CN118203589A - Application of ursodeoxycholic acid in preparing medicine for treating colorectal cancer combined with Fusobacterium nucleatum infection - Google Patents
Application of ursodeoxycholic acid in preparing medicine for treating colorectal cancer combined with Fusobacterium nucleatum infection Download PDFInfo
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- CN118203589A CN118203589A CN202410268835.9A CN202410268835A CN118203589A CN 118203589 A CN118203589 A CN 118203589A CN 202410268835 A CN202410268835 A CN 202410268835A CN 118203589 A CN118203589 A CN 118203589A
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- colorectal cancer
- ursodeoxycholic acid
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Abstract
The invention belongs to the field of new application discovery of clinical medicines, in particular to application of ursodeoxycholic acid in preparing medicines for treating colorectal cancer and infection of clostridium nucleatum, wherein the ursodeoxycholic acid can regulate lipid metabolism and reverse lipid accumulation caused by clostridium nucleatum; inhibiting expression of virulence factors; inhibit proliferation of tumor ball, and reduce size of tumor ball; significantly reducing the mouse DAI index; has good treatment effect on the tumor infected by clostridium nucleatum, and obviously reduces the number of the intestinal tumors of mice; reducing tissue inflammation infiltration, protecting intestinal crypt and villus; inhibiting tumor cell proliferation, reducing Ki-67 expression, increasing ZO-1 and MUC-2 expression, and remarkably reducing blood triglyceride level; reducing secretion of inflammatory factors, lowering concentration of related lipid metabolites, increasing secretion of bile acids, and concentration of part of secondary bile acids. Has wide application prospect.
Description
Technical Field
The invention belongs to the field of new application discovery of clinical medicines, and particularly relates to application of ursodeoxycholic acid in preparation of medicines for treating colorectal cancer combined with fusobacterium nucleatum infection.
Background
Colorectal cancer (Colorectal Cancer, CRC) is the third most common cancer, the second leading cause of cancer-related death worldwide. From abnormal crypts to tumors, various genetic and environmental factors lead to further development of CRC. Wherein, the interaction of intestinal microecology and intestinal mucosa barrier forms intestinal canal steady state, and the symbiotic effect with host influences the important functions of human nutrition digestion and absorption, energy metabolism, physiological function, immune regulation and the like. Altered microbiota are associated with early development of CRC and specific microbial species have been identified as tumor-causing drivers, chronic infection of pathogenic microorganisms and consequent inflammation contributing to tumor development and progression. Recent mouse studies indicate that intestinal flora may regulate local immune responses and thus affect chemotherapy and immunotherapy.
The most extensively studied clostridium nucleatum (Fusobacterium nucleatum, fn) among colorectal cancer pathogenic bacteria gradually increases in abundance from normal tissues to adenoma tissues to colorectal cancer, and the bacterium can promote proliferation, invasion and metastasis of colorectal cancer cells, further causes chemotherapy resistance, and the targeted clostridium nucleatum contributes to prognosis and management of patients. Clostridium nucleatum promotes self-renewal of colorectal cancer stem cell-like cells (CCSC) by manipulating cellular lipid accumulation, whereas non-CCSCs acquires the CCSC characteristic. Clostridium nucleatum infection promotes CCSC self-renewal by enhancing fatty acid oxidation to reduce lipid accumulation in CCSCs. In contrast, clostridium nucleatum infection increases lipid accumulation in non-CCSC by activating NF- κb to regulate CPT1 and FASN, promoting fatty acid formation. Lipids are deposited in the form of lipid droplets on which the number is degraded by MDM2, activating Notch signaling, thereby facilitating the acquisition of stem cell-like cellular features. Inhibition of NF- κB can inhibit Clostridium nucleatum infection and reduce lipid abundance in CCSCs. Thus, targeting fatty acid synthesis pathways is a potential strategy for treating CRC with clostridium nucleatum infection.
Ursodeoxycholic acid is generally a substance extracted from bear bile, and is used for cholesterol type cholelithiasis, forming and bile deficiency type steatorrhea, and also can be used for preventing drug-induced calculus formation and treating fatty dysentery (after ileectomy), can increase bile acid secretion, change bile components, reduce cholesterol and cholesterol fat in bile, and is favorable for gradually dissolving cholesterol in cholelithiasis, and is used for cholesterol calculus which is not suitable for operation treatment, but can not dissolve cholelithiasis, mixed calculus and X-ray opaque calculus. Researchers found that ursodeoxycholic acid also has an effect of treating liver cancer (CN 108815172 a), thyroid cancer (CN 108451958 a), but no literature discloses that it has an effect of treating colorectal cancer.
The inventor unexpectedly found in the research process that ursodeoxycholic acid can inhibit proliferation of colorectal cancer cells caused by clostridium nucleatum, inhibit deterioration of intestinal tumors caused by clostridium nucleatum, improve lipid metabolism disorder, and can be used for colorectal cancer patients infected by clostridium nucleatum.
Disclosure of Invention
In view of the above technical problems, a primary object of the present invention is to provide an application of ursodeoxycholic acid in preparing a medicament for treating colorectal cancer.
The second object of the present invention is to provide the use of ursodeoxycholic acid in the preparation of a medicament for inhibiting clostridium nucleatum infection.
A third object of the present invention is to provide the use of ursodeoxycholic acid in the preparation of a medicament for the treatment of colorectal cancer combined with fusobacterium nucleatum infection.
Preferably, the medicament is formulated into a pharmaceutical dosage form, the dosage form comprising: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal agents, granules, medicinal granules, pills, pellets, suspensions, wines, tinctures and drops.
Preferably, the medicament contains one or more pharmaceutically acceptable carriers or excipients.
The beneficial effects of the invention are as follows: ① The invention provides application of ursodeoxycholic acid in preparing a medicament for treating colorectal cancer; ② The invention also provides application of ursodeoxycholic acid in preparing a medicine for inhibiting clostridium nucleatum infection; ③ The invention also provides application of ursodeoxycholic acid in preparing a medicament for treating colorectal cancer combined with Fusobacterium nucleatum infection. ④ The ursodeoxycholic acid disclosed by the invention can regulate lipid metabolism, reduce the expression of NF- κB/FASN/CPT1 signal axis genes, and reverse lipid accumulation caused by clostridium nucleatum; inhibiting expression of FadA, fap2, fomA virulence factors; inhibit proliferation of tumor ball, and reduce size of tumor ball; significantly reducing the mouse DAI index; the composition has good treatment effect on the tumor infected by clostridium nucleatum, and the UDCA can obviously reduce the number of the intestinal tumors of mice; reducing tissue inflammation infiltration, protecting intestinal crypt and villus; inhibiting tumor cell proliferation, reducing Ki-67 expression, increasing ZO-1 and MUC-2 expression, and remarkably reducing blood triglyceride level; reducing secretion of inflammatory factors, lowering concentration of related lipid metabolites, increasing secretion of bile acids, and concentration of part of secondary bile acids. Has wide application prospect.
Drawings
FIG. 1UDCA activity against Clostridium nucleatum;
FIG. 2UDCA expression experiment of virulence factor against F.nucleatum;
FIG. 3UDCA anti-CRC cell activity;
FIG. 4UDCA fat accumulation assay against F.nucleatum infection of CRC cells;
FIG. 5UDCA gene expression experiments against F.nucleatum infected CRC cells;
FIG. 6UDCA protein expression assay against F.nucleatum infected CRC cells;
FIG. 7UDCA anti-F.nucleatum infection CRC blastoma assay;
FIG. 8 calculation of free energy of binding between UDCA and fatty acid synthase;
FIG. 9 mouse DAI index curve;
Figure 10 mouse tumor number statistics;
FIG. 11 mouse intestinal tissue HE staining;
FIG. 12 intestinal tissue immunohistochemistry of mice;
FIG. 13 mouse blood triglyceride assay;
FIG. 14 measurement of inflammatory factors in the blood of mice;
FIG. 15 mouse fecal metabolome analysis;
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below are commercially available unless otherwise specified.
Reagent and consumable: sodium chloride (national medicine group chemical Co., ltd.), peptone (Beijing cool pacing technology Co., ltd.), beef powder (Beijing cool pacing technology Co., ltd.), yeast powder (OXOID), glucose (national medicine group chemical Co., ltd.), na 2HPO4 (national medicine group chemical Co., ltd.), sodium acetate (national medicine group chemical Co., ltd.), tween80, K 2HPO4 (national medicine group chemical Co., ltd.), citric acid diamine (national medicine group chemical Co., ltd.), mgSO 4·7H2 O (national medicine group chemical Co., ltd.), MnSO 4·H2 O (national pharmaceutical group chemical company, ltd.), soytone (beijing cool pacing technologies, ltd.), 96-well plate (soxhlet biosciences, ltd.), gram staining kit (beijing soybu technologies, ltd.), MTT thiazole blue (beijing soybu technologies, ltd.), green streptomycin mixed solution (100×) (beijing soybu technologies, ltd.), fetal bovine serum (zhejiang biosciences, ltd.), RPMI-1640 medium (beijing soybu technologies, trypsin (HyClone), SYBR GREEN QPCR MASTER MI x (wuhansai-wil biosciences, ltd.), FASN Rabbit Monoclonal Antibody (Shanghai Biyun biotechnology Co., ltd.), rabbit anti-CTP 1 polyclonal antibody (Biotechnology Co., ltd.), NF- κ B p 65-Rabbit Polyclonal Antibody (Shanghai Biyun biotechnology Co., ltd.), phospho-NF- κ B p65 (Ser 536) Rabbit Polyclonal Antibody (Shanghai Biyun biotechnology Co., ltd.), recombinant human bFGF (Sierrai (Beijing) life science Co., ltd.), recombinant human EGF (Saerein (Beijing) life science and technology Co., ltd.), B-27 serum-free additive (Saint Biotechnology (Shanghai) Co., ltd.), insulin (Shanghai Biyun Biotechnology Co., ltd.), oil red O (Biotechnology (Shanghai) Co., ltd.), C57BL/6 mice (China department of agriculture, orchis, veterinarian research Co., ltd.), pET28 (a+) -FAS plasmid (Biotechnology (Shanghai) Co., ltd.), BL21 competent cells (Biotechnology (Shanghai) Co., ltd.), triglyceride (TG) assay kit (Nanj established biological engineering research Co., ltd.), cDNA reverse transcription kit (Wohsai Vir Biotechnology Co., ltd.), primer synthesis (Biotechnology (Shanghai)) BCA protein quantification kit (Beijing Soy Bao Biotechnology Co., ltd.), UDCA (Shanghai Milin Biotechnology Co., ltd.), DSS (Shanghai Milin Biotechnology Co., ltd.), AO M (Sigma), IL-8 and TNF-alpha ELISA kit (Beijing Soy Bao Biotechnology Co., ltd.), ZO-1 rabbit polyclonal antibody and MUC-2 rabbit polyclonal antibody (Biotechnology (Shanghai) Co., ltd.).
Instrument: ultra clean bench (Shanghai Shen An medical instruments Co., ltd.), centrifuge (Hunan Hennuo instruments Co., ltd.), vertical high pressure steam sterilizer (Shanghai Shen An medical instruments Co., ltd.), 680 type enzyme mark instrument (Bio-Rad Co., ltd.), constant temperature incubator (Tiste instruments Co., tianjin Co., ltd.), 2.5L sealed anaerobic incubator (Mitsubishi MGC Co., ltd., japan), 371 type cell incubator (America ThermoFisher Scientific Co.); low-speed refrigerated centrifuge L530R (xiang instrument centrifuge, inc.), CKX41 inverted microscope (o Li Basi, usa), NANODROP 2000 (sammer fisher technologies), ultrasonic cell pulverizer (nivea new biosciences, inc.), real-time quantitative PCR instrument (ABIQ) (sammer fisher technologies), chemiluminescent imaging system (shanghai life sciences, inc.), high-speed refrigerated centrifuge (xiang instrument centrifuge, inc.), data analysis Software (R Software), molecular docking analysis Software (Autod ock Vina), and metabolome analysis (shanghai easter biomedical technologies, inc.).
EXAMPLE 1UDCA against Clostridium nucleatum infection colorectal cancer
Selecting the medicine (UDCA), preparing 10mM mother liquor by using DMSO, testing the inhibition effect on clostridium nucleatum, testing the inhibition effect on CRC cells by using an MTT method, detecting the dryness of the CRC cells by using a tumor ball experiment, detecting the expression change of related genes by using a qPCR experiment, detecting the expression change of related proteins by using a Western Blot experiment, detecting the accumulation of fat by using oil red O staining, establishing a mouse plan as a model for testing the treatment effect of the UDCA, analyzing the action mechanism of the curative effect of the UDCA by using Autodock Vina, and analyzing the binding mode of the UDCA and fatty acid synthase.
1. Determination of antibacterial Activity
(1) Clostridium nucleatum (Fusobacterium nucleatum, china general microbiological culture collection center, CGMCC 1.2526) is cultured: 40mL EG liquid culture medium (without horse blood) is added into a conical flask, a small amount of bacterial colony is picked up in the culture medium by an inoculating loop, and the bacterial colony is placed in an anaerobic box for constant temperature culture at 37 ℃ until bacterial colony grows to a logarithmic phase for use.
(2) Ultraviolet spectrophotometry counting: taking bacteria in logarithmic growth phase, centrifuging at 4000 rpm for 5min, adding corresponding culture medium, re-suspending, measuring absorbance at 600nm wavelength, and adjusting OD value to about 1.0 for standby. Gradient dilution with medium.
(3) The drug was formulated as a 100, 200, 400 μm solution in liquid medium.
(4) The 96-well plate was taken, and the bacterial liquid was added to the wells, 3 multiple wells, 50. Mu.L per well. Then 50. Mu.L of the drug solution was added to each well. Liquid medium plus bacteria solution was used as negative control, liquid medium was used as blank control, and 0.2% dmso was used as solvent control. Placing in an anaerobic box (anaerobe), and culturing in an incubator at 37 ℃ for 24 hours.
(5) After incubation for 24 hours, absorbance was measured using a microplate reader at 600nm wavelength.
(6) Results are expressed as mean ± standard deviation. The inhibition ratio was calculated according to the following formula:
inhibition (%) = (OD negative control-OD experimental group-OD blank)/(OD negative control-OD blank)% x
As shown in fig. 1, ursodeoxycholic acid (UDCA) is capable of inhibiting proliferation of clostridium nucleatum and has concentration dependence.
2. QPCR experiment for inhibiting virulence factor of Fusobacterium nucleatum
Fusobacterium nucleatum is inoculated into a 6-hole plate, 1mL of bacterial liquid and 1mL of drug solution are added into each hole, anaerobic culture is carried out for 24 hours at 37 ℃, bacteria are collected, total RNA is extracted, cDNA synthesis is carried out, and the expression change of virulence genes is checked by utilizing a dye method qPCR aiming at virulence factors such as FadA, fap2, fomA and the like.
3. MTT assay for anti-colorectal cancer proliferation Activity
(1) Human colon cancer cells HCT116 (American type culture Collection ATCC CCL-247), human colorectal adenocarcinoma cells Caco-2 (American type culture Collection ATCC HTB-37) were treated with trypsin when the cells grew to 80%, and the cells were harvested. Centrifugation at 1000r/min for 5min, removal of supernatant, addition of RPMI-1640 medium, 96-well plates were used, and 100. Mu.L of cells (total 10,000 cells) were added per well. The cells were incubated at 37℃in a cell incubator (5% CO 2) for 24h.
(2) The drug concentration was 10. Mu.M in RPMI-1640 medium, and 0.1% DMSO-RPMI-1640 was used as a negative control. The culture supernatant from the 96-well plate was discarded and the drug solution (50, 100, 200, 400 μm) was added. The cells were incubated at 37℃in a cell incubator (5% CO 2) for 24h.
(3) Mu.L of MTT solution (0.5 mg/mL) was added to each well, the mixture was incubated in a 37℃cell incubator (5% CO 2) for 4 hours, the supernatant of the medium in the 96-well plate was discarded, 100. Mu.L of DMSO solution was added, and after shaking, the absorbance at 492nm was measured using an ELISA reader.
(4) Results are expressed as mean ± standard deviation. The inhibition ratio was calculated according to the following formula:
inhibition (%) = (OD negative control-OD experimental group-OD blank)/(OD negative control-OD blank)% x
As shown in fig. 3, ursodeoxycholic acid (UDCA) was able to inhibit colorectal cancer cell proliferation and had a concentration dependence, UDCA had a cell inhibition rate of more than 20% at 400 μm and a inhibition rate of more than 20% at 200 μm, and thus, a concentration of 100 μm was selected for the subsequent experiments to distinguish the inhibition of UDCA itself from the inhibition of UDCA on the carcinogenicity of clostridium nucleatum.
4. Oil red O dyeing experiment
(1) When human colon cancer cells HCT116 and human colorectal adenocarcinoma cells Caco-2 grew to 80%, the cells were harvested by trypsin treatment. Centrifuging at 1000r/min for 5min, discarding supernatant, adding RPMI-1640 culture medium, taking 6-well plate, adding 2mL per well, and adding 40 cells per well;
(2) After 24h of incubation, bacteria diluted with RPMI-1640 medium were added at a multiplicity of infection of 100, after 4h of incubation, medium containing the diabodies was added, after 2h of incubation, the drug was added (UDCA 100. Mu.M).
(3) After 48 hours incubation, cells were fixed with 4% paraformaldehyde for 30min, washed 3 times with PBS, stained with oil red 0 for 60 min, washed 3 times with PBS, and photographed.
As shown in fig. 3, fusobacterium nucleatum infected colorectal cancer cells can promote fat accumulation, and ursodeoxycholic acid (UDCA) treated can reverse fat accumulation.
QPCR experiment
(1) When human colon cancer cells HCT116 and human colorectal adenocarcinoma cells Caco-2 grew to 80%, the cells were harvested by trypsin treatment. Centrifuging at 1000r/min for 5min, discarding supernatant, adding RPMI-1640 culture medium, taking 6-well plate, adding 2mL per well, and adding 40 cells per well;
(2) After 24h of incubation, bacteria diluted with RPMI-1640 medium were added at a multiplicity of infection of 100, after 4h of incubation, medium containing the diabodies was added, after 2h of incubation, the drug was added (UDCA 100. Mu.M).
(3) After culturing for 48 hours, total RNA was extracted by adding TRIzol, and reverse transcription was performed to synthesize cDNA according to the procedure described.
(4) QPCR sample mixing was performed following the protocol described (pre-denaturation 95 ℃,30s; denaturation/annealing/extension 40 cycles, 95 ℃,10s,95 ℃,30 s).
As shown in FIG. 4, the expression of NF- κB/FASN/CPT1 signal axis gene was promoted after infection of colorectal cancer cells with Clostridium nucleatum, and the expression of the above-mentioned genes for fat synthesis and metabolism was reduced after UDCA treatment of the cells, thereby antagonizing the results of Clostridium nucleatum.
Western Blot experiments
(1) When human colon cancer cells HCT116 and human colorectal adenocarcinoma cells Caco-2 grew to 80%, the cells were harvested by trypsin treatment. Centrifuging at 1000r/min for 5min, discarding supernatant, adding RPMI-1640 culture medium, taking 6-well plate, adding 2mL per well, and adding 40 cells per well;
(2) After 24h of incubation, bacteria diluted with RPMI-1640 medium were added at a multiplicity of infection of 100, after 4h of incubation, medium containing the diabodies was added, after 2h of incubation, the drug was added (UDCA 100. Mu.M).
(3) After 48 hours of incubation, the lysate, phosphatase inhibitor, PMSF were added and the mixture was lysed on ice for 30min to extract total protein, which was quantified using BCA kit.
(4) SDS-PAGE gel electrophoresis was performed, primary and secondary antibodies were incubated and imaged using a chemiluminescent apparatus.
As shown in FIG. 5, the expression of NF- κB/FASN/CPT1 signaling axin was promoted after infection of colorectal cancer cells with Clostridium nucleatum, and the expression of the above-mentioned fat synthesis and metabolism protein was reduced after UDCA treatment of the cells, thereby antagonizing the results of Clostridium nucleatum.
7. Tumor ball experiment
(1) When human colon cancer cells HCT116 and human colorectal adenocarcinoma cells Caco-2 grew to 80%, the cells were harvested by trypsin treatment. Centrifuging at 1000r/min for 5min, discarding supernatant, adding RPMI-1640 culture medium, taking 6-well plate, adding 2mL per well, and adding 40 cells per well;
(2) After 24h adherence, bacteria diluted with RPMI-1640 medium were added, the multiplicity of infection was 100, after 4 hours incubation, medium containing double antibodies was added, after 2 hours incubation, cells were harvested by trypsin treatment. Centrifuging at 1000r/min for 5min, removing supernatant, adding stem cell culture medium, collecting ultra-low adsorption 24-well plate, adding 0.5mL per well, adding 3000 cells per well, and adding medicine (UDCA 100 μm).
(3) After 48 hours of incubation, observations were photographed.
As shown in fig. 7, after infection of colorectal cancer cells with clostridium nucleatum, the colorectal cancer cells can be promoted to form tumor balls (larger diameter), and after treatment of cells infected with bacteria by UDCA, proliferation of tumor balls can be inhibited, and the size of tumor balls can be reduced, thereby antagonizing the results of clostridium nucleatum.
8. Molecular docking experiments
(1) Downloading fasn protein structure (1 XKT) from https:// www.rcsb.org/website, and downloading UDCA chemical structural formula from https:// pubchem.
(2) The binding pattern of UDCA to FASN protein was analyzed using Autodock Vina and the free binding energy was calculated.
As shown in fig. 15, the molecular docking results of UDCA and FAS proteins showed that the binding energy score was-7.8, and that a nonpolar bond could be formed, thereby inhibiting the activity of the enzyme.
9. In situ colorectal cancer experiment in mice
(1) Mice were acclimatized for one week, and AOM (10 mg/kg) was intraperitoneally injected, after one week, 2% dss was given 2 weeks, then normal water was given 1 week, and three cycles were repeated.
(2) Mice in the control group normally drink water, lavage PBS, and mice in the Fn group lavage bacteria 1 x 10 9 CUF, and the treatment group lavage UDCA 150mg/kg while feeding Fn. Body weight, stool morphology, occult blood (colloidal gold method) were recorded weekly, disease activity index (DISEASE ACTIVITY index DAI), (body weight was not changed to 0, 1% -5% was 1 minute, 5% -10% was 2 minutes, 10% -20% was 3 minutes, and more than 20% was 4 minutes), stool viscosity (normal 0, soft stool was 1 minute, myxoid stool was 3 minutes, thin fluid stool was 3 minutes), and stool bleeding (colloidal gold test negative was 0 minutes, light purple was 1 minute, purple was 2 minutes, dark purple was 3 minutes, macroscopic blood stool was 4 minutes), i.e., dai= (body weight index+stool shape+bleeding condition)/3.
(3) Taking mouse blood to measure triglyceride content, and performing operation according to the instruction;
(4) The intestinal tracts of mice were taken for HE staining and paraffin sections were dewaxed to water: sequentially placing the slices into xylene I8 min-xylene II 8 min-absolute ethanol I6 min-absolute ethanol II 6min-95% alcohol 6min-85% alcohol 6min-75% alcohol 5 min-running water for flushing. Hematoxylin-stained nuclei: the slices are stained with Harris hematoxylin for 3-8min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for several seconds, rinsed with tap water, and returned to blue with running water. Eosin-stained cytoplasm: the slices are dyed in eosin dye solution for 1-3min. And (3) removing the water sealing piece: sequentially placing the slices into 75% alcohol 30s-85% alcohol 30s-95% alcohol 1min-95% alcohol II 2 min-absolute alcohol I5 min-absolute alcohol II 5 min-xylene I5 min-xylene II 7min, dehydrating and transparency, taking out the slices from the xylene, drying slightly, and sealing with neutral resin. Microscopic examination, image acquisition and analysis;
(5) Intestinal tissues were taken for FASN, ki67, ZO-1, MUC-2, and the procedure was as follows: dewaxing paraffin sections: (Paraffin sections were placed in an oven at 60℃for 1 hour before staining). P-xylene I, II, 10 min each, gradient alcohol: 100%,85%,70% each for 10 minutes; washing with distilled water for 5min, 2 times (placing in a shaker). Antigen retrieval: a0.01 mol/L sodium citrate solution was prepared and placed in boiling water for preheating. Placing the slices into a boiled sodium citrate solution for water bath for 30min, and cooling to room temperature after finishing; washed 3 times with PBS for 5min each. Hydrogen peroxide blocks endogenous peroxidases: 3%H 2O2, room temperature for 10 minutes (protected from light). PBS was washed 5min,3 times (on shaker). BSA blocking: the sections were removed from the dye vat, the back side of the sections and the surrounding tissue on the front side of the sections were wiped off, the pencil was circled, and 3% BSA blocking solution was added dropwise at 37℃for 60 minutes. Dripping primary antibody: the excess blocking solution was sucked off with filter paper, without washing, the primary antibody was directly added dropwise, and the mixture was placed in a refrigerator at 4℃overnight. PBS was washed 5min,3 times (on shaker). HRP-labeled secondary antibody was added dropwise, at 37 ℃, for 60 min, washed with PBS for 5min,3 times (on shaker). DAB color development: and (3) observing under a mirror for 3-5min, and stopping at proper time. Counterstaining the nuclei: counterstaining with hematoxylin for 3min, and washing with distilled water for 2 times; hydrochloric acid alcohol differentiated for 10 seconds. The blue is returned by washing with tap water for 10 minutes. Gradient alcohol dehydration: 80%,90%,100%,100%, each 5min. Xylene is transparent: i, II (xylene) each for 5min. Sealing piece: dropping 20 mu L of paraxylene and 20 mu L of neutral gum on the tissue block, covering a cover glass, and carrying out image acquisition under a microscope;
The oil red O staining procedure was as follows: freezing and slicing for hydration: the sections were allowed to stand at room temperature for 30min in sequence, and then washed 3 times with PBS (pH 7.4) for 5min each. Frozen sections were rinsed slightly in distilled water. Soaking in 60% isopropanol for 20-30s. Adding oil red O staining solution (capping), and hermetically staining for 10-15min. Color separation: the dye liquor was removed by a slight wash with 60% isopropanol. Placing in distilled water for slightly cleaning. Adding Mayer hematoxylin staining solution, and counterstaining for 1-2min. The 1% hydrochloric acid solution differentiated slightly. Rinsing with tap water for 10min. Placing in distilled water for slightly cleaning. The surrounding water was sucked dry with filter paper. And (5) sealing with glycerol. Image acquisition was performed under a microscope.
(6) Measuring the size and the statistics of the tumor;
(7) Taking mouse blood to measure inflammatory factors, and performing operation according to the instruction;
(8) The mouse feces were taken for non-targeted metabolome analysis, the specific process was as follows:
In the following operations, quality control samples (QC) were prepared from equal volumes of extracts of all samples mixed together. All extraction reagents were pre-chilled at-20 ℃. The analytical instrument for the experiment is a liquid-mass combination system consisting of Waters ACQUITY UPLC I-Class plus/Thermo QE ultra-high performance liquid tandem high resolution mass spectrometer.
Weigh 60mg of sample into a 1.5mL centrifuge tube, add two small steel balls and 600. Mu.L of methanol-water (V: V=4:1, containing mixed internal standard, 4. Mu.g/mL); precooling in a refrigerator at-40deg.C for 2min, and grinding in a grinder (60 Hz,2 min); ultrasonic extracting with ice water bath for 10min, standing at-40deg.C overnight; centrifuge for 10min (12000 rpm,4 ℃), aspirate 150. Mu.L of supernatant with syringe, filter with 0.22 μm organic phase pinhole filter, transfer to LC sample vial, store at-80℃until LC-MS analysis.
Chromatographic column: chromatographic column: ACQUITY UPLC HSS T3 (100 mm. Times.2.1 mm,1.8 um); column temperature: 45 ℃; mobile phase: mobile phase: a-water (0.1% formic acid), B-acetonitrile; flow rate: 0.35mL/min; sample injection volume: 2. Mu.L.
Mass spectrometry conditions ：Spray Voltage(V)(3800 -3000),Capillary Temperature(℃)(320),Au x gas heater temperature(℃)(350),Sheath Gas Flow Rate(Arb)(35),Aux gas flow rat e(Arb)(8),S-lens RF level(50),Mass range(m/z)(70-1050),Full ms resolution(70000),MS/MS resolution(17500),NCE/stepped NCE(10,20,40).
Data preprocessing prior to pattern recognition, the raw data was baseline filtered, peak identified, integrated, retention time corrected, peak aligned and normalized by metabonomics processing software Progenesis QI v 3.0.0, whose main parameters were the identification of ：precurs or tolerance：5ppm(HMDB+Lipidmaps)/10ppm(LuMet-Animal+METLIN);product tolera nce：10ppm(HMDB+Lipidmaps)/20ppm(LuMet-Animal+METLIN). compounds based on multiple dimensions of RT (retention time), exact mass number, secondary fragmentation and isotope distribution, and identification analysis using The Human Meta bolome Database (HMDB), LIPIDMAPS (v 2.3) and METLIN databases and LuMet-anal 3.0 local databases.
The extracted data is subjected to deletion value processing, 0 value replacement, score scoring screening, data merging and the like. Missing value processing and 0 value substitution: the ion peaks with deletion values of >50% in the group were deleted, and the remaining 0 values were replaced with half of the minimum value of all ion intensities of all samples. Score scoring screen: the qualitatively obtained compounds were screened according to a Score (Score) of the qualitative results, the screening criteria being 36 points (80 points full), and less than 36 points being considered inaccurate and deleted.
The multivariate statistical analysis firstly adopts an unsupervised Principal Component Analysis (PCA) to observe the overall distribution among samples and the stability of the whole analysis process, and then uses a supervised partial least squares analysis (PLS-DA) and an orthogonal partial least squares analysis (OPLS-DA) to distinguish the overall differences of the metabolic profiles among groups and find the differential metabolites among groups. .
As shown in fig. 9, the experiment of the in-situ colorectal cancer model of the mice shows that after clostridium nucleatum infection, the DAI index can be increased, and the DAI index of the mice can be obviously reduced by UDCA.
As shown in FIG. 10, after Clostridium nucleatum infection, tumor proliferation can be promoted, and UDCA has good therapeutic effect on Clostridium nucleatum infection tumor. UDCA can significantly reduce the number of intestinal tumors in mice.
As shown in fig. 11, HE staining showed that after clostridium nucleatum infection, inflammatory cell infiltration can be increased, intestinal tissue structure is destroyed, UDCA can reduce tissue inflammatory infiltration, and intestinal crypt and villus are protected.
As shown in FIG. 12, the result of immunohistochemistry shows that after the colorectal cancer cells are infected by clostridium nucleatum, the tumor proliferation can be promoted, the expression of Ki-67 is increased, the expression of ZO-1 and MUC-2 is reduced, the expression of Ki-67 is reduced by UDCA which can inhibit the proliferation of tumor cells, and the expression of ZO-1 and MUC-2 is increased.
As shown in fig. 13, UDCA treatment was able to significantly lower blood triglyceride levels.
As shown in FIG. 14, ELISA experiments show that after Clostridium nuclear infection, secretion of inflammatory factors TNF-alpha and IL-8 can be increased, and UDCA can reduce secretion of inflammatory factors.
As shown in FIG. 15, clostridium nucleatum infection can increase lipid metabolite production in blood, decrease related lipid metabolite concentration after UDCA treatment, increase bile acid secretion, and partial secondary bile acid concentration.
In summary, the invention provides application of ursodeoxycholic acid in preparing medicines for treating colorectal cancer, medicines for inhibiting clostridium nucleatum infection and medicines for treating colorectal cancer combined with clostridium nucleatum infection, and the ursodeoxycholic acid can regulate lipid metabolism and reduce the expression of NF- κB/FASN/CPT1 signal axis genes, and reverse lipid accumulation caused by clostridium nucleatum; inhibiting expression of FadA, fap2, fomA virulence factors; inhibit proliferation of tumor ball, and reduce size of tumor ball; significantly reducing the mouse DAI index; the composition has good treatment effect on the tumor infected by clostridium nucleatum, and the UDCA can obviously reduce the number of the intestinal tumors of mice; reducing tissue inflammation infiltration, protecting intestinal crypt and villus; inhibiting tumor cell proliferation, reducing Ki-67 expression, increasing ZO-1 and MUC-2 expression, and remarkably reducing blood triglyceride level; reducing secretion of inflammatory factors, lowering concentration of related lipid metabolites, increasing secretion of bile acids, and concentration of part of secondary bile acids. Has wide application prospect.
The foregoing description is directed to the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and all equivalent changes or modifications made under the technical spirit of the present invention should be construed to fall within the scope of the present invention.
Claims (5)
1. Application of ursodeoxycholic acid in preparing medicine for treating colorectal cancer is provided.
2. Application of ursodeoxycholic acid in preparing medicine for inhibiting clostridium nucleatum infection is provided.
3. Application of ursodeoxycholic acid in preparing medicine for treating colorectal cancer and fusobacterium nucleatum infection is provided.
4. A use according to any one of claims 1 to 3, wherein the medicament is in a pharmaceutical dosage form comprising: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal agents, granules, medicinal granules, pills, pellets, suspensions, wines, tinctures and drops.
5. The use according to claim 4, wherein: the medicine contains one or more than one pharmaceutically acceptable carrier or excipient.
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