CN113694202B - Application of ASS1 or BCKDK inhibitor in preparation of medicine for treating ulcerative colitis - Google Patents
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Abstract
The invention discloses application of an ASS1 or BCKDK inhibitor in preparation of a medicament for treating ulcerative colitis. The invention discovers that the transcription levels and protein levels of ASS1 and BCKDK are obviously increased in ulcerative colitis, and the ulcerative colitis can be obviously relieved by interfering the expression of the ASS1 or BCKDK gene. Therefore, ASS1 or BCKDK can be used as a drug action target for preparing drugs for preventing and treating ulcerative colitis, can be used as a drug action target for screening and discovering drugs for preventing and treating ulcerative colitis, and an inhibitor of ASS1 or BCKDK can be used for preparing drugs for preventing and treating ulcerative colitis, wherein: ASS1 refers to ASS1 gene or protein, BCKDK refers to BCKDK gene or protein; the inhibitory activity of the inhibitor is inhibition of the activity of ASS1 or BCKDK protein, or inhibition of the expression of ASS1 or BCKDK gene.
Description
The application is a divisional application of a patent application with the application number of 202010605346X and the invention name of application of ASS1 or BCKDK inhibitor in the preparation of a medicament for treating ulcerative colitis, which is filed on 29/06/2020.
Technical Field
The invention belongs to the field of medicines, and relates to application of an inhibitor of a known target in treatment of ulcerative colitis, in particular to application of an ASS1 or BCKDK inhibitor in preparation of a medicine for treating ulcerative colitis.
Background
Ulcerative Colitis (UC) is also called chronic nonspecific ulcerative colitis or idiopathic ulcerative colitis, is a common chronic intestinal disease, and is also a multifactorial and multilevel nonspecific inflammation with unknown reasons. Lesions are mainly in the mucosa and submucosa of the colon, and are distributed diffusely and continuously, and mostly affect the rectum and sigmoid colon. The main clinical manifestations include abdominal pain, diarrhea, mucopurulent bloody stool, tenesmus and the like, and some patients have extra-intestinal manifestations such as joint, hepatobiliary tube diseases and eye and skin injuries; the duration of the disease is prolonged and the severity is different; the age groups of onset are mainly 20-50 years old, but there is no significant sex difference. Epidemiological statistical data at home and abroad show that the incidence and the prevalence of UC are obviously increased, and are classified as one of the modern refractory diseases by the world health organization, and the etiology, the pathogenesis and the treatment medicine of UC are always research hotspots.
Argininosuccinate synthetase 1 (ASS 1) is an enzyme that catalyzes the synthesis of argininosuccinate from citrulline and aspartate, and an abnormal increase in its expression can lead to a significant increase in the blood nitrogen and other urea cycle by-products, such as arginine. Branched chain alpha-ketoacid dehydrogenase kinase (BCKDK) is a negative regulation kinase of a key complex enzyme (BCKDH) for catabolism of Branched Chain Amino Acid (BCAA), and accumulation of branched chain amino acid-leucine is easily caused by abnormal increase of BCKDK expression.
At present, no research has been published on the relationship of ASS1 or BCKDK to the treatment of ulcerative colitis.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides application of ASS1 or BCKDK inhibitor in preparation of a medicine for treating ulcerative colitis.
The above purpose of the invention is realized by the following technical scheme:
application of ASS1 or BCKDK as a drug action target in preparation of drugs for preventing and treating ulcerative colitis, wherein the ASS1 refers to ASS1 gene or protein, and the BCKDK refers to BCKDK gene or protein.
Application of ASS1 or BCKDK as a drug action target in screening and discovering drugs for preventing and treating ulcerative colitis, wherein the ASS1 refers to ASS1 gene or protein, and the BCKDK refers to BCKDK gene or protein.
The application of the inhibitor of ASS1 or BCKDK in the preparation of the medicine for preventing and treating ulcerative colitis is that the inhibitory activity of the inhibitor is to inhibit the activity of ASS1 or BCKDK protein or inhibit the expression of ASS1 or BCKDK gene.
Further, the inhibitor includes a small molecule compound, a nucleic acid molecule, and an expression vector comprising the nucleic acid molecule.
Has the advantages that:
the research of the invention discovers that the transcription levels and protein levels of ASS1 and BCKDK are obviously increased in ulcerative colitis, and the ulcerative colitis can be obviously relieved by constructing a target protein shRNA fragment through an adeno-associated virus vector to interfere the expression of the ASS1 or BCKDK gene. The person skilled in the art can determine that ASS1 or BCKDK can be used as a drug action target for preparing a drug for preventing and treating ulcerative colitis, can be used as a drug action target for screening and discovering a drug for preventing and treating ulcerative colitis, and an inhibitor of ASS1 or BCKDK can be used for preparing a drug for preventing and treating ulcerative colitis, wherein: ASS1 refers to ASS1 gene or protein, BCKDK refers to BCKDK gene or protein; the inhibitory activity of the inhibitor is inhibition of the activity of ASS1 or BCKDK protein, or inhibition of the expression of ASS1 or BCKDK gene.
Drawings
Fig. 1 is a line graph of body weights of each group of mice.
Fig. 2 shows the DAI scores of the mice in each group.
FIG. 3 shows the expression levels of IL-1. Beta., TNF-. Alpha.and IL-6 in the serum of each group of mice; wherein: a is the expression level of IL-1 beta in the serum of different groups of mice, B is the expression level of TNF-alpha in the serum of different groups of mice, and C is the expression level of IL-6 in the serum of different groups of mice.
FIG. 4 shows the results of colon length measurement of each group of mice.
FIG. 5 is a colon pathology section and histological scoring results for each group of mice; wherein: a is a colon pathological section picture of each group of mice, and B is a histological scoring result of each group of mice.
FIG. 6 shows the results of measurement of BCKDK, BCKDHA, P-BCKDHA protein expression levels and BCKDKmRNA relative expression levels in colon tissues of various groups of mice; wherein: a is a measurement result of detecting the relative expression level of BCKDK mRNA in each group of mouse colon tissues by Q-PCR, B is the protein expression condition of BCKDK in each group of mouse colon tissues detected by Westernblot, and C is the protein expression condition of BCKDHA and P-BCKDHA in each group of mouse colon tissues detected by Western blot.
FIG. 7 shows the results of Western blot for measuring the protein expression level of ASS1 and the relative expression level of ASS1 mRNA in colon tissues of various groups of mice; wherein A is a result of detecting ASS1 mRNA relative expression level in each group of mouse colon tissues by Q-PCR, and B is a result of detecting ASS1 protein expression level in each group of mouse colon tissues by Western blot.
FIG. 8 shows Westernblot analysis of protein expression of S6K and P-S6K in colon tissues of various groups of mice.
Detailed Description
The following detailed description will be given with reference to the accompanying drawings and examples, but the scope of the invention is not limited thereto.
1. Test materials and methods
1. Grouping, modeling and administering drugs
Materials: c57BL/6 type mice; dextran Sodium Sulfate (DSS); PBS buffer (pH 7.4);
the target gene is as follows: BCKDK-murine (Gene ID: 12041), ASS 1-murine (Gene ID: 11898) each Gene corresponds to a targeting site: BCKDK (ID: 12041) shRNA sequence: 5 '(CCTAGACAGACCTACTAAT) -3'; ASS1 (ID: 11898) shRNA sequence: 5 'GCCCAGATGTCCTTGAGAGAGAT-containing 3'.
Vector construction (see Haijiki Gene science and technology Co., ltd., supra):
adeno-associated viral vector information: pAAV-U6-shRNA-CMVbGlobin-eGFP-3Flag (GV 390).
Coating virus (see Haijiki Gene science and technology Co., ltd.):
the Geckie gene AAV Helper-Free System consists of three plasmids including a viral vector (carrying a target gene interference fragment: GV 390), a pAAV-RC vector (carrying a virus capsid protein, namely, serotype 9) and a pHelper vector (comprising a set of adenovirus genes VA, E2A and E4 necessary for AAV-293 cells to produce high-titer viruses).
Selecting C57BL6 type mice as experimental objects, purchasing from Qinglong mountain animal farm in Jiangning district of Nanjing, the week age is 8-12 weeks, adaptively feeding in animal experiment center of Chinese medicinal university for about one week, and performing experiment when the weight is about 20 g. Randomly divided into a blank group, a model group, an ASS1 virus interference group and a BCKDK virus interference group, and each group contains 10 mice.
The experimental period is 10 days, the blank group of mice drink normal water for 10 days, and other groups begin to drink 3% DSS aqueous solution for ten days from the first day to carry out Ulcerative Colitis (UC) modeling. The ASS1 virus interference group is administrated with the enveloped virus containing shRNA fragments interfering the expression of the ASS1 gene by intragastric administration once a day, and the virus titer is 1E +11. The BCKDK virus interference group is administered with the enveloped virus containing the shRNA segment interfering the BCKDK gene expression by intragastric administration for the first time, and the virus titer is 1E +11. The model group was modeled only and no drug was administered. Mice were killed five hours after the end of the last day experiment.
2. Body weight determination
The weights of the mice were weighed at the same time point every day on the first day of the mice starting the actual experiment, recorded in a table, and all weights were collated after the experiment on the 10 th day to make a line graph, and the disease severity of the mice was fed back by the weights.
3. Determination of Disease Activity Index (DAI) score
During the experiment, the change of the mouse body mass, the stool character and the hematochezia condition are recorded every day, and the Disease Activity Index (DAI) score is measured. DAI is obtained by adding weight loss rate fraction, stool character fraction and hematochezia fraction, and the score is between 0 and 12. The scoring criteria were as follows: 1) Weight loss rate score: no weight loss, score 0; reducing by 5-9% for 1 minute; the weight is reduced by 10 to 14 percent for 2 minutes; the weight is reduced by 15 to 20 percent and is divided into 3 minutes; the reduction is more than 20 percent and 4 minutes. 2) And (3) stool character scoring: normal, 0 point; loosening, 1 minute; half-forming for 2 min; not forming thin, 3 minutes; and (4) draining the water sample. 3) Stool and hematochezia scoring: negative for stool (-), score 0; weak positive (+) of hematochezia, 1 point; fecal blood positive (++), score 2; strong positive in stool blood (+++), score 3; bloody stool was visible with naked eyes, 4 points. At the end of the experiment on day 10, day 10 DAI scores were used as an index to reflect the severity of disease in mice.
4. Determination of serum inflammatory factor expression levels
Materials: IL-6 hypersensitive enzyme-linked immunosorbent (ELISA) kit; an IL-1 beta high-sensitivity enzyme-linked immunosorbent (ELISA) kit; TNF-alpha high-sensitivity enzyme-linked immunosorbent assay (ELISA) kit.
The method comprises the following steps: after the experiment on the 10 th day, the eye sockets of the mice were bled, placed in a serum tube, shaken evenly and stood. Centrifuging at 3500rpm/min for 10min, sucking the upper layer serum into 1.5mL EP tube, and storing in refrigerator at-80 deg.C for use without repeated freeze thawing. Measuring the temperature at the beginning and mixing uniformly.
1) Sample adding: a blank hole, a standard hole and a sample hole to be detected are respectively arranged. The blank wells are filled with 100 mu L of sample diluent, and the other wells are filled with 100 mu L of standard sample or sample solution to be detected respectively. And adding the sample to the bottom of the hole of the ELISA plate during sample adding, gently shaking and uniformly mixing, adding the film on the ELISA plate, and incubating for 1h at 37 ℃.
2) Discarding the liquid, and drying the liquid without washing. Add 100. Mu.L of the working solution A (prepared within one hour before use, add 99. Mu.L of the test dilution A to one. Mu.L of the test solution A, mix gently) into each well at 37 ℃ for 60min.
3) After incubation for 60min, the liquid in the wells was discarded, spin-dried, plate-washed 3 times, each time soaking for 2min,350 μ L/well, spin-dried.
4) Add 100. Mu.L of the working solution of detection solution B (same as the working solution of detection A) to each well, and incubate at 37 ℃ for 1h.
5) After incubation for 60min, the liquid in the wells was discarded, spin-dried, and the plate was washed 5 times, each time for 2min,350 μ L/well, spin-dried.
6) The substrate solution 90. Mu.L was added to each well in sequence, and the reaction was incubated at 37 ℃ for 15min in the dark.
7) The reaction was stopped by adding 50. Mu.L of stop solution to each well in sequence.
8) The optical density (OD value) of each well was measured at a wavelength of 450nm with a microplate reader.
5. Determination of colon Length
After the mice were sacrificed on day 10, the mice were dissected, the colon portion thereof was taken out, placed on white paper, and the length was measured with a ruler and recorded, with the colon length as one aspect to reflect the severity of the disease.
6. Colon pathology sectioning and histological scoring
Materials: 4% paraformaldehyde; EDTA (ethylene diamine tetraacetic acid); HE dye liquor; neutral resin; xylene; ethanol
The method comprises the following steps: after an experimental mouse is killed, cutting about 1cm of colon containing a diseased part, fixing the colon in 4% paraformaldehyde, decalcifying the colon by using Ethylene Diamine Tetraacetic Acid (EDTA), embedding the colon in paraffin, slicing the colon into 4-5 mu m (longitudinal cutting), and dewaxing the colon to water by using conventional xylene, ethanol of each grade and distilled water; and (5) HE staining. Hematoxylin staining nuclei and eosin staining cytoplasm; and sealing the neutral resin sheet, and observing pathological and histological changes by using an optical microscope. The histological scoring standard of the colon section is divided into 2 aspects of epithelial injury and mucositis cell infiltration: epithelial lesions were graded 5: grade 0, no damage; grade 1, a small number of goblet cells were lost; grade 2, loss of large numbers of goblet cells; grade 3, disappearance of a few crypts and loss of a large number of goblet cells; the large crypts of grade 4 disappear. Inflammatory infiltration was graded as 5: level 0, no infiltration; level 1, soaking to the bottom of the crypt; 2, infiltrating into a mucous membrane muscular layer; grade 3, a large amount of infiltration of the mucous membrane muscular layer is accompanied by edema; grade 4, infiltration into submucosa. The sum of the scores of the above 2 parts is the histological score.
7. Western blot for measuring protein expression levels of ASS1, BCKDK, BCKDHA and P-BCKDHA in colon tissues and Q-PCR for detecting relative expression conditions of ASS1 mRNA and BCKDK mRNA in colon tissues
Materials: trizol Reagent; isopropyl alcohol; chloroform; ethanol; M-MLV reverse transcriptase; dNTP; DEPC water; random primers; bovine Serum Albumin (BSA); extracting lysate from RIPA strong protein; PMSF; a BCA protein quantification reagent; pre-staining the marker with protein; a PVDF membrane; skimmed milk powder; ECL luminescent liquid; a GAPDH antibody; a BCKDK antibody; a BCKDHA antibody; a P-BCKDHA antibody;
the method comprises the following steps: after killing the mice, the colon tissues were taken and stored in a-80 ℃ refrigerator. The RNA extraction and Q-PCR operation steps are as follows:
1) Extracting total RNA of tissues:
(1) colon tissue was collected in an EP tube, 1mL Trizol was added, and the tissue was ground to a homogenate using a homogenizer (care was taken to work on ice to prevent RNA degradation at too high a temperature).
(2) 0.2mL of chloroform was added thereto, followed by vigorous shaking for 15 seconds and standing at room temperature for 3min.
(3) Centrifuge at 12000rpm for 15min at 4 ℃ and remove the supernatant into a new EP tube.
(4) Adding equal volume of isopropanol, mixing, and standing at-20 deg.C for 30min.
(5) Centrifugation was carried out at 12000rpm for 10min at 4 ℃ and the supernatant was discarded.
(6) The precipitate was washed by adding 1mL 75% ethanol (in DEPC water). Centrifuge at 12000rpm for 2min at 4 ℃ and discard the supernatant.
(7) Air-dry at room temperature for 3min, dry RNA precipitation.
(8) Appropriate amount of DEPC water was added to dissolve RNA sufficiently.
2) Reverse transcription
(1) To a 0.2mL EP tube, 16. Mu.L of total RNA, 10. Mu.L of random primer, water bath at 65 ℃ for 5min, and then ice bath for 3min were added.
(2) mu.L of 5 XM-MLVBuffer, 4. Mu.L of 10mM dNTPs, 2. Mu.L of M-MLV reverse transcriptase and 8. Mu.L of DEPC water were added. The total volume was 50. Mu.L.
(3) Reverse transcription conditions: 30 ℃ for 10min;42 ℃ for 1h;95 ℃ for 10min. The reaction solution was cDNA.
TABLE 1 amplification primer sequences for genes of interest
3) Fluorescent quantitative PCR reaction
Taking out cDNA as a template for fluorescence quantification, wherein the reaction system comprises the following steps:
reaction conditions are as follows: 95 ℃ for 5min; at 95 ℃ for 30s; primer annealing temperature, 30s;72 ℃ for 1min.
30 cycles of 10min at 72 ℃.
The experimental results were calculated using the 2- Δ Ct.
The Western blot method comprises the following steps: taking and weighing the preserved colon tissue, adding the weighed colon tissue according to the proportion of 0.1g tissue to 1mLRIPA, then homogenizing on ice by a homogenizer, cracking on ice for 30min, and swirling once every 5min to ensure that the tissue is fully cracked. Centrifugation was carried out at 12000rpm for 10min at 4 ℃ to collect the supernatant, and total protein concentration was measured using BCA quantification kit from Gegen. Add 5X protein Loading Buffer, boiling water bath for 5min, take out and stand for several minutes at room temperature to prepare for Loading.
1) BCA protein quantification:
(1) preparing a standard curve:
TABLE 2BSA Standard concentration preparation
(2) Preparing a working solution: the reagent a and the reagent B were thoroughly mixed at a volume ratio of 50.
(3) The determination method comprises the following steps: and (3) sucking 25 mu L of sample liquid into a 96-well plate, adding 200 mu L/well of BCA working solution, incubating for 30min at 37 ℃, and detecting absorbance at 590nm by using an enzyme-labeling instrument. The protein concentration of each sample was calculated over its linear range with reference to a standard curve.
2) Western Blot detection
(1) 10% SDS-PAGE of 30. Mu.g of the protein sample was performed.
(2) The wet-to-electrotransfer method transfers proteins on the gel to the PVDF membrane.
(3) The PVDF membrane was blocked at the blocking solution temperature for 2h.
(4) The membrane was washed with TBST, and primary antibody was added thereto and incubated overnight at 4 ℃.
(5) The membrane was washed 3 times with TBST for 10min each time. And adding a secondary antibody for incubation at room temperature for 1h.
(6) The membrane was washed 3 times with TBST for 10min each time. ECL luminous liquid and an exposure instrument.
Analyzing the development result by using gel image analysis software Quantity One, and taking the ratio of the optical density of the target protein band to the optical density of the internal reference band as a target protein expression quantitative index.
8. Western blot for measuring expression levels of S6K and P-S6K proteins in colon tissues
Materials: bovine Serum Albumin (BSA); extracting lysate from RIPA protein; PMSF; a BCA protein quantification reagent; pre-staining marker with protein; a PVDF membrane; skimmed milk powder; ECL luminescent liquid; a GAPDH antibody; an S6K antibody; P-S6K antibodies.
The method comprises the following steps: taking preserved colon tissue, weighing, adding according to the proportion of 0.1g tissue and 1mLRIPA, then homogenizing on ice by a homogenizer, cracking on ice for 30min, and vortexing once every 5min to ensure that the tissue is fully cracked. Centrifugation was carried out at 12000rpm for 10min at 4 ℃ to collect the supernatant, and total protein concentration was measured using BCA quantification kit from Gegen. Adding 5x protein Loading Buffer solution, boiling water bath for 5min, taking out and placing for several minutes at room temperature to prepare for Loading.
1) BCA protein quantification:
(1) preparing a standard curve:
TABLE 3 BSA Standard concentration preparation
(2) Preparing a working solution: the reagent a and the reagent B were thoroughly mixed at a volume ratio of 50.
(3) The determination method comprises the following steps: and (3) sucking 25 mu L of sample liquid into a 96-well plate, adding 200 mu L/well of BCA working solution, incubating for 30min at 37 ℃, and detecting absorbance at 590nm by using an enzyme-labeling instrument. The protein concentration of each sample was calculated over its linear range with reference to a standard curve.
2) Western Blot detection
(1) A30. Mu.g sample of the protein was subjected to 10% SDS-PAGE.
(2) The wet-to-electrotransfer method transfers proteins on the gel to the PVDF membrane.
(3) The PVDF membrane was blocked at the blocking solution temperature for 2h.
(4) The membrane was washed with TBST, and primary antibody was added thereto and incubated overnight at 4 ℃.
(5) The membrane was washed 3 times with TBST for 10min each time. And adding a secondary antibody for incubation at room temperature for 1h.
(6) The membrane was washed 3 times with TBST for 10min each time. ECL luminous liquid and an exposure instrument.
(7) And analyzing a developing result by using gel image analysis software Quantity One, and taking the ratio of the optical density of the target protein strip to the optical density of the internal reference strip as a target protein expression quantitative index.
9. Data processing
The results of the experiment are expressed as (Mean + -SEM) using Student's t for comparisons between the two groups and One-way ANOVA and post hoc tests for comparisons between the groups. * P <0.05 had statistical differences,. P <0.01 had statistically significant differences,. P <0.001 had statistically significant differences.
3. Results of the experiment
1. Results of body weight measurement
The body weight line graph of each group of mice is shown in fig. 1, the body weight of the blank group of mice in the experimental period has no obvious change, and compared with the blank group of mice, the body weight of the model group of mice in the experimental period has an obvious descending trend; compared with the model group, the weight reduction trend of mice in the ASS1 virus interference group and the BCKDK virus interference group in the experimental period is effectively controlled.
2. Disease Activity Index (DAI) score results
The DAI score results of each group of mice are shown in FIG. 2, and compared with the blank group, the DAI score of the model group of mice is obviously increased; the DAI scores of ASS1 virus-disrupted group and BCKDK virus-disrupted group mice were significantly reduced compared to the model group.
3. Measurement of serum inflammatory factor expression level
The expression levels of IL-1 beta, TNF-alpha and IL-6 in the serum of each group of mice are shown in figure 3, and compared with the blank group, the expression levels of IL-1 beta, TNF-alpha and IL-6 in the serum of the model group of mice are obviously increased; compared with the model group, the expression levels of IL-1 beta, TNF-alpha and IL-6 in the serum of mice in the ASS1 virus interference group and the BCKDK virus interference group are obviously reduced.
4. Colon Length determination
The colon length measurement result of each group of mice is shown in figure 4, and compared with the blank group, the colon length of the model group of mice is obviously reduced; compared with the model group, the colon length of mice in the ASS1 virus interference group and the BCKDK virus interference group is remarkably increased.
5. Colon pathology sectioning and histological scoring results
The results of colon pathological section and histological scoring of each group of mice are shown in fig. 5, compared with the blank group, the colon of the model group of mice has obvious lesion and enhanced inflammatory infiltration; compared with the model group, the colon lesion of the mice in the ASS1 virus interference group and the BCKDK virus interference group is obviously relieved, and the inflammatory infiltration is improved.
6. The results of measuring the expression levels of BCKDK mRNA, BCKDK, BCKDHA and P-BCKDHA proteins in colon tissues
The results of measuring BCKDK mRNA level and BCKDK, BCKDHA, P-BCKDHA protein expression levels in colon tissues of each group of mice are shown in FIG. 6, and it can be seen from the results: compared with the blank group, the BCKDK mRNA level and the BCKDK protein level of the model group are obviously increased, and the substrate P-BCKDHA/BCKDHA is also obviously increased, so that the activity of BCKDH enzyme is inhibited; compared with the model group, the BCKDK virus interference group can obviously reduce the BCKDK mRNA level and the BCKDK, P-BCKDHA/BCKDHA protein expression level, thereby activating the activity of the BCKDH enzyme.
7. ASS1 mRNA and protein expression level measurement results in colon tissue
The results of measuring the expression levels of ASS1 mRNA and ASS1 protein in the colon tissues of each group of mice are shown in fig. 7, and the expression levels of ASS1 mRNA and ASS1 protein in the colon tissues of the mice in the model group are significantly increased compared with those in the control group; compared with the model group, the ASS1 virus interference group mice have significantly reduced ASS1 mRNA level and ASS1 protein expression level in colon tissues.
8. Results of measurement of expression levels of S6K and P-S6K proteins in colonic tissue
The results of measuring the expression levels of S6K and P-S6K proteins in colon tissues of each group of mice are shown in FIG. 8, from which it can be seen that: compared with a control group, the level of protein P-S6K/S6K in colon tissues of mice in a model group is obviously up-regulated; compared with the model group, the level of protein P-S6K/S6K in the colon tissue of the mice in BCKDK and ASS1 virus interference groups is obviously reduced, which indicates that ulcerative colitis is effectively relieved.
The experimental results show that in ulcerative colitis, ASS1 and BCKDK mRNA levels and protein expression levels are obviously increased, and through inhibiting ASS1 or BCKDK gene levels, protein expression is reduced, so that ulcerative colitis can be obviously relieved. The person skilled in the art can determine that ASS1 or BCKDK can be used as a drug action target for preparing a drug for preventing and treating ulcerative colitis, can be used as a drug action target for screening and discovering a drug for preventing and treating ulcerative colitis, and an inhibitor of ASS1 or BCKDK can be used for preparing a drug for preventing and treating ulcerative colitis, wherein: ASS1 refers to ASS1 gene or protein, and BCKDK refers to BCKDK gene or protein; the inhibitory activity of the inhibitor is inhibition of the activity of ASS1 or BCKDK protein, or inhibition of the ASS1 or BCKDK gene level.
The above-described embodiments are intended to illustrate the material nature of the present invention, but those skilled in the art will recognize that the scope of the present invention should not be limited to such embodiments.
Claims (1)
- The application of an inhibitor of BCKDK in the preparation of a medicament for treating ulcerative colitis, wherein the inhibitory activity of the inhibitor is the inhibition of the activity of BCKDK protein or the inhibition of the expression of BCKDK gene; the inhibitor is a nucleic acid molecule or an expression vector comprising the nucleic acid molecule; the nucleic acid molecule sequence is: 5 'CCTAGACACTCCCTACAAT-3'.
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