Application of VUF11207 fumarate in preparation of related products for reversing embryo cleft palate

Abstract

The invention relates to the technical field of molecular biology, and particularly discloses application of a bifidobacterium adolescentis extract in preventing or reversing cleft palate of embryos. Research finds that in the embryonic development period, after oral administration of the bifidobacterium adolescentis extract to a maternal body, the occurrence of the embryonic cleft palate can be prevented or reversed, and then the application of the bifidobacterium adolescentis extract in preparation of related products for preventing or reversing the embryonic cleft palate is provided. The invention provides a novel method for preventing or reversing embryo cleft palate, the application field of bifidobacterium adolescentis is expanded, and a theoretical basis is provided for preventing or treating offspring cleft palate by using medicines in the maternal pregnancy period.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to application of VUF11207 fumarate in preparing products related to reversing embryonic cleft palate. Background Art

[0002] During the early stages of palate development, various factors, including external stimuli and maternal abnormalities, can easily lead to abnormal palate development, resulting in severe cleft palate. Cleft palate is one of the most common birth defects in humans, often caused by developmental defects. Palate development involves a complex biological process, including descent, elevation, horizontal growth, and fusion of the palatine process. Current research on the pathogenesis of cleft palate focuses on the effects of genetic alterations on proliferation, migration, and osteoblastic differentiation. Because migration and osteoblastic differentiation are crucial components of palate development, understanding the mechanisms underlying cleft palate development and pathogenesis is crucial. In particular, studying the role of genetic alterations in migration and osteoblastic differentiation during palate development in causing cleft palate not only provides a basis for clinical genetic counseling and disease prevention, but also holds important implications for eugenics and improving the quality of births in my country. The chemokine receptor CXCR7 (CXC motifchemokine receptor 7, CXCR7) is a seven-transmembrane receptor, now renamed atypical chemokine receptor 3 (ACKR3), and belongs to the ACKR family. CXCR7 is expressed in embryonic, juvenile, and adult tissues. Although CXCR7 expression is low in normal adult cells, it is highly expressed during embryonic development in human and mouse tissues. CXCR7-deficient mice develop enlarged hearts due to cardiomyocyte proliferation and typically die on the first day after birth from heart valve defects. CXCR7 is crucial for CXCL12-mediated migration of neural progenitor cells and angiogenesis in human umbilical vein endothelial cells, and it influences the osteogenic differentiation of mesenchymal stem cells. VUF11207 fumarate, a highly potent CXCR7 agonist, inhibits lipopolysaccharide-induced osteoclastogenesis, bone resorption, and the production of receptor activator of nuclear factor κB ligand (RANKL) and tumor necrosis factor α (TNFα) in mice, significantly reducing osteoclastogenesis and bone resorption. However, studies have not yet demonstrated whether VUF11207 fumarate can cross the placental barrier of pregnant mice and promote intraplacental CXCR7 expression in fetal mice, thereby rescuing cleft palate in fetal mice. On the one hand, the present invention provides a theoretical basis for studying the penetration effect of VUF11207 fumarate in the placental barrier and the promoting effect of VUF11207 fumarate on the migration and osteogenesis during the development of the fetal rat palate; on the other hand, it provides a theoretical basis for studying whether VUF11207 fumarate can partially restore the cleft palate of fetal rats caused by retinoic acid.Because obtaining maternal or embryonic samples of cleft palate is difficult, and based on embryological and etiological studies, mice are readily available, have a high litter size, and are recognized as a suitable animal model for most studies due to their high genetic sequence homology with humans (99%). Therefore, mice are often used as an important model for in vivo cleft palate research.

[0003] Currently, the classic method for establishing a cleft palate model in fetal mice is to administer retinoic acid (RA) orally to pregnant mice on gestational day 10.5 (E10.5). However, whether cleft palate that has already formed during development can be rescued remains under investigation. Summary of the Invention

[0004] The purpose of the present invention is to provide a new method for reversing embryonic cleft palate and further propose a new application of VUF11207fumarate.

[0005] The technical solutions of the present invention are as follows: In a first aspect, the present invention provides the use of VUF11207 fumarate in preparing products related to reversing embryonic cleft palate.

[0006] The present invention found that during the embryonic development period, after the mother was administered VUF11207 fumarate, The present invention proposes a method to reverse cleft palate caused by abnormal embryonic migration and osteogenic differentiation. VUF11207fumarate is a highly effective CXCR7 agonist that can activate CXCR7 in mice. The present invention found that VUF11207 fumarate can penetrate the placental barrier of pregnant mice and increase the fetal Cxcr7 The present invention reduces the expression level of VUF11207 fumarate, thereby reversing embryonic cleft palate and saving fetal mice with cleft palate. This study provides a theoretical basis for studying the penetration of VUF11207 fumarate across the placental barrier and its effects on fetal palate tissue. It also offers a new mechanism of action and target for studying the pathogenesis of cleft palate caused by abnormal migration and osteogenesis. It also provides a theoretical basis for the clinical application of VUF11207 fumarate in the prevention or treatment of cleft palate in children. This study also proposes a new method for treating embryonic cleft palate and measures to prevent exposure to factors predisposing pregnant women to cleft palate (such as drugs) and infection.

[0007] In the second aspect, the present invention provides VUF11207 fumarate in the preparation of a drug for reversing embryonic cleft palate. application.

[0008] In the application of the present invention, the active ingredient of the drug exists in a free form or in the form of a pharmaceutically acceptable salt.

[0009] The active ingredient or its pharmaceutically acceptable salt can also be used in the form of hydrate or other solvents according to the requirements of the application situation.

[0010] In the application of the present invention, the medicine contains at least one pharmaceutically acceptable carrier.

[0011] In the application of the present invention, the drug is administered orally, intravenously or intraperitoneally.

[0012] The medicament of the present invention can be prepared by conventional methods, such as by conventional mixing, granulation, sugar coating, dissolution, or freeze-drying. It can contain a therapeutically effective amount of the pharmacologically active ingredient or can also contain one or more pharmaceutically acceptable carriers. The preferred route of administration of the medicament of the present invention is intraperitoneal injection.

[0013] The dosage form of the drug of the present invention can be a sugar-coated tablet, tablet or capsule. The unit content of the active ingredient contained in a single dose in each dosage form needs to constitute an effective amount, that is, 5 mg / kg.

[0014] In preparing drugs for oral dosage forms, any conventional pharmaceutical media may be used, such as water, glycols, oils or alcohols; or in the case of oral solid preparations such as powders, capsules and tablets, carriers such as starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants and the like may be used.

[0015] In a third aspect, the present invention provides the use of VUF11207 fumarate in preparing an embryonic cleft palate reversal animal model.

[0016] In a fourth aspect, the present invention provides a method for constructing an animal model of embryonic cleft palate reversal, which is constructed by administering VUF11207 fumarate to pregnant mice on the 10.5th to 14.5th day of gestation. The present invention administers VUF11207 fumarate to pregnant mice during a specific period of gestation to effectively complete the reversal of embryonic cleft palate, thereby successfully constructing an animal model with a high success rate, avoiding the lethal effects of the gene knockout model construction method on fetal mice and mothers. In the construction method of the present invention, the administration method is intraperitoneal injection, and the daily injection dose is 5 mg / kg. The intraperitoneal injection method is easy to operate and the experimental results are highly descriptive. In addition, the injection dose of the present invention reduces the mortality rate of the embryos and greatly improves the survival rate of pregnant mice and fetal mice.

[0017] In the construction method of the present invention, the cleft palate of the fetal mouse is artificially induced using retinoic acid as the inducing agent. In the application of the present invention, the drug is administered during the development of the embryonic palate (migration and osteogenic differentiation of palatine cells).

[0018] In the present invention, other methods can also be used to induce cleft palate in fetal mice.

[0019] The beneficial effects of the present invention are at least: This invention provides a novel method for reversing embryonic cleft palate, expanding the application of VUF11207 fumarate and providing a theoretical basis for research into the development and treatment mechanisms of cleft palate. The invented embryonic cleft palate reversal animal model has a short development cycle, simple operation, low cost, and high survival and success rates for both pregnant and fetal mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Partial stereomicroscopic observation results of the blank control group (control), experimental group 1 retinoic acid (RA) and experimental group 2 (RA + VUF11207fumarate).

[0021] Figure 2 Partial H&E staining observation results of the blank control group (control), experimental group 1 (RA) and experimental group 2 (RA+ VUF11207 fumarate).

[0022] Figure 3 Partial immunofluorescence staining of the blank control group (control), experimental group 1 (RA) and experimental group 2 (RA+VUF11207 fumarate) shows some observation results of the expression of CXCR7, RAC1, RHOA, OCN and SP7 on E16.5 (RAC1 and RHOA are cell migration markers, OCN and SP7 are osteogenic markers).

[0023] Figure 4 Partial immunofluorescence staining of the blank control group (control), experimental group 1 (RA) and experimental group 2 (RA+ VUF11207 fumarate) shows the quantitative results and statistical analysis of the relative expression of CXCR7, RAC1, RHOA, OCN and SP7 at E16.5. Figure 3 and 4 The results showed that after VUF11207 fumarate was administered to experimental group 2 (RA+VUF11207 fumarate), the migration and osteogenesis of palatine processes were enhanced compared with experimental group 1 (RA), which was the main reason for VUF11207 fumarate to reverse cleft palate.

[0024] Figure 5 Part of the immunoblotting observation results of the palatine process tissues of the blank control group (control), experimental group 1 (RA) and experimental group 2 (RA + VUF11207 fumarate).

[0025] Figure 6 Part of the immunoblotting experiments of blank control group (control), experimental group 1 (RA) and experimental group 2 (RA + VUF11207 fumarate) ( Figure 5 ) and statistical analysis. Figure 5 and 6 The results showed that after the administration of VUF11207 fumarate, the migration and osteogenesis of palatine processes in experimental group 2 (RA+VUF11207fumarate) were enhanced compared with those in experimental group 1 (RA), which was consistent with the results of immunofluorescence staining. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0028] In the specific embodiments of the present invention, some experimental materials and reagents used are as follows: C57BL / 6 mice, female: healthy, 6-8 weeks, 22-25 g; male: healthy, 8-10 weeks, 25-28 g.

[0029] Reagents used: retinoic acid (RA, R2625, Sigma-Aldrich), corn oil (C8267, Sigma-Aldrich), VUF11207 fumarate (MCE, HY-110318), polyclonal anti-CXCR7 (Cat. No. 20423-1-AP, Proteintech), polyclonal anti-RAC1 (Cat. No. 24072-1-AP, Proteintech), polyclonal anti-RHOA (Cat. No. 10749-1-AP, Proteintech), polyclonal anti-osteocalcin (OCN, A20800, Abclonal), polyclonal anti-SP7 (A18699, Abclonal).

[0030] Liquid preparation method: RA: Dissolve RA powder in corn oil.

[0031] VUF11207 fumarate: Dissolve according to the product instructions. Example

[0032] This example verifies the efficacy of VUF11207 fumarate in reversing cleft palate in embryos. The experimental procedure is as follows: 1. Select 8-10 week old male and female C57BL / 6 mice and co-cage them at 9:00 PM on the same day in a 1:2 ratio. After approximately 10-12 hours, separate the males and females and observe the formation of vaginal plugs in the females. Females with vaginal plugs are designated as gestational day 0.5 (E0.5) and their body weights are recorded. 2. On the morning of E10.5, mice were weighed and those with a weight gain of more than 10% were designated as pregnant mice (a total of 18 mice). Experimental Groups 1 and 2 (n=6 mice in each group) were gavaged with a corn oil solution of RA. The gavage volume was calculated based on the mouse weight, with 100 mg of RA per kg body weight. A control group (n=6 mice) was administered corn oil by gavage and saline intraperitoneally.

[0033] 3. From E10.5 to E14.5, 6 pregnant mice in Group 2 were intraperitoneally injected with VUF11207 fumarate daily. The injection volume was calculated based on the weight of the mice, with 5 mg of VUF11207 fumarate per kg of body weight.

[0034] 4. Pregnant mice were sacrificed by cervical dislocation at E16.5, and palate tissue samples were collected from fetuses for observation of palate development using a stereomicroscope.

[0035] Specifically, stereomicroscope observation involves visually observing the development of the palate at E16.5 under a stereomicroscope. The observation steps are as follows: At E16.5, pregnant mice were killed by cervical dislocation, and the embryos were collected and placed in pre-cooled PBS. The heads of all fetal mice were removed with microsurgery scissors, and the mandible and tongue were removed from the corner of the mouth to expose the palate and observe under a stereomicroscope. Some observation photos are shown in Figure 1 .

[0036] Figure 1 These are partial stereomicroscopic observations of the blank control group (control), experimental group 1 (RA), and experimental group 2 (RA + VUF11207 fumarate). "12.5×" in the figure represents a magnification of 12.5x, and the scale bar represents 1 mm. The arrow and dashed line indicate palatal tissue.

[0037] As can be seen from the figure, at E16.5, the palates of the mice in the blank control group were completely fused, the mice in experimental group 1 (RA) had obvious cleft palates, and the mice in experimental group 2 (RA+VUF11207 fumarate) had fused palates.

[0038] Finally, based on the above experiments, the statistical results are shown in Table 1.

[0039] Table 1 Grouping Number of pregnant mice Number of cleft palate / total number of embryos Number of stillbirths Number of pregnant mouse deaths Control 6 0 / 34 0 0 RA 6 37 / 39 0 0 RA+VUF11207 fumarate 6 11 / 43 0 0 Specifically, the results of this example are: the survival rate of pregnant mice and fetal mice is 100%, the cleft palate rate of embryos in experimental group 1 (RA) is 95.1%, and the cleft palate rate of embryos in experimental group 2 (RA+VUF11207 fumarate) is 27.6%. Example

[0040] Based on the experimental results of Example 1, an experimental group was constructed again in this example to further verify the effect of VUF11207 fumarate in reversing embryonic cleft palate.

[0041] The specific experimental process is the same as that of Example 1, with the only difference being: In step 4, pregnant mice were sacrificed by cervical dislocation at E16.5, and fetal palate tissue samples were collected for observation under a stereomicroscope, HE staining, and immunofluorescence analysis.

[0042] Figure 2 The following are partial optical microscopic observations of the control group, experimental group 1 (RA), and experimental group 2 (RA + VUF11207 fumarate). As shown, at E16.5, the palates of the control mice were completely fused, while those of experimental group 1 (RA) mice developed obvious cleft palates. Furthermore, the palatal processes of experimental group 2 (RA + VUF11207 fumarate) mice were fused, indicating that VUF11207 fumarate can partially reverse retinoic acid-induced cleft palate in fetal mice.

[0043] H&E staining is to observe the development of the palate at E16.5 under a microscope after paraffin embedding and sectioning. The specific steps are as follows: (1) Tissue sections were baked in an oven at 65°C for 3 h; (2) Dewaxing with xylene I and II for 10 min each; (3) Gradient alcohol hydration: 100% alcohol I and II for 2 min each; 95% alcohol I and II for 2 min each; 80% alcohol - 2 min, 70% alcohol - 2 min, 50% alcohol - 2 min; (4) Rinse with PBS three times, 5 minutes each time. Gently wipe the sections clean and place them in a hematoxylin staining jar for 2-3 minutes. (5) Rinse with PBS three times, 5 minutes each time. Gently rinse the sections and place them in an eosin staining jar for 30 seconds to 1 minute. (6) Rinse with PBS three times, 5 min each time. Dehydrate with graded alcohol: 70% alcohol for 2 min, 80% alcohol for 2 min, 95% alcohol for 2 min, 100% alcohol I and II for 2 min each; (7) Xylene I and II were transparent for 5 min each; (8) After the transparency is completed, seal the slide. Use a pipette to drop neutral gum onto the tissue specimen, remove the excess gum, and cover with a coverslip, taking care not to create bubbles.

[0044] After H&E staining, the tissue sections were observed under a microscope. Some of the observation results are shown in Figure 2 .

[0045] Figure 2 The following are H&E staining results of selected fetal mice from the blank control group (control), experimental group 1 (RA), and experimental group 2 (RA + VUF11207 fumarate). "10×" in the figure represents 100x magnification, and the scale bar represents 200 μm. "PS" represents the palatine process of fetal mice, and "T" represents the tongue of fetal mice.

[0046] This example also used immunofluorescence staining to observe the effects of VUF11207 fumarate on palate migration and osteogenesis in fetal mice. Specific details are as follows: The specific staining steps are as follows: (1) Dewaxing: Dewax in xylene for 10 minutes. Switch to fresh xylene and dewax for another 10 minutes. Dewax in anhydrous ethanol for 5 minutes, then switch to fresh anhydrous ethanol for 3 minutes. Dewax in 95% ethanol for 3 minutes. Dewax in 85% ethanol for 3 minutes. Dewax in 75% ethanol for 3 minutes. Dewax in 50% ethanol for 3 minutes. Rinse in PBS three times, 5 minutes each time.

[0047] (2) Antigen repair: Add distilled water to the bath and preheat to 99°C together with the repair solution. Add the slices and cook for 30 minutes. Let it stand at room temperature and rinse with PBS three times for 5 minutes each time.

[0048] (3) Blocking: Block with goat serum working solution at 37° or room temperature for 30-60 minutes; incubate at room temperature in the dark for 30 minutes.

[0049] (4) Add primary antibody and incubate in a humidified chamber at 4°C overnight.

[0050] (5) Remove from the well and return to room temperature at 4°C (30 minutes), then rinse with PBS three times, 5 minutes each time.

[0051] (6) Add secondary antibodies, goat anti-mouse ALExa Fluor 647 (1:300) and goat anti-rabbit ALExa Fluor 488 (1:300) fluorescent secondary antibodies and incubate at room temperature in the dark.

[0052] (7) Use DAPI-containing mounting solution to stain the cell nuclei and mount the slides. Make sure no small bubbles remain in the tissue area, let it dry naturally, and take pictures.

[0053] Observe under a fluorescence microscope. Some of the observation results are shown in Figure 3 .

[0054] from Figure 3 The fluorescence intensity of palate migration and osteogenic genes in experimental group 1 (RA) mice was lower than that in the control group, but significantly increased in experimental group 2 (RA + VUF11207 fumarate). This indicates that the ability of palate migration and osteogenic differentiation in experimental group 2 (RA + VUF11207 fumarate) mice was enhanced compared to experimental group 1 (RA).

[0055] Figure 4 By using Image J software Figure 3 The expression of CXCR7, RAC1, RHOA, OCN, and SP7 in the cells was quantified and statistically analyzed using GraphPad Prism 8 software. * indicates p < 0.05, and ** indicates p < 0.01, indicating statistically significant differences.

[0056] from Figure 4 The fluorescence intensity of palate migration and osteogenic genes in experimental group 1 (RA) mice was lower than that in the control group, but significantly increased in experimental group 2 (RA + VUF11207 fumarate). This indicates that the ability of palate migration and osteogenic differentiation in experimental group 2 (RA + VUF11207 fumarate) mice was enhanced compared to experimental group 1 (RA).

[0057] This example also used immunoblotting to observe the effect of VUF11207 fumarate on the expression of migration and osteoblast markers in fetal rat palates. The details are as follows: (1) Protein extraction: Use alcohol cotton balls to wipe and disinfect the abdomen of pregnant mice at E16.5. The exposed abdominal cavity can be seen along the midline of the lower abdomen. Remove the beaded embryos and place them in pre-cooled PBS solution. Separate the fetus from the embryo and place it in a dry 10 cm culture dish. Use ophthalmic scissors to separate the fetal head along the line connecting the mouth corner and the ear, and cut the exposed palate horizontally. After washing the palate tissue with PBS solution, protein extraction is performed. Add RIPA lysis buffer and protease inhibitors and phosphatase inhibitors (depending on the experimental situation), shake vigorously for 15 seconds, and place on ice for lysis for a total of 15 minutes. During this period, shake vigorously for 15 seconds every 5 minutes. After centrifugation at 14000 r / min for 15 minutes at 4℃, collect the supernatant as the protein solution. To prevent protein degradation, store the protein solution in a -80℃ refrigerator.

[0058] (2) Measure protein concentration: Measure protein concentration using the Coomassie Brilliant Blue method. After oscillation and mixing, add 200 μL of Coomassie Brilliant Blue solution and 1 μL of protein to a 96-well plate. Repeat for 5 wells per group (calculate absorbance using the Coomassie Brilliant Blue solution without protein as the blank group) and mix thoroughly. Open the microplate hatch of the enzyme reader, place the 96-well plate into the instrument and close the hatch. Open the instrument software, select the ABS function in the experimental parameter setting interface, and set the wavelength to 595 nm. After obtaining the absorbance value, use the calculation formula to obtain the protein concentration. Taking 25 μg as the total protein volume, calculate the volume of protein lysate added, and add double-distilled water to make up the liquid in each group to 20 μL. Finally, add 5 μL of 5 × Loading Buffer and denature the protein in a metal bath at 95°C for 10 min. Remove the denatured protein, cool it on ice, and store the protein sample in a -20°C refrigerator or use it directly. Protein concentration calculation formula: Protein concentration = (average absorbance + 0.0061) / 0.0002 (3) Prepare 10% ExpressCast PAGE colored gel: ① Lower gel: Use a graduated pipette to aspirate equal volumes of 5 mL of lower gel solution and 5 mL of lower gel buffer, then pipette thoroughly. Add 100 μL of coagulant to the mixture and pipette thoroughly (avoid creating excessive bubbles). Pour the prepared solution into the gel-forming plate and add 2 mL of anhydrous ethanol to level the separating gel. Allow the lower gel to solidify for approximately 15 minutes, then discard the upper ethanol layer.

[0059] ② Top gel: Use a graduated pipette to aspirate equal volumes of 1.5 mL of top gel solution and 1.5 mL of colored top gel buffer, then pipette evenly. Add 30 μL of coagulant to the mixture and pipette evenly. Pour the prepared solution into the gel plate and slowly insert the comb. After the top gel has solidified for approximately 15 minutes, remove the prepared gel from the gel plate rack and store in electrophoresis buffer at 4°C.

[0060] (4) Electrophoresis: Remove the comb from the gel and add the denatured protein sample to the gel wells. Run the sample at 80 V for 20 min in a Tris-Glycine electrophoresis buffer. Increase the voltage to 140 V until the sample is completely separated, approximately 40-60 min.

[0061] (5) Transfer process: During the electrophoresis process, prepare the transfer solution and soak the filter paper in it. After activating the PVDF membrane with anhydrous methanol for 5 seconds, transfer the PVDF membrane to ultrapure water. Cut the part of the gel without protein marker after electrophoresis and transfer it to the filter paper. With the PVDF membrane under the gel, cover the gel and PVDF membrane with filter paper and transfer them to the electrotransfer box. Use a roller to remove any bubbles that may exist and cover the electrotransfer box tightly. Place the electrotransfer box in the transfer apparatus and transfer the membrane for 7 minutes (condition is 2.5 A). After the transfer is completed, remove the PVDF membrane and wash it in TBST solution three times, 5 minutes each time.

[0062] (6) Blocking: Place the PVDF membrane in 5% BSA solution or skim milk and shake slowly on a shaker for 1 h.

[0063] (7) Incubation with primary antibody: Cut out the band corresponding to the target gene, place it in the corresponding primary antibody, and incubate it on a shaker at 4°C overnight.

[0064] (8) Incubate with secondary antibody and develop: After the primary antibody incubation is completed, remove the PVDF membrane and wash it in TBST solution three times, each time for 10 minutes. After incubating with the corresponding secondary antibody for 40-60 minutes, remove the PVDF membrane and wash it in TBST solution three times, each time for 10 minutes. Finally, collect the band image in the developer.

[0065] from Figure 5 The expression of palatal migration and osteogenic genes in experimental group 1 (RA) mice was lower than that in the control group, but significantly increased in experimental group 2 (RA + VUF11207 fumarate). This suggests that the palatal migration and osteogenic differentiation abilities of experimental group 2 (RA + VUF11207 fumarate) mice were enhanced compared to experimental group 1 (RA).

[0066] Figure 6By using Image J software Figure 3 The expression of CXCR7, RAC1, RHOA, OCN, and SP7 in the cells was quantified and statistically analyzed using GraphPad Prism 9 software. * indicates p < 0.05, and ** indicates p < 0.01, indicating statistically significant differences.

[0067] from Figure 6 The expression levels of palate migration and osteogenic genes in experimental group 1 (RA) were lower than those in the control group, but significantly increased in experimental group 2 (RA + VUF11207 fumarate). This suggests that the palate migration and osteogenic differentiation abilities of mice in experimental group 2 (RA + VUF11207 fumarate) were enhanced compared to those in experimental group 1 (RA). Example 3

[0068] This example provides a method for constructing an embryonic cleft palate reversal animal model of the present invention.

[0069] 1. Co-cage female mice with a male ratio of 2:1 overnight. Separate them the next day and observe the formation of vaginal plugs in female mice. Female mice with vaginal plugs observed are marked as gestational day 0.5 (E0.5) and weighed. 2. On E10.5, the female mice were weighed and those with a weight gain of less than 10% were excluded. Those with a weight gain of more than 10% were gavaged with retinoic acid in corn oil (RA, 100 mg / kg) and designated as the RA group to establish a fetal cleft palate model. 3. Subsequently, mice in the RA group were intraperitoneally injected with VUF11207 fumarate (5 mg / kg) once daily from E10.5 to E14.5 (the RA + VUF11207 fumarate group), establishing a rescue model. On E16.5, after measuring the survival rate of the pregnant mice, the pregnant mice were sacrificed, the embryos were removed, and the palate development of the fetuses was observed under a stereomicroscope. The success rate of cleft palate reversal in the embryos was calculated. The specific criterion for successful cleft palate reversal in the embryos was the absence of a cleft palate. Specifically, the results of this example showed a 100% survival rate and a 74.4% success rate for cleft palate reversal in the embryos.

[0070] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Application of VUF11207 fumarate in the preparation of products related to reversing embryonic cleft palate.

2. Application of VUF11207 fumarate in the preparation of drugs for reversing embryonic cleft palate.

3. The use according to claim 2, characterized in that The active ingredient of the drug exists in free form or in the form of a pharmaceutically acceptable salt.

4. The use according to claim 2 or 3, characterized in that The medicament contains at least one pharmaceutically acceptable carrier.

5. The use according to any one of claims 2 to 4, characterized in that: The drug is administered orally, intravenously or intraperitoneally.

6. The use according to any one of claims 2 to 5, characterized in that: The drug is administered early in the development of the embryonic palate.

7. Application of VUF11207 fumarate in the preparation of embryonic cleft palate reversal animal model.

8. A method for constructing an embryonic cleft palate reversal animal model, characterized in that: This was constructed by administering VUF11207 fumarate to pregnant mice carrying fetuses with cleft palate from day 10.5 to day 14.5 of gestation.

9. The construction method according to claim 8, characterized in that: The administration method is intraperitoneal injection, and the daily injection dose is 5 mg / kg.

10. The construction method according to claim 8 or 9, characterized in that: The cleft palate of the fetal rat is formed by artificial induction, and the inducing agent used is retinoic acid.

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