Application of And-1 expression promoter in preparation of medicine for preventing and treating ulcerative colitis
By filtering the use of AND-1 expression in ulcerative colitis and screening AND-1 as a drug target target, it solves the lack of application of AND-1 in existing technology. Symptoms and the effect of discovering new drugs and logo.
Patent Information
- Application Number
- CN202411553602.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-02
- Publication Date
- 2025-01-28
AI Technical Summary
The prior art has failed to effectively utilize And-1 in the prevention and treatment of ulcerative colitis, and its pathogenesis in ulcerative colitis is not completely clear.
By using AND-1 to express the promotion of AND-1 expression in the preparation of drugs, and to screen the AND-1 as a drug target, it is found that drugs and diagnosis, treatment or prognosis of ulcerative colitis are found.
It is found that the level of expression of AND-1 is significantly reduced in ulcerative colitis. Tap or silence and silently will aggravate the disease. Expressive expression can improve the symptoms of ulcerative colitis, providing new drug development and diagnostic signs The possibility.
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Figure CN119367543A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medicine and relates to the application of an And-1 expression promoter in preparing a medicine for preventing and treating ulcerative colitis. Background Art
[0002] Ulcerative colitis (UC) is a chronic nonspecific inflammatory bowel disease (IBD) caused by multiple factors, characterized by recurrent and remitted mucosal inflammation, starting from the rectum and affecting the entire colon. Clinically, it is mainly manifested by abdominal pain, diarrhea, bloody stools, and weight loss. In severe cases, it can lead to colon cancer. Its pathogenesis is not yet fully understood and is mostly related to environmental, immune, genetic, and intestinal microbial factors. Currently, drug therapy and surgical treatment are the main treatments.
[0003] Acidic nucleoplasmic DNA-binding protein (And-1), also known as WDHD1 (WD Repeat And HMG-Box DNA Binding Protein 1), is a natural chimeric protein. And-1 interacts with the eukaryotic DNA helicase complex and DNA polymerase α, and participates in DNA replication; And-1 can also interact with the DNA damage repair protein CtIP to promote homologous recombination repair of DNA double-strand breaks.
[0004] Currently, there are no reports on the role of And-1 in the prevention and treatment of ulcerative colitis. Summary of the invention
[0005] The first purpose of the present invention is to provide an application of an And-1 expression promoter in the preparation of a drug for preventing and treating ulcerative colitis, wherein the And-1 expression promoter refers to a substance that promotes the expression of And-1; the second purpose is to provide an application of And-1 as a drug target in screening and discovering drugs for preventing and treating ulcerative colitis, wherein the And-1 refers to an And-1 gene or protein; the third purpose is to provide an application of And-1 as a drug target in screening and discovering markers for diagnosis, treatment or prognosis of ulcerative colitis, wherein the And-1 refers to an And-1 gene or protein.
[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:
[0007] Use of an And-1 expression promoter in the preparation of a drug for preventing and treating ulcerative colitis; wherein the And-1 expression promoter refers to a substance that promotes the expression of And-1.
[0008] Preferably, the And-1 expression promoter is a nucleotide sequence promoting And-1 expression or a vector containing the nucleotide sequence, or a small molecule compound, polypeptide or polysaccharide promoting And-1 expression.
[0009] The application of And-1 as a drug target in screening and discovering drugs for preventing and treating ulcerative colitis; wherein the And-1 refers to the And-1 gene or protein.
[0010] The use of And-1 as a drug target in screening and discovering markers for diagnosis, treatment or prognosis of ulcerative colitis; wherein the And-1 refers to the And-1 gene or protein.
[0011] Beneficial effects:
[0012] The present invention found that the expression level of And-1 is significantly decreased in ulcerative colitis, knocking out or silencing And-1 will significantly aggravate ulcerative colitis, and overexpressing And-1 can significantly improve ulcerative colitis. Based on this, those skilled in the art can determine that the And-1 expression promoter can be used to prepare drugs for preventing and treating ulcerative colitis, and the And-1 gene or protein can be used as a drug target for screening and discovering drugs for preventing and treating ulcerative colitis and for screening and discovering markers for diagnosis, treatment or prognosis of ulcerative colitis; wherein, the And-1 expression promoter refers to a substance that promotes the expression of And-1, which can be a nucleotide sequence that promotes the expression of And-1 or a vector containing the nucleotide sequence, or a small molecule compound, polypeptide or polysaccharide that promotes the expression of And-1. The prior art has not disclosed the technical solution of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 For WT and And-1 △IEC The results of the relative expression levels of And-1 mRNA and And-1 protein in the colon tissues of each group of mice; A is the protein expression of And-1 in the colon tissues of each group of mice detected by Western blot, and B is the relative expression level of And-1 mRNA in the colon tissues of each group of mice detected by Q-PCR;
[0014] Figure 2 For WT and And-1 △IEC Line graph of weight change rate of each group of mice;
[0015] Figure 3 For WT and And-1 △IEC DAI score results of each group of mice;
[0016] Figure 4 For WT and And-1 △IECActual colon images and length measurement results of each group of mice; A is the actual colon image of each group of mice, and B is the statistical graph of colon length of each group of mice;
[0017] Figure 5 For WT and And-1 △IEC Colon pathological section results of each group of mice;
[0018] Figure 6 For WT and And-1 △IEC The expression levels of TNF-α, IL-1β, and IL-6 in the colon tissues of each group of mice;
[0019] Figure 7 is the expression level of And-1 protein in colon tissues of each group of AAV-Vec and AAV-And-1 mice;
[0020] Figure 8 It is a line graph of the weight change rate of each group of AAV-Vec and AAV-And-1 mice;
[0021] Fig. 9 DAI score results of each group of AAV-Vec and AAV-And-1 mice;
[0022] Fig.10 The actual colon images and length measurement results of each group of AAV-Vec and AAV-And-1 mice; A is the actual colon image of each group of mice, and B is the statistical graph of the colon length of each group of mice;
[0023] Fig.11 These are the results of colon pathological sections of each group of AAV-Vec and AAV-And-1 mice. DETAILED DESCRIPTION
[0024] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.
[0025] Target gene cds sequence (And-1 (Gene ID: 218973)):
[0026]
[0027] 1. Experimental Materials
[0028] 1. Experimental Animals
[0029] Healthy female C57BL / 6 wild-type mice (WT) and Villin-cre mice aged 8-10 weeks were purchased from Shanghai Model Organisms Technology Co., Ltd. And-1 (Gene ID: 218973) conditional knockout mice (And-1 flox / fiox ) was commissioned by Shanghai Model Organisms Technology Co., Ltd. to construct; all types of mice were backcrossed to a 99% homologous C57BL / 6 background. Mice were raised in the Animal Experiment Center of Nanjing University of Chinese Medicine under standard temperature (20±2℃) and 12h light / dark cycle, given normal sterile feed, and free access to water. All mice were adapted to the above conditions for at least 7 days before the start of the experiment, and the experiment was carried out when the body weight reached about 20g.
[0030] 2. Experimental reagents
[0031] Table 1 Main reagents
[0032]
[0033]
[0034] 2. Experimental Methods
[0035] 1. Construction of intestinal epithelial-specific And-1 knockout mice
[0036] And-1 flox / fiox Mice were mated with Villin-cre mice to obtain F1 heterozygous mice And-1 flox / -,Villin-cre The F1 generation of male and female mice were mated together to obtain And-1 flox / fiox Female mice homozygous for Villin-cre (And-1 flox / flox,Villin-cre ), the mouse is referred to as And-1 △IEC Mouse.
[0037] 2. Construction of AAV-mediated And-1 overexpression mice in mouse colon tissue
[0038] Adeno-associated virus vector information: pAAV-CMV-CBH-GFP. The CDS region of the mouse target gene And-1 (Gene ID: 218973) was synthesized and constructed into the pAAV-CMV-CBH-GFP vector to obtain the And-1 expression vector. The virus packaging selected the serum type 9 AAV with high transduction efficiency for intestinal tissue. The virus packaging system consists of three plasmids, the And-1 expression vector, pHelper and pAAV-RC9. The construction of the And-1 overexpression vector and the virus packaging were completed by Zhenjiang Huamao Biotechnology Co., Ltd.
[0039] Healthy female C57BL / 6 wild-type mice aged 4-5 weeks were used to construct And-1 overexpression mice. The blank vector group (AAV-Vec) mice were injected with the control adeno-associated virus pAAV-CMV-CBH-GFP via tail vein, and the overexpression group (AAV-And-1) mice were injected with the overexpression adeno-associated virus AAV9-CMV-And-1 via tail vein.
[0040] Virus titer ≥2.5×10 12 vg / ml, the injection dose is 2×10 11 vg / mouse, and DSS model was established three weeks after virus injection.
[0041] 3. Construction of mouse ulcerative colitis model
[0042] 3.1 Modeling
[0043] Healthy female wild-type mice and knockout mice aged 8-10 weeks were housed in the Animal Experiment Center of Nanjing University of Chinese Medicine. The living conditions were standard temperature (20±2℃) and 12h light / dark cycle. They were given normal sterile feed and had free access to water. All mice were adapted to these conditions for at least 7 days before the start of the experiment, and the experiment was performed when their body weight reached about 20g. They were randomly divided into a wild-type mouse control group, a wild-type mouse model group, an And-1 gene intestinal epithelium-specific knockout mouse control group, and a model group.
[0044] The mice in the blank vector group (AAV-Vec) were randomly divided into a blank vector control group (AAV-Vec control) and a blank vector model group (AAV-Vec DSS). The mice in the overexpression group (AAV-And-1) were randomly divided into an overexpression control group (AAV-And-1control) and an overexpression model group (AAV-And-1DSS).
[0045] The modeling period was 7 days. The mice in the control group drank distilled water freely, and the mice in the model group drank 3% DSS aqueous solution for 7 days. The modeling was stopped on the 7th day.
[0046] 3.2 Weight Record
[0047] On the first day of the formal experiment, the weight of each group of mice was recorded at the same time period every day. After the end of the experiment on the 7th day, all weight data were sorted out and the weight change curve of mice was calculated using the following formula: weight change on the nth day = (weight on the nth day / weight on the 0th day) × 100%, and a line graph of the weight change of mice was made.
[0048] 3.3DAI score determination
[0049] During the experiment, the fecal characteristics and blood in the stool of the mice were recorded and scored every day. The disease activity index (DAI) was obtained by adding the percentage of weight loss, the fecal characteristics score and the blood in the stool score. The scoring criteria are shown in Table 2.
[0050] Table 2 DAI score table
[0051] Fraction Weight loss rate Stool characteristics Blood in stool 0 <1％ normal Negative blood in stool 1 1％-5％ Loose Weak positive blood in stool 2 5％-10％ Semi-formed thin Positive blood in stool 3 10％-20％ No shape Strong positive blood in stool 4 >20％ Watery diarrhea Visible blood in stool
[0052] 3.4 Colon length measurement
[0053] After the mice were killed on the 7th day, they were dissected and the colon section from the anus to the cecum was taken and placed on white A4 paper. The length of the colon was measured with a ruler and the data was recorded.
[0054] 4. Histopathological sections and tissue staining analysis
[0055] (1) Sampling: The colon tissue was fixed with a fixative for more than 24 hours. The tissue was removed from the fixative and trimmed flat with a scalpel in a fume hood. The trimmed tissue and the corresponding label were placed in a dehydration box.
[0056] (2) Dehydration and wax dipping: Place the dehydration box in a dehydrator and dehydrate with graded alcohols in sequence. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, paraffin I melted at 65°C for 1 hour, paraffin II melted at 65°C for 1 hour, paraffin III melted at 65°C for 1 hour.
[0057] (3) Embedding: Embed the wax-soaked tissue in an embedding machine. First, place the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and place it into the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool in a -20℃ freezer. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block.
[0058] (4) Sectioning: Place the trimmed wax block in a -20℃ freezer to cool, then place the cooled wax block in a paraffin slicer to slice it to a thickness of 4μm. Float the slices on 40℃ warm water on the slicer to flatten the tissue, pick up the tissue with a glass slide, and bake the slices in a 60℃ oven. After the wax is baked dry, take it out and store it at room temperature for later use.
[0059] (5) HE staining: The sections were stained with hematoxylin solution for 3-5 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with bluing solution, rinsed with running water, dehydrated with 85% and 95% graded alcohol for 5 minutes each, and finally stained with eosin solution for 5 minutes.
[0060] (6) Sealing: Seal the slides with neutral gum, then examine under a microscope and analyze and collect images.
[0061] 5. Real-time fluorescence quantitative PCR reaction
[0062] (1) Sample processing
[0063] Cut 10-20 mg of tissue into an enzyme-free centrifuge tube, add 1 mL of lysis buffer LB and homogenate beads, and homogenize. Let stand for 5 minutes to allow the ribosomes to completely dissociate.
[0064] (2) RNA extraction
[0065] ① Add 200 μL of chloroform to the above lysate, vortex for 15 seconds, and incubate at room temperature for 3 minutes.
[0066] ②12000rpm, 4℃, centrifuge for 15min. After centrifugation, the solution is divided into three layers: the upper colorless aqueous phase, the white middle phase, and the lower red organic phase. Pipette 400μL of the upper aqueous phase into a new centrifuge tube.
[0067] ③Add half volume of anhydrous ethanol and mix well.
[0068] ④The mixture was transferred to the adsorption column, centrifuged at 12000rpm for 45s, and the waste liquid was discarded.
[0069] ⑤ Add 500 μL of deproteinized solution PL, centrifuge at 12000 rpm for 45 seconds, and discard the waste liquid.
[0070] ⑥Add 500 μL of rinsing solution W*, centrifuge at 12000 rpm for 45 seconds, and discard the waste liquid. Repeat this step once.
[0071] ⑦ Empty column, centrifuge at 12000rpm for 2min.
[0072] ⑧ Transfer the adsorption column to a 1.5 mL enzyme-free centrifuge tube, carefully add 50 μL RNase-free water to the adsorption column, let it stand for 2 minutes, and centrifuge at 12000 rpm for 1 minute to obtain the RNA solution.
[0073] (3) Genomic DNA removal
[0074] ① Use a spectrophotometer to detect the concentration of RNA solution, pipette 1 μg RNA sample solution into an RNase-free eight-row centrifuge tube, add RNase-free water to 8 μL, and add 2 μL gDNA Wiper. Incubate at 42°C for 2 minutes.
[0075] (4) Reverse transcription
[0076] ① Add the following reagents to the reaction system in the previous step.
[0077] Table 3 Reverse transcription system
[0078] 10×RTMix 2μL HiScriptⅢEnzymeMix 2μL Oligo(dT)20VN 1μL Random hexamers 1μL <![CDATA[RNase-freeH2O]]> 4μL
[0079] ②37℃, 15min; 85℃, 5sec. Samples were stored at -20℃.
[0080] (5) Real-time fluorescence quantitative PCR reaction
[0081] ① Prepare the reaction system as follows:
[0082] Table 4 QPCR reaction system
[0083] SYBRGreenMix 10μL cDNA 2μL Primer 0.5 μL each <![CDATA[RNase-freeH2O]]> 7μL
[0084] ② The amplification program adopted a three-step method: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 sec; annealing at 55-60°C for 20 sec; extension at 72°C for 20 sec (40 cycles); the melting curve stage parameters were the instrument default settings.
[0085] ③ Use the ΔΔCT method to analyze and process the obtained data.
[0086] (6) The primer sequences involved in QPCR are as follows
[0087] Table 5 Primer sequences
[0088]
[0089]
[0090] 6. Western Blotting
[0091] (1) Tissue protein extraction
[0092] Cut about 20 mg of colon tissue, place it in a sterilized enzyme-free EP tube, add homogenate beads, add RIPA lysis buffer (PMSF, protease inhibitors and phosphatase inhibitors have been added in advance) at a ratio of 1 mg / 10 μL, and homogenize. Lyse on ice for 30 minutes, centrifuge at 13500 rpm, 4°C for 15 minutes, and collect the supernatant, which is the total protein solution.
[0093] (2) Histone quantification:
[0094] The BSA standard system configuration is as follows:
[0095] Table 6 BSA standard system
[0096] Vial Dilution volume (μL) 2mg / mL BSA volume (μL) BSA final concentration (μg / mL) A 0 30 2000 B 9 21 1400 C 12 18 1200 D 15 15 1000 E 21 9 600 F 24 6 400 G 27 3 200 H 49 1 40 I 30 0 0 = Blank
[0097] ② Prepare BCA working solution: add 1 volume of BCA reagent B to 50 volumes of BCA reagent A (A:B=50:1) and mix thoroughly.
[0098] ③ Determination method: Take 2.5μL of the sample to be tested and 22.5μL of diluent and add them to a 96-well plate. Add 200μL BCA working solution to each well and shake for 30s to mix thoroughly. Incubate at 37℃ for 30min. Detect the absorbance at a wavelength of 570nm on the microplate reader.
[0099] Draw a standard curve based on the absorbance of the BSA standard, calculate the sample protein concentration based on the standard curve and the dilution multiple of the sample, add 5× Loading Buffer, boil for 5 minutes, take out and place at room temperature for a few minutes in preparation for sample loading, and store in a -20℃ refrigerator.
[0100] (3) Electrophoresis
[0101] 30 μg protein was subjected to 10% SDS-PAGE electrophoresis at a constant voltage of 150 V for 1 h.
[0102] (4) Transfer
[0103] The proteins on the gel were transferred to the NC membrane at a constant current of 400 mA for 0.5 h.
[0104] (5) Closed
[0105] Block with rapid blocking solution for 10 min and wash the membrane three times.
[0106] (6) Apply primary antibody
[0107] The corresponding bands were cut out and placed in an antibody incubation box, and incubated with primary antibody at 4°C overnight.
[0108] (7) Apply secondary antibody
[0109] The membrane was washed three times, incubated with secondary antibody at room temperature for 1 h, and washed three times.
[0110] (8) Exposure
[0111] The bands were exposed with ECL exposure solution, and the results were analyzed with Image J software for semi-quantitative analysis of protein bands.
[0112] 7. Data processing
[0113] All data were processed using GraphPad Prism 8.0 software, and the data between the two groups were analyzed using t-test. The significance of multiple groups of samples was tested by Two-way ANOVA. All data are expressed as mean ± SEM. *p < 0.05 is statistically different, **p < 0.01 is significantly statistically different, and ***p < 0.01 is extremely significantly statistically different.
[0114] 3. Experimental Results
[0115] 1. WT and And-1 △IEC Expression of And-1 protein and mRNA levels in mouse colon
[0116] The results of the determination of And-1 protein and mRNA levels in the colon tissues of each group of mice are shown in Figure 1 Compared with the wild-type control group (WT control), the And-1 knockout control group (And-1 △IEC The expression level of And-1 in the colon of control mice was significantly decreased, indicating that the And-1 low-expression And-1 in the colon was successfully constructed. △IEC Mice. The wild-type model group (WTDSS) and the wild-type control group (WT control) and the And-1 knockout model group (And-1 △IEC DSS) and And-1 knockout control group (And-1 △IEC By comparing the expression level of And-1 in the colon of UC mice with serotype-controlled mice, we found that the expression level of And-1 was significantly decreased in UC mice.
[0117] 2. WT and And-1 △IEC Mouse body weight measurement results
[0118] The body weight of mice in each group is shown in the figure Figure 2 Compared with the wild-type control group (WT control), the body weight of the wild-type model group (WT DSS) mice showed a significant downward trend on the third day of the experiment; compared with the And-1 knockout control group (And-1 △IEC Compared with the And-1 control group, the And-1 knockout model group (And-1△IEC The weight of the mice in the wild-type DSS group also showed a significant downward trend on the third day of the experiment; this result indicated that the DSS-induced ulcerative colitis model was successfully established. Compared with the downward trend in the weight of the mice in the wild-type model group (WT DSS), the And-1 knockout model group (And-1 △IEC The weight loss trend was more obvious in DSS) mice, indicating that And-1 knockout would aggravate the weight loss of ulcerative colitis mice.
[0119] 3. WT and And-1 △IEC Results of mouse DAI score determination
[0120] The DAI scores of mice in each group are shown in Figure 3 Compared with the wild-type control group (WT control), the DAI score of the wild-type model group (WTDSS) mice was significantly increased; compared with the And-1 knockout control group (And-1 △IEC Compared with the And-1 control group, the And-1 knockout model group (And-1 △IEC The DAI scores of the And-1 knockout model group (And-1) were significantly increased compared with the wild-type model group (WTDSS) mice; this result indicates that the DSS-induced ulcerative colitis model was successfully established. △IEC The increase trend of DAI scores in DSS) mice was more obvious, indicating that And-1 knockout would aggravate the disease activity index of ulcerative colitis mice.
[0121] 4. WT and And-1 △IEC Actual image and length measurement of mouse colon
[0122] The colon length results of each group of mice are as follows Figure 4 Compared with the wild-type control group (WT control), the colon of the wild-type model group (WT DSS) mice was significantly shortened; compared with the And-1 knockout control group (And-1 △IEC Compared with the And-1 control group, the And-1 knockout model group (And-1 △IEC The colon of the And-1 knockout model group (And-1) was significantly shortened compared with the wild-type model group (WTDSS) mice. △IEC The shortening of the colon was more obvious in mice with ulcerative colitis (DSS), indicating that And-1 knockout aggravates colon damage in mice with ulcerative colitis.
[0123] 5. WT and And-1 △IEC Mouse colon pathology section results
[0124] The results of colon HE of mice in each group are as follows Figure 5 As shown. Wild-type control group (WT control), And-1 knockout control group (And-1 △IEC The colon glands of the And-1 knockout mice were neatly arranged and normal in shape, with no obvious abnormalities in the tissues. Compared with the wild-type control group (WT control), the crypt structure of the wild-type model group (WTDSS) mice was deformed, and inflammatory cell infiltration was observed; compared with the And-1 knockout control group (And-1 △IEC Compared with the And-1 control group, the And-1 knockout model group (And-1 △IEC DSS) mice showed obvious destruction of colon epithelium, deformation and destruction of crypt structure, thickening of base, detachment and necrosis of colon epithelial cells, and infiltration of a large number of inflammatory cells. △IEC The colon pathological damage of DSS mice was significantly more severe. The above results indicate that And-1 knockout can aggravate the colon pathological damage of ulcerative colitis mice.
[0125] 6. WT and And-1 △IEC Results of inflammatory factor expression levels in mouse colon
[0126] The expression results of inflammatory factors in the colon tissue of each group of mice are as follows Figure 6 As shown, after 7 days of 3% DSS treatment, the wild-type model group (WTDSS) mice and the And-1 knockout model group (And-1 △IEC The expression levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in the colon tissue of mice treated with DSS were significantly increased, and the And-1 knockout model group (And-1 △IEC The expression of pro-inflammatory factors in the wild-type DSS mice was significantly higher than that in the wild-type model group (WTDSS) mice. The above results indicate that And-1 knockout aggravates the inflammatory symptoms of the colon in ulcerative colitis mice.
[0127] 7. Results of And-1 protein expression in colon tissues of AAV-Vec and AAV-And-1 mice
[0128] The results of And-1 protein expression level determination in colon tissue of each group of mice are as follows Figure 7 As shown, the expression level of And-1 protein in the colon of mice in the overexpression group (AAV-And-1) was significantly increased, indicating that mice with And-1 overexpression in colon tissue were successfully constructed.
[0129] 8. Line graph of weight change rate of each group of AAV-Vec and AAV-And-1 mice
[0130] The body weight of mice in each group is shown in the figure Figure 8 As shown, there was no significant change in the body weight of the blank vector control group (AAV-Vec control) and the overexpression control group (AAV-And-1control) mice during the experimental period; compared with the blank vector control group (AAV-Vec control) and the overexpression control group (AAV-And-1control) mice, the body weight of the blank vector model group (AAV-Vec DSS) and the overexpression model group (AAV-And-1DSS) mice showed a significant downward trend, indicating that the DSS-induced ulcerative colitis model was successfully established; compared with the decreasing trend of the body weight of the blank vector model group (AAV-Vec DSS) mice, the decreasing trend of the overexpression model group (AAV-And-1DSS) was significantly improved, indicating that And-1 overexpression helps to alleviate the weight loss of ulcerative colitis model mice.
[0131] 9. DAI scores of AAV-Vec and AAV-And-1 mice
[0132] The DAI scores of mice in each group are shown in Fig. 9 As shown, compared with the blank vector control group (AAV-Vec control) and overexpression control group (AAV-And-1control) model group mice, the DAI scores of the blank vector model group (AAV-Vec DSS) and overexpression model group (AAV-And-1DSS) mice were significantly increased, indicating that the DSS-induced ulcerative colitis model was successfully established; compared with the increasing trend of the DAI scores of the blank vector model group (AAV-Vec DSS) mice, the increasing trend of the DAI scores of the overexpression model group (AAV-And-1DSS) mice was significantly improved, indicating that And-1 overexpression helps to improve the disease activity index of ulcerative colitis model mice.
[0133] 10. Actual colon images and length measurement results of each group of AAV-Vec and AAV-And-1 mice
[0134] The colon length results of each group of mice are as follows Fig.10 As shown, compared with the blank vector control group (AAV-Vec control) and overexpression control group (AAV-And-1control) model group mice, the colon of the blank vector model group (AAV-Vec DSS) and overexpression model group (AAV-And-1DSS) mice was significantly shortened, indicating that the DSS-induced ulcerative colitis model was successfully established; compared with the shortening degree of the colon of the blank vector model group (AAV-Vec DSS) mice, the shortening degree of the overexpression model group (AAV-And-1DSS) was significantly improved, indicating that And-1 overexpression helps to improve colon damage in ulcerative colitis model mice.
[0135] 11. Colon pathology sections of AAV-Vec and AAV-And-1 mice
[0136] The results of colon HE of mice in each group are as follows Fig.11 As shown. The colon glands of mice in the blank vector control group (AAV-Vec control) and the overexpression control group (AAV-And-1control) were neatly arranged, with normal morphology and no obvious abnormalities in the tissues. Compared with the blank vector control group (AAV-Vec control), the crypt structure of mice in the blank vector model group (AAV-Vec DSS) disappeared, and a large number of inflammatory cells were observed to infiltrate; compared with the blank vector model group (AAV-Vec DSS), the colon epithelial inflammation of mice in the overexpression model group (AAV-And-1DSS) was milder, without a large number of cell necrosis. The results show that And-1 overexpression helps to improve colon pathological lesions in mice with ulcerative colitis model.
[0137] In summary:
[0138] The expression level of And-1 is significantly decreased in the colon tissue of mice with ulcerative colitis. Knockout or silencing of And-1 will significantly aggravate ulcerative colitis, and overexpression of And-1 can significantly improve the symptoms of ulcerative colitis. Based on this, those skilled in the art can determine that the And-1 expression promoter can be used to prepare drugs for preventing and treating ulcerative colitis, and the And-1 gene or protein can be used as a drug target for screening and discovering drugs for preventing and treating ulcerative colitis and for screening and discovering markers for diagnosis, treatment or prognosis of ulcerative colitis; wherein, the And-1 expression promoter refers to a substance that promotes the expression of And-1, which can be a nucleotide sequence that promotes the expression of And-1 or a vector containing the nucleotide sequence, or a small molecule compound, polypeptide or polysaccharide that promotes the expression of And-1.
[0139] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.
Claims
1. Use of an And-1 expression promoter in the preparation of a drug for preventing and treating ulcerative colitis; wherein: The And-1 expression promoter refers to a substance that promotes the expression of And-1.
2. The use according to claim 1, characterized in that: The And-1 expression promoter is a nucleotide sequence that promotes the expression of And-1 or a vector containing the nucleotide sequence.
3. The use according to claim 1, characterized in that: The And-1 expression promoter is a small molecule compound, polypeptide or polysaccharide that promotes And-1 expression.
4. Application of And-1 as a drug target in screening and discovering drugs for the prevention and treatment of ulcerative colitis; The And-1 refers to the And-1 gene or protein.
5. Application of And-1 as a drug target in screening and discovering markers for diagnosis, treatment or prognosis of ulcerative colitis; The And-1 refers to the And-1 gene or protein.