Use of a trifluoromethyl vinyl ester compound in the preparation of a drug for treating ulcerative colitis

Abstract

The present invention belongs to the field of medicine and discloses the use of a trifluoromethyl vinyl ester compound in the preparation of a drug for treating ulcerative colitis. The compound has the chemical structure shown in Formula 1: The present invention reveals that the compound has the effect of treating ulcerative colitis. The present invention uses a mouse ulcerative colitis model and an LPS-induced macrophage RAW264.7 inflammation model to observe a series of tests such as the therapeutic effect of the novel trifluoromethyl vinyl ester compound BLF-16 on ulcerative colitis, and explores the therapeutic effect of compound BLF-16 on dextran sulfate sodium-induced ulcerative colitis in mice. It is first revealed that compound BLF-16 can treat ulcerative colitis and has good medicinal prospects.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and relates to the use of a trifluoromethyl vinyl ester compound in the preparation of anti-ulcerative colitis drugs. Specifically, it relates to the use of a trifluoromethyl vinyl ester compound BFL-16 in the preparation of anti-ulcerative colitis drugs and in the preparation of drugs for inhibiting lipopolysaccharide-induced macrophage inflammation. Background Art

[0002] When the body is stimulated by foreign pathogens, it will cause the excessive release of various inflammatory mediators and the over-activation of inflammatory cells. As an important part of the body's defense against foreign invasion, macrophages will produce a large amount of NO after being activated by foreign stimuli, further inducing the production of inflammatory cytokines. Therefore, inhibiting the release of NO and inflammatory cytokines by macrophages is beneficial to the prevention or treatment of inflammation. Inappropriate or excessive inflammation will damage tissues and cause various diseases, such as common cardiovascular diseases, autoimmune diseases and malignant tumors.

[0003] Ulcerative colitis (UC) is an idiopathic intestinal inflammatory disease mainly involving the rectal and colonic mucosa, characterized by chronic, recurrent attacks and unknown etiology. The clinical manifestations are mainly diarrhea, abdominal pain, mucous bloody stools, etc.; it can be accompanied by various autoimmune diseases, such as erythema nodosum, arthritis, ankylosing spondylitis, sclerosing cholangitis, autoimmune hemolytic anemia, etc. The incidence and prevalence of this disease show an obvious increasing trend in China.

[0004] At present, it is considered that the main pathogenesis is the imbalance of colonic mucosal immune function and the increased secretion of pro-inflammatory cytokines, leading to an inflammatory cascade amplification reaction. Commonly used therapeutic drugs include aminosalicylate drugs, glucocorticoids, immunosuppressants and some traditional Chinese medicines, but there is still no ideal drug so far. Developing drugs that treat both the symptoms and the root causes and are safe and effective has positive practical significance.

[0005] The compound trifluoromethyl vinyl ester BLF-16 (Formula 1) is a prodrug structure formed by the combination of a molecule of trifluoromethyl ketone compound and a molecule of non-steroidal anti-inflammatory drug (NSAID) ibuprofen. It is reported that trifluoromethyl ketone compounds have a variety of biological activities, including anti-inflammatory, anti-neurodegenerative diseases, and anti-cardiovascular diseases. No other applications have been found. Therefore, studying the therapeutic potential of new compounds in the preparation of anti-ulcerative colitis drugs provides more options for future human colitis patients. Summary of the Invention

[0006] The purpose of the present invention is to provide a new use of the compound BLF-16, and the structure of the compound BLF-16 is shown as the following formula:

[0007]

[0008] Use of a trifluoromethyl vinyl ester compound in the preparation of a drug for treating ulcerative colitis.

[0009] Furthermore, in addition to pharmaceutically acceptable salts, hydrates or solvates, the drug for treating ulcerative colitis further contains pharmaceutical excipients. The pharmaceutical excipients refer to conventional pharmaceutical excipients, such as one or a combination of two of solvents, disintegrants, suspending agents, preservatives, colorants and binders.

[0010] Furthermore, the dosage form of the drug for treating ulcerative colitis is selected from the following group: liquid preparations, solid preparations, semi-solid preparations, gas preparations.

[0011] Furthermore, the dosage form of the drug for treating ulcerative colitis is selected from the following group: oral dosage forms, such as capsules, tablets, powders, granules, oral liquids; topical dosage forms for the skin, nasal mucosa dosage forms, rectal dosage forms.

[0012] Furthermore, solution-type, colloid-type, emulsion-type, and suspension-type preparations prepared from the drug for treating ulcerative colitis.

[0013] Furthermore, the drug for treating ulcerative colitis is a drug for improving clinical symptoms of the disease, such as improving body weight, reducing the disease activity index, and increasing the colon length.

[0014] Furthermore, the drug for treating ulcerative colitis is a drug for reducing the level of pro-inflammatory factors and increasing the level of anti-inflammatory cytokines.

[0015] Furthermore, the drug for treating ulcerative colitis has the effect of reducing the expression of inflammation-related proteins such as inducible NO synthase, matrix metalloproteinase, and pro-inflammatory interleukin.

[0016] Furthermore, the trifluoromethyl vinyl ester compound can be formulated into a 0.5% sodium carboxymethylcellulose suspension.

[0017] Furthermore, the preparation method of the sodium carboxymethylcellulose suspension of the trifluoromethyl vinyl ester compound is as follows: Weigh 5 g of CMC-Na, take 1000 mL of deionized water, add CMC-Na in small amounts several times, and stir while adding to obtain a 0.5% sodium carboxymethylcellulose (CMC-Na) solution; Weigh 10 mg of BLF-16 and dissolve it thoroughly in 0.1 mL of the 0.5% CMC-Na solution to obtain a solution with a concentration of 100 mg / mL, store it in a laboratory low-temperature refrigerator at 4 °C. The solution is stable and effective within 14 days, and repeated freezing and thawing should be avoided.

[0018] Furthermore, the method for using the sodium carboxymethyl cellulose suspension of the trifluoromethyl vinyl ester compound is as follows: daily intragastric administration to mice, with a dose of 10 mg / kg / d, a dosing volume of 0.1 mL / 20 g, once a day, administered one day in advance, and continuously administered for 7 days.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] (1) The present invention uses compound BLF-16 as the active ingredient.

[0021] (2) Compound BLF-16 has good in vitro and in vivo anti-ulcerative colitis activity, shows dose-dependent inhibition of the production of lipopolysaccharide-induced macrophage inflammatory factors, and has good medicinal prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Effect of compound BLF-16 on the body weight change of DSS (dextran sulfate sodium)-induced BALB / c mice, *P < 0.05, compared with the DSS group;

[0023] Figure 2 Effect of compound BLF-16 on the DAI index change of DSS (dextran sulfate sodium)-induced BALB / c mice, *P < 0.05, compared with the DSS group;

[0024] Figure 3 Effect of compound BLF-16 on the colon length change of DSS (dextran sulfate sodium)-induced BALB / c mice, *P < 0.05, compared with the DSS group;

[0025] Figure 4 Effect of compound BLF-16 on the organ index of DSS (dextran sulfate sodium)-induced BALB / c mice, *P < 0.05, compared with the DSS group;

[0026] Figure 5 Effect of compound BLF-16 on the IL-4 content in the colon tissue of DSS (dextran sulfate sodium)-induced BALB / c mice, *P < 0.05, compared with the DSS group;

[0027] Figure 6 Effect of compound BLF-16 on the IL-1β content in the colon tissue of DSS (dextran sulfate sodium)-induced BALB / c mice, *P < 0.05, compared with the DSS group;

[0028] Figure 7 Effect of compound BLF-16 on the iNOS protein expression in the colon tissue of DSS (dextran sulfate sodium)-induced BALB / c mice, *P < 0.05, compared with the DSS group;

[0029] Figure 8 、 Figure 9 Effect of compound BLF-16 on the expression of IL-1β protein in the colon tissues of DSS (dextran sulfate sodium)-induced BALB / c mice;

[0030] Figure 10 Effect of compound BLF-16 on the expression of MMP-2 protein in the colon tissues of DSS (dextran sulfate sodium)-induced BALB / c mice;

[0031] Figure 11 Effect of compound BLF-16 on the viability of LPS (lipopolysaccharide)-induced RAW264.7 macrophages;

[0032] Figure 12 Effect of compound BLF-16 on the NO release rate of LPS (lipopolysaccharide)-induced RAW264.7 macrophages, *P < 0.05, compared with the LPS group;

[0033] Figure 13 Effect of compound BLF-16 on the expression of iNOS protein in LPS (lipopolysaccharide)-induced RAW264.7 macrophages, *P < 0.05, compared with the LPS group;

[0034] Figure 14 Effect of compound BLF-16 on the secretion of IL-1β by LPS (lipopolysaccharide)-induced RAW264.7 macrophages, *P < 0.05, compared with the LPS group;

[0035] Figure 15 Effect of compound BLF-16 on the immunofluorescence localization of iNOS in LPS (lipopolysaccharide)-induced RAW264.7 macrophages. The iNOS fluorescence staining was enhanced in the LPS model group, and the expression of iNOS in the BLF-16 treatment group was weakened compared with the LPS group. Detailed implementation mode

[0036] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments, but the specific embodiments cited do not limit the present invention in any way.

[0037] The test methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials are all commercially available unless otherwise specified.

[0038] I. Preparation of the suspension preparation of compound BLF-16 for gastrointestinal administration

[0039] Weigh 5 g of CMC-Na, take 1000 mL of deionized water, add CMC-Na in small amounts multiple times while stirring, and a 0.5% sodium carboxymethyl cellulose (CMC-Na) solution can be obtained.

[0040] Weigh 10 mg of BLF-16 and dissolve it thoroughly in 0.1 mL of 0.5% CMC-Na solution to obtain a solution with a concentration of 100 mg / mL. Place it in a laboratory low-temperature refrigerator at 4°C for storage. The solution is stable and effective within 14 days, and repeated freezing and thawing should be avoided.

[0041] II. Intragastric administration of compound BLF-16 preparation improves dextran sulfate sodium (DSS)-induced ulcerative colitis in mice.

[0042] Experimental materials

[0043] (1) Reagents: DSS (dextran sulfate sodium) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0044] (2) Animals: Specific pathogen-free (SPF) male BALB / c mice, weighing 18–22 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0045] (3) Instruments: Tabletop high-speed refrigerated centrifuge, Thermo Fisher Scientific; Analytical balance, Mettler Toledo Instruments Co., Ltd.

[0046] (4) Drug solutions: 0.5% sodium carboxymethyl cellulose suspension of BLF-16; 4% DSS aqueous solution.

[0047] Animal experiment method: Fast the mice for 24 h without water restriction before the experiment. Divide 15 mice into 3 groups evenly, with 5 mice in each group, namely the control group (control group), the DSS-induced group (DSS group), and the BLF-16 treatment group (BLF-16 group). The DSS group and the BLF-16 group were given free access to 4% DSS in drinking water for 7 days. The blank control group was given ordinary tap water throughout the experiment. The BLF-16 group was administered intragastrically daily at a dose of 10 mg / kg / d, and the administration volume was 0.1 mL / 20 g, once a day, and the drug was administered one day in advance. Weigh the mice daily, measure relevant data, sacrifice the mice on the 8th day of the experiment, and take samples for relevant experiments.

[0048] Example 1: BLF-16 improves DSS (dextran sulfate sodium)-induced ulcerative colitis in mice.

[0049] Experimental method: Weigh the mice in each group daily during the animal experiment and draw a weight change curve; calculate the disease activity index (DAI) of the mice daily and measure the colon length of each group of mice after sacrificing and taking samples at the end of the experiment.

[0050] Experimental results: The mice in the BLF-16 treatment group had the least weight loss, indicating that BLF-16 could inhibit the weight loss caused by DSS-induced ulcerative colitis( Figure 1 ). The mice in the BLF-16 treatment group had the lowest disease activity index (DAI), suggesting that BLF-16 could inhibit the disease activity of DSS-induced ulcerative colitis( Figure 2 ). The colon length of the mice in the BLF-16 treatment group was longer than that of the mice in the DSS-induced ulcerative colitis group, indicating that BLF-16 inhibited the colon shortening caused by DSS-induced ulcerative colitis. The differences in the effects among the normal mice, DSS-induced mice, and BLF-16-treated mice were statistically significant( Figure 3 ). The organ index of the mice in the BLF-16 group showed the same trend as that of the control group mice and the opposite trend as that of the model group mice, indicating that BLF-16 could enhance the immunity of mice( Figure 4 ).

[0051] Example 2: BLF-16 inhibits the production of inflammatory factors in mice with DSS (dextran sulfate sodium)-induced ulcerative colitis.

[0052] (1) BLF-16 inhibits the production of the pro-inflammatory factor IL-1β and promotes the production of the anti-inflammatory factor IL-4 in the serum of mice with dextran sulfate sodium-induced ulcerative colitis.

[0053] Experimental method: At the end of the animal experiment, blood was collected by enucleating the eyeballs and placed in a 1.5 mL EP tube. After standing for 30 min, it was centrifuged at 1500 rpm for 15 min, and the supernatant was taken. According to the instructions, the contents of IL-1β and IL-4 in the serum were measured using an ELISA kit.

[0054] Experimental results: The ELISA experimental results showed that in the DSS-induced mice treated with BLF-16, the content of IL-1β in the serum was lower than that in the DSS-induced mice, and the content of IL-4 was higher than that in the DSS-induced mice. There were statistically significant differences among the groups.

[0055] The experiment showed that BLF-16 could effectively inhibit the increase in IL-1β induced by DSS, indicating that BLF-16 could inhibit the rise of pro-inflammatory factors during ulcerative colitis; BLF-16 could effectively inhibit the decrease in IL-4 induced by DSS, indicating that BLF-16 could promote the rise of anti-inflammatory factors during ulcerative colitis( Figure 5-6 ).

[0056] Example 3: BLF-16 can reduce the expression of inflammation-related proteins iNOS, IL-1β, and MMP-2 in the colon tissue of mice with ulcerative colitis.

[0057] Experimental method: At the end of the animal experiment, the colon tissues of mice in each group were taken, and the colon tissues were lysed with tissue grinding solution. The protein concentration was measured using the BCA protein assay. Equal amounts of protein (30 - 55 μg) were separated by 8 - 15% SDS - PAGE gel and then blotted onto a polyvinylidene fluoride membrane (PVDF). The PVDF membrane was blocked with 5% non - fat milk in TBST and incubated with anti - iNOS, IL - 1β, MMP - 2 antibodies (diluted 1:1000) and β - actin (diluted 1:10000) overnight at 4°C. The antibodies were detected for 1 hour with horseradish peroxidase (HRP) - conjugated secondary antibodies (diluted 1:5000), and the antibodies were observed using ECL luminescent solution (Beyotime). The band intensity was measured using ImageJ software.

[0058] Experimental results: BLF - 16 decreased the expression of inflammation - related proteins iNOS, IL - 1β, and MMP - 2. The experimental results showed that LF - 16 inhibited ulcerative colitis by decreasing the expression of inflammation - related proteins iNOS, IL - 1β, and MMP - 2 in colon tissues ( Figure 7-10 ).

[0059] Example 4: Compound BLF - 16 inhibits the inflammatory response of LPS (lipopolysaccharide) - induced RAW264.7 macrophages.

[0060] Materials and instruments

[0061] (1) Materials: Compound BLF - 16 was synthesized by the chemical laboratory itself; mouse RAW264.7 macrophage cell line was purchased from Cyagen Biosciences (Shanghai) Co., Ltd.; DMEM medium was purchased from Beijing Solarbio Science & Technology Co., Ltd.; fetal bovine serum (FBS) was purchased from Dingguo Biological Co., Ltd.; lipopolysaccharide (LPS) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; thiazolyl blue (MTT) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; nitric oxide (NO) detection kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; iNOS antibody was purchased from Abbkine Biological Technology Co., Ltd.; mouse IL - 1β ELISA kit was purchased from Andygene Biological Technology Co., Ltd.; β - actin antibody was purchased from Bioworld Biological Technology Co., Ltd.; HRP - goat anti - rabbit secondary antibody was purchased from Abbkine Biological Technology Co., Ltd.; HRP - goat anti - mouse secondary antibody was purchased from Abbkine Biological Technology Co., Ltd.

[0062] (2) Instruments: Analytical balance, Mettler Toledo Instruments Co., Ltd.; Constant temperature water bath, Shanghai Yiheng Scientific Instruments Co., Ltd.; Desktop high-speed refrigerated centrifuge, Thermo Fisher Scientific; Carbon dioxide cell incubator, Thermo Fisher Scientific; Microplate reader, Thermo Fisher Scientific; Electrophoresis apparatus, Beijing Liuyi Biotechnology Co., Ltd.; Developing cassette, Guangdong Yuehua Medical Devices Factory Co., Ltd.; 96-well plate, Beijing Solarbio Science & Technology Co., Ltd.

[0063] Cell culture: RAW264.7 macrophages were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 μg / mL penicillin. Cells in the logarithmic phase were seeded at a density of 5×10 4 cells / well in cell culture dishes (100 mm) and cultured overnight in a humidified incubator at 37°C and 5% CO2 for subsequent experiments.

[0064] Drug concentration treatment: Compound BLF-16 was dissolved in 0.1% cell-grade dimethyl sulfoxide to prepare non-toxic concentrations for testing its performance. The concentrations of BLF-16 were 25 μmol / L, 50 μmol / L, and 100 μmol / L.

[0065] Example 5: Verification of the non-toxicity of BLF-16 to cells.

[0066] Experimental method: When RAW264.7 cells grew to the logarithmic growth phase, they were seeded at a density of 1×10 4 cells / well into 96-well culture plates, and 200 μL of cell suspension was added to each well. Six replicates were set for each group, and each experiment was repeated at least 3 times. After culturing for 12 h, the original medium was discarded. A normal control group (DMEM medium), an inflammatory model group (LPS 1 μg / mL), and drug treatment groups of BLF-16 (25, 50, 100 μmol / L) were set up respectively. When RAW264.7 cells grew to the logarithmic phase, BLF-16 was added to the cells and pre-incubated for 2 h, then LPS (lipopolysaccharide) 1 μg / mL was added, and after continued culturing for 24 h, 20 μL of MTT (5 mg / mL) solution was added to each well, and the cells were incubated in the cell incubator for another 4 h. The supernatant was discarded, 150 μL of DMSO was added to each well, and the mixture was placed on an oscillator to completely dissolve the crystals. The absorbance value was measured at 490 nm.

[0067] According to the formula:

[0068]

[0069] Experimental results: The effects of different concentrations of BLF-16 on the viability of RAW264.7 macrophages showed no significant differences, indicating that BLF-16 was non-toxic to RAW264.7 macrophages ( Figure 11)。

[0070] Example 6: BLF-16 dose-dependently inhibits the production of NO in LPS-induced RAW264.7 macrophages.

[0071] Experimental method: The content of N0 was detected by the Griess method. When RAW264.7 cells grew to the logarithmic growth phase, they were inoculated into a 96-well culture plate at a density of 1X10 4 / well, and 200 μL of cell suspension was added to each well. Three replicate wells were set for each group, and each experiment was repeated at least 3 times; after culturing for 12 h, the original culture medium was discarded. After adding BLF-16 to the cells and pre-incubating for 2 h, 1 μg / mL of LPS (lipopolysaccharide) was added, and the cells were cultured for another 24 h. After the culture was completed, the supernatant of the cell culture fluid was collected, and the content of NO was measured using a NO detection kit. 50 μL of the supernatant was spread on a 96-well plate, and an equal amount of Griess reagent was added thereto, and then the absorbance at 540 nm was measured using an enzyme-linked immunosorbent assay reader. A standard curve (0-100 μM) was drawn using sodium nitrite (NaN02) standard, and the content of NO was calculated according to the standard curve.

[0072] Experimental results: BLF-16 can significantly inhibit the secretion of NO in the supernatant of LPS-induced RAW264.7 macrophages, and shows a dose-dependent manner ( Figure 12 )。

[0073] Example 7: BLF-16 dose-dependently inhibits the expression of the inflammation-related protein iNOS in LPS-induced RAW264.7 macrophages.

[0074] Experimental method: RAW264.7 cells were washed twice with PBS after being treated with BLF-16 (25-100 μM) in the presence of LPS (1 μg / mL) for 24 h, and then lysed in ice-cold RIPA lysis buffer supplemented with 1% protease inhibitor. The protein concentration was measured using the BCA protein assay. Equal amounts of protein (30-55 μg) were separated by 8-15% SDS-PAGE gel and then blotted onto a polyvinylidene difluoride membrane (PVDF). The PVDF membrane was blocked with 5% non-fat milk in TBST and incubated with anti-iNOS antibody (1:1000 dilution) and β-actin (1:10000 dilution) overnight at 4 °C. The antibody was detected with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution) for 1 h, and the antibody was observed using ECL luminescent solution (Beyotime). The band intensity was measured using ImageJ software.

[0075] Experimental results: BLF-16 can significantly inhibit the expression level of iNOS protein in LPS-induced RAW264.7 macrophages, and shows a dose-dependent manner ( Figure 13)。

[0076] Example 8: BLF-16 dose-dependently inhibits the secretion of inflammatory cytokine IL-1β induced by LPS in RAW264.7 macrophages.

[0077] Experimental method: When RAW264.7 cells grew to the logarithmic phase, they were inoculated in 96-well plates at a density of 1×10 4 cells / well, with 200 μL of cell suspension in each well. At least 3 replicates were set for each group, and each experiment was repeated at least 3 times. After culturing for 12 h, the original culture medium was discarded. After adding BLF-16 to the cells and pre-incubating for 2 h, 1 μg / mL of LPS (lipopolysaccharide) was added, and the cells were cultured for another 24 h. After the culture was completed, the supernatant of the cell culture medium was collected, and the content of cytokine IL-1β was measured using an IL-1β ELISA detection kit. According to the instructions of the ELISA kit, the content of the cytokine was detected.

[0078] Experimental results: BLF-16 can significantly inhibit the secretion of inflammatory factor IL-1β in the supernatant of RAW264.7 macrophages induced by LPS (lipopolysaccharide), and shows a dose-dependence ( Figure 14 )。

[0079] Example 9: BLF-16 dose-dependently inhibits the immunofluorescence expression of inflammatory-related protein iNOS induced by LPS in RAW264.7 macrophages.

[0080] Experimental method: The localization of iNOS protein in RAW264.7 cells. When RAW264.7 cells grew to the logarithmic growth phase, at 1×10 5Inoculate at a density of cells per well into a 6-well culture plate with glass slides, add 2 mL of cell suspension to each well, set 3 replicate wells for each group, and repeat each experiment at least 3 times. After culturing for 12 h, discard the original culture medium. Add BLF-16 to the cells and pre-incubate for 2 h, then add 1 μg / mL of LPS (lipopolysaccharide) and continue culturing for 24 h. After the culture is completed, remove the cell supernatant. Gently wash the 6-well plate with the glass slides on which the cells have adhered well 3 times with PBS for 1 min each time. Fix the slides with 4% paraformaldehyde for 20 min, wash the slides with PBS 3 times for 3 min each time. Permeabilize with 0.5% Triton X-100 (prepared with PBS) at room temperature for 20 min, wash the slides with PBS 3 times for 3 min each time. Drop 2% BSA on the slides and block at room temperature for 30 min. Remove the blocking solution without washing, drop a sufficient amount of diluted primary antibody (iNOS 1:1000) on each slide, and incubate overnight at 4°C. Remove the primary antibody, wash the slides with PBS 3 times for 3 min each time, drop the diluted fluorescent secondary antibody, add the fluorescent secondary antibody, and incubate on a shaker at room temperature for 1 h (pay attention to avoiding light). Wash the sections with PBS 3 times for 3 min each time. (Note: From the addition of the fluorescent secondary antibody, all subsequent operation steps should be carried out as much as possible in a darker place.) Drop DAPI and incubate in the dark for 5 min to stain the nuclei of the specimens, and wash away the excess DAPI with PBS 3 times for 3 min each time. Blot the liquid on the slides with absorbent paper, mount the slides with a mounting medium containing an anti-fluorescence quencher, and then observe and collect images under a fluorescence microscope.

[0081] Experimental results: BLF-16 can significantly inhibit the expression level of iNOS protein in LPS (lipopolysaccharide)-induced RAW264.7 macrophages, and shows a dose-dependent manner ( Figure 15 ).

[0082] The content not described in detail in the specification of the present invention belongs to the prior art well-known to those skilled in the art. Although the illustrative specific embodiments of the present invention are described above for the convenience of those skilled in the art to understand the present invention, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art in this technical field, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

Claims

1. Use of a trifluoromethyl vinyl ester compound in the preparation of an anti-ulcerative colitis drug, characterized in that, The trifluoromethyl vinyl ester compound is BLF-16, and its structure is shown as follows:

2. Use of a trifluoromethyl vinyl ester compound as described in claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that, The anti-ulcerative colitis drug further comprises a pharmaceutical excipient.

3. Use of a trifluoromethyl vinyl ester compound as described in claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that, The anti-ulcerative colitis drug is any one of liquid preparations, solid preparations, semi-solid preparations, and gas preparations.

4. Use of a trifluoromethyl vinyl ester compound as described in claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that, The dosage form of the anti-ulcerative colitis drug is any one of oral dosage forms, skin dosage forms, nasal mucosa dosage forms, and rectal dosage forms.

5. Use of a trifluoromethyl vinyl ester compound as described in claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that, The clinical symptoms of the disease improved by the anti-ulcerative colitis drug include: improving body weight, reducing the disease activity index, and increasing the colon length.

6. Use of a trifluoromethyl vinyl ester compound as described in claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that, The anti-ulcerative colitis drug is a drug that reduces the level of pro-inflammatory factors and increases the level of anti-inflammatory cytokines.

7. Use of a trifluoromethyl vinyl ester compound as described in claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that, The anti-ulcerative colitis drug has the effect of reducing the expression of inflammation-related proteins: inducible NO synthase, matrix metalloproteinase, and pro-inflammatory interleukin.

8. Use of a trifluoromethyl vinyl ester compound as described in claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that, The trifluoromethyl vinyl ester compound is formulated into a 0.5% sodium carboxymethylcellulose suspension.

9. Use of a trifluoromethyl vinyl ester compound as described in claim 8 in the preparation of a drug for treating ulcerative colitis, characterized in that, The trifluoromethyl vinyl ester compound is a 0.5% sodium carboxymethylcellulose suspension, and its usage method in mice is: intragastric administration daily, with a dose of 10 mg / kg / d.

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