Application of GLS1 inhibitor in relieving ulcerative colitis

Abstract

The invention relates to the technical field of biological medicine, and discloses application of a GLS1 inhibitor BPTES in relieving ulcerative colitis and a medicine for relieving ulcerative colitis, and the active ingredient of the medicine comprises BPTES. The invention proves that the inhibitor BPTES of the glutaminase 1 (GLS1) can obviously relieve clinical symptoms of mouse ulcerative colitis, improve intestinal mucosal barrier damage, reduce the level of inflammation in a mouse body and inhibit excessive activation of a Wnt / beta-catenin signal channel; likewise, the regulation of glutamine metabolism is expected to become a new treatment direction of UC.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to the use of a GLS1 inhibitor in alleviating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a digestive tract disease that occurs in the colorectal area, has insidious symptoms, and has a high recurrence rate and canceration rate. It mainly invades the colon mucosa, causing mucosal damage and intestinal histological changes. It is generally believed that the pathogenesis of UC is related to multiple factors such as environment, diet, intestinal flora imbalance, immune response disorder, mucosal and epithelial barrier defects, and genetic tendency. However, the initial driving factors of UC are still unclear. It may be driven by intestinal epithelial barrier dysfunction leading to intestinal mucosal inflammatory response, or driven by intestinal mucosal lamina propria inflammatory response, which leads to intestinal epithelial barrier destruction. Ultimately, the two promote each other and cause UC disease to continue to progress.

[0003] Under normal circumstances, the intestinal barrier function is mainly maintained by the intestinal epithelial barrier and the mucus barrier on top of the intestinal epithelial cells. The number of goblet cells in the colonic epithelium of UC patients decreases, resulting in reduced mucin synthesis, and then reduced mucus barrier thickness and mucus barrier dysfunction. After the mucus barrier is destroyed, the direct interaction between the intestinal flora and the intestinal epithelium increases, leading to intestinal epithelial cell apoptosis and reduced synthesis of tight junctions (TJs) between epithelial cells, such as ZO-1, occludin and claudins, and then intestinal epithelial barrier damage. In addition to reduced synthesis, structural abnormalities of TJs-related proteins are also involved in the process of intestinal epithelial barrier destruction in UC patients. When the intestinal epithelial barrier is destroyed and excessive intestinal content antigens enter the lamina propria, the lamina propria tolerance mechanism fails, the pro-inflammatory response of immune cells is no longer inhibited, and chemokines are secreted to recruit immune cell infiltration, thereby exacerbating the inflammatory response process.

[0004] The Wnt / β-catenin signaling pathway is a widely functional regulatory network that plays an important role in many different types of cancer and metabolic diseases. The β-catenin degradation complex is composed of APC, Axin, casein kinase 1 (CK-1) and glycogen synthase kinase (GSK-3β), which is essential for the phosphorylation of β-catenin and subsequent degradation by the ubiquitin-proteasome pathway. When Wnt is not activated, the complex rapidly degrades free β-catenin in the cytoplasm, keeping it at a low level; when the Wnt pathway is activated, the degradation complex is inhibited, β-catenin in the cytoplasm accumulates and moves into the nucleus to form a complex with TCF / LEF transcription factors, inducing the transcription of downstream genes such as c-Myc and Cyclin D1. Dejure et al. reported that c-Myc is a key transcription factor that promotes glutamine metabolism in HCT116 colon cancer cells. In addition, c-Myc also enhances mitochondrial GLS expression and glutamine metabolism in B lymphoma cells, human prostate cancer cells and lymphoblasts.

[0005] Studies have shown that the renewal of the intestinal epithelial barrier is regulated by the Wnt / β-catenin signaling pathway. The Wnt signaling pathway is involved in the regulation of multiple physiological and pathological processes, including embryonic development, cell proliferation, and tissue repair. In intestinal tissue, Wnt protein is a necessary condition for intestinal stem cell proliferation and epithelial cell renewal. Blocking the Wnt signal transmitted to the intestinal mucosal crypts in mice leads to the inhibition of intestinal stem cell proliferation, accompanied by damage to the intestinal epithelial barrier. It is worth noting that the continuous activation of the Wnt pathway can lead to excessive proliferation of epithelial cells and increase the risk of colorectal adenomas or colorectal cancer, which is consistent with the characteristics of increased expression of Wnt signaling pathway-related proteins in colon cancer patients.

[0006] Glutaminase (GLS) hydrolyzes glutamine to produce glutamate and ammonia, which is the rate-limiting step of glutamine degradation in the intestine. There are two subtypes of GLS: kidney-type glutaminase (GLS1) and liver-type glutaminase (GLS2). The expression of GLS2 is mainly limited to the liver, brain, pituitary gland and pancreas, while GLS1 is widely distributed in extrahepatic tissues and is most active in the kidney, small intestinal villus enterocytes, and rapidly dividing cells in the innate and acquired immune systems. Studies have found that BPTES, an inhibitor of GLS1, can inhibit myocardial hypertrophy and cardiac fibrosis, while overexpression of GLS1 can aggravate dextran sodium sulfate (DSS)-induced colitis in mice. Therefore, GLS1 may be an important target for promoting inflammatory damage.

[0007] Although glutamine not only plays an important regulatory role in inflammation under catabolic conditions, but also regulates intestinal permeability and intestinal epithelial barrier, it is still unclear whether glutamine catabolism has an effect on UC; therefore, studying the effect of GLS1 inhibitors on ulcerative colitis by inhibiting glutamine decomposition provides potential significance for finding effective drugs for the treatment of UC. Summary of the invention

[0008] The purpose of the present invention is to provide a use of a GLS1 inhibitor in alleviating ulcerative colitis in view of the above problems.

[0009] In order to achieve its purpose, the present invention adopts the following technical solution:

[0010] The first aspect of the present invention provides use of a GLS1 inhibitor in the preparation of a medicament for alleviating ulcerative colitis.

[0011] In the application technology scheme, the GLS1 inhibitor is the glutaminase allosteric inhibitor BPTES.

[0012] In the application technology scheme, the GLS1 inhibitor has at least one of the following effects:

[0013] (1) Alleviate or relieve the clinical symptoms of ulcerative colitis;

[0014] (2) reduce the disease activity index;

[0015] (3) Improve intestinal mucosal barrier damage, increase the number of goblet cells and reduce the infiltration of inflammatory cells;

[0016] (4) Promote the repair of the intestinal epithelial barrier;

[0017] (5) Reduce the mRNA expression levels of proinflammatory cytokines and chemokines.

[0018] In the application, the BPTES inhibits the over-activation of the Wnt / β-catenin signaling pathway, thereby alleviating ulcerative colitis.

[0019] In the application, the BPTES promotes the expression of TJ-related proteins, reduces the permeability of colon tissue and promotes the repair of the intestinal epithelial barrier.

[0020] In the application, the TJ-related proteins include ZO-1, occludin, claudin 1 and claudin2.

[0021] In the application, the pro-inflammatory cytokines include IL-6 and MCP-1; the chemokines include CXCL1 and CXCL2.

[0022] The second aspect of the present invention provides a drug for alleviating ulcerative colitis, wherein the active ingredient of the drug comprises BPTES.

[0023] Preferably, the medicament is a pharmaceutical composition.

[0024] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0025] The beneficial effects of the present invention are:

[0026] The present study confirmed that BPTES, an inhibitor of glutaminase 1 (GLS1), can significantly alleviate the clinical symptoms of ulcerative colitis in mice, improve intestinal mucosal barrier damage, reduce the level of inflammation in mice and inhibit the over-activation of the Wnt / β-catenin signaling pathway; it also showed that BPTES has a protective effect on UC, providing a new option for the prevention and treatment of UC. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of mouse grouping and weight changes in the present application example, wherein Figure 1 (A) is a schematic diagram of mouse grouping. Figure 1 (B) is a statistical chart of body weight changes of mice in each group;

[0028] Figure 2 is a graph of the length of the colon tissue of each group of mice in the examples of this application, wherein Figure 2 (A) is a schematic diagram of the mouse intestine. Figure 2 (B) is a numerical statistical graph of mouse intestinal length;

[0029] Figure 3 Statistical graph of DAI scores of mice in each group in the present application example;

[0030] Figure 4 Schematic diagram of colon tissue sections and pathological scores of each group of mice in the present application example, wherein Figure 4 (A) is a microscopic image of HE-stained mouse colon sections. Figure 4 (B) is a statistical graph of histopathological scores of mouse colon section samples;

[0031] Figure 5 is a schematic diagram of the mRNA expression levels of pro-inflammatory cytokines and chemokines in tissue samples of each group;

[0032] Figure 6 This is the expression result of ZO-1, occludin, claudin 1 and claudin 2 detected by WB. Figure 6 (A) is the protein band diagram, Figure 6 (B, C, D, E) are statistical graphs of the quantitative results of each protein;

[0033] Figure 7 : is a graph showing the results of immunohistochemistry experiments on mouse tissue sections in each group in the examples of the present application;

[0034] Figure 8 This is the result of WB detection of β-catenin, c-Myc and Cyclin D1 expression. Figure 8 (A) is the protein band diagram, Figure 8 (B, C, D) are statistical graphs of the quantitative results of each protein. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0037] Example 1

[0038] The research method of the present invention is as follows: first, 32 male C57BL / 6J mice were randomly and evenly divided into 4 groups: a normal control group (Control group), a drug control group (BPTES group), a model group (DSS group) and a drug treatment group (DSS+BPTES group). The first two groups used drinking water, and the latter two groups used 3% DSS. The drug group was intraperitoneally injected with 12.5 mg / kg of BPTES every day; the general state, weight change, stool morphology and fecal occult blood of the mice were recorded every day to calculate the disease activity index; hematoxylin-eosin (HE staining) was used to detect the colon pathological state; the mRNA expression levels of IL-6, MCP-1, CXCL1 and CXCL2 in the tissues of each group of mice were detected; the expression and distribution of ZO-1, occludin and claudin1 in the colon tissues were detected by immunohistochemistry (IHC) technology; the expression of ZO-1, occludin, claudin 1, claudin2, β-catenin, c-Myc and Cyclin in each group of mice were detected. The protein expression level of D1.

[0039] Results: Compared with the model group, the body weight of mice treated with BPTES increased, the stool morphology was normal, the fecal occult blood decreased, and the DAI score decreased (P<0.05); the colon pathological state was alleviated (P<0.05); the mRNA expression levels of IL-6, MCP-1, CXCL1 and CXCL2 in the tissues were significantly decreased (P<0.05); the protein expression levels of ZO-1, occludin, claudin 1 and claudin 2 in the colon tissues were significantly upregulated, while the protein expression levels of β-catenin, c-Myc and Cyclin D1 were significantly downregulated (P<0.0).

[0040] The specific experimental process is as follows:

[0041] 1. Materials

[0042] 1.1 Experimental animals

[0043] Animal source: C57BL / 6J SPF grade mice (male) were purchased from the Experimental Animal Center of Chongqing Medical University. The breeding conditions were as follows: daily light and night alternation of 12 hours, room temperature of 24±2℃, humidity of 40%-70%, free access to water, regular replacement of feed, water bottles and sterile bedding.

[0044] Experimental feeding: Male C57BL / 6J mice of 6-8 weeks old and weighing 20-22 g were purchased from the Experimental Animal Center of Chongqing Medical University. Before the experiment, the mice were kept in a room without specific pathogens, with a day / night cycle adjusted to 12 h and environmental conditions set (temperature: 20-25 ° C, relative humidity: 50 ± 5%), and were kept for 1 week to adapt to the experimental environment; these mice were fed with standard laboratory feed and free drinking water during the feeding period, and feed, water bottles and sterile bedding were changed regularly. The experimental procedures were approved by the Animal Protection and Utilization Committee of Chongqing Medical University and met the ethical standards of animal experiments.

[0045] 1.2 Main experimental reagents and instruments

[0046]

[0047]

[0048]

[0049] For other specific conditions not specified, the experiments were carried out according to conventional conditions or those recommended by the manufacturer. Reagents or instruments used without indicating the manufacturer were conventional products that can be purchased commercially.

[0050] 2. Methods

[0051] 2.1 Construction of animal model

[0052] Thirty-two male C57BL / 6J mice aged 6-8 weeks, weighing 18-22 g, were randomly divided into 4 groups: Control group, BPTES group, DSS group and DSS+BPTES group, with 8 mice in each group; after the mice were adapted to the groups, mice in the BPTES group and DSS+BPTES group were intraperitoneally injected with BPTES according to their body weight, and the other two groups were intraperitoneally injected with solvent (10% DMSO+90% corn oil) according to their body weight; the DSS group and DSS+BPTES group drank 3% DSS, and the drinking water was changed every two days, and the other two groups drank normal water. This intervention method was used for 7 days. The specific grouping is shown in Table 1.1.

[0053] The intraperitoneal injection concentration of BPTES was 12.5 mg / kg body weight, and the intraperitoneal injection was performed at 10 μL / g body weight.

[0054] The weight changes of mice were recorded, blood in stool was observed, and fecal occult blood was detected at the same time every day. The disease activity index (DAI) was used to score the mice. The specific scoring rules are shown in Table 1.2.

[0055] After 7 days, the mice were killed, their blood was collected, and the colorectum of each group of mice was dissected and the length was recorded; the colorectal tissue 1 cm away from the anus was fixed, and the remaining colorectal tissue was immediately frozen in liquid nitrogen and stored in a -80°C refrigerator for later use.

[0056] Table 1.1 Experimental animal grouping table

[0057]

[0058] Table 1.2 Disease Activity Index Assessment Criteria

[0059]

[0060] 2.2 Sample collection preparation

[0061] Prepare a sterile operating table, surgical forceps, tissue scissors, EP tubes, cameras, ice boxes, filter paper, physiological saline, 10% formaldehyde, and liquid nitrogen. Perform the entire operation aseptically. The main samples collected include: mouse blood, colorectal tissue, and feces. After collection, mark them and store them at -80℃ for later use.

[0062] 2.3 Mouse serum sample collection

[0063] When collecting samples after modeling, the mice were anesthetized and blood was collected from their orbits and collected in 1.5 mL EP tubes. After centrifugation at 4°C, 5,000 rpm for 20 min, the supernatant was collected in a new EP tube and stored at -80°C for later use.

[0064] 2.4 H&E staining

[0065] 2.4.1 Tissue section preparation

[0066] 1) Tissue fixation: The colorectal tissue of the mouse 1 cm away from the anus was fixed with 4% paraformaldehyde for more than 24 hours;

[0067] 2) Tissue dehydration: Remove the fixed tissue, rinse with running water, and dehydrate in 70%, 80%, 90% and 100% ethanol for 1 hour;

[0068] 3) Tissue transparency: Take out the dehydrated tissue and transparentize it in xylene I and xylene II for 30 minutes respectively;

[0069] 4) Paraffin embedding: Take out the transparent tissue and immerse it in wax cups I, II and III for 1 hour respectively; pour the melted wax into the paraffin frame, quickly put the wax-immersed tissue into the middle of the paraffin frame, and slowly flatten it until a clear wax film is formed on the surface, and then cool it;

[0070] 5) Preparation of tissue paraffin sections: Use a paraffin slicer to cut the tissue into 4 μM thick wax slices, gently place them in warm water for spreading, and use a glass slide to pick up the intact tissue. Then, place the glass slide in a 50°C oven for 5 hours to obtain tissue paraffin sections.

[0071] 2.4.2 Hematoxylin-eosin staining (HE)

[0072] 1) Dewaxing: Dewaxing the paraffin sections in xylene I and xylene II for 20 min.

[0073] 2) Hydration: Rehydrate the dewaxed sections in 100%, 95%, 85%, 75% ethanol and tap water for 5 min.

[0074] 3) Hematoxylin staining: Place the tissue sections in hematoxylin for 1 minute; observe the coloration of the cell nuclei under an optical microscope, then differentiate with 1% hydrochloric acid ethanol and LiCO3 in sequence; rinse in running water for 5 minutes to turn blue;

[0075] 4) Eosin staining: Place the tissue sections in eosin for staining for 1 minute; observe the cytoplasmic staining under an optical microscope and then rinse with tap water;

[0076] 5) Dehydration and sealing: Dehydrate the tissue sections in 75%, 85%, 95% and 100% ethanol for 5 minutes; place them in xylene III and xylene IV for 5 minutes; seal the sections with neutral gum;

[0077] 6) Photography: Take photos under an upright microscope and calculate the histopathological score of each sample according to the histopathological scoring standard (see reference 1).

[0078] 2.4.3 Immunohistochemistry (IHC)

[0079] 1) Dewaxing: Dewaxing the paraffin sections in xylene I and xylene II for 20 min.

[0080] 2) Hydration: The dewaxed sections were rehydrated in 100%, 95%, 85% and 75% ethanol for 5 min, and then washed with PBS for 5 min × 3 times;

[0081] 3) Antigen repair: Place the tissue sections in boiling sodium citrate antigen repair solution and heat for 15 minutes, keeping the antigen repair solution boiling during the process. After repair, cool naturally to room temperature and then wash with PBS for 5 minutes × 3 times;

[0082] 4) Removal of endogenous peroxidase: add 50 μL of endogenous peroxidase, incubate in a humidified box in the dark for 10 min, and then wash with PBS for 5 min × 3 times;

[0083] 5) Blocking: Add 50 μL of non-specific staining blocking agent and incubate in a humidified box in the dark for 20 min;

[0084] 6) Incubate with primary antibody: dilute the antibody according to the antibody instructions, add 50 μL of the diluted antibody, incubate in a suitable wet box for 12 to 16 hours, and then wash with PBS for 5 minutes × 3 times;

[0085] 7) Incubate with secondary antibody: add 50 μL of secondary antibody-biotin-labeled goat anti-mouse / rabbit IgG polymer, incubate in a wet box in the dark for 20 min, and then wash with PBS for 5 min × 3 times;

[0086] 8) Add 50 μL of streptavidin-peroxidase, incubate in a wet box in the dark for 10 min, and then wash with PBS for 5 min × 3 times;

[0087] 9) Prepare DAB at a ratio of 1:20 (must be prepared before use), add 50 μL of DAB and immediately observe the color development time under a microscope. The color development time of each sample should be consistent, and then place it in PBS to stop the color development;

[0088] 10) Place the sections in hematoxylin for 2 minutes, then rinse with tap water to reverse the blue;

[0089] 11) Color separation: Place the sections in hydrochloric acid ethanol and lithium carbonate for 3 seconds, then rinse with PBS;

[0090] 12) Dehydration and sealing: Dehydrate the tissue sections in 75%, 85%, 95% and 100% ethanol for 5 min; place in xylene III and xylene IV for 5 min; seal the sections with neutral gum;

[0091] 13) Photography: Take photos under an upright microscope and calculate the histopathological score of each sample according to the tissue immunohistochemistry scoring standard (see reference 1).

[0092] 2.5 Colorectal tissue protein concentration

[0093] 1) BCA protein concentration standard curve: detect the OD562 value at each concentration gradient according to the instructions and draw a standard curve;

[0094] 2) Sample dilution: Pipette 5 μL of colorectal tissue lysate and add 195 μL of PBS to dilute 40 times, and mix the sample thoroughly;

[0095] 3) Prepare BCA protein detection solution: according to the number of samples × the number of duplicate wells = the number of sample wells, add 200 μL BCA protein detection solution to each well to obtain the required total volume of BCA protein detection solution, and prepare it according to the ratio of A solution: B solution = 50:1;

[0096] 4) Sample addition: Take 25 μL of the protein lysis dilution solution and add it to the wells of the enzyme label strip. Set up 3 replicate wells for each sample and add 200 μL of BCA protein detection solution in sequence to avoid bubbles;

[0097] 5) Incubation: Incubate at 37°C for 30 min in the dark;

[0098] 6) Detection: Turn on the microplate reader 10 minutes in advance to preheat the instrument. Detect the OD562 value immediately after incubation. Finally, calculate the protein concentration of the colorectal tissue lysate based on the BCA protein concentration standard.

[0099] 2.6 Western Blot Experiment

[0100] 1) Total protein extraction of tissue: 30 mg of colorectal tissue was weighed for each sample, washed with PBS and placed in a 1.5 mL centrifuge tube, 300 μL of tissue lysis buffer (the ratio of RIPA lysis buffer to protease inhibitors, phosphatase inhibitors, and EDTA was 50:1:1:1) was added, and then the tube was fully ground with a grinding rod on ice and fully broken using ultrasound. After the ultrasound was completed, the tube was placed on ice for lysis for 10 min, and then centrifuged at 12,000 rpm and 4°C for 15 min, and the supernatant was collected in a new centrifuge tube.

[0101] 2) Protein quantification and denaturation: Use the BCA protein quantification kit to determine the total protein concentration of each sample. Take 80 μL of protein lysate in a new centrifuge tube, add 20 μL of 5× protein loading buffer, mix well, and denature in a 100°C metal bath for 5 min. The sample can be aliquoted and stored at -80°C together with the remaining protein lysate.

[0102] 3) Electrophoresis: Load the sample onto a 10-well, 1 mm thick SDS-PAGE gel, add 1× electrophoresis buffer, concentrate gel at 60 V for 30 min, separate gel at 100 V for 80 min;

[0103] 4) Transfer: PVDF membrane (0.45μm) was activated with methanol for 30s, then immersed in electrotransfer solution, separation gel of corresponding width was cut according to molecular weight, and transfer clips were placed in the order of black transfer plate, sponge, filter paper, separation gel, PVDF membrane, filter paper, sponge, and transparent transfer plate to avoid bubbles. Then the "sandwich" clips were placed in the electrotransfer tank, 1× electrotransfer solution was added, and the membrane was transferred in an ice bath. According to the molecular weight, 220V constant voltage was used for the corresponding time.

[0104] 5) Blocking: Take out the PVDF membrane in the "sandwich" clip, prepare 5% skim milk powder with 1×TBST, and block at room temperature for 2 hours;

[0105] 6) Primary antibody: dilute the primary antibody in an appropriate ratio with primary antibody diluent, incubate the PVDF membrane with the primary antibody overnight at 4°C, and then wash the membrane three times with 1× TBST, each time for 10 min;

[0106] 7) Secondary antibody: dilute the secondary antibody in an appropriate ratio with 1×TBST, incubate the PVDF membrane with the secondary antibody at room temperature for 2 h, and then wash the membrane three times with 1×TBST, each time for 10 min;

[0107] 8) Development: Prepare the developer according to the ECL developer instructions, immerse it on the PVDF membrane, and then immediately develop it with a BIO-RAD chemiluminescence imager and take photos for record; use Image J-Win64 to quantify the protein bands using the total protein normalization method, and the calculation formula is: normalization factor (× lane) = total lane protein amount (× lane) / reference lane protein amount, normalized target protein amount (× lane) = target protein amount (× lane) / normalization factor (× lane).

[0108] 2.7 Real-time quantitative polymerase chain reaction (RT-qPCR)

[0109] 2.7.1 Extraction of total RNA from tissues

[0110] Sample preparation: According to the instructions of the rapid RNA extraction kit, take ≤10 mg of tissue, add 300 μL Buffer QLS lysis buffer to a 1.5 mL enzyme-free EP tube, and grind it on ice with a grinder; after sufficient grinding, centrifuge at 4°C and 12000 rpm for 5 min, and aspirate the supernatant into a 1.5 mL enzyme-free EP tube; add an equal volume of anhydrous ethanol to the above supernatant and mix thoroughly; immediately transfer the mixed solution to the Quick RNA Mini Column adsorption column, centrifuge at 4°C and 12000 rpm for 2 min; discard the filtrate, add 700 μL Buffer QWB, and centrifuge at 4°C and 12000 rpm for 2 min; install the adsorption column to a new Collection Tube (collection tube), centrifuge at 4°C, 12000rpm for 2min; install the adsorption column into a new enzyme-free EP tube, add 50μL enzyme-free water, let it stand at room temperature for 3min, and centrifuge at 4°C, 12000rpm for 2min; the obtained eluate is the total tissue RNA, which is stored at -80°C for future use.

[0111] 2.7.2 Determination of total RNA concentration in tissues

[0112] 1) Pipette 1 μL of enzyme-free water to clean the detection port of the micro-spectrophotometer and wipe it dry with filter paper;

[0113] 2) Pipette 1 μL of enzyme-free water to adjust the zero and wipe the detection port with filter paper;

[0114] 3) Take 1 μL RNA sample for measurement and wipe the detection port with filter paper;

[0115] 4) After all RNA samples have been tested, 1 μL of enzyme-free water was taken to clean the test port and the test port was wiped dry with filter paper.

[0116] 2.7.3 RNA conversion to cDNA

[0117] Component name volume 5×gDNA Clean Reaction Mix 2μL Total RNA - RNase free water Up to 10μL

[0118] Among them: Due to inconsistent RNA concentration, the amount added here is variable. After adding RNA, it is supplemented with water to 10 μL. The reaction procedure is: 42°C, 2min, and then transferred to 4°C for storage.

[0119] 2) Step 2: Reverse transcription reaction, add the following reagents in sequence according to the instructions:

[0120] Component name volume 5×EVO M-MLV RT Reaction Mic 4μL RNase free water 6μL The first step reaction solution 10μL Total volume 20μL

[0121] Reaction procedure: 37°C, transfer to 85°C for 15 min, transfer to 4°C for 5 s, and store the obtained cDNA sample at -80°C for later use.

[0122] 2.7.4 cDNA amplification

[0123] 1) Add the following reagents to the cDNA sample in sequence according to the instructions:

[0124] Component name volume Forward Primer (10μM) 0.4μL Reverse Primer (10μM) 0.4μL RNase free water 3.2μL SYBRⅡ 5μL cDNA samples 1μL Total volume 10μL

[0125] Reaction program: 95°C, 30s, cycle once, then transfer to 95°C, 5s, then 60°C, 30s, cycle 40 times, then transfer to 4°C for storage.

[0126] 2) The primer sequences are as follows:

[0127]

[0128] 3. Data Statistical Analysis

[0129] The experimental data were statistically analyzed using GraphPad Prism 9.0.0 and IBM SPSS Statistics 26 software. Since the sample size was small, the Shapiro-Wilk test was used to test whether the data followed a normal distribution. If yes, one-way ANOVA and two-way ANOVA were used to compare the differences among the four groups. All data were expressed as mean ± SD, and P < 0.05 indicated statistical significance.

[0130] 4. Results

[0131] 4.1 BPTES alleviates clinical symptoms in DSS-induced UC mice

[0132] Mice were grouped as Figure 1 (A) shows the statistical graph of mouse weight changes. Figure 1 As shown in (B), during the adaptive feeding period, all mice had normal diet, drinking water, urination and defecation, and steady weight gain.

[0133] Depend on Figure 1(B) It can be seen that during the experiment, the status of the Control group was similar to that during the adaptive feeding period; the weight of the mice in the DSS group decreased slightly when the solvent was intraperitoneally injected on the first day, and increased in the next two days, reaching a peak weight on the third day; then they began to lose weight, their stools gradually became soft, their activities decreased, their hair gloss decreased, their food intake decreased, and their stools gradually became bloody; on the sixth day of model induction, the mice had watery and bloody stools; the DSS+BPTES group drank DSS freely and was given the glutaminase 1 inhibitor BPTES intraperitoneally every day. The weight change trend of the mice in the first three days was similar to that of the DSS group; their weight increased steadily from the second day to the fifth day; their weight decreased slightly in the next two days, but they were significantly heavier than the DSS group; the mice had slight bloody stools in the next two days, their activity and mental state were average, their stools became soft, but watery stools were rare; compared with the DSS group, the symptoms of the mice were significantly improved, the degree of weight loss was reduced, the mental state and activity of the mice improved, and the degree of bloody stools and diarrhea were reduced.

[0134] After the modeling was completed, the mice in each group were killed, blood was collected, and the segment from the ileocecal region to the anus of the mice was taken out and spread on a scaled plate. The appearance of the mouse intestine is shown in the figure below. Figure 2 (A) shows the numerical statistics of mouse intestinal length. Figure 2 (B) shown.

[0135] Depend on Figure 2 It can be seen that the entire intestine of the mice in the Control group had no bloody stools, and hard feces were scattered at the end of the colon; the intestine of the mice in the BPTES group was no different from that of the Control group; the intestine of the mice in the DSS group was significantly shortened, the ileocecal part became smaller, and more loose and bloody stools were seen in the cecum and colorectum; compared with the DSS group, the intestinal shortening of the mice in the DSS+BPTES group was improved, and brown soft stools were seen in the intestinal cavity.

[0136] Therefore, it can be concluded that BPTES can alleviate the symptoms of DSS-induced colitis in mice.

[0137] 4.2 BPTES reduces the DAI score of DSS-induced UC mice

[0138] The weight, fecal viscosity and fecal occult blood of mice were observed to calculate the disease activity index; the results are as follows Figure 3 shown.

[0139] Depend on Figure 3It can be seen that the DAI score of mice in the Control group was always low throughout the modeling process. On the first day, due to the influence of intraperitoneal injection of drugs and solvents, the scores of mice in the other three groups were slightly higher, but they all decreased on the second and third days. In the following days, the scores of mice in the DSS group gradually increased, especially on the fifth day, when the increase was large. In contrast, the scores of mice in the DSS+BPTES group recovered. Therefore, it can be concluded that BPTES can reduce the DAI scores of DSS-induced UC mice.

[0140] 4.3BPTES improves colon pathology in DSS-induced UC mice

[0141] Microscopic image of mouse colon section stained with HE Figure 4 (A) shows the statistical diagram of the histopathological scores of each sample. Figure 4 As shown in (B), Figure 4 It can be seen that the colon mucosa of the Control group was intact in shape, without obvious edema and inflammatory infiltration; the BPTES group was no different from the Control group; the mucosa of the DSS group was partially or even mostly missing, and the colon mucosal structure was significantly damaged, with a large number of inflammatory cells infiltrating and invading the submucosa, edema of the submucosa, disappearance of colonic crypts, and reduction of goblet cells; the colon mucosa and crypts of the DSS+BPTES group were relatively intact, and the inflammatory cell infiltration and goblet cell loss were improved; therefore, it can be concluded that BPTES can alleviate the pathological state of DSS-induced colitis in mice.

[0142] 4.4BPTES improves inflammatory response in DSS-induced UC mice

[0143] In order to study the anti-inflammatory effect of BPTES, the expression of inflammatory cytokines and chemokines in the colon tissues of each group of mice was analyzed by qRT-PCR detection technology; the expression levels of each factor were as follows Figure 5 (A, B, C, D) as shown.

[0144] Depend on Figure 5 It can be seen that the mRNA expression levels of IL-6, MCP-1, CXCL1 and CXCL2 in mice in the DSS group were significantly increased, while after administration of BPTES, their mRNA expression decreased.

[0145] Therefore, it can be concluded that BPTES can alleviate the inflammatory response induced by DSS in mice, thereby playing a therapeutic role in UC.

[0146] 4.5BPTES improves intestinal epithelial barrier disruption in DSS-induced UC mice

[0147] The intestinal epithelial barrier is an important component of intestinal barrier function. Tight junction-related proteins form a complete monolayer between the intestinal epithelium to prevent harmful bacteria and pathogenic antigens in the intestinal cavity from entering the submucosal layer and inducing inflammatory responses. The goblet cells in the colonic epithelium of UC patients are reduced, resulting in reduced mucin synthesis, and then reduced mucus barrier thickness and mucus barrier dysfunction. After the mucus barrier is destroyed, the direct interaction between the intestinal flora and the intestinal epithelium increases, leading to intestinal epithelial cell apoptosis and reduced synthesis of tight junctions (TJ) between epithelial cells, such as ZO-1, occludin and claudins, and then intestinal epithelial barrier damage. In addition to reduced synthesis, structural abnormalities of TJs-related proteins are also involved in the process of intestinal epithelial barrier destruction in UC patients.

[0148] Therefore, the protein expression levels of tight junction-related proteins ZO-1, occludin, claudin 1, and claudin 2 were detected by Western Blot experiments to explore the effect of BPTES on the intestinal epithelial barrier of UC mice; the Western blotting results are shown in Figure 6 As shown in (A, B, C, D, E), Figure 6 (A) is the WB protein band diagram, Figure 6 (B, C, D, E) are statistical graphs of the quantitative results of each protein.

[0149] Depend on Figure 6 It can be seen that the protein expression levels of ZO-1, occludin, claudin 1 and claudin 2 in the DSS group were significantly lower than those in the Control group and BPTES group, while BPTES intervention could significantly increase their protein expression levels.

[0150] Immunohistochemistry was further used to detect the expression and distribution of ZO-1, occludin and claudin 1. The IHC results are as follows: Figure 7 As shown, combined Figure 6 and Figure 7 It can be seen that the expression levels of ZO-1, occludin and claudin 1 in mice in the DSS group were reduced, and their structure and distribution were disordered, while BPTES intervention could increase the expression levels of the two and promote the restoration of their structure and distribution to normal.

[0151] Therefore, it can be concluded that BPTES can improve intestinal epithelial barrier disruption in DSS-induced UC mice.

[0152] 4.6BPTES improves Wnt / β-catenin signaling pathway activation in DSS-induced UC mice

[0153] The Wnt signaling pathway is of great significance in the proliferation and differentiation of intestinal mucosal epithelial cells. The proliferation of epithelial cells is the key to the repair of the intestinal mucosal barrier in UC. The β-catenin degradation complex is composed of APC, Axin, casein kinase 1 (CK-1) and glycogen synthase kinase (GSK-3β). This complex is essential for the phosphorylation of β-catenin and the subsequent degradation by the ubiquitin-proteasome pathway. However, enhanced β-catenin activity and prolonged epithelial proliferation may lead to the occurrence of UC-related colon cancer. When Wnt is not activated, the complex rapidly degrades free β-catenin in the cytoplasm to keep it at a low level; when the Wnt pathway is activated, the degradation complex is inhibited, β-catenin in the cytoplasm accumulates and moves into the nucleus to form a complex with TCF / LEF transcription factors, inducing the transcription of downstream genes such as c-Myc and Cyclin D1.

[0154] It is not clear whether the Wnt signaling pathway is involved in the anti-colitis effect of BPTES, but the continuous activation of the Wnt pathway can lead to excessive proliferation of epithelial cells and increase the risk of colorectal adenoma or colorectal cancer, which is consistent with the increased expression of Wnt signaling pathway-related proteins in colon cancer patients. Therefore, we used Western Blot and IHC experiments to demonstrate whether the Wnt signaling pathway is involved in the anti-colitis effect of BPTES. The results are shown in the figure. Figure 8 shown.

[0155] Depend on Figure 8 It can be seen that the protein levels of β-catenin, c-Myc and Cyclin D1 in the colon tissues of mice in the DSS group were upregulated, and the use of the GLS1 inhibitor BPTES significantly reversed this result, suggesting that BPTES may exert its anti-colitis effect by inhibiting the overactivation of the Wnt / β-catenin signaling pathway.

[0156] In summary, the present invention used DSS to induce the establishment of a UC mouse model. Compared with the control group, the mice in the DSS group showed a significant decrease in weight and an increased trend in DAI scores, and the colon was significantly shortened. Colon pathological HE staining showed obvious destruction of the colon mucosal structure and a large number of inflammatory cell infiltration, which were basically consistent with the clinical UC pathological phenotype, indicating that the DSS-induced UC mouse model was successfully established in this study.

[0157] After using the GLS1 inhibitor BPTES, the weight loss trend, DAI score increase trend and colon shortening of UC mice were significantly alleviated. At the same time, the gene expression levels of IL-6, MCP-1, CXCL1 and CXCL2 in the colon tissue of UC mice were significantly reduced, indicating that BPTES exerts good anti-inflammatory activity in the treatment of UC mice.

[0158] Under physiological conditions, goblet cells are abundant in the intestinal mucosa and have normal secretory functions. The mucus layer formed by their secretion is rich in antimicrobial peptides, which can effectively isolate the intestinal flora from the contact with epithelial cells and prevent infection. One of the pathological characteristics of UC is the reduction of goblet cells, damage to the mucus layer, and increase in the adhesion of mucus and epithelium, which further leads to amplified intestinal inflammation and aggravated tissue damage. DSS-induced UC mice do have chemical barrier damage. The present invention confirms that the intervention of BPTES can restore the damage to the colon mucosal structure, increase the number of goblet cells and reduce the infiltration of inflammatory cells; this shows that BPTES significantly improves the chemical barrier damage caused by DSS to UC mice.

[0159] The maintenance of intestinal epithelial barrier function depends on the normal content and structure of tight junctions (TJs) between intestinal epithelial cells. The loss of TJs-related proteins is an important factor in epithelial barrier damage, which leads to increased intestinal permeability and promotes bacterial translocation. In the DSS-induced UC mouse model, the expression of TJ-related proteins ZO-1, occludin, claudin 1 and claudin 2 were all downregulated, indicating that the epithelial barrier was damaged. After BPTES intervention, the expression of TJ-related proteins was significantly promoted and the permeability of colon tissue was reduced. Further, through colon tissue immunohistochemistry, BPTES can not only promote the increase of ZO-1, occludin and claudin 1 expression, but also promote the restoration of its mucosal structure to normal, thereby restoring intestinal epithelial barrier function. These data prove that BPTES can promote the repair of intestinal epithelial barrier.

[0160] Finally, the present invention also explores the molecular mechanism by which BPTES inhibits colon inflammation in UC mice and promotes intestinal epithelial barrier recovery. Activation of the Wnt / β-catenin signaling pathway is an important prerequisite for mucosal epithelial repair, but because the intestinal epithelium of UC patients is damaged and repaired multiple times under the influence of chronic inflammation, this may cause enhanced β-catenin activity and prolonged epithelial proliferation, leading to the occurrence and development of UC-related colon cancer. Therefore, preventing excessive activation of the Wnt / β-catenin signaling pathway in UC patients is beneficial to reducing the risk of UC malignancy. After BPTES intervention in UC mice, the protein expression levels of β-catenin, c-Myc and Cyclin D1 were significantly downregulated, suggesting that BPTES may play an anti-inflammatory and colon mucosal barrier repairing role through the Wnt / β-catenin signaling pathway.

[0161] Therefore, the use of drugs containing the GSL1 inhibitor BPTES plays an important role in alleviating ulcerative colitis and provides a new option for the prevention and treatment of UC.

[0162] Reference 1 mentioned in this application is as follows:

[0163] [1]JANG Y J,KIM W K,HAN D H,et al.Lactobacillus fermentum speciesameliorate dextran sulfate sodium-induced colitis by regulating the immuneresponse and altering gut microbiota[J].Gut microbes,2019,10(6):696-711.

Claims

1. Application of GLS1 inhibitors in the preparation of drugs for relieving ulcerative colitis.

2. The use according to claim 1, characterized in that: GLS1 inhibitor is the allosteric glutaminase inhibitor BPTES.

3. The use according to claim 2, characterized in that: The GLS1 inhibitor has at least one of the following effects: (1) Alleviate or relieve the clinical symptoms of ulcerative colitis; (2) reduce the disease activity index; (3) Improve intestinal mucosal barrier damage, increase the number of goblet cells and reduce the infiltration of inflammatory cells; (4) Promote the repair of the intestinal epithelial barrier; (5) Reduce the mRNA expression levels of proinflammatory cytokines and chemokines.

4. The use according to claim 2, characterized in that: in, The BPTES inhibits the over-activation of the Wnt / β-catenin signaling pathway, thereby playing the role of alleviating ulcerative colitis.

5. The use according to claim 2, characterized in that: in, The BPTES promotes the expression of TJ-related proteins, reduces the permeability of colon tissue and promotes the repair of the intestinal epithelial barrier.

6. The use according to claim 5, characterized in that: The TJ-related proteins include ZO-1, occludin, claudin 1 and claudin 2.

7. The use according to claim 2, characterized in that: in, The pro-inflammatory cytokines include IL-6 and MCP-1; the chemokines include CXCL1 and CXCL2.

8. A drug for relieving ulcerative colitis, characterized in that: Its active ingredients include BPTES.

9. The drug according to claim 8, characterized in that: The medicine is a pharmaceutical composition.

10. The drug according to claim 9, characterized in that: The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

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