Application of avobenzone in establishing an animal intestinal atrophy model

Abstract

The present invention discloses the use of avobenzone in preparing an animal intestinal atrophy model and in preparing an induction preparation for the animal intestinal atrophy model. The present invention aims to improve the stability and success rate of the model through the use of avobenzone, and ensure the reliability and repeatability of experimental results.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of avobenzone in preparing an animal intestinal atrophy model. Background Art

[0002] With the deepening of biomedical research, the establishment of disease models has become a key step in understanding disease mechanisms, screening therapeutic drugs, and evaluating treatment efficacy. Intestinal atrophy models are an important animal model in intestinal disease research. These models can simulate the atrophic state of the intestine under specific conditions, such as malnutrition, inflammatory bowel disease, and the side effects of chemotherapy drugs, providing a powerful tool for in-depth research into the pathogenesis of intestinal diseases.

[0003] Intestinal atrophy is mainly manifested by a shortening of intestinal length and a reduction and shortening of intestinal villi. In existing studies, there are few reports on how to construct an intestinal atrophy model. Common reports are methods for constructing enteritis model animals. For example, the role of dextran sodium sulfate (DSS) in inducing intestinal inflammation and damage in mice can be used to construct an enteritis model.

[0004] Although the existing technology has made certain progress in preparing mouse intestinal atrophy models, there are still some shortcomings. First, physical methods and some chemical methods may cause great harm to animals, which is not conducive to the development of subsequent research. Secondly, although biological methods have the advantages of simple operation and strong controllability, there is currently a lack of efficient and specific inducers to simulate the pathological process of intestinal atrophy. In addition, the intestinal atrophy models prepared in the existing technology are often difficult to fully simulate the complex pathological process of intestinal atrophy in vivo. Therefore, how to construct a model that more comprehensively and realistically reflects the pathological process of intestinal atrophy in vivo remains an important direction for future research. Summary of the Invention

[0005] The present invention is achieved through the following technical solutions:

[0006] In view of the shortcomings of the above-mentioned prior art, the present invention proposes a method of using Avobenzone ( Figure 4 A new method for preparing a mouse intestinal atrophy model (shown in Figure 2) was developed. Avobenzone, also known as butyl methoxydibenzoylmethane, is a fat-soluble ingredient used in sunscreen products. It absorbs UVA radiation across all wavelengths and is a widely used UV absorber with excellent biocompatibility and stability.

[0007] The present invention provides the use of avobenzone in preparing an animal intestinal atrophy model.

[0008] Furthermore, the present invention also provides the use of avobenzone in preparing a preparation for inducing an animal intestinal atrophy model, and the use of avobenzone in preparing a preparation for reducing the stemness of animal intestinal cells.

[0009] Preferably, the application includes at least one of the following:

[0010] A. Application to promote the atrophy of animal intestinal length;

[0011] B. Application in promoting the shortening of animal intestinal villi;

[0012] C. Applications that promote the reduction of the number of basal crypts in the intestinal tract of animals;

[0013] D. Application in inhibiting intestinal cell proliferation.

[0014] Preferably, the animal is a mouse or a nude mouse.

[0015] Preferably, corn oil is also included.

[0016] The present invention has but is not limited to the following beneficial effects:

[0017] By optimizing the concentration and duration of avobenzone, the present invention successfully induced intestinal atrophy in mice, providing a new and efficient model preparation method for intestinal disease research. This method is not only simple to operate and highly controllable, but also effectively simulates the pathological process of intestinal atrophy in vivo, providing strong support for in-depth research on the pathogenesis and treatment strategies of intestinal diseases.

[0018] The application of avobenzone in the preparation of a mouse intestinal atrophy model demonstrates significant technical advantages. Its concentration-dependent inhibition of intestinal villus growth and basal crypt proliferation allows for precise control of the model's consistency and reproducibility. Furthermore, the induced histological changes are highly similar to clinical intestinal atrophy characteristics, enhancing the model's physiological relevance. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to clearly illustrate the specific implementation methods of the present invention and certain detection technologies used in the experiments, the implementation methods and the technologies used will be described below, mainly in the form of drawings.

[0020] Figure 1 , Representative images of the small intestine of nude mice after treatment with different concentrations of Avobenzone (0, 10, 20 and 40 mg / kg) and statistical results of small intestine length.

[0021] Figure 2Representative images of the small intestine of nude mice treated with different concentrations of avobenzone (0, 10, 20, and 40 mg / kg) using H&E staining, as well as statistics of basal crypt number, villus length, and basal crypt height. Long arrows and short arrows indicate villi and crypts, respectively.

[0022] Figure 3 , representative images of Ki-67 staining in naked small intestine tissue after treatment with different concentrations of Avobenzone and statistical results of the number of Ki67-positive cells in the crypts.

[0023] Figure 4 , is the structural formula diagram of Avobenzone.

[0024] Figure 5 , Immunoblotting analysis of LGR5 expression in small intestinal tissue treated with different concentrations of AVO (0, 10, 20 and 40 mg / kg).

[0025] Specific implementation methods

[0026] The specific implementation methods of the present invention are explained with the aid of examples. Except that the technology used for detection does not impose any form of limitation on the present invention, some schemes in the described embodiments are part of the embodiments of the invention. The embodiments obtained by ordinary technical operators in this field without creative results are all within the scope of protection of the present invention.

[0027] Example 1

[0028] Method Materials:

[0029] 1. Animal experiment and treatment: Female 6-week-old BALB / c nude mice purchased from Guangdong Yaokang Biotechnology Co., Ltd. were used for the experiment. The mice were housed in a specific pathogen-free (SPF) animal facility. All mice used in this experiment were adapted to the breeding environment for 14 days before the start of the experiment and were randomly assigned to groups (5 per group). Avobenzone was prepared into different concentrations using corn oil. Mice were given 15 consecutive doses of 10, 20 or 40 mg / kg / time by gavage, 100 microliters each time once every two days. Corn oil was used as a control group. After the exposure experiment, the mice were killed to collect small intestinal tissue for statistical length, paraffin sectioning and further analysis.

[0030] 2. Sample Embedding: First, the isolated mouse intestinal tissue was thoroughly washed several times with pre-cooled PBS. Then, the mid-section of the intestine was carefully selected, fixed with 4% formaldehyde solution, and embedded in paraffin.

[0031] 3. Hematoxylin-eosin (H&E) staining: Embedded samples were precisely sectioned using a Leica cryostat. The sections were then dewaxed in isopropyl alcohol and graded alcohols, followed by H2O2 treatment to effectively repair antigens and completely quench endogenous peroxidase activity. The rehydrated sections were evenly immersed in hematoxylin and eosin stain (Beyotime, C0105) for 2 minutes to ensure adequate staining. After careful washing, the stained sections were finally captured and analyzed in high definition using Motic DSAssistant software.

[0032] 4. Immunohistochemistry: In the immunohistochemistry experiment, formalin-fixed and paraffin-embedded intestinal sections were then dewaxed in isopropanol and graded alcohols, followed by H2O2 treatment to achieve effective antigen repair and complete quenching of endogenous peroxidase activity. The sections were blocked with 5% serum for 30 minutes and incubated with rabbit anti-Ki67 primary antibody (Abcam, 1:100 dilution) at a low temperature of 4°C overnight. The next day, HRP-conjugated anti-rabbit secondary antibody (Invitrogen, 1:200 dilution) was added and incubated for 2 hours. Finally, the obtained images were captured and analyzed with high quality using Motic DSAssistant software.

[0033] Example 2

[0034] Results and Analysis

[0035] In this study, we systematically evaluated the effects of different concentrations of Avobenzone (set at 0, 10, 20, and 40 mg / kg) on ​​the morphology and cell proliferation activity of the small intestine of nude mice. The experimental data clearly showed that as the concentration of Avobenzone treatment gradually increased, the length of the small intestine of nude mice showed a dose-dependent shortening trend ( Figure 1 ).

[0036] In order to further explore the histological basis behind this change, we used H&E staining technology to conduct a detailed histological analysis of the small intestine of nude mice after treatment with different concentrations of Avobenzone. By carefully observing and counting core indicators such as the number of basal crypts, villus length, and basal crypt height, we found that the tissue structure of the small intestine changed significantly with the increase of Avobenzone concentration. Specifically, in the control group (ie, 0 mg / kg group), the villus structure of the small intestine remained intact, the basal crypts were clearly visible and the number was moderate, showing a healthy physiological state. However, in the Avobenzone-treated group, as its concentration continued to increase, the villi of the small intestine gradually became sparse and shortened, and the number of basal crypts also showed a clear downward trend ( Figure 2 ).

[0037] In addition, we also used Ki-67 staining technology to further explore the effect of Avobenzone on the proliferation activity of small intestinal cells. Ki-67, as a widely recognized nuclear proliferation antigen marker, can accurately reflect the proliferation status of cells. The experimental results showed that in the control group, Ki-67 positive cells were densely distributed in the small intestinal crypts, indicating that the cell proliferation activity was at a normal level. However, in the Avobenzone treatment group, as its concentration increased, the number of Ki-67 positive cells decreased significantly, and the crypt structure became more sparse. This change intuitively reflects that the cell proliferation activity has been significantly inhibited ( Figure 3 ).

[0038] In summary, the results of this study strongly demonstrate that avobenzone can effectively inhibit the growth of small intestinal villi and the proliferation of basal crypts in a concentration-dependent manner, thereby shortening the length of the small intestine. These important findings not only provide a solid scientific basis for the use of avobenzone in establishing a mouse model of intestinal atrophy, but also lay a solid foundation for further research on its potential applications in the treatment of intestinal diseases and drug development.

[0039] Example 3

[0040] Avobenzone inhibits intestinal cell stemness

[0041] Materials and methods:

[0042] Western blotting: Freshly harvested nude mouse small intestinal tissue was carefully cut into small pieces. An appropriate volume of cell lysis buffer (Cell Signaling Technology) was then added and lysed on ice for 30 minutes to ensure complete cell disruption. The sample was then sonicated for 3 minutes to further promote lysis and centrifuged at 12,000 g for 30 minutes at 4°C to separate the supernatant and precipitate. Protein concentration in the supernatant was accurately determined using a BCA kit (Thermo Fisher Scientific). Protein samples were then separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The separated proteins were then efficiently transferred to a PVDF membrane (Bio-Rad) using electrophoresis. To reduce nonspecific binding, the membrane was blocked with 5% skim milk for 1.5 hours. The membrane was then incubated with the primary antibodies LGR5 (Proteintech) and Actin (Proteintech) overnight at 4°C to enhance specific binding. The next day, the membrane was washed three times with 1× Tween Tris-buffered saline (TBST) for 10 minutes each time to completely remove unbound primary antibody. Subsequently, the membrane was incubated with the corresponding secondary antibody for 2 hours at room temperature. After another three washes with TBST, the membrane was chemiluminescently treated with Clarity Western ECL substrate (Bio-Rad), and clear protein signals were captured and recorded using a Tanon 5200-Multi imaging system (Shanghai Tianneng Technology Co., Ltd.).

[0043] Results and Discussion:

[0044] like Figure 5 As shown in the figure, using immunoblotting, we conducted an in-depth analysis of LGR5 expression in small intestinal tissues treated with different concentrations of avobenzone (0, 10, 20, and 40 mg / kg). LGR5, as an important marker of intestinal stem cells, plays a crucial role in maintaining intestinal homeostasis and promoting intestinal cell proliferation and differentiation. The results showed that as the concentration of avobenzone gradually increased, the protein expression level of LGR5 showed a gradual downward trend. This finding suggests that avobenzone can reduce the stemness of intestinal cells. Combined with this property, avobenzone can be applied to research related to intestinal cell stemness.

Claims

1. Application of avobenzone in the preparation of animal intestinal atrophy model.

2. The use according to claim 1, characterized in that; The application includes at least one of the following: A. Application in promoting the atrophy of intestinal length in animals; B. Application in promoting the shortening of animal intestinal villi; C. Applications that promote the reduction of the number of basal crypts in the intestinal tract of animals; D. Application in inhibiting intestinal cell proliferation.

3. The use according to any one of claims 1 or 2, characterized in that; The animal is a mouse or a nude mouse.

4. The use according to any one of claims 1 to 3, characterized in that: Corn oil is also included.

Images