阅读说明：本技术 Ass1或bckdk抑制剂在制备治疗溃疡性结肠炎的药物中的应用 (Application of ASS1 or BCKDK inhibitor in preparation of medicine for treating ulcerative colitis ) 是由 刘史佳 周伟 陈玉根 陈选青 徐民民 卢晓云 于 2020-06-29 设计创作，主要内容包括：本发明公开了ASS1或BCKDK抑制剂在制备治疗溃疡性结肠炎的药物中的应用。本发明发现,在溃疡性结肠炎中,ASS1和BCKDK转录水平、蛋白水平显著升高,通过干扰ASS1或BCKDK基因的表达,可以显著缓解溃疡性结肠炎。因此,ASS1或BCKDK可以作为药物作用靶点用于制备防治溃疡性结肠炎的药物,可以作为药物作用靶点用于筛选发现防治溃疡性结肠炎的药物,ASS1或BCKDK的抑制剂可以用于制备防治溃疡性结肠炎的药物,其中：ASS1指ASS1基因或蛋白,BCKDK指BCKDK基因或蛋白；抑制剂的抑制活性为抑制ASS1或BCKDK蛋白的活性,或抑制ASS1或BCKDK基因的表达。(The invention discloses application of ASS1 or BCKDK inhibitor in preparation of a medicine for treating ulcerative colitis. The invention discovers that the transcription level and the protein level of ASS1 and BCKDK are obviously increased in ulcerative colitis, and the ulcerative colitis can be obviously relieved by interfering the expression of ASS1 or BCKDK genes. Therefore, 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.)

The application of an inhibitor of BCKDK in preparing a medicament for preventing and treating ulcerative colitis, wherein the inhibitory activity of the inhibitor is the activity of inhibiting BCKDK protein or the expression of BCKDK gene.

2. The use of claim 1, the inhibitor comprising a small molecule compound, a nucleic acid molecule, and an expression vector comprising the nucleic acid molecule.

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), also known as chronic nonspecific ulcerative colitis or idiopathic ulcerative colitis, is a common chronic intestinal disease and is a multifactorial, multilayered, unexplained nonspecific inflammation. 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 of onset is 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(ASS1) 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 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 study 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 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 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 alleviated by constructing a target protein shRNA fragment through an adeno-associated virus vector and interfering the expression of ASS1 or BCKDK genes. 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 body weight line graph 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 colon pathological section picture of each group of mice, and B is 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 Q-PCR detection of the relative expression level of BCKDK mRNA in colon tissues of each group of mice, B is a Westernblot detection of protein expression of BCKDK in colon tissues of each group of mice, and C is a Western blot detection of protein expression of BCKDHA and P-BCKDHA in colon tissues of each group of mice.

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 each group of mice; wherein A is a result of detecting the relative expression level of ASS1 mRNA in colon tissues of each group of mice by Q-PCR, and B is a result of detecting the protein expression level of ASS1 in colon tissues of each group of mice by Western blot.

FIG. 8 shows Westernblot assay for protein expression of S6K and P-S6K in colon tissue of each group of mice.

Detailed Description

The following detailed description of the present invention is provided in connection with the accompanying drawings and examples, but not intended to limit the scope of the invention.

Experimental 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-mouse (Gene ID:12041), ASS 1-mouse (Gene ID:11898) Each Gene corresponds to a targeting site: BCKDK (ID: 12041) shRNA sequence: 5'-CCTAGACACTCCCTACAAT-3', respectively; ASS1 (ID: 11898) shRNA sequence: 5'-GCCCAGATGTCCTTGAGAT-3' are provided.

Vector construction (see Haiziki Gene science and technology, Inc.:

adeno-associated viral vector information: pAAV-U6-shRNA-CMVbGlobin-eGFP-3Flag (GV 390).

Enveloped virus (see Haijiki Gene science and technology, Inc.:

the Geckie gene AAV Helper-Free System consists of three plasmids including a viral vector (carrying a target gene interference fragment: GV390), 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 blank group, model group, ASS1 virus interference group and BCKDK virus interference group, each group consisting of 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. ASS1 virus interference group was first administered by gavage with an enveloped virus containing shRNA fragments interfering with ASS1 gene expression at a virus titer of 1E + 11. The BCKDK virus interference group is administered with the enveloped virus containing shRNA fragments interfering BCKDK gene expression by intragastric administration for the first time a day, 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. The DAI is obtained by adding a weight loss rate score, a stool character score and a stool blood score, and the score is between 0 and 12. The scoring criteria were as follows: 1) weight loss rate score: no weight loss, score 0; lightening by 5-9 percent for 1 minute; lightening by 10-14 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; positive in stool blood (++), 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: an 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 a refrigerator at-80 deg.C for use to avoid repeated freeze thawing. Measuring the temperature at the beginning and mixing uniformly.

1) Sample adding: respectively setting a blank hole, a standard hole and a sample hole to be measured. 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 addition, slightly shaking and uniformly mixing, adding a 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 60 min.

3) After incubation for 60min, the liquid in the wells is discarded, the wells are dried, the plate is washed for 3 times, the soaking is carried out for 2min each time, and the soaking is carried out at 350 mu L/well and the drying is carried out.

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 1 h.

5) After incubation for 60min, the liquid in the wells is discarded, the wells are dried, the plate is washed for 5 times, the soaking is carried out for 2min each time, and the drying is carried out at a rate of 350 mu L/well.

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 absence of light.

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; a 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 on 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 for nuclei and eosin staining for cytoplasm; and sealing the neutral resin sheet, and observing pathological and histological changes by using an optical microscope. The colon section histological scoring standard is divided into 2 aspects of epithelial injury and mucositis cell infiltration: epithelial lesions were graded 5: grade 0, no damage; grade 1, small goblet cells were lost; grade 2, loss of a large number 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, infiltrating 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 tissue, and Q-PCR for detecting relative expression conditions of ASS1 mRNA and BCKDK mRNA in colon tissue

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 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, 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:

first, colon tissue was collected in an EP tube, 1mL Trizol solution was added, and the tissue was ground to a homogenized state with a homogenizer (care was taken to work on ice to prevent RNA degradation at too high a temperature).

② adding 0.2mL chloroform, shaking vigorously for 15s, and standing for 3min at room temperature.

③ centrifuging at 12000rpm for 15min at 4 ℃, and taking the supernatant and transferring the supernatant into a new EP tube.

Fourthly, adding isopropanol with the same volume, mixing the mixture gently and evenly, and placing the mixture for 30min at the temperature of minus 20 ℃.

Fifthly, centrifuging at 12000rpm for 10min at 4 ℃, and discarding the supernatant.

Sixthly, adding 1mL of 75% ethanol (prepared by DEPC water) to wash and precipitate. Centrifuge at 12000rpm for 2min at 4 ℃ and discard the supernatant.

Seventhly, drying the RNA precipitate for 3min at room temperature.

Adding appropriate amount of DEPC water to fully dissolve RNA.

2) Reverse transcription

Add 16. mu.L total RNA, 10. mu.L random primer, water bath at 65 ℃ for 5min, then ice bath for 3min to 0.2mL EP tube.

② adding 5x M-MLVBuffer 10 uL, 10mM dNTPs 4 uL, M-MLV reverse transcriptase 2 uL, DEPC water 8 uL. The total volume was 50. mu.L.

③ reverse transcription conditions: 30 ℃ for 10 min; 42 ℃ for 1 h; 95 ℃ for 10 min. 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 is as follows:

reaction conditions are as follows: 95 ℃ for 5 min; at 95 ℃ for 30 s; primer annealing temperature, 30 s; 72 ℃ for 1 min.

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 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. 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:

preparing a standard curve:

TABLE 2BSA Standard concentration preparation

Preparing a working solution: the reagent A and the reagent B were mixed well in a volume ratio of 50: 1.

Measuring method: 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

30 μ g of protein sample was subjected to 10% SDS-PAGE.

② the wet-to-electric transfer method transfers the protein on the gel to the PVDF membrane.

And thirdly, sealing the PVDF membrane for 2h at the sealing liquid temperature.

And fourthly, washing the membrane by TBST, adding primary antibody for incubation, and keeping the temperature at 4 ℃ overnight.

TBST washing the membrane for 3 times, 10min each time. And adding a secondary antibody for incubation at room temperature for 1 h.

Sixthly, washing the membrane for 3 times of 10min by TBST. 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 determination of 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; the P-S6K antibody.

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. Add 5X protein Loading Buffer, boiling water bath for 5min, take out room temperature and stand for several minutes to prepare for Loading.

1) BCA protein quantification:

preparing a standard curve:

TABLE 3 BSA Standard concentration preparation

Preparing a working solution: the reagent A and the reagent B were mixed well in a volume ratio of 50: 1.

Measuring method: 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

30 μ g of protein sample was subjected to 10% SDS-PAGE.

② the wet-to-electric transfer method transfers the protein on the gel to the PVDF membrane.

And thirdly, sealing the PVDF membrane for 2h at the sealing liquid temperature.

And fourthly, washing the membrane by TBST, adding primary antibody for incubation, and keeping the temperature at 4 ℃ overnight.

TBST washing the membrane for 3 times, 10min each time. And adding a secondary antibody for incubation at room temperature for 1 h.

Sixthly, washing the membrane for 3 times of 10min by TBST. ECL luminous liquid and an exposure instrument.

Analysis of the development result by using gel image analysis software Quantity One, and using the ratio of the optical density of the target protein strip to the optical density of the internal reference strip as the target protein expression quantitative index.

9. Data processing

The results of the experiments are expressed as (Mean + -SEM) and comparisons between two groups are performed using Student's t and between groups using One-wayANOVA and post hoc tests. P <0.05 had statistical differences, # P <0.01 had statistically significant differences, and # P <0.001 had statistically significant differences.

Third, experimental results

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 result of the DAI score of each group of mice is shown in FIG. 2, and the DAI score of the model group of mice is obviously increased compared with that of the blank group; 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 measurement results

The results of colon length measurement of each group of mice are shown in fig. 4, and compared with the blank group, the colon length of the model group of mice is significantly reduced; compared with the model group, the colon length of mice in the ASS1 virus interference group and the BCKDK virus interference group is significantly increased.

5. Colon pathology sectioning and histological scoring results

The results of colon pathology sections and histological scoring of the mice in each group are shown in fig. 5, compared with the blank group, the colon of the mice in the model group has obvious lesion and the inflammatory infiltration is enhanced; 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 BCKDK mRNA level, BCKDK, BCKDHA, P-BCKDHA protein expression level in colon tissue

The results of measuring BCKDK mRNA level and BCKDK, BCKDHA, P-BCKDHA protein expression level in colon tissues of each group of mice are shown in FIG. 6, from which it can be seen that: 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. Results of measuring ASS1 mRNA and protein expression level 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 compared with the control group, the expression levels of ASS1 mRNA and ASS1 protein in the colon tissues of the mice in the model group are remarkably increased; compared with the model group, the ASS1 virus interfered the ASS1 mRNA level and the ASS1 protein expression level in the colon tissue of mice in the group to be significantly reduced.

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 the protein P-S6K/S6K in colon tissues of the mice in the model group is remarkably up-regulated; compared with the model group, the levels of the protein P-S6K/S6K in the colon tissues of mice in BCKDK and ASS1 virus interference groups are obviously reduced, which indicates that the ulcerative colitis is effectively alleviated.

The experimental results show that in ulcerative colitis, ASS1, BCKDK mRNA level and protein expression level are obviously increased, and through inhibiting ASS1 or BCKDK gene level, 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, 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 ASS1 or BCKDK gene level.

The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.