Application of gambogic acid in preparation of medicine for treating ulcerative colitis

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of gambogic acid in preparation of a medicine for treating ulcerative colitis. The gambogic acid provided by the invention can be used for preparing a programmed necrosis inhibitor and a medicine for treating ulcerative colitis, and the gambogic acid is beneficial to improving cell morphology, increasing cell viability and improving symptoms of ulcerative colitis caused by DSS. Chemical structure of gambogic acid: # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of gambogic acid in the preparation of a medicine for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory intestinal disease with an unclear etiology. It may be related to multiple factors such as genetics, immunity, environment, and infection. It is more common in people with a family history of the disease, long-term smokers, and those who eat a high-fat diet, and has a low mortality rate. Clinical symptoms include diarrhea, abdominal pain, mucus, pus, and blood in the stool. It is not contagious, and severe cases may be complicated by intestinal perforation, massive bleeding, and cancer. According to statistics, up to 25%-30% of UC patients require colectomy, and more than 20% of patients with inflammatory bowel disease will develop colitis-related colorectal cancer within 30 years. At present, UC treatment drugs mainly include aminosalicylic acid, hormones, immunosuppressants, etc., but there are problems such as high drug resistance, many adverse reactions, and increased tumor risk. Therefore, UC has become a social public medical problem that needs to be solved urgently, and it is of great significance to find new drugs that can effectively prevent and treat UC.

[0003] At present, the drugs used to prevent and treat ulcerative colitis mainly include aminosalicylic acid, glucocorticoids and biological agents, but there are adverse reactions such as drug dependence, cytotoxicity and liver and kidney damage, and relapse is easy after drug withdrawal. Traditional Chinese medicine has the advantages of stable efficacy, low recurrence rate, good patient compliance and few side effects, and has been widely used in clinical practice. Garcinia acid (GA) is a flavonoid compound that has been found in G. hanburyi resin and has anticancer activity. However, whether it has an effect on ulcerative colitis remains a blank. Summary of the invention

[0004] In order to develop a safe and effective drug for treating ulcerative colitis, the present invention provides the use of gambogic acid in the preparation of a drug for treating ulcerative colitis. In the present invention, gambogic acid can be used to prepare a programmed necrosis inhibitor and a drug for treating ulcerative colitis. Gambogic acid is beneficial for improving cell morphology, increasing cell activity, and improving the symptoms of ulcerative colitis caused by DSS.

[0005] The present invention provides the use of gambogic acid in the preparation of a drug for treating ulcerative colitis. The chemical structure of the gambogic acid is as follows:

[0006]

[0007] After treating the DSS-induced UC model in mice with garcinia acid, it was found that the model mice recovered significantly, the DAI score decreased, the blood in stool and stool quality improved, the colon length increased, the intestinal permeability decreased, the submucosal edema of the mouse colon was alleviated, the loss of mucosal epithelial cells and goblet cells decreased, the inflammatory infiltration improved, and the FITC fluorescence intensity showed that the intestinal permeability decreased, and its effect was equivalent to that of the Nec-1 group. It shows that garcinia acid treatment can significantly improve intestinal inflammation and intestinal barrier dysfunction caused by DSS. There is no significant effect on heart, liver, and kidney function. Garcinia acid treatment is safe and has no side effects. It can be used to prepare drugs for the treatment of ulcerative colitis.

[0008] Furthermore, the drug contains gambogic acid as the only active ingredient.

[0009] Furthermore, the gambogic acid is used to prepare a programmed necrosis inhibitor.

[0010] Furthermore, the gambogic acid is used to prepare a drug for treating ulcerative colitis caused by programmed necrosis.

[0011] Furthermore, the gambogic acid is used to prepare an inflammation inhibitor.

[0012] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the pharmaceutically acceptable excipient is any one or more combinations of sodium alginate, chitosan, polyvinyl alcohol, glutamine, formaldehyde dextran, adipic acid dihydrazide, Tween 60, lauramidopropyl betaine, gardenia glycoside and gardenia oil.

[0014] Furthermore, the dosage form of the drug includes any one of solution, powder, granule, tablet, capsule, aerosol, ointment or film.

[0015] Furthermore, the drug has the effects of inhibiting programmed necrosis of intestinal epithelial cells, improving the symptoms of ulcerative colitis caused by DSS, and improving the intestinal barrier dysfunction caused by DSS.

[0016] Furthermore, the improvement of the symptoms of ulcerative colitis caused by DSS is manifested as: restoring body weight, improving blood in stool and stool consistency, increasing colon length, reducing colon submucosal edema, improving inflammatory infiltration, and reducing intestinal permeability.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The garcinia acid in the present invention can be used to prepare a programmed necrosis inhibitor and a drug for treating ulcerative colitis. The garcinia acid is beneficial to improving the morphology of intestinal epithelial cells, increasing cell activity, and improving the symptoms of ulcerative colitis caused by DSS. The garcinia acid treats ulcerative colitis by inhibiting the "heat toxin" of intestinal epithelial cell necrosis, which is in line with the theory of "clearing heat toxins to eliminate evil" in traditional Chinese medicine.

[0019] After treatment with garcinia acid, the weight of the DSS-induced UC model mice recovered significantly, the DAI score decreased, the stool blood and stool quality improved, the colon length increased, the intestinal permeability decreased, the submucosal edema of the mouse colon was alleviated, the loss of mucosal epithelial cells and goblet cells decreased, the inflammatory infiltration improved, and the FITC fluorescence intensity showed that the intestinal permeability decreased, and its effect was equivalent to that of the Nec-1 group. Treatment with garcinia acid can significantly improve intestinal inflammation and intestinal barrier dysfunction caused by DSS. There was no significant effect on heart, liver, and kidney function. Treatment with garcinia acid is safe and has no side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 To evaluate the anti-necroptosis activity of garcinia acid in vitro;

[0022] In the figure, A shows the effect of gambogic acid on the morphology of HT-29 cells, wherein the first row shows the cell state at 0 h of TSZ induction, the second row shows the cell morphology at 12 h of TSZ induction, the first column shows DMSO treatment, and the second column shows treatment with 5 μM gambogic acid solution;

[0023] B shows the effects of different concentrations of gambogic acid on the cell activity of HT-29 cell necroptosis model induced by TSZ and the effects of different concentrations of gambogic acid on the cytotoxicity of HT-29 cells, where DMSO means that HT-29 cells were directly added with different concentrations of gambogic acid, and TSZ means that TSZ model was established and different concentrations of gambogic acid were added;

[0024] C is the curve of the half-effective dose of garcinia acid.

[0025] Figure 2 The effect of gambogic acid on the key proteins of necroptosis is p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, and MLKL;

[0026] In the figure, A shows the effects of different concentrations of gambogic acid on key proteins of necroptosis;

[0027] B shows the effects of different exposure times of garcinia acid on key proteins of necroptosis.

[0028] Figure 3 The effect of gambogic acid on ulcerative colitis in mice;

[0029] In the figure, A is a DSS-induced mouse ulcerative colitis model and drug administration flow chart;

[0030] B is the effect of gambogic acid on the body weight of ulcerative colitis model mice;

[0031] C is a representative picture of the effect of gambogic acid on the colon of ulcerative colitis model mice;

[0032] D is the effect of gambogic acid on the DAI score of ulcerative colitis model mice;

[0033] E is the effect of gambogic acid on the colon length of ulcerative colitis model mice on the 13th day;

[0034] F is the effect of gambogic acid on the FITC fluorescence intensity of ulcerative colitis model mice;

[0035] G is the effect of gambogic acid on colon pathology in ulcerative colitis model mice.

[0036] Figure 4 The effect of gambogic acid on intestinal inflammation and intestinal barrier function in ulcerative colitis model mice;

[0037] In the figure, A is an immunofluorescence analysis of the effect of gambogic acid on the distribution and expression of intestinal barrier protein ZO-1 in DSS-induced ulcerative colitis mice;

[0038] B is an immunofluorescence analysis of the effect of gamboge acid on the distribution and expression of intestinal barrier protein Occludin in DSS-induced ulcerative colitis mice

[0039] C is a representative PAS staining image of colon tissue under different treatments;

[0040] D is the distribution and expression of Muc-2 in colon tissue determined by immunofluorescence;

[0041] E is the effect of Western blot analysis on the expression of Zo-1 and Occludin;

[0042] F is the effect of gambogic acid on serum TNF-α content in DSS-induced ulcerative colitis mice determined by ELISA, **P<0.05 compared with the DSS group;

[0043] Figure 5The effect of gambogic acid on necroptosis of intestinal epithelial cells in ulcerative colitis model mice;

[0044] In the figure, A is the effect of protein and phosphorylation level expression of RIPK1 / 3 and MLKL, key proteins of necroptosis, analyzed by Western blot;

[0045] B is the distribution and expression of p-RIPK3 in colon tissue determined by immunofluorescence;

[0046] C: Immunofluorescence analysis of the distribution and expression of p-MLKL in colon tissue.

[0047] Figure 6 The effects of gambogic acid treatment on liver and kidney;

[0048] In the figure, A shows the effect of garcinia acid on alanine aminotransferase in mouse blood;

[0049] B is the effect of gambogic acid on aspartate aminotransferase in mouse blood

[0050] C is the effect of gambogic acid on serum creatinine in mice

[0051] D is the effect of gambogic acid on blood urea nitrogen in mice

[0052] E is the pathological effects of garcinia acid on the heart, liver and kidney of mice. DETAILED DESCRIPTION

[0053] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0054] Example 1: Application of gambogic acid in the preparation of a drug for treating ulcerative colitis.

[0055] 1. Experimental Materials and Methods

[0056] 1. Experimental Materials

[0057] (1) Experimental reagents: Dextran sulfate sodium was purchased from Beijing Coolbo Technology Co., Ltd., with a molecular weight of 36000-50000 and a product number of CD4421. Garcinia acid was purchased from Taosu Biochemical Technology Co., Ltd., with a product number of T6185, a purity of 98.38%, and a batch number of 150301. Necrostain-1, a necroptosis inhibitor, abbreviated as Nec-1, with a product number of S8037, was purchased from Selleck. Human-TNF-alpha was purchased from Suzhou Jinan Protein Technology Co., Ltd., with a product number of C008; SM-164 Hydrochloride was purchased from Med Chem Express, with a product number of HY-15989A; z-VAD-fmk was purchased from Taosu Biochemical Technology Co., Ltd., with a product number of T6013. Cell Signaling Technology provided anti-ripk1 with catalog number 3493S, anti-HSP70 with catalog number 4872, anti-EGFR with catalog number 4267, and human-specific anti-phospho-RIPK1 with catalog number 65746S. Hangzhou Bailing Biotechnology Co., Ltd. provided mouse-specific anti-phospho-RIPK1 with catalog number BX60008. Abcam provided anti-RIPK3 / p-RIPK3 with catalog number ab209384 and mouse-specific anti-phospho-RIPK3 with catalog number ab195117. RIPK3 polyclonal antibody with catalog number 17563-1-A and Anti-MLKL with catalog number 66675-1-Ig were obtained from Proteintech. Abcam provided Anti-MLKL with catalog number ab184718 and human-specific anti-phospho-MLKL with catalog number ab187091; mouse-specific anti-phospho-MLKL was from Beijing Bioson Biotechnology Co., Ltd. with catalog number bsm-54104R. Wuhan Saiweier Biotechnology Co., Ltd. provided Occludin with catalog number GB111401 and ZO-1 body with catalog number GB111402. Anti-GAPDH was purchased from Abcam with catalog number ab181602. BCA protein detection kit was purchased from Kangwei Century Biotechnology Co., Ltd. with catalog number CW0014S. CellTiter-Lumi TM The luminescence detection kit was purchased from Bio-Technology Co., Ltd.

[0058] (2) Experimental cells: HT-29 cell line was purchased from Wuhan Pronocell Life Science Co., Ltd., catalog number: CL-0118. Cells were cultured in growth medium (Hampnocell Life Science Co., Ltd., CM-0118) supplemented with 10% fetal bovine serum (BI, C04001-500) and 100 U / mL penicillin / streptomycin (Gibco, 15140-122) at 37°C and 5% CO2.

[0059] (3) Experimental animals: Animal experiments were approved by the Animal Care and Use Committee of Henan University of Traditional Chinese Medicine (IACUC-S202403126). Male C57BL / 6J mice aged 6–7 weeks were obtained from Liaoning Changsheng Biotechnology Co., Ltd. These mice were housed under temperature- and humidity-controlled conditions with a 12-h dark-light cycle and provided with a standard dry diet and tap water ad libitum.

[0060] 2. Effect of GA on TSZ-induced necroptosis in HT-29 cells

[0061] Evaluation of in vitro anti-necroptosis activity: HT-29 cells were seeded in 96-well plates, with 10,000 cells per well, and each group was repeated 3 times. DMSO group, TSZ group, DMSO+administration group (DMSO+GA) and TSZ+administration group (TSZ+GA) were set up, where DMSO+administration group and TSZ+administration group both included different concentrations of gambogic acid, which was dissolved in DMSO and diluted to concentrations of 5μM, 2.5μM, 1.25μM, 0.625μM, 0.3125μM, 0.15μM, and 0.075μM. After 12 hours, the cells in the TSZ group and TSZ+administration group were pretreated with 10nM SM-164 hydrochloric acid and 20μM caspase inhibitor z-VAD-fmk for 30 minutes, and then stimulated with 20ng / mL h-TNF-α for 12 hours. After 12 hours, the cell culture plate was taken out and equilibrated at room temperature for 10 minutes. Add 100 μl CellTiter-Lumi TM Luminescence detection reagent. Oscillate at room temperature for 2 minutes to promote cell lysis. Incubate at 25°C for 10 minutes to stabilize the luminescence signal. TM The cell viability was determined by luminescence detection kit, and the luminescence readings were recorded using SpectraMax M5 microplate reader. The cell activity was calculated according to formula (1), and the model was considered successful when the cell activity in the TSZ group was 10%-20%. The cytotoxicity was calculated according to formula (2). The cell status was observed and recorded using a fluorescence microscope. After calculating the cell activity of each drug-treated group, Prism software was used to calculate the EC of gambogic acid to improve TSZ-induced necroptosis in HT-29 cells. 50 value.

[0062] Formula (1): Cell activity = (TSZ group or TSZ+administration group) luminescence reading / (DMSO group) luminescence reading × 100%.

[0063] Formula (2): Cytotoxicity = [1-(DMSO+administration group) luminescence reading / (DMSO group) luminescence reading] × 100%.

[0064] 3. Effects of Garcinia Acid on Cell Necrotic Apoptosis Proteins

[0065] TSZ-induced necroptosis model of HT-29 cells was obtained. Garcinia acid was diluted with DMSO to 1.25 μM, 2.5 μM and 5 μM gambogic acid solutions, and then HT-29 cells were incubated with different concentrations of gambogic acid solutions for 0-6 h.

[0066] The effects of GA on the key proteins of necroptosis, p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, and MLKL, were determined by WB. The specific process is as follows:

[0067] (1) Protein sample preparation

[0068] Discard the supernatant of the treated cells and place them on ice. Wash twice with pre-cooled PBS, add an appropriate amount of cell lysis buffer, incubate on ice for 10 minutes, collect the cells with a cell scraper into a 1.5mL EP tube, and centrifuge at 4℃12000RPM for 5 minutes. Take the supernatant, add 5× protein electrophoresis buffer and boil for 7 minutes.

[0069] (2) Protein concentration determination

[0070] According to the instructions of the BCA protein concentration determination kit, set up the standard curve, add the sample to be tested, and set up one secondary well for each group. Add 200μL of BCA working solution to each well, incubate in a 55℃ shaking incubator for 15min, measure the absorbance at a wavelength of 562nm on an ELISA reader, and obtain the protein concentration based on the standard curve and the absorbance of each group. Calculate the loading volume of each group based on the equal amount of protein loading.

[0071] (3) Gel preparation

[0072] Place the washed and dried glass plate on the gel preparation rack, check for leaks, prepare the lower gel (8%-12%) according to the SDS-PAGE separation gel composition table, add 200μL of isopropanol to remove bubbles and flatten the gel surface. After the lower gel solidifies, absorb the isopropanol with absorbent paper, prepare the upper gel, and then insert the comb. After the gel solidifies, set aside.

[0073] (4) Electrophoresis

[0074] Load the sample according to the sample volume calculated according to the protein concentration, and first use 80V constant voltage electrophoresis until the protein marker runs away. After each group of samples runs to the same horizontal line, adjust the voltage to 120V.

[0075] (5) Transfer

[0076] Transfer the protein molecules on the SDS-PAGE gel to a solid phase carrier nitrocellulose filter membrane (NC membrane) that matches the gel size, cover the NC membrane with filter paper and sponge in turn, and then put it into the transfer tank. Add ice cubes to the black electrotransfer tank at the negative end of the gel side, and electrotransfer at a constant current of 250mA for 60-90min according to the molecular weight.

[0077] (6) Closed

[0078] The NC membrane after electrotransfer was terminated was taken out, and was quickly placed in Ponceau red dye solution for staining, and then rinsed with ultrapure water and placed in 5% skim milk for blocking for 30 minutes.

[0079] (7) Incubation with primary antibody

[0080] The membrane was washed with TBST solution on a shaker for 5 min × 4 times, and the primary antibody (anti-p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, MLKL antibody) was diluted in 1% skim milk, placed on a PVDF membrane corresponding to the molecular weight, and incubated overnight in a 4°C refrigerator with slow shaking.

[0081] (8) Incubation with secondary antibody and scanning

[0082] After incubation with enzyme-labeled secondary antibody for 1 h, the membrane was scanned using an imaging system (Bio-Rad, Hercules, USA), and the optical density of the bands was analyzed using Image-J software.

[0083] 4. Construction, grouping and drug administration of DSS-induced UC mouse model

[0084] Twenty-four 6-7 week old male C57BL / 6J mice weighing 18-22 g were selected and randomly divided into four groups, namely control group (Con), DSS treatment group (DSS), garcinia acid treatment group (GA), and Nec-1 treatment group (Nec-1), with 6 mice in each group. The 6 mice in the control group were given standard food and water, and the other groups were given 2.5% DSS by mass to induce UC for 7 consecutive days. After 7 days of DSS modeling, the 18 ulcerative colitis model mice that were successfully modeled were treated according to different groups, and the specific treatments were as follows:

[0085] Control group (Con): 0.2 mL of normal saline was administered intragastrically once a day for 7 days.

[0086] DSS-treated group (DSS): 0.2 mL of normal saline was administered intragastrically once a day for 7 days.

[0087] Garcinia acid treatment group (GA): 5 mg / kg of garcinia acid was intragastrically administered once a day for 7 days. The solvent of garcinia acid was 5% DMSO + 95% CMCna.

[0088] The specific operation is as follows: 10 mg of gambogic acid is dissolved in 1 ml of DMSO to prepare a mother solution, 100 uL of the mother solution is taken each time, and then 1900 uL of 0.5% CMCna is added to mix into a gambogic acid solution for intragastric administration.

[0089] Nec-1 treatment group (Nec-1): Nec-1 5 mg / kg was intraperitoneally injected once a day for 7 days. The solvent of Nec-1 was 5% DMSO + 95% saline;

[0090] The specific operation is as follows: 10 mg Nec-1 is dissolved in 1 ml DMSO to prepare a mother solution, 100 uL of the mother solution is taken each time, and then 1900 uL of normal saline is added to mix into a Nec-1 solution for intraperitoneal injection.

[0091] The "%" in 95% CMCna represents the volume fraction, and CMCna is sodium carboxymethyl cellulose; the "%" in 95% normal saline represents the volume fraction.

[0092] 5. Effect of Garcinia Acid on DSS-Induced Mouse UC Model

[0093] According to the above experimental process, the general conditions (such as activity, sensitivity of reaction, perianal condition, etc.) of the mice in the control group (Con), DSS-treated group (DSS), gambogic acid-treated group (GA) and Nec-1-treated group (Nec-1) were observed every day during the DSS modeling and drug administration. The body weight was measured, and the stool texture and occult blood in the stool were recorded to score the disease activity index (DAI) (Table 1).

[0094] Table 1 DAI scoring criteria

[0095]

[0096] On the 13th day of the experiment, the mice were euthanized and blood, colon, heart, liver and kidney tissue samples were collected. The blood was centrifuged at 3000RPM for 10 minutes and the supernatant was taken.

[0097] ① General condition: During the modeling and drug administration period, the general condition of the mice (such as activity, sensitivity of response, perianal condition, etc.) was observed every day, the body weight was weighed, the stool texture and occult blood in the stool were recorded for DAI scoring, and the colon length of the mice was measured and recorded.

[0098] ② Colon tissue pathology: Colon tissue of mice with more serious ulcers, about 1 cm, was obtained, fixed with 4% paraformaldehyde, routinely paraffin-embedded, sectioned, and stained with HE. Pathological changes were observed under an optical microscope and photographed.

[0099] 6. Effects of Garcinia Acid on Intestinal Barrier Function and Inflammation in Mice

[0100] ① Intestinal barrier function test: After fasting, mice were gavaged with 600 mg / kg of FITC-dextran (Sigma, FD4, molecular weight: 3000-5000) 4 hours before euthanasia. Blood samples were obtained and centrifuged, and then the supernatant was diluted with PBS at a ratio of 1:5. The SpectraMax M5 fluorescence method was then used to measure the FITC fluorescence intensity in the serum, with an excitation wavelength of 488 nm and an emission wavelength of 525 nm to evaluate the intestinal permeability of mice;

[0101] ② After the paraffin tissue was sectioned, dewaxed, transparent, and antigen repaired, it was blocked with BSA for 1 hour and incubated with primary antibodies (ZO-1, Occludin-1, Muc-2) at 4°C overnight. The expression and distribution of ZO-1 and Occludin-1 in colon tissue were detected by immunofluorescence technology. We commissioned Wuhan Saiweier Technology Co., Ltd. to perform the test. The specific process is as follows:

[0102] Dewax the paraffin sections to water: put the sections into environmentally friendly dewaxing solution I for 10 minutes-environmentally friendly dewaxing solution II for 10 minutes-environmentally friendly dewaxing solution III for 10 minutes-anhydrous ethanol I for 5 minutes-anhydrous ethanol II for 5 minutes-anhydrous ethanol III for 5 minutes-distilled water for washing; during the repair process, the buffer should be prevented from evaporating excessively and the sections should not be dried. After the repair is completed, cool naturally. Place the slides in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time; circle serum blocking: After the sections are slightly dried, use a tissue pen to draw circles around the tissue, add BSA (the primary antibody is from goat and is blocked with 10% donkey serum, and the primary antibody from other sources is blocked with 3% BSA), and block for 30 minutes. Add primary antibody: Add the prepared primary antibody (ZO-1, Occludin-1, Muc-2), and the sections are placed flat in a humidified box and incubated overnight at 4°C. Add secondary antibody: Place the slides in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time. Add the corresponding secondary antibody and incubate at room temperature in the dark for 50 minutes. DAPI counterstaining of cell nuclei: Place the slide in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time. Add DAPI staining solution and incubate at room temperature in the dark for 10 minutes. Quench tissue autofluorescence: Place the slide in PBS (PH7.4) and wash 3 times on a decolorizing shaker, 5 minutes each time. Add autofluorescence quencher B solution for 5 minutes and rinse with running water for 10 minutes. Seal: Seal with anti-fluorescence quenching sealant. Collect images: DAPI excitation wavelength 330-380nm, emission wavelength 420nm; 488 excitation wavelength 465-495nm, emission wavelength 515-555nm; CY3 excitation wavelength 510-560nm, emission wavelength 590nm; CY5 excitation wavelength 608-648nm, emission wavelength 672-712nm.

[0103] The results showed that the cell nucleus in the DAPI channel was blue and the positive cell nucleus in the CY3 channel was red.

[0104] ③ Intestinal inflammation level: ELISA method was used to detect the expression level of serum TNF-α inflammatory factor according to the kit instructions. The specific experimental steps are as follows:

[0105] a. Take out the kit before use and equilibrate to room temperature. Prepare the standard curve according to the instructions and dilute the analytical buffer and washing solution to the working concentration, and dilute the biotin conjugate or calibration diluent to the working concentration for later use;

[0106] b. Dilute the collected plasma 10-fold with sample buffer for later use;

[0107] c. Sample loading: Wash the sample plate 3 times with washing solution before loading. First, add 50 μL of sample buffer to each well, then add 50 μL of standard curve and diluted sample, and add 50 μL of biotin conjugate to each well. Cover the ELISA plate with film and place it in a shaking heater at 22℃ 400RPM for 2 hours to prepare HRP conjugate.

[0108] e. After 2 hours, wash 5 times with washing solution, add 100 μL of HRP conjugate to each well, cover the plate with film, and shake the heater at 22℃ 400RPM for 1 hour;

[0109] f. After 1 hour, wash 5 times with washing solution, cover the ELISA plate with 100 μL TMB matrix solution, and incubate at 22°C for 30 min;

[0110] g. After 30 min, 100 μL of stop solution was added and the absorbance at 450 nm was measured by an ELISA instrument. The TNF-α content of each group was calculated according to the standard curve.

[0111] ④ The mouse colon was placed in liquid nitrogen, and the colon tissue was ground using a grinder. The colon tissue was lysed in 1 mL of RIPA lysis buffer (CWBIO, Jiangsu, China), ground at 2000 rpm for 10 min, and centrifuged at 12000 rpm for 10 min at 4 ° C. The supernatant was the total protein, and the protein concentration was determined using the BCA protein assay kit (CWBIO, Jiangsu, China), and the loading buffer was boiled. Subsequently, the protein samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). Subsequently, the membrane was treated with 5% BSA for 2 hours and then incubated overnight at 4 ° C with different primary antibodies (ZO-1, Occludin, Muc-2) at a dilution of 1:1000. After incubation with enzyme-labeled secondary antibodies for 1 hour, the membrane was scanned using an imaging system (Bio-Rad, Hercules, USA), and the optical density of the bands was analyzed using Image-J software.

[0112] ⑤ Take colon tissue for PAS staining to detect changes in mouse goblet cells. We commissioned Wuhan Saiweier Technology Co., Ltd. to perform the test. The specific operation is as follows:

[0113] a. Dewaxing of paraffin sections: put the sections into environmentally friendly dewaxing solution I for 20 minutes, environmentally friendly dewaxing solution II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and 75% alcohol for 5 minutes, and then wash with tap water. Rewarming and fixing of frozen sections: take the frozen sections out of the -20° refrigerator and restore them to room temperature, fix them with tissue fixative for 15 minutes, and then rinse with running water.

[0114] b. Stain the sections in PAS staining solution B for 10-15 minutes, wash with tap water, and wash twice with distilled water;

[0115] c. Immerse the sections in PAS staining solution A for 25-30 minutes, protect from light, and rinse with running water for 5 minutes;

[0116] d. Stain the sections with PAS staining solution C for 30 seconds, wash with tap water, differentiate with hydrochloric acid solution, wash with tap water, blue with ammonia solution, and rinse with running water.

[0117] e. Dehydration and sealing: The slices were placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene for 5 min, and xylene II for 5 min to make them transparent, and then sealed with neutral gum.

[0118] f. Microscope examination, image acquisition and analysis.

[0119] 7. Effect of Garcinia Acid on Necroptosis of Mouse Intestinal Epithelial Cells

[0120] ① Wuhan Saiweier Technology Co., Ltd. was commissioned to conduct the test. After the paraffin tissue was sectioned, dewaxed, transparent, and antigen repaired, it was blocked with BSA for 1 hour and then incubated with primary antibodies (p-RIPK3, p-MLKL) at 4°C overnight. The expression and distribution of p-RIPK3 and p-MLKL in colon tissue were detected by immunofluorescence technology;

[0121] ② The mouse colon was placed in liquid nitrogen, and the colon tissue was ground using a grinder. The colon tissue was lysed in 1 mL of RIPA lysis buffer (CWBIO, Jiangsu, China), ground at 2000 rpm for 10 min, and centrifuged at 12000 rpm for 10 min at 4 ° C. The supernatant was the total protein, and the protein concentration was determined using the BCA protein assay kit (CWBIO, Jiangsu, China), and the loading buffer was boiled. Subsequently, the protein samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). Subsequently, the membrane was treated with 5% BSA for 2 hours and then incubated overnight at 4 ° C with different primary antibodies (p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, MLKL) at a dilution of 1:1000. After incubation with secondary antibodies for 1 hour, the membrane was scanned using an imaging system (Bio-Rad, Hercules, USA), and the optical density of the bands was analyzed using Image-J software.

[0122] 8. Effects of Garcinia Acid on the Heart, Liver and Kidney of DSS-Induced UC Mice

[0123] The DSS-induced ulcerative colitis model was used. After the mice were euthanized, the heart, liver and kidney tissues of the mice were fixed with 4% paraformaldehyde, embedded in paraffin, sliced, and stained with hematoxylin and eosin (HE staining). The blood liver function (blood alanine aminotransferase, aspartate aminotransferase) and renal function (creatinine, urea nitrogen) levels of the mice were detected using a kit (Nanjing Jiancheng Kit).

[0124] 2. Experimental Results

[0125] 1. Effect of gambogic acid on TSZ-induced necroptosis activity of HT-29 cells

[0126] The results are as follows Figure 1 As shown in the results, garcinia acid can improve cell swelling and rupture morphology, and inhibit the expression of key proteins of programmed necrosis in a dose- and time-dependent manner. Figure 1 As shown in A, HT-29 cells showed swelling and rupture after TSZ stimulation, while intervention with gambogic acid improved cell morphology. At the same time, gambogic acid significantly increased cell activity in a dose-dependent manner. EC 50 =1.48μM( Figure 1 B and Figure 1 C); in addition, garcinia acid has no significant effect on the cell activity of human colon cells, and at an effective dose, garcinia acid has no cytotoxicity.

[0127] 2. Effect of Garcinia Acid on Cell Necroptosis

[0128] TSZ-induced necroptosis model of HT-29 cells was obtained, and garcinia acid was diluted with DMSO to 1.25μM, 2.5μM and 5μM garcinia acid solutions, and then HT-29 cells were incubated with different concentrations of garcinia acid solutions for 0-6h. The effect of GA on the key proteins of necroptosis p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, and MLKL was determined by WB.

[0129] The results are as follows Figure 2 As shown, garcinia acid inhibited necroptosis in a dose- and time-dependent manner.

[0130] 3. Protective effect of garcinia acid on DSS-induced ulcerative colitis (UC) model in mice

[0131] To confirm the protective effect of GA on ulcerative colitis, a DSS-induced ulcerative colitis model in mice was used.

[0132] The results are as follows Figure 3 As shown in the results, gambogic acid can significantly improve the symptoms of ulcerative colitis induced by DSS, and the weight of mice treated with gambogic acid was significantly restored compared with the model group ( Figure 3B), the DAI score decreases ( Figure 3 D), blood in stool and stool consistency improved, colon length increased ( Figure 3 C and Figure 3 HE staining results showed that the submucosal edema of the mouse colon was reduced, the loss of mucosal epithelial cells and goblet cells was reduced, and the inflammatory infiltration was improved ( Figure 3 G), FITC fluorescence intensity indicates decreased intestinal permeability ( Figure 3 F), its effect was equivalent to that of the Nec-1 group.

[0133] 4. Effects of Garcinia Acid on Intestinal Inflammation and Intestinal Barrier Function in UC Model Mice

[0134] The results are as follows Figure 4 As shown in Figure 2, the expression and distribution of ZO-1 and Occludin-1 in colon tissue increased after treatment with garcinia acid ( Figure 4 A and Figure 4 B), PAS results showed an increase in colonic goblet cells ( Figure 4 C), the serum inflammatory factor TNF-α content decreased ( Figure 4 F), while increasing the distribution and expression of intestinal mucus protein MUC-2 ( Figure 4 D), indicating that garcinia acid can significantly improve intestinal inflammation and intestinal barrier dysfunction caused by DSS.

[0135] 5. Effect of Garcinia Acid on Necroptosis of Intestinal Epithelial Cells in UC Model Mice

[0136] A DSS-induced mouse ulcerative colitis model was used. Western blotting was used to determine the expression of key proteins in colonic necroptosis, and immunofluorescence was used to determine the distribution and expression of p-RIPK3 and p-MLKL.

[0137] The results are as follows Figure 5 As shown in the figure, compared with the DSS model group, GA can reduce the expression of p-RIPK3 and p-MLKL in colon tissue ( Figure 5 A) and distribution ( Figure 5 B and Figure 5 C).

[0138] 6. The effects of garcinia acid treatment on the liver and kidneys.

[0139] The results are as follows Figure 6 As shown in the results, the serum liver function and kidney function of mice were tested, and the results showed that the levels of alanine aminotransferase, aspartate aminotransferase, creatinine and urea nitrogen in the serum of mice after administration of garcinia acid were not significantly different from those of the control group (P>0.05). HE results showed that after administration of garcinia acid, there was no obvious pathological damage to the heart, liver and kidney of mice. Garcinia acid treatment is safe and has no side effects.

[0140] In summary, Garcinia acid has the effects of inhibiting programmed necrosis, improving the symptoms of DSS-induced ulcerative colitis, and improving the intestinal barrier dysfunction caused by DSS. Improving the symptoms of DSS-induced ulcerative colitis includes: restoring the weight of mice, improving blood in stool and stool quality, increasing colon length, reducing submucosal edema in the colon of mice, improving inflammatory infiltration, and reducing intestinal permeability. In addition, Garcinia acid treatment has no significant effect on heart, liver, and kidney function, and is safe and has no side effects.

[0141] It should be noted that GA (5 mg / kg) in the accompanying drawings of the specification refers to the gambogic acid-treated group in the present invention.

[0142] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0143] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of gambogic acid in the preparation of a drug for treating ulcerative colitis, characterized in that: The chemical structure of the garcinia acid is as follows:

2. The use of gambogic acid according to claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that: The drug contains gambogic acid as the only active ingredient.

3. The use of gambogic acid according to claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that: The gambogic acid is used for preparing a programmed necrosis inhibitor.

4. The use of gambogic acid according to claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that: The gambogic acid is used for preparing medicine for treating ulcerative colitis caused by programmed necrosis.

5. The use of gambogic acid according to claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that: The gambogic acid is used for preparing inflammation inhibitor.

6. The use of gambogic acid according to claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that: The drug also includes pharmaceutically acceptable excipients.

7. The use of gambogic acid according to claim 6 in the preparation of a drug for treating ulcerative colitis, characterized in that: The pharmaceutically acceptable excipient is any one or more combinations of sodium alginate, chitosan, polyvinyl alcohol, glutamine, formaldehyde dextran, adipic acid dihydrazide, Tween 60, lauramide propyl betaine, gardenia glycoside and gardenia oil.

8. The use of gambogic acid according to claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that: The dosage form of the drug includes any one of solution, powder, granule, tablet, capsule, aerosol, ointment or film.

9. The use of gambogic acid according to claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that: The drug has the effect of inhibiting programmed necrosis of intestinal epithelial cells, and also has the effect of improving the symptoms of ulcerative colitis caused by DSS and improving the intestinal barrier dysfunction caused by DSS.

10. The use of gambogic acid according to claim 9 in the preparation of a drug for treating ulcerative colitis, characterized in that: The improvement of the symptoms of ulcerative colitis caused by DSS is manifested in: improving blood in stool and stool consistency, increasing colon length, improving inflammatory infiltration, and reducing intestinal permeability.

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