Use of gls1 inhibitors in the alleviation of ulcerative colitis

Abstract

The application relates to the field of biological medicine, and discloses application of a GLS1 inhibitor BPTES in relieving ulcerative colitis and a medicine for relieving ulcerative colitis, wherein the active ingredient of the medicine comprises BPTES. The application proves that the inhibitor BPTES of glutaminase 1 (GLS1) can obviously relieve the clinical symptoms of ulcerative colitis of mice, improve the destruction of intestinal mucosal barrier, reduce the inflammation level in the body of the mice, and inhibit the overactivation of the Wnt / beta-catenin signal path; and also proves that 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 technical field of biological medicine, in particular to the application of GLS1 inhibitor in relieving ulcerative colitis. BACKGROUND

[0002] Ulcerative colitis (UC) is a digestive tract disease that occurs in the colorectal site, has hidden symptoms, high recurrence rate and canceration rate, mainly invades the colonic mucosa, causes 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 imbalance, mucosal and epithelial barrier defects, and genetic predisposition, but the initial driving factor of UC is not clear. It may be driven by intestinal epithelial barrier dysfunction to cause intestinal mucosal inflammatory response, or by intestinal mucosal lamina propria inflammatory response to drive and cause intestinal epithelial barrier destruction, and finally both promote each other to cause continuous progression of UC disease.

[0003] Under normal circumstances, the intestinal barrier function is mainly maintained by the intestinal epithelial barrier and the mucus barrier at the top of the intestinal epithelial cells. The number of goblet cells in the colonic epithelium of UC patients is reduced, leading to reduced synthesis of mucin, and then the thickness of the mucus barrier is reduced, the mucus barrier dysfunction occurs, and after the mucus barrier is destroyed, the direct interaction between the intestinal flora and the intestinal epithelium is increased, leading to apoptosis of intestinal epithelial cells, reduced synthesis of tight junctions (TJs) such as ZO-1, occludin and claudins between epithelial cells, and then the intestinal epithelial barrier is damaged. In addition to reduced synthesis, structural abnormalities of TJs related proteins also participate in the process of intestinal epithelial barrier destruction in UC patients. When the intestinal epithelial barrier is damaged, too much intestinal content antigen enters the lamina propria, the tolerance mechanism of the lamina propria fails, and the pro-inflammatory response of immune cells is no longer inhibited, and immune cells are recruited by chemokines secreted by the immune cells to infiltrate, and then the inflammatory response process is aggravated.

[0004] Wnt / β-catenin signaling pathway is a widely functional regulatory network, which plays an important role in various 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 the subsequent ubiquitin-proteasome pathway degradation. In the non-activated state, Wnt rapidly degrades the free β-catenin in the cytoplasm, keeping it at a low level; when the Wnt pathway is activated, the degradation complex is inhibited, and the cytoplasmic β-catenin accumulates and enters 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 the metabolism of glutamine in HCT116 colon cancer cells, in addition, c-Myc also enhances the expression of mitochondrial GLS 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, and the Wnt signaling pathway is involved in the regulation of many physiological and pathological processes, including embryonic development, cell proliferation, tissue repair, etc. In the intestinal tissue, Wnt protein is a necessary condition for the proliferation of intestinal stem cells and the renewal of epithelial cells. Blocking Wnt signals that transmit to the crypt of the intestinal mucosa in mice leads to inhibition of the proliferation of intestinal stem cells, 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, increasing the risk of colorectal adenoma or colorectal cancer. This is consistent with the characteristics of increased expression of Wnt signaling pathway-related proteins in patients with colon cancer.

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

[0007] Although glutamine not only has important regulatory effects on inflammation under catabolic conditions, but also regulates intestinal permeability and intestinal epithelial barrier, it is not clear whether glutamine catabolism has an effect on UC; therefore, studying the effect of GLS1 inhibitor inhibition of glutamine catabolism on ulcerative colitis provides potential significance for finding effective drugs for treating UC. SUMMARY

[0008] The purpose of the present application is to provide a GLS1 inhibitor for use in alleviating ulcerative colitis in order to solve the above problems.

[0009] In order to achieve the purpose of the present application, the technical solution adopted by the present application is:

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

[0011] In the application technical solution, the GLS1 inhibitor is a glutaminase allosteric inhibitor BPTES.

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

[0013] (1) reducing or alleviating the clinical symptoms of ulcerative colitis;

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

[0015] (3) improving the destruction of the intestinal mucosal barrier, increasing the number of goblet cells and reducing the infiltration of inflammatory cells;

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

[0017] (5) reducing the mRNA expression levels of pro-inflammatory cytokines and chemokines.

[0018] The application, wherein the BPTES inhibits the overactivation of the Wnt / β-catenin signaling pathway, thereby exerting the effect of alleviating ulcerative colitis.

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

[0020] The application, wherein the TJ-related proteins include ZO-1, occludin, claudin 1 and claudin 2.

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

[0022] The second aspect of the present application provides a medicine for relieving ulcerative colitis, wherein an active ingredient of the medicine comprises BPTES.

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

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

[0025] The present application has the following beneficial effects:

[0026] The present application has proved that the inhibitor BPTES of glutaminase 1 (GLS1) can significantly relieve the clinical symptoms of ulcerative colitis in mice, improve the destruction of intestinal mucosal barrier, reduce the inflammation level in mice and inhibit the overactivation of Wnt / β-catenin signaling pathway, which also indicates that BPTES has a protective effect on UC and provides a new selection direction for the prevention and treatment of UC. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Fig. 1 is a schematic diagram of the grouping of mice and the change in body weight in the embodiments of the present application, wherein Figure 1 Fig. 1(A) is a schematic diagram of the grouping of mice, Figure 1 Fig. 1(B) is a statistical diagram of the change in body weight of mice in each group;

[0028] Figure 2 Fig. 2 is a diagram of the colon tissue length of mice in each group in the embodiments of the present application, wherein Figure 2 Fig. 2(A) is a schematic diagram of the intestinal tract of mice, Figure 2 Fig. 2(B) is a statistical diagram of the length of the intestinal tract of mice;

[0029] Figure 3 Fig. 3 is a statistical diagram of the DAI score of mice in each group in the embodiments of the present application;

[0030] Figure 4 Fig. 4 is a schematic diagram of the colon tissue section and histopathology score of mice in each group in the embodiments of the present application, wherein Figure 4 Fig. 4(A) is a microscope diagram of the HE staining of the colon section of mice, Figure 4 Fig. 4(B) is a statistical diagram of the histopathology score of the colon section sample of mice;

[0031] Figure 5 Fig. 5 is a diagram of the expression level of proinflammatory cytokines and chemokines mRNA in the tissue sample of each group;

[0032] Figure 6 Fig. 6 is a diagram of the expression results of WB detection of ZO-1, occludin, claudin 1 and claudin 2, wherein Figure 6 Fig. 6(A) is a protein band diagram, Figure 6 Fig. 6(B, C, D, E) are statistical diagrams of the quantification results of each protein;

[0033] Figure 7 are the immunohistochemistry experimental results of each group of mice tissue slices in the embodiments of the present application;

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

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

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

[0037] Example 1

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

[0039] Results: compared with the model group, after treatment with BPTES, the body weight of the mice increased, the fecal morphology was normal, the fecal occult blood was reduced, and the DAI score decreased (P<0.05); the colon pathological state was relieved (P<0.05); the mRNA expression levels of IL-6, MCP-1, CXCL1 and CXCL2 in the tissues were significantly reduced (P<0.05); the protein expression levels of ZO-1, occludin, claudin 1 and claudin 2 in the colon tissues were significantly up-regulated, while the protein expression levels of β-catenin, c-Myc and Cyclin D1 were significantly down-regulated (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 level mice (male) were purchased from Chongqing Medical University Experimental Animal Center, and the feeding conditions were as follows: 12 hours of light and dark alternation per day, room temperature 24±2℃, humidity 40%-70%, free drinking water, and regular replacement of feed, drinking water bottles and sterile bedding.

[0044] Experimental feeding: 6-8 week old male C57BL / 6J mice weighing 20-22g were purchased from Chongqing Medical University Experimental Animal Center. Before the experiment, the mice were raised in a specific pathogen-free room, the day / night cycle was adjusted to 12h and the environmental conditions were set (temperature: 20-25℃, relative humidity: 50±5%), and they were raised 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 the feed, drinking water bottles and sterile bedding were replaced regularly, the experimental procedure was approved by the Chongqing Medical University Animal Protection and Utilization Committee and met the animal experiment ethical standards.

[0045] 1.2 Main experimental reagents and instruments

[0046]

[0047]

[0048]

[0049] Other specific conditions not specified were carried out according to the conventional conditions or the manufacturer's recommended conditions, and the reagents or instruments not specified by the manufacturer were conventional products that could be purchased on the market.

[0050] 2. Methods

[0051] 2.1 Construction of animal model

[0052] Thirty-two 6-8 week old male C57BL / 6J mice weighing 18-22g were randomly divided into four groups: Control group, BPTES group, DSS group and DSS+BPTES group, 8 mice in each group; After the mice were grouped, the BPTES group and the DSS+BPTES group were injected intraperitoneally with BPTES according to their body weight, and the other two groups were injected intraperitoneally with solvent (10% DMSO+90% corn oil) according to their body weight; the DSS group and the DSS+BPTES group drank 3% DSS, which was replaced every two days, and the other two groups drank normal water; the intervention lasted for 7 days in this way, and the specific grouping is shown in Table 1.1.

[0053] BPTES was administered intraperitoneally at a concentration of 12.5 mg / kg body weight, and was administered intraperitoneally at 10 μL / g body weight.

[0054] The body weight of the mice was recorded, and the presence of blood in the stool was observed and detected, and the disease activity index (DAI) score was determined, according to the disease activity index (DAI) score, as shown in Table 1.2.

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

[0056] Table 1.1 Experimental animal grouping table

[0057]

[0058] Table 1.2 Disease activity index evaluation criteria

[0059]

[0060] 2.2 Sample collection preparation

[0061] A sterile operating table, surgical forceps, tissue scissors, an EP tube, a camera, an ice box, filter paper, physiological saline, 10% formaldehyde, liquid nitrogen, and a whole sterile operation were prepared, and the main samples collected included: mouse blood, colorectal tissue, and feces, which were labeled and stored at -80°C for standby.

[0062] 2.3 Mouse serum sample collection

[0063] When the samples were collected at the end of modeling, the mice were anesthetized and blood was collected from the orbit, and was collected in a 1.5 mL EP tube. After centrifugation at 4°C, 5,000 rpm for 20 min, the supernatant was collected into a new EP tube and stored at -80°C for standby.

[0064] 2.4 H&E staining

[0065] 2.4.1 Tissue section preparation

[0066] 1) Tissue fixation: The colorectal tissue of the mice 1 cm from the anus was fixed with 4% paraformaldehyde, and the fixation time was more than 24 h;

[0067] 2) Tissue dehydration: The fixed tissue was removed and washed with running water, and was gradiently dehydrated in 70%, 80%, 90%, and 100% ethanol for 1 h, respectively;

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

[0069] 4) Paraffin embedding: Take out the transparent tissue, and respectively in wax cup I, wax cup II and wax cup III for 1 h; pour the melted wax into the paraffin frame, quickly put the waxed tissue into the middle position of the paraffin frame, and slowly flatten it, and then cool it after the surface has obvious wax film;

[0070] 5) Tissue paraffin section preparation: cut the tissue into 4 μM thickness of wax sheet with a paraffin section machine, and gently put it into warm water for sectioning, and then use a glass slide to pick up the intact tissue. Then put the glass slide in a 50 °C oven for 5 h to obtain the tissue paraffin section.

[0071] 2.4.2 Hematoxylin-eosiin staining (HE)

[0072] 1) De-waxing: Put the paraffin section into xylene I and xylene II for 20 min;

[0073] 2) Hydration: Put the de-waxed section into 100%, 95%, 85%, 75% ethanol and tap water for 5 min;

[0074] 3) Hematoxylin staining: Put the tissue section into hematoxylin for 1 min; after observing the cell nucleus coloration under an optical microscope, differentiate it with 1% hydrochloric acid ethanol and LiCO3; and then wash it with tap water for 5 min to remove the blue color;

[0075] 4) Eosin staining: Put the tissue section into eosin for 1 min; after observing the cytoplasm coloration under an optical microscope, wash it with tap water;

[0076] 5) Dehydration and mounting: Put the tissue section into 75%, 85%, 95% and 100% ethanol for 5 min; and then put it into xylene III and xylene IV for 5 min; and then mount it with neutral balsam;

[0077] 6) Photography: Take a photograph under a microscope, and calculate the histopathology score of each sample according to the histopathology score standard (see reference 1).

[0078] 2.4.3 Immunohistochemistry (IHC)

[0079] 1) De-waxing: Put the paraffin section into xylene I and xylene II for 20 min;

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

[0081] 3) Antigen retrieval: The tissue sections were heated in a boiling sodium citrate solution for 15 min, during which the solution was kept boiling, and then naturally cooled to room temperature, and then washed with PBS for 5 min x 3 times;

[0082] 4) Endogenous peroxidase blocking: 50 μL of endogenous peroxidase was added, and the sections were incubated in a humidified chamber for 10 min, and then washed with PBS for 5 min x 3 times;

[0083] 5) Blocking: 50 μL of non-specific staining blocking agent was added, and the sections were incubated in a humidified chamber for 20 min;

[0084] 6) Incubation of primary antibody: The antibody was diluted according to the manufacturer's instructions, 50 μL of the diluted antibody was added, and the sections were incubated in a suitable humidified chamber for 12-16 h, and then washed with PBS for 5 min x 3 times;

[0085] 7) Incubation of secondary antibody: 50 μL of secondary antibody-biotin-labeled goat anti-mouse / rabbit IgG polymer was added, and the sections were incubated in a humidified chamber for 20 min, and then washed with PBS for 5 min x 3 times;

[0086] 8) 50 μL of streptavidin-peroxidase was added, and the sections were incubated in a humidified chamber for 10 min, and then washed with PBS for 5 min x 3 times;

[0087] 9) DAB was prepared at a ratio of 1:20 (freshly prepared), 50 μL of DAB was added, and the color development time was observed under a microscope, and the color development time of each sample should be consistent, and then the color development was terminated by placing the sample in PBS;

[0088] 10) The sections were stained with hematoxylin for 2 min, and then rinsed with tap water to reverse the blue color;

[0089] 11) Color separation: The sections were placed in hydrochloric acid ethanol and lithium carbonate for 3 s, and then rinsed with PBS;

[0090] 12) Dehydration and mounting: The tissue sections were sequentially dehydrated in 75%, 85%, 95% and 100% ethanol for 5 min, sequentially transparentized in xylene III and xylene IV for 5 min, and then mounted with neutral balsam;

[0091] 13) Photography: The samples were photographed under a microscope, and the histopathological scores of the samples were calculated according to the histological immunohistochemical scoring standard (see reference 1).

[0092] 2.5 Concentration of colorectal tissue proteins

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

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

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

[0096] 4) Sample addition: take 25 μL of protein lysate dilution solution and add it to the enzyme-labeled strip well, set 3 duplicate wells for each sample, and then add 200 μL of BCA protein detection solution, avoiding bubbles;

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

[0098] 6) Detection: turn on the enzyme-labeled instrument 10 min in advance and preheat the instrument, immediately detect the OD562 value after incubation, and finally calculate the protein concentration of the colorectal tissue lysate according to the BCA protein concentration standard curve.

[0099] 2.6 Western Blot experiment

[0100] 1) Tissue total protein extraction: each sample was weighed 30 mg of colorectal tissue, washed with PBS, and then placed in a 1.5 mL centrifuge tube, 300 μL of tissue lysis buffer (RIPA lysis buffer, protease inhibitor, phosphatase inhibitor, EDTA ratio 50:1:1:1) was added, then ground on ice with a grinder and broken up with ultrasound, after ultrasonic completion, placed on ice for 10 min, then centrifuged at 12000 rpm, 4°C for 15 min, 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 5x protein loading buffer, mix well, and denature at 100°C metal bath for 5 min, can be divided and stored together with the remaining protein lysate at -80°C.

[0102] 3) Electrophoresis: 10 wells, 1 mm thick SDS-PAGE gel, add 1x electrophoresis liquid, concentrate gel 60V 30min, separation gel 100V 80min;

[0103] 4) Transfer: PVDF membrane (0.45 pm) was activated with methanol for 30 s, then soaked in the transfer buffer, the separation gel was cut to the corresponding width according to the molecular weight, the transfer clamp was placed in the order of black transfer plate, sponge, filter paper, separation gel, PVDF membrane, filter paper, sponge, transparent transfer plate to avoid air bubbles, then the "sandwich" clamp was placed in the transfer tank, 1x transfer buffer was added, and the transfer was carried out in ice bath, and the transfer was carried out according to the molecular weight using 220V constant voltage for the corresponding time.

[0104] 5) Blocking: the PVDF membrane in the "sandwich" clamp was taken out, 5% skimmed milk was prepared with 1x TBST, and the blocking was carried out at room temperature for 2h;

[0105] 6) Primary antibody: the primary antibody was diluted with the primary antibody diluent at an appropriate ratio, the PVDF membrane was incubated with the primary antibody at 4°C overnight, then the membrane was washed with 1x TBST for 3 times, each for 10 min;

[0106] 7) Secondary antibody: the secondary antibody was diluted with 1x TBST at an appropriate ratio, the PVDF membrane was incubated with the secondary antibody at room temperature for 2h, then the membrane was washed with 1x TBST for 3 times, each for 10 min;

[0107] 8) Development: the developing solution was prepared according to the ECL developing solution instruction, and was immersed on the PVDF membrane, then immediately developed with the BIO-RAD chemiluminescence imager, and photographed. The protein bands were quantified using Image J-Win64 with total protein normalization method, and the calculation formula was: normalization factor (x lane) = total lane protein amount (x lane) / reference lane protein amount, and the normalized target protein amount (x lane) = target protein amount (x lane) / normalization factor (x 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 quick RNA extraction kit instructions, take ≤10 mg of tissue, add 300 μL Buffer QLS lysis solution to a 1.5 mL enzyme-free EP tube, and grind on ice with a grinder; after thorough grinding, centrifuge at 4°C, 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 a Quick RNA Mini Column adsorption column, centrifuge at 4°C, 12000 rpm for 2 min; discard the filtrate, add 700 μL Buffer QWB, and centrifuge at 4°C, 12000 rpm for 2 min; install the adsorption column into a new collection tube, centrifuge at 4°C, 12000 rpm for 2 min; install the adsorption column into a new enzyme-free EP tube, add 50 μL of enzyme-free water, and stand at room temperature for 3 min, then centrifuge at 4°C, 12000 rpm for 2 min; the resulting eluate is the total RNA of the tissue, which is stored at -80°C for later use.

[0111] 2.7.2 Determination of the concentration of total RNA from tissues

[0112] 1) Aspirate 1 μL of enzyme-free water to clean the microspectrophotometer detection port and wipe dry with filter paper;

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

[0114] 3) Aspirate 1 μL of RNA sample for determination and wipe the detection port dry with filter paper;

[0115] 4) After all the RNA samples have been detected, aspirate 1 μL of enzyme-free water to clean the detection port and wipe the detection port dry with filter paper.

[0116] 2.7.3 Reverse transcription of RNA to cDNA

[0117] Component Name Volume 5x gDNA Clean Reaction Mix 2 μL Total RNA - RNase free water Up to 10 μL

[0118] Wherein: due to the inconsistency of RNA concentration, the amount added here can be variable, and after adding RNA, make up to 10 μL with water; reaction procedure: 42°C, 2 min, then 4°C.

[0119] 2) Second step: reverse transcription reaction, according to the instructions, add the following reagents in order:

[0120] Component Name Volume 5x EVO M-MLV RT Reaction Mix 4 μL RNase free water 6 μL Reaction solution for first step 10 μL Total volume 20 μL

[0121] Reaction procedure: 37°C, 15 min, then 85°C, 5 s, then 4°C, resulting cDNA sample stored at -80°C for later use.

[0122] 2.7.4 cDNA amplification

[0123] 1) The following reagents were added to the cDNA sample in order according to the instructions:

[0124] Component Name Volume Forward Primer (10 μM) 0.4 μL Reverse Primer (10 μM) 0.4 μL RNase free water 3.2 μL SYBR II 5 μL cDNA sample 1 μL Total volume 10 μL

[0125] Reaction procedure: 95°C, 30s, cycle 1, then 95°C, 5s, then 60°C, 30s, cycle 40, then 4°C storage.

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

[0127]

[0128] 3. Statistical analysis of data

[0129] The experimental data were statistically analyzed using GraphPad Prism 9.0.0 and IBM SPSS Statistics 26 software; since 32 belongs to small sample size, Shapiro-Wilk test was used to test whether the data conform to normal distribution; if it does, one-way ANOVA and two-way ANOVA are used to compare the differences between the four groups; all data are expressed as mean ± SD, P < 0.05 indicates statistical significance.

[0130] 4. Results

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

[0132] The mice were grouped as shown in Figure 1 (A), and the statistical graph of the change in body weight of the mice is shown in Figure 1 (B), during the adaptive feeding period, all mice had normal diet, normal drinking water, normal stool and urine, and steady weight gain.

[0133] From Figure 1(B) can be known that the state of Control group is similar to that during adaptive feeding during the experiment; the body weight of DSS group mice slightly decreases when they are injected with solvent in the first day, and the body weight increases in the following two days, and reaches the peak in the third day; then the body weight begins to decrease, the stool gradually becomes soft, the activity decreases, the hair luster decreases, the food intake decreases, and the stool gradually becomes bloody; on the sixth day of model induction, the mice have watery and bloody stool; the DSS+BPTES group freely drinks DSS, and is injected with the glutamine enzyme 1 inhibitor BPTES every day, and the change trend of the body weight of the mice in the first three days is similar to that of the DSS group; the body weight steadily increases from the second day to the fifth day; then the body weight slightly decreases in the following two days, but is obviously heavier than that of the DSS group; the mice have slight blood in stool in the following two days, the activity and mental state are normal, the stool becomes soft, but the watery stool is rarely seen; compared with the DSS group, the symptoms of the mice are obviously improved, the degree of body weight decrease is reduced, the mental state and activity of the mice are improved, and the blood in stool and diarrhea are reduced.

[0134] After modeling, the mice in each group were sacrificed, the blood was collected, and the ileocecal part to the anal part of the mice was taken out and laid on a scale plate, the appearance state of the intestinal tract of the mice is shown in Figure 2 (A), and the numerical statistical graph of the intestinal tract length of the mice is shown in Figure 2 (B).

[0135] It can be known from Figure 2 that the Control group mice have no blood stool in the whole intestinal tract, and the colon end has scattered hard stool; the intestinal tract of the BPTES group mice is similar to that of the Control group; the intestinal tract of the DSS group mice is obviously shortened, the ileocecal part is small, and there are more watery and soft stool and blood stool in the cecum and colorectum; compared with the DSS group, the intestinal tract of the DSS+BPTES group mice is improved, and brown soft stool is 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 body weight, stool consistency and fecal occult blood of the mice were observed to calculate the disease activity index; the results are shown in Figure 3 .

[0139] It can be known from Figure 3It can be seen that the DAI scores of mice in the Control group remained very 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 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 larger. 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 HE-stained mouse colon section Figure 4 (A) shows the statistical graph 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, with no obvious edema or 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. A large number of inflammatory cells infiltrated and invaded the submucosa, the submucosa was edematous, the colonic crypts disappeared, and the goblet cells decreased; 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 the 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) 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 their mRNA expression decreased after administration of BPTES.

[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] Intestinal epithelial barrier is an important part of intestinal barrier function. The tight junction associated proteins form a complete monolayer between the intestinal epithelium, preventing harmful bacteria and pathogenic antigens in the intestinal lumen from entering the submucosal layer to induce inflammatory response. In UC patients, goblet cells in the colon epithelium are reduced, leading to reduced synthesis of mucin, and then the thickness of the mucus barrier is reduced, the mucus barrier dysfunction occurs, and the destruction of the mucus barrier increases the direct interaction of intestinal flora and intestinal epithelium, leading to intestinal epithelial cell apoptosis, reduced synthesis of tight junction (TJ) such as ZO-1, occludin and claudins between epithelial cells, and then the intestinal epithelial barrier is damaged. In addition to reduced synthesis, structural abnormalities of TJs associated proteins also participate in the process of intestinal epithelial barrier destruction in UC patients.

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

[0149] It can be seen from Figure 6 that the protein expression levels of ZO-1, occludin, claudin 1 and claudin 2 in DSS group mice were significantly lower than those in Control group and BPTES group, and the protein expression levels were significantly increased after BPTES intervention.

[0150] Further, the expression and distribution of ZO-1, occludin and claudin 1 were detected by immunohistochemical experiment, and the results of IHC are as shown in Figure 7 , in combination with Figure 6 and Figure 7 It can be seen that the expression levels of ZO-1, occludin and claudin 1 in DSS group mice are reduced, and their structure and distribution are disordered, while BPTES intervention can increase the expression levels of the two and promote the structure and distribution to return to normal.

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

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

[0153] Wnt signaling pathway plays an important role in the proliferation and differentiation of intestinal mucosal epithelial cells, and 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). The complex is crucial for the phosphorylation of β-catenin and the subsequent ubiquitin-proteasome pathway degradation. However, the increase of β-catenin activity and the prolongation of epithelial proliferation period may lead to the occurrence of UC-related colon cancer. In the case of non-activation of Wnt, the complex rapidly degrades the free β-catenin in the cytoplasm, keeping it at a low level. When the Wnt pathway is activated, the degradation complex is inhibited, and the cytoplasmic β-catenin accumulates and enters the nucleus to form a complex with the TCF / LEF transcription factor, inducing the transcription of downstream c-Myc and Cyclin D1 genes.

[0154] It is not clear whether Wnt signaling pathway is involved in the anti-colitis effect of BPTES, but the persistent activation of 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 characteristics of increased expression of Wnt signaling pathway-related proteins in colon cancer patients. Therefore, we demonstrated whether Wnt signaling pathway is involved in the anti-colitis effect of BPTES by Western Blot and IHC experiments, and the results are shown in the following figure. Figure 8

[0155] As can be seen from Figure 8 , the protein levels of β-catenin, c-Myc and Cyclin D1 in the colon tissues of DSS group mice are all up-regulated, and this result is significantly reversed after using the inhibitor BPTES of GLS1, which suggests that BPTES may play an anti-colitis role by inhibiting the overactivation of Wnt / β-catenin signaling pathway.

[0156] In summary, the present application adopts DSS to induce the establishment of a UC mouse model. Compared with the control group, the DSS group mice show a significant trend of weight loss and DAI score increase, and the colon shortening is obvious. The colon pathological HE staining shows that the colon mucosa structure is obviously destroyed and a large number of inflammatory cells infiltrate, which is basically consistent with the clinical UC pathological phenotype, indicating that the DSS-induced UC mouse model is successfully constructed in the present study.

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

[0158] ​Under physiological conditions, goblet cells are abundant in the intestinal mucosa, and their secretory function is normal. The mucus layer formed by their secretion is rich in antibacterial peptides, which can effectively isolate the contact between intestinal flora and epithelial cells and prevent infection. One of the pathological characteristics of UC is the reduction of goblet cells, the damage of mucus layer, and the increase of adherent bacteria of mucus and epithelium, which further leads to the amplification of intestinal inflammation and aggravation of tissue damage. The destruction of the chemical barrier does exist in DSS-induced UC mice, and the present application proves that the intervention of BPTES can restore the destruction of the colonic mucosa structure, increase the number of goblet cells and reduce the infiltration of inflammatory cells; this indicates that BPTES significantly improves the chemical barrier damage caused by DSS in 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 absence of TJ-related proteins is an important factor for the damage of epithelial barrier, which leads to the increase of intestinal permeability and promotes the translocation of flora. In the DSS-induced UC mouse model, the expression of TJ-related proteins ZO-1, occludin, claudin 1 and claudin 2 is down-regulated, which indicates that the epithelial barrier is damaged. After the intervention of BPTES, the expression of TJ-related proteins is significantly promoted, and the permeability of colonic tissue is reduced. Further, the immunohistochemistry of colonic tissue shows that BPTES can not only promote the expression of ZO-1, occludin and claudin 1, but also promote the recovery of mucosal structure, thereby restoring the function of intestinal epithelial barrier. These data prove that BPTES can promote the repair of intestinal epithelial barrier.

[0160] Finally, the present application also discusses the molecular mechanism of BPTES in inhibiting colonic inflammation and promoting the recovery of intestinal epithelial barrier in UC mice. The activation of Wnt / β-catenin signaling pathway is an important prerequisite for the repair of mucosal epithelium. However, due to the repeated damage and repair of intestinal epithelium in UC patients under the influence of chronic inflammation, it may cause the enhancement of β-catenin activity, the prolongation of epithelial proliferation period, and the occurrence and development of UC-related colon cancer. Therefore, preventing the overactivation of Wnt / β-catenin signaling pathway in UC patients is beneficial to reduce the risk of UC malignancy. After the intervention of BPTES in UC mice, the protein expression levels of β-catenin, c-Myc and Cyclin D1 are significantly down-regulated, which suggests that BPTES may play a role in anti-inflammation and repair of colonic mucosal barrier through Wnt / β-catenin signaling pathway.

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

[0162] The reference 1 mentioned in the present application is as follows:

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

Claims

1. Use of a GLS1 inhibitor in the preparation of a medicament for alleviating ulcerative colitis, characterized in that: The GLS1 inhibitor is the glutaminase allosteric inhibitor BPTES.

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