The method for constructing an animal model of liver fibrosis described in the first aspect is used to construct a model, the effects of candidate drugs on the model are analyzed, and candidate drugs with therapeutic effects are screened.
[0021] Compared with the prior art, the present invention has at least the following beneficial effects: This invention discovers that adenine can be effectively used to construct liver fibrosis models, stably inducing liver fibrosis. Furthermore, a method for constructing animal models of liver fibrosis was designed, including a specific feeding process, to achieve rapid and stable construction of animal models of liver fibrosis without the need for procedures such as gavage. This is of great significance for studying the pathogenesis of the disease and screening drugs. Attached Figure Description
[0022] Figure 1 Image showing the results of Masson staining of liver tissue sections.
[0023] Figure 2 Image showing the results of TGF-β1 immunostaining on liver tissue sections.
[0024] Figure 3 Image showing the results of α-SMA immunostaining on a liver tissue section. Detailed Implementation
[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.
[0026] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.
[0027] Example 1 This embodiment constructs an animal model of liver fibrosis.
[0028] Six- to eight-week-old SD rats were placed in an SPF-grade environment with free access to food and water. The room temperature was controlled at (21±2)℃ and the relative humidity at (50±15)%. Adenine (manufacturer: Maclean, catalog number A6279) was added to the normal diet (manufacturer: Xiehe, catalog number XT19031, including maintenance diet and 10% sucrose) to prepare a maintenance diet containing adenine with a final concentration of 0.25% (0.25% adenine diet: maintenance diet ratio 89.75% + 0.25% adenine (Ade) + 10% sucrose). The rats were allowed free access to food.
[0029] Example 2 This embodiment constructs an animal model of liver fibrosis.
[0030] Six- to eight-week-old SD rats were placed in an SPF-grade environment with free access to food and water. The room temperature was controlled at (21±2)℃ and the relative humidity at (50±15)%. Adenine was added to the diet to a final concentration of 0.5% (0.5% adenine diet: maintenance diet ratio 89.5% + 0.5% adenine + 10% sucrose). Sufficient feed was provided, and the rats were allowed free access to food.
[0031] Example 3 This embodiment constructs an animal model of liver fibrosis.
[0032] Six- to eight-week-old SD rats were placed in an SPF-grade environment with free access to food and water. The room temperature was controlled at (21±2)℃ and the relative humidity at (50±15)%. Adenine was added to the diet to a final concentration of 0.75% (0.75% adenine diet: maintenance diet ratio 89.25% + 0.75% adenine + 10% sucrose). Sufficient feed was provided, and the rats were allowed free access to food.
[0033] Test case (1) After feeding SD rats for 1 week (1W) and 2 weeks (2W) in each example, livers were taken for embedding, sectioning and fibrosis pathological staining. The specific process included: Masson staining: First, dewax paraffin sections until hydrated. Hematoxylin is then used to stain cell nuclei, followed by a mixture of Ponceau S and Acid Fructose to stain muscle fibers and cytoplasm. Phosphomolybdic acid or phosphotungstic acid is then used to differentiate the colors and enhance contrast. Finally, aniline blue or brilliant green is used to stain collagen fibers. After differentiation, dehydration, and clearing, the sections are mounted with neutral resin. After staining, collagen fibers appear blue (aniline blue) or green (brilliant green), muscle fibers and cytoplasm appear red, and cell nuclei appear bluish-black. This method is commonly used to observe tissue fibrosis or scar formation.
[0034] Immunohistochemical staining: First, paraffin sections are dewaxed, hydrated, and subjected to antigen retrieval (e.g., thermal retrieval or enzyme retrieval) to expose the antigenic epitopes. Then, endogenous enzyme activity is eliminated using a peroxidase inhibitor, and non-specific binding sites are blocked with serum. Specific primary antibody is added for incubation, allowing the antibody to bind to the target protein, followed by incubation with a secondary antibody containing a labeled enzyme (e.g., HRP) or fluorescein. Finally, a chromogenic substrate (e.g., DAB) is added to produce a visible precipitate, and the cell nuclei are counterstained with hematoxylin. After dehydration and clearing, the sections are mounted. The results can be observed under a microscope; the target protein appears brown (due to DAB staining) or shows a fluorescent signal, enabling protein localization analysis.
[0035] Masson staining results are as follows Figure 1 As shown, liver fibrosis gradually worsened over time (blue); the 0.75% Ade group showed significant blue collagen fiber deposition around blood vessels at 2 weeks (indicated by yellow arrows), indicating the most severe fibrosis; the 0.5% Ade group showed only mild blue collagen deposition at 2 weeks; and the 0.25% Ade group showed the least fibrotic changes at 2 weeks. This demonstrates that the present invention specifically regulates the dosage of adenine agents to efficiently promote liver collagen deposition and accelerate fibrosis progression. TGF-β1 and α-SMA immunostaining results are shown below. Figure 2 and Figure 3 As shown, in the TGF-β1 detection, the 0.75% Ade group showed obvious brownish-red positive staining at 2 weeks (indicated by yellow arrows), and the staining intensity and range were significantly higher than those in the 0.25% and 0.5% Ade groups; the 0.5% Ade group showed only weak staining, and the 0.25% Ade group showed very little positive signal. In the α-SMA detection, a large number of brownish-red α-SMA-positive myofibroblasts appeared around the blood vessels at 2 weeks in the 0.75% Ade group (indicated by yellow arrows), indicating significant myofibroblast activation; the 0.5% Ade group showed only a small number of positive cells, and the 0.25% Ade group showed the weakest positive signal. This indicates that the targeted regulation of high-dose adenine dosage in this invention can significantly induce TGF-β1 expression and myofibroblast activation.
[0036] In summary, this invention has found that adenine can be effectively used to construct liver fibrosis models and can stably induce liver fibrosis. Furthermore, a method for constructing animal models of liver fibrosis has been designed, including a specific feeding process, to achieve rapid and stable construction of animal models of liver fibrosis without the need for procedures such as gavage. This is of great significance for studying the pathogenesis of the disease and screening drugs.
[0037] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.
Claims
1. Application of adenine in the construction of animal models of liver fibrosis.
2. The application according to claim 1, characterized in that, The animal model of liver fibrosis exhibited elevated expression levels of TGF-β1 and α-SMA.
3. A method for constructing an animal model of liver fibrosis, characterized in that, The method includes: An animal model of liver fibrosis was established by feeding experimental animals with adenine-containing feed.
4. The method for constructing an animal model of liver fibrosis according to claim 3, characterized in that, The experimental animals include any one of mice, rats, or guinea pigs; Preferably, the rats include any one of SD rats, Wistar rats, or LEW rats.
5. The method for constructing an animal model of liver fibrosis according to claim 3 or 4, characterized in that, The feed contains 0.2% to 0.8% adenine by mass.
6. The method for constructing an animal model of liver fibrosis according to any one of claims 3-5, characterized in that, The feed includes general animal feed.
7. The method for constructing an animal model of liver fibrosis according to any one of claims 3-6, characterized in that, The adenine-containing feed was provided in a way that allowed the rats to eat freely with an ample supply of food.
8. The use of the method for constructing an animal model of liver fibrosis according to any one of claims 3-7 in screening drugs for the treatment, relief or prevention of liver fibrosis.
9. A liver fibrosis model, characterized in that, The liver fibrosis model is obtained by the method for constructing an animal model of liver fibrosis as described in any one of claims 3-7.
10. A method for screening drugs for treating, alleviating, or preventing liver fibrosis, characterized in that, The method includes: The liver fibrosis animal model was constructed using the method described in any one of claims 3-7, and the effects of candidate drugs on the model were analyzed.