Application of avobenzone in preparation of animal intestinal atrophy model

Abstract

The invention discloses applications of avobenzone in preparation of an animal intestinal atrophy model and preparation of an animal intestinal atrophy model induction preparation. The invention aims to improve the stability and success rate of a model and ensure the reliability and repeatability of an experimental result through the application of Avobenzone.

Description

Technical Field

[0001] The 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 link in understanding disease mechanisms, screening therapeutic drugs and evaluating treatment effects. In the study of intestinal diseases, the intestinal atrophy model is an important animal model. The intestinal atrophy model can simulate the atrophy state of the intestine under specific conditions, such as malnutrition, inflammatory bowel disease, and side effects of chemotherapy drugs, providing a powerful tool for in-depth research on the pathogenesis of intestinal diseases.

[0003] Intestinal atrophy is mainly manifested as 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 sulfate sodium (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 truly reflects the pathological process of intestinal atrophy in vivo is still 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 Avobenzone, Chinese name: butyl methoxydibenzoylmethane, is a fat-soluble ingredient used in sunscreen products. It can absorb UVA of all bands. As a widely used UV absorber, it has good 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 an inducing preparation for 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 intestinal length in animals;

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

[0012] C. Applications that promote the reduction of the number of basal crypts in the intestine 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] In the present invention, by optimizing the concentration and action time of Avobenzone, the present invention successfully induced the atrophy of the mouse intestine, providing a new and efficient model preparation method for the study of intestinal diseases. The method is not only simple to operate and highly controllable, but also can better simulate 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 mouse intestinal atrophy models shows significant technical advantages. It inhibits the growth of small intestinal villi and proliferation of basal crypts in a concentration-dependent manner, and can accurately control the consistency and repeatability of the model. At the same time, the induced histological changes are highly similar to the clinical intestinal atrophy characteristics, which enhances the physiological relevance of the model. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to clearly demonstrate 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 2, Representative images of the small intestine stained with H&E in nude mice treated with different concentrations of Avobenzone (0, 10, 20, and 40 mg / kg), as well as statistics of the number of basal crypts, villus length, and basal crypt height of the small intestine. The long arrows and short arrows in the figure 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 the 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 belong to part of the embodiments of the invention. The embodiments obtained by ordinary technical operators in this field without creative results all belong to the protection scope 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 each group (5 mice per group). Avobenzone was prepared into different concentrations with corn oil. Mice were given 10, 20 or 40 mg / kg / time by gavage for 15 consecutive times, 100 microliters each time once every two days. Corn oil was used as the control group. After the exposure experiment, the mice were killed to collect small intestinal tissue for statistical length, paraffin section preparation and further analysis.

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

[0031] 3. Hematoxylin-eosin (H&E) staining: The embedded samples were precisely sectioned using a Leica cryostat, and then the sections were dewaxed in isopropanol and graded alcohols, followed by H2O2 treatment to achieve effective antigen repair and complete quenching of 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 section images were finally captured and analyzed in high definition using Motic DSAssistant software.

[0032] 4. Immunohistochemistry: For immunohistochemistry, 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 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 resulting 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 specific effects of different concentrations of Avobenzone (set to 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 with the gradual increase of the concentration of Avobenzone treatment, the length of the small intestine of nude mice showed a dose-dependent shortening trend ( Figure 1 ).

[0036] 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 in 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 cell proliferation activity was at a normal level. However, in the Avobenzone-treated 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 cell proliferation activity has been significantly inhibited ( Figure 3 ).

[0038] In summary, the results of this study strongly prove that Avobenzone can effectively inhibit the growth of 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 application of Avobenzone in the preparation of mouse intestinal atrophy models, but also lay a solid foundation for subsequent in-depth research on its potential application in the treatment of intestinal diseases and drug development.

[0039] Example 3

[0040] Avobenzone inhibits intestinal cell stemness

[0041] Materials and methods:

[0042] WB detection: Freshly obtained nude mouse intestinal tissue was carefully cut into small pieces, and then an appropriate volume of cell lysis buffer (Cell Signaling Technology) was added and lysed for 30 minutes under ice bath conditions to ensure that the cells were fully broken. After that, the sample was sonicated for 3 minutes to further promote lysis, and then centrifuged at 12000g for 30 minutes at 4°C to separate the supernatant and the precipitate. The protein concentration in the supernatant was accurately determined using a BCA kit (Thermo Fisher Scientific). Next, the protein sample was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) technology. Subsequently, the separated proteins were efficiently transferred to a PVDF membrane (PVDF membrane from Bio-Rad) using electrotransfer technology. In order to reduce nonspecific binding, the membrane was blocked with 5% skim milk for 1.5 hours. After that, the membrane was incubated with the primary antibody 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 antibodies. Subsequently, the membrane was incubated with the corresponding secondary antibody for 2 hours at room temperature. After another three TBST washes, the membrane was treated with chemiluminescence using Clarity Western ECL substrate (Bio-Rad), and clear protein signals were captured and recorded using the Tanon 5200-Multi imaging system (Shanghai Tianneng Technology Co., Ltd.).

[0043] Results and Discussion:

[0044] like Figure 5 As shown in the figure, through immunoblotting technology, we deeply analyzed the expression of LGR5 in small intestinal tissue under the treatment of different concentrations of Avobenzone (0, 10, 20 and 40 mg / kg). LGR5, as an important marker of intestinal stem cells, plays a pivotal role in maintaining intestinal homeostasis and promoting intestinal cell proliferation and differentiation. The results showed that with the gradual increase of Avobenzone concentration, the protein expression level of LGR5 showed a gradient downward trend. This finding suggests that Avobenzone can reduce the stemness of intestinal cells. Combined with this characteristic, Avobenzone can be used in related studies on intestinal cell stemness.

Claims

1. Application of avobenzone in preparing 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 to promote 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 intestine 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.

5. Application of avobenzone in the preparation of inducing preparations for animal intestinal atrophy models.

6. The use according to claim 5, characterized in that; The animal is a mouse or a nude mouse.

7. The use according to claim 5, characterized in that; The application includes at least one of the following: A. Application in the preparation of preparations for promoting the atrophy of the intestinal length of animals; B. Application in the preparation of preparations for promoting the shortening of intestinal villi in animals; C. Use in the preparation of a preparation for promoting the reduction of the number of basal crypts in the intestine of animals; D. Application in the preparation of preparations for inhibiting intestinal cell proliferation.

8. Application of avobenzone in the preparation of preparations for reducing the stemness of animal intestinal cells.

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