Pgk1 application of target in preparing or screening medicine for treating gastrointestinal tract diseases
1, Pgk1 as a target in screening drugs for preventing or treating gastrointestinal diseases, wherein the drugs take Pgk1 as a target and promote the expression of Pgk 1.
2, Pgk1 protein, or recombinant plasmid expressing Pgk1 protein, or recombinant probiotic bacteria expressing Pgk1 protein, or a compound promoting Pgk1 protein expression in the preparation of medicaments for preventing or treating gastrointestinal diseases.
3. The use of claim 2, wherein the recombinant plasmid for expressing Pgk1 protein is obtained by introducing a gene encoding Pgk1 protein into an expression vector comprising pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pPIC9K, pHIL-S1, pPICZ alpha, pYAM75P, PNZ8149-usp 45.
4. The use of claim 3, wherein the expression vector is pET-28a, the recombinant plasmid for expressing Pgk1 protein is pET-28a-Pgk1, and the gene sequence of pET-28a-Pgk1 is shown in SEQ ID NO. 3.
5. The use of claim 2, wherein the recombinant probiotic bacteria expressing Pgk1 protein is obtained by transforming a recombinant plasmid expressing Pgk1 protein into probiotic bacteria, or integrating a gene encoding Pgk1 protein into probiotic bacteria, and the probiotic bacteria include Escherichia coli (Nissle 1917), Bacillus, lactococcus, butyric acid bacillus, Lactobacillus, Bifidobacterium, Actinomyces, Pichia pastoris.
6. The use according to claim 2, wherein the compound promoting Pgk1 protein expression comprises quinazoline derivatives represented by the following formula (I), or pharmaceutically acceptable salts thereof, or tautomers thereof, or racemates thereof, or enantiomers thereof, or diastereomers thereof, or solvates thereof, or esters thereof, or prodrugs thereof, or pharmaceutical compositions thereof as active ingredients,
wherein:
R1and R2Are respectively selected from hydrogen, amino, hydroxyl and C1-6Straight chain alkyl, C1-6Substituted alkyl, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C1-6Alkyl acyl, aryl acyl, C6-10Aryl radical, C5-6Cycloalkyl, or R1And R2Together with the nitrogen atom to which they are attached form a 5-or 6-membered ring; the substituted alkyl group comprises 1-3 hydroxyl and/or halogen substituted alkyl groups;
R3and R4Are respectively selected from hydrogen, halogen and C1-6Alkyl radical, C1-6Haloalkyl, C2-6Alkenyl radical, C2-6Alkynyl, nitrile group, nitro group, amino group, hydroxyl group and C1-6Alkoxy radical, C1-6Alkanoyloxy, C1-6Alkanoylamino, C1-6Alkanoylamino, arylacylamino, saturated or unsaturated 5-membered carbocyclyl or heterocyclyl, saturated or unsaturated 6-membered carbocyclyl or heterocyclyl, saturated or unsaturated 5-membered carbocyclyloxy or heterocyclyloxy, saturated or unsaturated 6-membered carbocyclyloxy or heterocyclyloxyBase, C1-6An alkyl acyl group; or R3And R4Together with the ring atoms to which they are attached form a 5-or 6-membered carbocyclic/heterocyclic ring;
R5and R6Are respectively selected from hydrogen, halogen, nitrile group, nitryl, amino, hydroxyl and C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Alkanoylamino, C1-6Haloalkyl, C2-6Alkenyl radical, C2-6Alkynyl, C1-6Alkanoylamino, C1-6Alkanoylamino, arylacylamino, saturated or unsaturated 5-membered carbocyclyl or heterocyclyl, saturated or unsaturated 6-membered carbocyclyl or heterocyclyl, saturated or unsaturated 5-membered carbocyclyloxy or heterocyclyloxy, saturated or unsaturated 6-membered carbocyclyloxy or heterocyclyloxy, C1-6An alkyl acyl group;
R7selected from hydrogen, hydroxy, nitro, C1-6Alkyl radical, C1-6Haloalkyl, C1-6Alkoxy-substituted alkyl, C3-6Cycloalkyl, spirocycloalkyl, bridged cycloalkyl, substituted or unsubstituted phenyl or heterocyclyl, wherein the substituent of said substituted phenyl or heterocyclyl comprises C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Haloalkyl, nitro, amino, nitrile, halogen, C1-6Thioether or its oxidation products sulfone, sulfoxide.
7. The use of claim 6, wherein the quinazoline derivative comprises prazosin, terazosin, doxazosin, alfuzosin and tramazosin.
8. The use according to any one of claims 1 to 7, wherein the gastrointestinal disorder comprises peptic ulcer, inflammatory bowel disease.
9. The use according to claim 8, wherein the peptic ulcer comprises gastric ulcer, duodenal ulcer, retrobulbar ulcer, pyloric ulcer, composite ulcer and kissing ulcer; the inflammatory bowel disease comprises colitis, ulcerative colitis and Crohn's disease.
10. The use of claim 2, wherein Pgk1 protein, or a recombinant plasmid expressing Pgk1 protein, or a recombinant probiotic bacterium expressing Pgk1 protein, or a compound promoting expression of Pgk1 protein is added with a pharmaceutically acceptable carrier to make any one of tablets, injections, suspensions, capsules, and granules.
Technical Field
Gastrointestinal diseases are one of the most common diseases of human beings, including esophagus, stomach, small intestine, colon, rectum and the like, and the common main symptoms comprise rhythmic and periodic upper abdominal pain, diarrhea, hunger abdominal pain, acid regurgitation, fever, black stool and bloody stool, gastrointestinal bleeding, intestinal obstruction and the like. The most common of these include swallowing disorders, gastric ulcers, duodenal ulcers, complex ulcers, gastroparesis, delayed gastric emptying, Irritable Bowel Syndrome (IBS), and Inflammatory Bowel Disease (IBD). Ulcer formation is caused by various factors, such as excessive gastric acid secretion, helicobacter pylori infection, and decreased gastric mucosal protection. Delayed gastric emptying and bile reflux, gastrointestinal peptide action, genetic factors, pharmaceutical factors, environmental factors, mental factors, and the like are all associated with the occurrence of peptic ulcers. It is presently believed that gastric ulcer formation is more heavily focused on weakening of the gastric mucosal barrier and increasing gastrin secretion, while duodenal ulcer formation is more heavily focused on increasing total parietal cell volume. In addition, excessive drinking, irregular eating, long-term mental stress, and long-term administration of non-steroidal anti-inflammatory drugs (such as aspirin), glucocorticoids, clopidogrel, etc., are all associated with the formation of gastric and duodenal ulcers. Inflammatory bowel disease may be caused by organisms such as bacteria, fungi, viruses, parasites, protozoa, etc., and may also be caused by allergies and physicochemical factors. According to the etiology, the Disease can be divided into specific inflammatory lesions and non-specific inflammatory lesions, wherein the former is infectious Colitis, ischemic Colitis, pseudomembranous Colitis and the like, and the latter mainly comprises Ulcerative Colitis (UC) and Crohn's Disease (CD); the clinical manifestations mainly include weight loss, tenesmus, diarrhea, abdominal pain and even bloody stool. The ulcerative colitis is limited to the mucosa and submucosa of large intestine, and the disease usually affects the rectum and gradually spreads to the whole colon. The types of western medicines mainly used for treating gastrointestinal diseases at present comprise gastrointestinal motility promoting medicines, spasmolytic medicines, antiemetic medicines, peptic ulcer medicines, gastric mucosa protective agents, digestive aids, microecologics and the like. The medicines for treating peptic ulcer mainly comprise a proton pump inhibitor, an H2-receptor antagonist, a bismuth preparation, prostaglandins and the like, and although symptoms can be improved and relieved, the medicines are difficult to completely cure and are easy to repeatedly attack. The pathogenesis of the colitis is not completely clarified, the commonly used medicines for clinical treatment comprise amino salicylic acid preparations, glucocorticoid, immunosuppressant and the like, the symptoms of the colitis can be controlled by short-term use, but the cure rate is extremely low, various adverse reactions can be induced by long-term use, the problems of relapse after stopping the medicine and the like can be solved, and serious patients can cause canceration. Therefore, the development of a new drug for treating gastrointestinal diseases is a technical problem which needs to be solved urgently at present.
Phosphoglycerate kinase 1 (Pgk 1) is a key metabolic enzyme in the glycolytic pathway, and is capable of catalyzing the conversion of 1, 3-diphosphoglycerate (1, 3-BPG) to 3-Phosphoglycerate (3-PG) and producing the first ATP in the glycolytic pathway, thus playing an important role in cellular energy metabolism. Pgk1 are expressed at high levels in a variety of cancer cells, and recent studies have shown that Pgk1 is closely related to the development and progression of tumors; pgk1 phosphorylation and acetylation occur at multiple sites, mitochondrial and nuclear translocation occur under specific conditions, and further tricarboxylic acid (TCA) cycle metabolism is inhibited, glycolytic activity is enhanced directly or indirectly, and Warburg effect of cancer cells is enhanced, and proliferation and growth of cancer cells are promoted. However, there is no document disclosing the use of the protein transcribed from the Pgk1 gene in the treatment of gastrointestinal diseases.
Moreover, the quinazoline derivative is mainly used for treating cardiovascular and cerebrovascular diseases, wherein the cerebrovascular diseases comprise cerebral thrombosis, cerebral ischemia, cerebral infarction and the like, and the cardiovascular diseases comprise myocardial infarction, myocardial ischemia, myocardial injury, coronary heart disease, angina pectoris, heart failure and the like. Are used for the treatment and/or prevention of sepsis and its complications, and for the activation of new targets; also can be used for preparing medicines for treating and/or preventing hyperglycemia, cerebral thrombosis and complications thereof. However, there is no prior document disclosing the use of said quinazoline derivatives for the treatment of gastrointestinal disorders.
The invention unexpectedly discovers that Pgk1, recombinant plasmid or recombinant bacteria expressing Pgk1 and drug activating Pgk1 expression can effectively inhibit cell death, enhance and improve the integrity of mucous membrane, promote the improvement of tissue morphology, have obvious curative effect on peptic ulcer and inflammatory bowel disease and have good clinical application prospect.
Disclosure of Invention
In view of the above technical problems, the primary object of the present invention is to provide the use of Pgk1 as a target for preparing or screening drugs for treating gastrointestinal diseases, and to provide drugs for treating gastrointestinal diseases. The method specifically comprises the following steps:
in a first aspect, the invention provides an application of Pgk1 as a target in screening a medicine for preventing or treating gastrointestinal diseases, wherein the medicine takes Pgk1 as the target and promotes the expression of Pgk 1.
Preferably, the amino acid sequence of Pgk1 is shown in SEQ ID NO. 1.
Preferably, the gastrointestinal disease comprises peptic ulcer, inflammatory bowel disease.
Preferably, the peptic ulcer comprises gastric ulcer, duodenal ulcer, retrobulbar ulcer, pyloric ulcer, composite ulcer, kissing ulcer; the inflammatory bowel disease comprises colitis, ulcerative colitis and Crohn's disease.
Preferably, the peptic ulcer is gastric ulcer and the inflammatory bowel disease is ulcerative colitis.
In a second aspect, the invention provides an application of Pgk1 protein, or a recombinant plasmid expressing Pgk1 protein, or a recombinant probiotic bacterium expressing Pgk1 protein, or a compound promoting Pgk1 protein expression in preparing a medicament for preventing or treating gastrointestinal diseases.
Preferably, the recombinant plasmid for expressing Pgk1 protein is obtained by inserting gene encoding Pgk1 protein into expression vector
Preferably, the expression vector includes, but is not limited to, pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pPIC9K, pHIL-S1, pPICZ alpha, pYAM75P, PNZ8149-usp 45.
Preferably, the expression vector is pET-28a, the recombinant plasmid for expressing Pgk1 protein is pET-28a-Pgk1, and the gene sequence of pET-28a-Pgk1 is shown in SEQ ID NO. 3.
Preferably, the recombinant probiotic bacteria expressing Pgk1 protein is obtained by transforming recombinant plasmid expressing Pgk1 protein into probiotic bacteria, or integrating gene encoding Pgk1 protein into probiotic bacteria.
Preferably, the probiotic bacteria include, but are not limited to, Escherichia coli (Nissle) Nissle 1917, probiotic bacillus, lactococcus, butyric acid bacillus, lactobacillus, bifidobacterium, actinomycetes, pichia pastoris.
Preferably, the probiotic is (Escherichia coli) Nissle 1917.
Preferably, the compound promoting the expression of Pgk1 protein comprises quinazoline derivatives shown in the following formula (I), or pharmaceutically acceptable salts thereof, or tautomers thereof, or internal racemates thereof, or enantiomers thereof, or diastereomers thereof, or solvates thereof, or esters thereof, or prodrugs thereof, or pharmaceutical compositions thereof as active ingredients,
wherein:
R1and R2Are respectively selected from hydrogen, amino, hydroxyl and C1-6Straight chain alkyl, C1-6Substituted alkyl, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C1-6Alkyl acyl, aryl acyl, C6-10Aryl radical, C5-6Cycloalkyl, or R1And R2Together with the nitrogen atom to which they are attached form a 5-or 6-membered ring; the substituted alkyl group comprises 1-3 hydroxyl and/or halogen substituted alkyl groups;
R3and R4Are respectively selected from hydrogen, halogen and C1-6Alkyl radical, C1-6Haloalkyl, C2-6Alkenyl radical, C2-6Alkynyl, nitrile group, nitro group, amino group, hydroxyl group and C1-6Alkoxy radical, C1-6Alkanoyloxy, C1-6Alkanoylamino, C1-6Alkanoylamino, arylacylamino, saturated or unsaturated 5-membered carbocyclyl or heterocyclyl, saturated or unsaturated 6-membered carbocyclyl or heterocyclyl, saturated or unsaturated 5-membered carbocyclyloxy or heterocyclyloxy, saturated or unsaturated 6-membered carbocyclyloxy or heterocyclyloxy, C1-6An alkyl acyl group; or R3And R4Together with the ring atoms to which they are attached form a 5-or 6-membered carbocyclic/heterocyclic ring;
R5and R6Are respectively selected from hydrogen, halogen, nitrile group, nitryl, amino, hydroxyl and C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Alkanoylamino, C1-6Haloalkyl, C2-6Alkenyl radical, C2-6Alkynyl, C1-6Alkanoylamino, C1-6Alkanoylamino, arylacylamino, saturated or unsaturated 5-membered carbocyclyl or heterocyclyl, saturated or unsaturated 6-membered carbocyclyl or heterocyclyl, saturated or unsaturated 5-membered carbocyclyloxy or heterocyclyloxy, saturated or unsaturated 6-membered carbocyclyloxy or heterocyclyloxy, C1-6An alkyl acyl group;
R7selected from hydrogen, hydroxy, nitro, C1-6Alkyl radical, C1-6Haloalkyl, C1-6Alkoxy radicalSubstituted alkyl, C3-6Cycloalkyl, spirocycloalkyl, bridged cycloalkyl, substituted or unsubstituted phenyl or heterocyclyl, wherein the substituent of said substituted phenyl or heterocyclyl comprises C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Haloalkyl, nitro, amino, nitrile, halogen, C1-6Thioether or its oxidation products sulfone, sulfoxide.
Preferably, R7 is selected from C1-6Alkyl radical, C1-6Haloalkyl, C1-6Alkoxy-substituted alkyl, C3-6Cycloalkyl, spirocycloalkyl, bridged cycloalkyl, substituted or unsubstituted phenyl or heterocyclyl, wherein the substituent of said substituted phenyl or heterocyclyl comprises C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Haloalkyl, nitro, amino, nitrile, halogen, C1-6Thioether or its oxidation products sulfone, sulfoxide.
Preferably, the quinazoline derivative comprises prazosin, terazosin, doxazosin, alfuzosin and tramazosin.
Preferably, the quinazoline derivative is terazosin.
Preferably, the gastrointestinal disease comprises peptic ulcer, inflammatory bowel disease.
Preferably, the peptic ulcer comprises gastric ulcer, duodenal ulcer, retrobulbar ulcer, pyloric ulcer, composite ulcer, kissing ulcer; the inflammatory bowel disease comprises colitis, ulcerative colitis and Crohn's disease.
Preferably, the peptic ulcer is gastric ulcer and the inflammatory bowel disease is ulcerative colitis.
Preferably, Pgk1 protein, or recombinant plasmid expressing Pgk1 protein, or recombinant probiotic expressing Pgk1 protein, or compound promoting expression of Pgk1 protein is added with pharmaceutically acceptable carrier to prepare any one of tablets, injections, suspensions, capsules and granules.
The invention has the beneficial effects that: the recombinant probiotics is obtained by constructing Pgk1 gene recombinant plasmid pET-28a-Pgk1 and transferring the plasmid into probiotics, and the recombinant probiotics can effectively inhibit cell death, enhance and improve the integrity of mucous membrane, promote the improvement of tissue morphology, remarkably reduce the weight reduction percentage of colitis mice, reduce the improvement of disease activity, relieve the pathological injury of colon tissue, improve the shortening condition of colon and have remarkable curative effect on peptic ulcer and inflammatory bowel disease; the quinazoline derivative can activate Pgk1 expression, can effectively inhibit cell death, enhance and improve mucous membrane integrity, promote tissue morphology improvement, remarkably reduce the weight reduction percentage of colitis mice, reduce disease activity improvement, relieve colon tissue pathological injury, improve colon shortening, has remarkable curative effect on peptic ulcer and inflammatory bowel disease, has 20-80 times higher activity than the existing clinical medicaments, and can be clinically popularized and applied; the invention finds that Pgk1 is taken as a target spot and can be used for preparing or screening medicines for treating gastrointestinal diseases; the invention also changes the conventional administration mode (intramuscular injection or intravenous injection) of the protein medicament, applies the protein medicament to the treatment of gastrointestinal tract diseases in an oral administration mode, solves the technical problem which is not solved for a long time in the field, and obtains remarkable effect.
Drawings
FIG. 1 shows the results of PCR reaction of DH5 alpha strain transformed with recombinant plasmid;
FIG. 2 shows the results of PCR reaction of recombinant plasmid transformation (Escherichia coli) Nissle 1917 resistant bacteria;
FIG. 3 is a plan view of the damage of the recombinant probiotics to gastric mucosa of a gastric ulcer mouse;
FIG. 4 effect of recombinant probiotics expressing Pgk1 protein on ulcer index, ulcer area and ulcer inhibition rate of gastric ulcer mice, wherein,###p<control group 0.001 vs;*p<0.05、**p<0.01 and***p<0.001vs. ethanol group;
FIG. 5 shows the colon injury of mice with ulcerative colitis caused by recombinant probiotics expressing Pgk1 protein;
FIG. 6 Effect of recombinant Probiotics expressing Pgk1 protein on ulcerative colitis DAI score and colon Length, wherein###p<0.001vs. blank control;**p<0.01 and***p<0.001vs.Da set of SS models;
FIG. 7 Effect of terazosin on GES-1 cell survival, wherein,###p<control group 0.001 vs;***p<0.001vs. ethanol group;
FIG. 8 Effect of terazosin on the activity of GES-1 cells Pgk1, wherein,#p<control group 0.05 vs;*p<0.05vs. ethanol group;
figure 9 effect of terazosin on gastric ulcer mice, wherein,###p<control group 0.001 vs;**p<0.01 and***p<0.001vs. ethanol group;
FIG. 10 cytotoxic and cell viability assay of terazosin on Caco-2 cells, wherein,###p<control group 0.001 vs;*p<0.05 and**p<0.01vs.H2O2group (d);
FIG. 11 Effect of terazosin on Caco-2 cell Pgk1 activity, wherein,##p<control group 0.01 vs;*p<0.05vs.H2O2group (d);
FIG. 12 the effect of terazosin on colitis mice body weight and DAI score, wherein,###p<control group 0.001 vs;*p<0.05 and**p<dss group 0.01 vs;
FIG. 13 effect of terazosin on colon and colon length in colitis mice, wherein,###p<control group 0.001 vs;*p<dss group 0.05vs.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following embodiments further describe the present invention in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The reagent materials and the like used in the following examples are commercially available unless otherwise specified.
Escherichia coli (Escherichia coli) DH 5. alpha. as described in the following examples was purchased from Kyoto Kogyo gold Biotech Co., Ltd; escherichia coli (Escherichia coli) Nissle 1917 was purchased from Germany (trade name Mutaflor); the recombinant plasmid pET-28a-EGFP is purchased from vast Ling plasmid platform; absolute ethanol was purchased from Tianjin Damao chemical reagent; beta-lactose (beta-lactose, 70% beta-lactose and 30% alpha-lactose) was purchased from mclin biotechnology limited; the PCR primers used were purchased from Biotechnology engineering (Shanghai) GmbH; the bacterial plasmid DNA extraction kit is purchased from AXYGEN company; the PrimeSTAR HS (Premix, 2X), T4 DNA ligase was purchased from Takara; the DNA endonucleases BamHI, EcoRII were purchased from NEB of Beijing;
the LB (Luria-Bertani) liquid culture medium formula is as follows: 1% tryptone, 0.5% yeast extract, 0.5% NaCl; the LB solid medium comprises the following components in percentage by weight: 1% tryptone, 0.5% yeast extract, 0.5% NaCl, 2% agar.
The KM mice described in the following examples are kunming mice; experimental animal KM mice, provided by Lanzhou veterinary research institute of Chinese academy of agricultural sciences, with license number SCXK (Glycine) -2020-.
In the present invention, alkyl means a straight-chain and branched-chain saturated and unsaturated alkyl group or an aliphatic group having a specific number of carbon atoms; the alkoxy refers to saturated and unsaturated alkyl-O-groups with specified carbon atoms; the said carboxylate group-substituted alkyl group means a saturated or unsaturated alkyl group having a specified number of carbon atoms-COOCHX(X＝0-3)-a group; the thioether refers to saturated and unsaturated alkyl-S-groups with specified carbon atoms; the oxidation product sulfone refers to saturated and unsaturated alkyl groups with specific carbon atoms; the oxidation product sulfoxide refers to saturated and unsaturated alkyl groups with specific carbon atoms; the "halogen" or "halo" means fluorine, chlorine, bromine or iodine as a substituent, and when a halogen atom is used as a substituent, the number of substitution thereof on the same carbon atom may be one, two or three; the aryl group includes phenyl, substituted phenyl (where substituted phenyl includes one, two or three groups: C)1-6Saturated and unsaturated alkyl radicals, C1-6Saturated and unsaturated alkoxy, phenoxy, nitrile, nitro, amino, or halogen, etc.); the term "heterocycle" as used herein denotes stable 5-to 7-membered heterocycles which may be saturated or unsaturated and consist of carbon atoms and optionally 1 to 4 heteroatoms selected from N, O and S, wherein the nitrogen and sulfur heteroatoms may be selectively oxidized and the nitrogen heteroatom may be selectively quaternized, preferably 6-membered heterocycles such as pyridine, piperidine, pyrazine, piperazine, morpholine or thiomorpholine and the like.
The GES-1 cells described in the following examples are human gastric mucosal cells, used to mimic the in vitro gastric ulcer cell model, purchased from Wuhan Proxel Biotech, Inc.; the Caco-2 cells are human colorectal adenocarcinoma cells, are used for simulating an in vitro colitis cell model and are purchased from Wuhan Punaoxi Biotech Co., Ltd; cimetidine tablet (Cimetidine, CIM) also known as Cimetidine, available from shanghai yibai balance pharmaceutical ltd; the histamine H2 receptor antagonist is mainly used for inhibiting gastric acid secretion, can obviously inhibit basic and night gastric acid secretion, can also inhibit the gastric acid secretion caused by the stimulation of histamine, depsipeptide gastrin, insulin, food and the like, reduces the acidity, has the effects of preventing and protecting erosive gastritis caused by chemical stimulation, and has obvious curative effects on erosive gastric ulcer and upper gastrointestinal hemorrhage. Thus, cimetidine was used as a positive drug in examples 2 and 4; the Dextran sodium Sulfate is used for modeling of mouse colitis, and the Dextran Sodium Sulfate (DSS) has MW of 40000 and is purchased from Aladdin Biotechnology Ltd; the sulfasalazine enteric-coated tablet (SASP) is purchased from Shanghai Xinyi balance pharmaceutical industry Co., Ltd, and has the following indications: (1) ulcerative colitis treats mild to moderate ulcerative colitis; can be used as adjuvant therapy for severe ulcerative colitis. Can also be used for maintaining and treating ulcerative colitis in remission stage; (2) crohn's disease is used to treat active Crohn's disease, particularly those patients with colon involvement; (3) rheumatoid arthritis and juvenile rheumatoid arthritis (polyarticular type) in which rheumatoid arthritis is not significantly effective for salicylic acids or other non-steroidal anti-inflammatory drugs; therefore, the sulfasalazine enteric-coated tablets in example 3 and example 5 were used as positive drugs.
The data processing method described in the following embodiments is: statistical analysis was performed using SPSS23.0 software, data are expressed as (x. + -.s), and comparisons between groups were performed pairwise using One-way ANOVA and LSD-t. p <0.05 was considered statistically significant.
Example 1 preparation of recombinant plasmid expressing Pgk1 and recombinant probiotic expressing Pgk1
1. Primer synthesis
According to the nucleotide sequence SEQ ID NO.2 of the gene Pgk1, comparing with a pET-28a plasmid map, designing a specific primer, inserting restriction enzyme cutting sites BamHI and EcoRI into the primer, wherein the specific information is shown in table-1, and the primer is synthesized by Shanghai biological engineering GmbH; the primers were dissolved in sterile deionized water to a concentration of 10. mu. mol/L.
TABLE 1 primer information and Synthesis
2. Construction of recombinant plasmid
The pET-28a-Pgk1 plasmid was synthesized by Kingzhi Biotechnology, Inc.
3. Transformation of (Escherichia coli) DH5 alpha bacterium with recombinant plasmid
Taking out DH5 alpha competent cells from a refrigerator at the temperature of minus 80 ℃, and placing the cells on an ice box for 10 to 20 minutes to melt the cells; taking out 50 μ L of competent cells, adding 5 μ L of plasmid, gently dialing with fingers for several times, mixing, and standing the mixture on ice for 25 min; after the heat shock at 42 ℃ for 45s, the cells are quickly placed back on ice and kept stand for 2min, 0.5mL of LB liquid medium (without antibiotics) at room temperature is added, the cells are cultured at 37 ℃ for 1h under the shaking of 200rpm, 200 mu L of the bacterial liquid is taken and coated on a flat plate containing 15 mu g/mL of kanamycin, the culture dish is inverted after the bacterial liquid is completely absorbed by the culture medium, and the cells are cultured for 12h at 37 ℃.
4. Successful colony verification of heat shock transformation
The single clone was picked up, amplified and cultured in a liquid medium containing 15. mu.g/mL kanamycin, and subjected to PCR verification and enzyme digestion verification. As a result of the verification, as shown in FIG. 1, the target band had a molecular weight of 1254bp, and the verification was correct (the position indicated by the arrow in the figure is the target band).
5. Extraction of recombinant plasmid DNA
Transferring the bacterial strain successfully transferred into the recombinant plasmid into an LB culture medium for amplification culture, wherein the culture conditions are as follows: the speed was 200rpm and the temperature was 37 ℃. Plasmids were extracted using a plasmid miniprep kit from AXYGEN.
6. Preparation of chemically competent cells of Escherichia coli (Escherichia coli) Nissle 1917
The stock E.coli (Escherichia coli) Nissle 1917(Mutaflor) strain was removed from the-80 ℃ freezer, picked up a small amount of the stock solution using a sterilized pipette tip, streaked onto LB solid medium, and cultured overnight in a 37 ℃ incubator: picking a wet and smooth single colony from an LB solid culture medium, putting the single colony into an LB culture solution, and culturing for 12h at 37 ℃; transferring the bacterial liquid into a sterilized centrifugal tube precooled on ice when the OD 600 value of the bacterial liquid is 0.3-0.5, carrying out ice bath for 30min, and centrifuging for 10min at 1520g under the condition of 4 ℃; the supernatant was discarded and 500. mu.L of pre-cooled CaCl was added2The solution (sterile filtered) gently suspends the cells, and then centrifuged at 1520g for 10min at 4 ℃; repeating the previous step once; the supernatant was discarded and 500. mu.L of precooled CaCl was added2The solution (sterile filtered) is carefully suspended to form a competent cell suspension; the prepared competent cell suspension was directly used for transformation experiments. The unused competent cells were added with the same volume of sterilized 20% glycerol, mixed well, dispensed into 1.5mL centrifuge tubes (100. mu.L per tube), and stored in a refrigerator at-80 ℃.
7. The recombinant plasmid was transferred into Escherichia coli (Escherichia coli) Nissle 1917 strain
Chemically competent cells (Escherichia coli) Nissle 1917 were removed from a-80 ℃ freezer and placed on an ice-box for 10-20 minutes to thaw. Taking out 50 μ L of competent cells, adding 5 μ L of plasmid, gently dialing with finger for several times, mixing, and standing on ice for 25 min; after heat shock at 42 ℃ for 45s, the cells were quickly returned to ice and allowed to stand for 2min, 0.5mL of room-temperature LB liquid medium (containing no antibiotics) was added, the cells were cultured at 37 ℃ with shaking at 200rpm for 1h, 200. mu.L of the above-mentioned bacterial solution was applied to a plate containing 15. mu.g/mL kanamycin, and after the bacterial solution was completely absorbed by the medium, the plate was inverted and cultured at 37 ℃ for 12 h.
8. Successful colony verification of heat shock transformation
The single clone was picked up, amplified and cultured in a liquid medium containing 15. mu.g/mL kanamycin, and then verified by PCR and enzyme digestion. The experimental results are shown in fig. 2, and it was verified that Pgk 1-expressing recombinant Escherichia coli (Nissle) Nissle 1917 probiotic was correctly obtained.
It should be noted that the method of the present invention is not the only limiting method for constructing Pgk 1-expressing recombinant probiotics, any recombinant plasmid expressing Pgk1 is constructed by a conventional technical means, and the recombinant plasmid expressing Pgk1 is transformed into Escherichia coli (Escherichia coli) Nissle 1917 strain or other probiotics by a conventional transformation operation, so that Pgk 1-expressing recombinant probiotics can be obtained by screening.
Example 2 therapeutic Effect of recombinant Probiotics expressing Pgk1 on gastric ulcer mice
Preparation of KM mouse alcoholic gastric ulcer model
48 male KM mice (20-25g) were housed in laboratory animal houses of Lanzhou university, and after one week of acclimatization, the mice were randomly divided into 6 groups of 8 mice each, and the grouping and dose were as follows:
blank control group: gavage to give 0.25mL of 11.1mg/mL (m/v) beta-lactose solution; ethanol control group: 0.25mL of absolute ethyl alcohol is given for intragastric administration; positive control group: cimetidine, 80mg/kg/day, administered intragastrically; (Escherichia coli) Nissle 1917 group: gavage 0.25mL (Escherichia coli) Nissle 1917 original bacteria; pET-28a-EGFP group: gavage 0.25mL of recombinant Escherichia coli (Escherichia coli) Nissle 1917 probiotics containing recombinant plasmid pET-28 a-EGFP; pET-28a-Pgk1 group: gavage 0.25mL of Pgk1 expressing recombinant Escherichia coli (Escherichia coli) Nissle 1917 probiotic.
Each group of mice was pre-dosed for 5 days (bacterial count 1X 10)9CFU, 11.1mg/ml beta-milkSugar solution preparation), the drug is administered once again 2h before molding, the anhydrous ethanol is gavaged 2h later, the experiment is ended 2h later after stimulation, and serum and gastric tissue are separated to measure various indexes.
2. Ulcer area, ulcer index and inhibition rate detection
Taking out stomach, cutting along the greater curvature of stomach, washing to remove the content, observing gastric mucosa ulcer, measuring the transverse diameter and longitudinal diameter of ulcer with ruler, and multiplying the two to obtain ulcer area (mm)2) As shown in equation 1; the ulcer area of the entire stomach tissue was then calculated to calculate the ulcer inhibition ratio (%), see equation 2. Meanwhile, the average value of the sum of the ulcer points of each group of mice is taken as the ulcer index (healing score 0, superficial mucosal erosion score 1, deep ulcer or transmural necrosis score 2, perforation or penetrating ulcer score 3)
Ulcer area (mm2) ═ maximum length of ulcer perpendicular to maximum width of maximum length (1)
3. Results of the experiment
After the mice of the above groups died, the stomach tissue was rapidly collected, the morphological change and ulcer damage were observed, and the ulcer area and ulcer index were statistically analyzed, and the experimental results are shown in fig. 3 and 4. As can be seen from FIG. 3, the blank control group mice had normal gastric tissue macroscopic morphology and no obvious hemorrhagic lesions, while the ethanol model group mice had obvious hemorrhagic lesions in the gastric tissue, with the most severe ulcers, indicating that the ethanol gastric ulcer model was successfully prepared; compared with the ethanol model group, the negative control pET-28a-EGFP treatment group does not show obvious ulcer protection; the (Escherichia coli) Nissle 1917 original bacteria control group can improve the damage degree of gastric mucosa, reduce ulcer area (p <0.01), and the ulcer inhibition rate reaches 51.91%; the ulcer inhibition rate of clinical cimetidine is 46.67%; the ulcer inhibition rate of recombinant Escherichia coli (Escherichia coli) Nissle 1917 probiotics expressing Pgk1 is as high as 70.00 percent, and is obviously higher than that of original bacteria (Escherichia coli) Nissle 1917 and cimetidine. The results show that the recombinant probiotics expressing Pgk1 has a remarkable gastric ulcer prevention and protection effect and a stronger gastric ulcer protection effect, and indicate that Pgk1 protein has a remarkable gastric ulcer prevention or treatment effect; the Pgk1 gene is further shown to be a new target for screening prevention or treatment of gastric ulcer, and provides a new strategy for treatment of gastric ulcer.
EXAMPLE 3 therapeutic Effect of recombinant Probiotics expressing Pgk1 on ulcerative colitis mice
1. Experimental grouping and gavage dosing:
48 male KM mice at 8 weeks of age were divided randomly into 6 groups by weight and the doses were as follows:
normal Control group (Control, oral administration of equal volume of 11.1mg/ml beta-lactose solution); DSS model control group (DSS, oral administration of an equal volume of 11.1mg/ml β -lactose solution); a group of positive drugs (SASP, orally administered sulfasalazine, 80 mg/kg/day); (Escherichia coli) Nissle 1917 group: gavage 0.25mL (Escherichia coli) Nissle 1917 original bacteria; pET-28a-EGFP group: gavage 0.25mL of recombinant Escherichia coli (Escherichia coli) Nissle 1917 probiotics containing recombinant plasmid pET-28 a-EGFP; pET-28a-Pgk1 group: gavage 0.25mL of Pgk1 expressing recombinant Escherichia coli (Escherichia coli) Nissle 1917 probiotic. The above administration groups were each prepared from 11.1mg/ml β -lactose solution.
2. Preparation of ulcerative colitis model
Preparing 4% DSS distilled water solution, freely drinking DSS water solution for molding mice in a DSS model control group, freely drinking DSS water solution for molding mice in a DSS + each bacteria treatment group, and simultaneously adding 0.25mL of bacteria solution, wherein the bacteria density is 1 multiplied by 109A CFU; mice in the group of DSS + SASP freely drink DSS aqueous solution for molding and are simultaneously given SASP of 80 mg/kg/day; the mice in the blank control group freely drink distilled water, all the mice are kept on conventional feed, when bloody diarrhea or bloody stool occurs, the aqueous solution containing DSS is changed into conventional drinking water, the drug treatment is continued, and after 2 hours of the last administration on the eighth day, serum and colon tissues are separated for standby.
3. Detection index of ulcerative colitis
Disease Activity Index (DAI) score: the mice in each group were scored for weight loss rate, fecal characteristics and fecal occult blood by reference to literature methods.
DAI ═ 3 (weight loss + fecal characteristics + fecal occult blood)/3
Evaluation of colon injury: after each group of mice died, the mouse colon was isolated, the ileum was cut from the junction of the ileum and colon, cut again near the anus, the fascia was isolated outside the colon to allow the colon to extend completely, the length of the mouse colon from the point back to the colon to the anus was measured with a ruler, and recorded by photography.
4. Analysis of results
The results of the effect of the recombinant probiotics on colon morphology and colon length of colitis mice: after the mice in each group were sacrificed, the colon tissues were rapidly removed, the morphological changes were observed, the lengths thereof were measured, and the statistical analysis was performed on each group of data, and the experimental results are shown in fig. 5 and fig. 6B. Compared with the blank control group, the length of the colon of the DSS model group is remarkably shortened (p <0.001), which indicates that the DSS model is successfully prepared; compared with a DSS model control group, the original bacterium (Escherichia coli) Nissle 1917 and the negative control pET-28a-EGFP can slightly improve the colonic edema and shorten the colonic edema, but have no significant difference; meanwhile, the effect of pET-28a-EGFP is not different from that of the original bacterium, which shows that the transferred target gene EGFP has no treatment effect on ulcerative colitis; the recombinant Escherichia coli (Escherichia coli) Nissle 1917 probiotics expressing Pgk1 can remarkably improve colonic edema and shortening conditions, and has statistical difference (p <0.001), namely, Pgk1 protein has the effect of preventing or treating ulcerative colitis; and Pgk1 gene can be used as a new target for screening prevention or treatment of ulcerative colitis.
The effect of the recombinant probiotics on the disease activity index of the ulcerative colitis mouse is as follows: in the experimental process, the weight, the stool viscosity, the hematochezia and the occult blood of the mouse are monitored, and SPSS23.0 software is used for single-factor analysis of variance in data statistics. As can be seen from a in fig. 6, the DAI score of the DSS model group showed a significant upward trend (p <0.001) compared to the blank control group, indicating that the DSS model was successfully prepared; compared with a model control group, the sulfasalazine administration group, (Escherichia coli) Nissle 1917 administration group, pET-28a-EGFP administration group and pET-28a-Pgk1 administration group all can obviously reduce DAI scores (p is less than 0.05), and the (Escherichia coli) Nissle 1917 resistant bacteria and the recombinant probiotics expressing Pgk1 are preliminarily shown to have treatment effects on ulcerative colitis.
The DAI score, the colon morphology and the colon length are determined, so that the recombinant probiotics expressing the Pgk1 protein has a treatment effect on DSS-induced ulcerative colitis, and has no obvious toxic or side effect, namely, the Pgk1 protein has the effect of preventing and/or treating ulcerative colitis; and Pgk1 gene can be used as a new target for screening prevention or treatment of ulcerative colitis.
The results of the example 2 and the example 3 are combined to show that the recombinant probiotics expressing the Pgk1 protein can obviously inhibit gastric ulcer and ulcerative colitis and can be used for treating gastrointestinal diseases.
Example 4 therapeutic Effect of terazosin on gastric ulcer mice
Experimental materials and sources: GES-1 cells were purchased from wuhan ponuosai biotechnology limited; RMPI 1640 medium, pancreatin, double antibody and fetal calf serum were purchased from BI Biotechnology Ltd; pgk1 antibodies were purchased from doctor warrior; terazosin (Terazosin, TZ, HPLC ≥ 98%) from Aladdin Biotechnology Ltd; absolute ethanol, Tianjin Damao chemical reagents; cimetidine Capsules (CIM) were purchased from Special pharmaceutical groups, Inc.
Experimental animals: 8 week-old SPF-class Kunming (KM) male mice, 18-22 g in body weight, without any drug before the experiment, were purchased from Lanzhou veterinary institute, national academy of agricultural sciences. The experimental animals are adaptively raised for one week in an environment with 24-26 ℃ and 12h/12h of alternate diurnal rules, the animals are fed with diet and freely drink distilled water, and then the experiments are carried out in groups.
1. In vitro cell model
MTT method for detecting cell viability analysis: GES-1 cells were cultured in RMPI 1640 medium containing 10% fetal bovine serum and 1% diabody. In order to induce the cell death model, the experiment uses absolute ethyl alcohol as an inducer to establish a GES-1 cell death modelThe specific method comprises the following steps: GES-1 cells in logarithmic growth phase were taken at 1X 104The cell density of each well is planted on a 96-well plate, after the cells are attached to the wall, a blank group, a blank + TZ (100nmol/L) group, a blank + TZ (10nmol/L), an ethanol model group (0.5mmol/L), an ethanol + TZ (100nmol/L) group and an ethanol + TZ (10nmol/L) are set, after the intervention of terazosin medicine is carried out for 24 hours, a culture medium containing 0.5mmol/L ethanol is changed for stimulation for 24 hours, the cell activity is detected by an MTT method, and the survival rate is calculated.
Effect of terazosin on Pgk1 activity: GES-1 cells in logarithmic growth phase were taken at 1X 106Cell density per well spread at 6cm2After cells adhere to the cell culture dish, a blank group, a blank + TZ (10nmol/L) group, an ethanol model group (0.5mmol/L) and ethanol + TZ (10nmol/L) are set, terazosin is added for intervention for 24 hours, a culture medium containing 0.5mmol/L ethanol is used for stimulation for 24 hours, and then SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis is carried out on the protein cleaved on ice.
2. In vivo animal model
Preparation of KM mouse alcoholic gastric ulcer model: 40 KM male mice (20-25g) were bred in laboratory animal houses of university of traditional Chinese medicine in Gansu, and after the mice were bred adaptively for one week, the mice were randomly divided into 4 groups of 10 mice each, and the grouping condition and the dosage are shown in Table 2;
TABLE 2 experimental groups of KM Male mice and the dose administered to each group
Group of
Dosage to be administered
Blank control group
0.3mL per gavage normal saline
Ethanol model group
0.3 mL/gavage absolute ethyl alcohol
Cimetidine positive group
80mg/kg/day cimetidine is administrated by intragastric administration, and after 2 hours, 0.3 mL/intragastric administration of absolute ethyl alcohol
TZ administration group
1mg/kg/day TZ intragastric administration, and after 2 hours, 0.3 mL/intragastric absolute ethyl alcohol
The experiment was terminated after 2h of stimulation of the groups of mice, and serum and gastric tissue were isolated to determine various indices.
Ulcer area, ulcer index and inhibition rate: taking out stomach, cutting along the greater curvature of stomach, washing to remove the content, observing gastric mucosa ulcer, measuring the transverse diameter and longitudinal diameter of ulcer with ruler, and multiplying the two to obtain ulcer area (mm)2) As shown in equation 1; the ulcer area of the entire stomach tissue was then calculated to calculate the ulcer inhibition ratio (%), see equation 2. Meanwhile, the average value of the sum of the ulcer points of each group of mice is taken as the ulcer index (healing score 0, superficial mucosal erosion score 1, deep ulcer or transmural necrosis score 2, perforation or penetrating ulcer score 3)
Ulcer area (mm2) ═ maximum length of ulcer perpendicular to maximum width of maximum length (1)
3. Results of the experiment
The activity analysis of terazosin cells is determined by an MTT method, the experimental result is shown in figure 7, compared with a blank group, the blank + TZ (100nm) group and the blank + TZ (10nm) group have no obvious difference, which shows that terazosin has no toxic effect on GES-1 cells and can not promote cell proliferation under the concentration of 10nmol/L and 100 nmol/L; compared with the blank group, the cell survival rate of the Ethanol group is remarkably reduced (p is less than 0.001), which indicates that the Ethanol-induced cell death model is successfully established; compared with the Ethanol group, the Ethanol + TZ (100nm) group and the Ethanol + TZ (10nm) group can obviously improve the cell survival rate, and the terazol can inhibit cell death caused by Ethanol (p is less than 0.001).
Effect of terazosin on Pgk1 activity as shown in fig. 8, terazosin was able to significantly up-regulate the expression level of Pgk1 protein (p <0.05), indicating that terazosin is able to activate Pgk 1.
The experimental results of terazosin on gastric tissue morphology, ulcer index, ulcer area and ulcer inhibition rate of gastric ulcer mice are shown in fig. 9. The macro morphology of the stomach tissue of the normal group of mice is normal, no obvious hemorrhagic lesion appears, the hemorrhagic lesion of the stomach tissue of the ethanol model group of mice is obvious, the ulcer is the most serious, and the success of the preparation of the ethanol gastric ulcer model is shown; compared with an ethanol model group, the administration groups of terazosin and cimetidine can obviously improve the damage degree of gastric mucosa and reduce ulcer area, and the ulcer inhibition rate (81.57%) of the terazosin is higher than that (69.91%) of clinical Cimetidine (CIM).
The ulcer index, ulcer area, ulcer inhibition rate and WB result determination show that the terazosin activating and expressing Pgk1 protein has a prevention effect on alcoholic gastric ulcer, and has no obvious toxic or side effect, namely Pgk1 protein has the effect of preventing and/or treating gastric ulcer; and the Pgk1 gene can be used as a new target for screening prevention and/or treatment of gastric ulcer.
Example 5 therapeutic Effect of terazosin on colitis mice
Experimental materials and sources: caco-2 cells were purchased from Wuhan Punuoise Life technologies, Inc.; RMPI 1640 medium, pancreatin, double antibody and fetal calf serum were purchased from BI Biotechnology Ltd; pgk1 antibodies were purchased from doctor warrior; terazosin (Terazosin, TZ, HPLC ≥ 98%) from Aladdin Biotechnology Ltd; sulfasalazine (SASP) enteric tablets were purchased from shanghai yinyiwei balance pharmaceutical limited; dextran Sulfate Sodium (DSS), MW 40000, available from Aladdin Biotechnology Ltd.
Experimental animals: 8 week-old SPF-class Kunming (KM) male mice weigh 18-22 g, are not treated with any drugs before the experiment, and are purchased from Lanzhou veterinary research institute of Chinese academy of agricultural sciences. The experimental animals are adaptively raised for one week in an environment with 24-26 ℃ and 12h/12h of alternate diurnal rules, the animals are fed with diet and freely drink distilled water, and then the experiments are carried out in groups.
1. In vitro cell model
Terazosin cytotoxicity assay: taking Caco-2 cells in logarithmic growth phase at 1 × 104The cell density of each well is planted in a 96-well plate, after the cells adhere to the wall, a blank group, a blank + TZ (1 mu mol/L) group, a blank + TZ (100nmol/L) group and a blank + TZ (10nmol/L) group are set, and after terazosin with different concentrations acts on the cells for 24 hours, the cytotoxicity is detected by using an MTT method.
Terazosin cell viability assay: taking Caco-2 cells in logarithmic growth phase at 1 × 104Planting the cells in 96-well plate, setting blank group and H after the cells adhere to the wall2O2Group (500. mu. mol/L), H2O2+TZ(1μmol/L)H2O2+ TZ (100nmol/L) group and blank + TZ (10nmol/L), after different concentrations of terazosin had been applied to the cells for 24h, with a solution containing 500. mu. mol/L H2O2After the cells were stimulated for 24 hours in the medium of (1), the cell viability was examined by the MTT method.
Effect of terazosin on Pgk1 activity: caco-cells were taken at 1X 10 in logarithmic growth phase6Cell density per well spread at 6cm2A cell culture dish, after the cells adhere to the wall, a blank group, a blank + TZ (10nmol/L) group and H are set2O2Model group (0.5mmol/L), and H2O2+ TZ (10nmol/L), after adding terazosin medicine to intervene for 24h, it is changed to contain 500 μmol/L H2O2After 24h of stimulation with the medium, proteins were cleaved on ice for SDS-PAGE Western blot analysis.
2. In vivo animal model
30 KM male mice were randomly divided into 5 groups of 6 mice each, and the groups and administration were as shown in Table 3.
TABLE 3 experimental groups of KM Male mice and the dose administered to each group
Group of
Dosage to be administered
Blank Control group (Control)
Free drinking of distilled water
Blank control group + TZ group
Distilled water is freely drunk and is 4mg/kg/day TZ
DSS model control group
4% m/v DSS free drink
Positive control group (DSS + SASP)
4% m/v DSS free drink +80mg/kg/day SASP
DSS + TZ group
4% m/v DSS free drink +4mg/kg/day TZ
Preparing 4% DSS distilled water solution, freely drinking DSS water solution for molding a DSS model control group mouse, freely drinking DSS water solution for molding a DSS + TZ group mouse, and simultaneously administering 4mg/kg/day terazosin; mice in the group of DSS + SASP freely drink DSS aqueous solution for molding and are simultaneously given SASP of 80 mg/kg/day; the mice in the blank control group freely drink distilled water, the mice in the blank control group and the TZ group freely drink distilled water and simultaneously give 4mg/kg/day of terazosin, and all the mice are kept on the conventional feed for 7 days of continuous molding. After 2h of the last administration on the seventh day, serum and colon tissue were isolated for use.
And (3) detection of clinical indexes of ulcerative colitis:
weight loss: calculated as percent (%) of weight loss in each mouse, no weight loss was scored as 0 point, 1% -5% weight loss was scored as 1 point, 6% -10% weight loss was scored as 2 points, 11% -15% weight loss was scored as 3 points, and greater than 15% weight loss was scored as 4 points.
Stool consistency: normal stool scores 0 points, loose stools 2 points, and diarrhea 4 points.
Hematochezia and occult blood conditions: normal stool scores 0 points, occult blood scores 1 points (judged according to occult blood detection results), and visual bloody stool scores 3 points.
The mean value of the weight loss condition, stool consistency, hematochezia and occult blood condition is the DAI. The three mean values are the DAI.
Measurement of colon length: after sacrifice, the colon of the mouse is isolated, the ileum is cut from the junction of the ileum and colon, cut again near the anus of the colon, the fascia is separated from the outside of the colon, the colon is allowed to extend completely, the length of the colon of the mouse from the location of the colon returning to the anus is measured with a ruler and recorded by taking a picture.
3. Results of the experiment
The results of the cytotoxicity experiments for MTT assay terazosin are shown in FIG. 10, panel A. Compared with a blank group, the blank + TZ (1 mu M), the blank + TZ (100nm) and the blank + TZ (10nm) groups have no obvious difference, which shows that terazosin has no toxic effect on Caco-2 cells and can not promote cell proliferation under the concentrations of 1 mu mol/L, 100nmol/L and 10 nmol/L;
the results of cell viability analysis of terazosin are shown in fig. 10, B, H, compared to blank2O2Significant decrease in group cell survival (p)<0.01), indicating H2O2Successfully establishing a cell death model; and H2O2Model group comparison, H2O2+TZ(1μmol/L)、H2O2The + TZ (100nmol/L) group and the blank + TZ (10nmol/L) group can obviously improve the cell survival rate, and the results show that the terazol can inhibit H2O2Oxidative stress-induced cell death (p)<0.05)。
Effect of terazosin on Pgk1 activity as shown in fig. 11, terazosin was able to significantly up-regulate the expression level of Pgk1 protein (p <0.05), indicating that terazosin is able to activate Pgk 1.
The effect of terazosin on colitis mice body weight and disease activity index is shown in figure 12. Compared with a blank control group, the blank control group plus the TZ administration group shows no statistical difference in the body weight index and the DAI scoring index, and the result shows that the terazosin has no toxic or side effect on normal mice; compared with a blank control group, the weight of the DSS model group is reduced, the DAI score shows a remarkable rising trend (p is less than 0.001), and the DSS model is successfully prepared; compared with a DSS model control group, each administration group can obviously improve weight loss and DAI (p is less than 0.05 and p is less than 0.01), and the curative effect of terazosin is stronger than that of sulfasalazine which is clinically used.
The effect of terazosin on colon morphology and colon length in colitis mice is shown in figure 13. Compared with a blank control group, the length of the colon of mice in the blank control group plus TZ administration group has no obvious change, which shows that the quinazoline derivative terazosin has no toxic or side effect on normal mice; compared with the blank control group, the length of the colon of the DSS model group is remarkably shortened (p <0.001), which indicates that the DSS model is successfully prepared; compared with a DSS model control group, each administration group can obviously improve the colonic edema and the shortening condition, has obvious difference (p is less than 0.05), and the curative effect of the quinazoline derivative terazosin is stronger than that of the sulfasalazine clinically used.
The weight difference, DAI score, colon morphology and colon length determination and WB result determination show that the terazosin activating to express Pgk1 protein has treatment effect on ulcerative colitis, and has no obvious toxic or side effect, namely Pgk1 protein has the effect of preventing and/or treating colitis; and Pgk1 gene can be used as a new target for screening colitis prevention and/or treatment.
The results of examples 4 and 5 show that the drug promoting the expression of Pgk1 protein can significantly inhibit gastric ulcer and ulcerative colitis, and can be used for treating gastrointestinal diseases.
Sequence listing
<110> Lanzhou university
Application of <120> Pgk1 in preparation or screening of medicine for treating gastrointestinal diseases by using target spot
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 417
<212> PRT
<213> human source (human)
<400> 1
Met Ser Leu Ser Ala Leu Leu Thr Leu Ala Leu Leu Ala Val Leu Gly
1 5 10 15
Leu Ala Val Val Met Ala Val Ala Pro Ala Val Pro Met Leu Ala Ala
20 25 30
Gly Ile Thr Ala Ala Gly Ala Ile Leu Ala Ala Val Pro Ser Ile Leu
35 40 45
Pro Cys Leu Ala Ala Gly Ala Leu Ser Val Val Leu Met Ser His Leu
50 55 60
Gly Ala Pro Ala Gly Val Pro Met Pro Ala Leu Thr Ser Leu Gly Pro
65 70 75 80
Val Ala Val Gly Leu Leu Ser Leu Leu Gly Leu Ala Val Leu Pro Leu
85 90 95
Leu Ala Cys Val Gly Pro Gly Val Gly Leu Ala Cys Ala Ala Pro Ala
100 105 110
Ala Gly Ser Val Ile Leu Leu Gly Ala Leu Ala Pro His Val Gly Gly
115 120 125
Gly Gly Leu Gly Leu Ala Ala Ser Gly Ala Leu Val Leu Ala Gly Pro
130 135 140
Ala Leu Ile Gly Ala Pro Ala Ala Ser Leu Ser Leu Leu Gly Ala Val
145 150 155 160
Thr Val Ala Ala Ala Pro Gly Thr Ala His Ala Ala His Ser Ser Met
165 170 175
Val Gly Val Ala Leu Pro Gly Leu Ala Gly Gly Pro Leu Met Leu Leu
180 185 190
Gly Leu Ala Thr Pro Ala Leu Ala Leu Gly Ser Pro Gly Ala Pro Pro
195 200 205
Leu Ala Ile Leu Gly Gly Ala Leu Val Ala Ala Leu Ile Gly Leu Ile
210 215 220
Ala Ala Met Leu Ala Leu Val Ala Gly Met Ile Ile Gly Gly Gly Met
225 230 235 240
Ala Pro Thr Pro Leu Leu Val Leu Ala Ala Met Gly Ile Gly Thr Ser
245 250 255
Leu Pro Ala Gly Gly Gly Ala Leu Ile Val Leu Ala Leu Met Ser Leu
260 265 270
Ala Gly Leu Ala Gly Val Leu Ile Thr Leu Pro Val Ala Pro Val Thr
275 280 285
Ala Ala Leu Pro Ala Gly Ala Ala Leu Thr Gly Gly Ala Thr Val Ala
290 295 300
Ser Gly Ile Pro Ala Gly Thr Met Gly Leu Ala Cys Gly Pro Gly Ser
305 310 315 320
Ser Leu Leu Thr Ala Gly Ala Val Thr Ala Ala Leu Gly Ile Val Thr
325 330 335
Ala Gly Pro Val Gly Val Pro Gly Thr Gly Ala Pro Ala Ala Gly Thr
340 345 350
Leu Ala Leu Met Ala Gly Val Val Leu Ala Thr Ser Ala Gly Cys Ile
355 360 365
Thr Ile Ile Gly Gly Gly Ala Thr Ala Thr Cys Cys Ala Leu Thr Ala
370 375 380
Thr Gly Ala Leu Val Ser His Val Ser Thr Gly Gly Gly Ala Ser Leu
385 390 395 400
Gly Leu Leu Gly Gly Leu Val Leu Pro Gly Val Ala Ala Leu Ser Ala
405 410 415
Ile
<210> 2
<211> 1254
<212> DNA
<213> human source (human)
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actgtggtcc tgaaagcagc aagaagtatg ctgaggctgt cactcgggct aagcagattg 1200
tgtggaatgg tcctgtgggg gtatttgaat gggaagcttt tgcccgggga accaaagctc 1260
tcatggatga ggtggtgaaa gccacttcta ggggctgcat caccatcata ggtggtggag 1320
acactgccac ttgctgtgcc aaatggaaca cggaggataa agtcagccat gtgagcactg 1380
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gcaatattta gggatccgcg acccatttgc tgtccaccag tcatgctagc catatggctg 1500
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ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc 1620
ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta cgccggacgc 1680
atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc 1740
accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt 1800
atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc 1860
cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag 1920
tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg 1980
tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac 2040
gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa 2100
ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct 2160
gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg 2220
cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc 2280
tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga 2340
agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa 2400
ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc 2460
gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga 2520
cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt 2580
agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct 2640
cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg 2700
ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc 2760
caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg 2820
tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc 2880
gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct 2940
gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa 3000
aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 3060
attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 3120
ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct 3180
tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga 3240
ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg 3300
gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa 3360
tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga 3420
agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt 3480
tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac 3540
gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa 3600
tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca 3660
ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg 3720
tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata 3780
ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc 3840
gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc 3900
cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta 3960
tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg 4020
cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc 4080
gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt 4140
gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg 4200
ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa 4260
ctatgcggca tcagagcaga ttgtactgag agtgcaccat atatgcggtg tgaaataccg 4320
cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac 4380
tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 4440
cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 4500
aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 4560
gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 4620
agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 4680
cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 4740
cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 4800
ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 4860
gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 4920
tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 4980
acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 5040
tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 5100
attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 5160
gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa aactgtctgc 5220
ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc 5280
taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga 5340
taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga 5400
gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag 5460
actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc 5520
tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga 5580
agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt 5640
gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca 5700
ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa 5760
tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga 5820
ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt 5880
aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat 5940
cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata 6000
tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt 6060
ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 6120
gggttccgcg cacatttccc cgaaaagtgc cacctgaaat tgtaaacgtt aatattttgt 6180
taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg 6240
gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt 6300
ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct 6360
atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt 6420
gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa 6480
agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc 6540
tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc 6600
tacagggcgc gtcccattcg cca 6623
