Application of uridine in preparation of medicine for preventing and treating ulcerative colitis

Abstract

The invention belongs to the field of biological medicines, and particularly discloses application of uridine in preparation of medicines for preventing and treating ulcerative colitis, including application of uridine in preparation of medicines for preventing and treating ulcerative colitis in acute stage, application of uridine in preparation of medicines for preventing and treating ulcerative colitis in remission stage and the like. Uridine can improve the symptoms of DSS-induced acute experimental colitis model mice, has a good treatment effect, and improves the weight of the model mice; experimental results indicate that a high-dose group of uridine has a good treatment effect, the mechanism and specificity of uridine up-regulate expression and activity of colon local SIRT1 protein, further inhibit phosphorylation activation of an NF-kappa B signal channel, regulate secretion of inflammatory factors and expression of tight junction protein, repair intestinal mucosal barrier while moderately regulating immunity, and improve the immunity of a human body. The systemic inflammatory response is effectively relieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the prevention and treatment of ulcerative colitis, especially the application of uridine in the preparation of drugs for preventing and treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is an autoimmune disease characterized by chronic intestinal inflammation and mucosal damage, and its incidence shows an upward trend globally. Although existing UC treatment drugs (including aminosalicylates, glucocorticoids, and biological agents, etc.) provide effective clinical treatment for many patients, some people still have problems of insufficient treatment response or drug tolerance, and there is an urgent need to develop new treatment strategies. As an endogenous nucleoside substance with relatively low toxicity, uridine has been proven to have pharmacological effects such as anti-inflammatory, antioxidant, and metabolic regulation, but its anti-inflammatory mechanism of action is not yet clear. Previous studies have shown that uridine can significantly increase the ratio of NAD⁺ to NADH, thereby regulating cell metabolism. This discovery can provide a breakthrough point for explaining the pharmacological mechanism of uridine.

[0003] NAD⁺ and NADH are key coenzymes in numerous redox reactions related to energy metabolism. The activity of NAD⁺-dependent deacetylase - silent information regulator 1 (SIRT1) is regulated by the NAD⁺ / NADH ratio. At the same time, SIRT1 plays a crucial role in delaying cell senescence, regulating metabolic processes, coping with oxidative stress, inhibiting inflammatory responses, and improving the cell microenvironment. In recent years' drug research, SIRT1 is one of the important targets in the research of some diseases (including metabolic diseases, immune diseases, and nervous system diseases, etc.).

[0004] Currently, SIRT1 has been proven to be able to weaken the body's inflammatory response by inhibiting the activation of the NF-κB pathway. Feeding tilapia with a diet rich in uridine can enhance the expression level of SIRT1 mRNA in tilapia liver tissue. However, in this study, the effects of uridine on the protein activity and expression level of SIRT1 have not been experimentally verified. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide the application of uridine in the preparation of drugs for preventing and treating ulcerative colitis in view of the deficiencies of the prior art, so as to solve the problems raised in the above background art.

[0006] In order to achieve the above object of the invention, the technical solution adopted by the present invention is as follows:

[0007] The application of uridine in the preparation of drugs for preventing and treating ulcerative colitis.

[0008] Preferably, the ulcerative colitis is in the acute phase.

[0009] Preferably, the ulcerative colitis is in remission.

[0010] Specifically, the uridine can improve the pathological damage of the colon tissue.

[0011] Specifically, the uridine can inhibit the secretion of pro-inflammatory factors in the colon.

[0012] Specifically, the pro-inflammatory factors in the colon are IL-1β and / or IL-6.

[0013] Specifically, the uridine can restore the colon mucosal barrier.

[0014] Specifically, the uridine can promote the expression and activity of SIRT1 protein in the colon.

[0015] Specifically, the uridine can inhibit the phosphorylation activation of the NF-κB signaling pathway.

[0016] Specifically, the dosage form of the drug is selected from any one of tablets, capsules, injections or enteric-coated preparations.

[0017] Beneficial effects:

[0018] (1) The present invention first reveals the mechanism of action of uridine in preventing and treating ulcerative colitis. Through in vivo and in vitro experiments, it is verified that it regulates the inflammatory response and repairs the intestinal mucosal barrier function through the SIRT1 / NF-κB pathway, providing a theoretical basis for the development of safe and effective new UC treatment drugs.

[0019] (2) The present invention verifies that uridine can improve the symptoms of mice in the DSS-induced acute experimental colitis model, has a good therapeutic effect, and increases the body weight of the model mice; the experimental results suggest that the high-dose group of uridine has a better therapeutic effect, and its mechanism is related to specifically up-regulating the expression and activity of SIRT1 protein in the colon, thereby inhibiting the phosphorylation activation of the NF-κB signaling pathway, while regulating the secretion of inflammatory factors and the expression of tight junction proteins, repairing the intestinal mucosal barrier while moderately regulating the immune response, and effectively alleviating the systemic inflammatory response. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0021] Figure 1 It is a perianal view of a mouse in an embodiment of the present invention.

[0022] Figure 2 It is a graph of the body weight and body weight percentage (body weight on the day / initial body weight) of mice in an embodiment of the present invention.

[0023] Figure 3It is a graph of the colon length and the ratio of intestinal weight to intestinal length of mice in the embodiment of the present invention.

[0024] Figure 4 It is a graph of the spleen coefficient and spleen of mice in the embodiment of the present invention.

[0025] Figure 5 It is a graph of the DAI score of mice in the embodiment of the present invention.

[0026] Figure 6 It is a HE staining graph of mouse colon tissue in the embodiment of the present invention.

[0027] Figure 7 It is a graph of the mRNA expression level of inflammatory factors in mouse colon tissue in the embodiment of the present invention.

[0028] Figure 8 It is a graph of the content of serum inflammatory factors of mice in the embodiment of the present invention.

[0029] Figure 9 It is a graph of the tight junction protein and mRNA level in mouse colon tissue in the embodiment of the present invention.

[0030] Figure 10 It is a graph of the expression level and activity of SIRT1 protein in mouse colon tissue in the embodiment of the present invention.

[0031] Figure 11 It is a graph of the phosphorylation of NF-κB in mouse colon tissue in the embodiment of the present invention.

[0032] Figure 12 It is a graph of the mRNA expression levels of inflammatory factors IL-1β, IL-6, TNF-α and COX-2 in RAW264.7 cells in the embodiment of the present invention.

[0033] Figure 13 It is a graph of the contents of inflammatory factors IL-1β and IL-6 in the supernatant of RAW264.7 cell culture medium in the embodiment of the present invention.

[0034] Figure 14 It is a graph of the expression level of SIRT1 protein in RAW264.7 cells in the embodiment of the present invention.

[0035] Figure 15 It is a graph of the phosphorylation of NF-κB in RAW264.7 cells in the embodiment of the present invention. Detailed implementation manners

[0036] According to the following embodiments, the present invention can be better understood.

[0037] Example 1

[0038] The present invention provides the application of uridine in the preparation of drugs for preventing and treating ulcerative colitis. The specific experimental steps are as follows:

[0039] S1. Randomly divide several male SPF - level C57BL / 6J mice into a control group, a model group, a sulfasalazine group, a low - dose uridine group, a medium - dose uridine group, and a high - dose uridine group;

[0040] S2. Establish an acute experimental colitis model in mice using dextran sulfate sodium for a total of 17 days;

[0041] S3. Monitor the body weight and fecal traits of mice every day, and evaluate the therapeutic effect of uridine on acute experimental colitis in mice by comparing disease activity index scores, colon histological injury index scores, etc.;

[0042] S4. Use the enzyme - linked immunosorbent assay method to detect the concentration levels of IL - 1β, IL - 6, and TNF - α in the plasma of mice, and use the Western blot method to detect the protein expression levels of intestinal adhesion proteins Occludin and ZO - 1. Use the RT - qPCR method to detect the mRNA expression levels of IL - 1β, IL - 6, TNF - α, Claudin - 5, Occludin, and ZO - 1 in colon tissues. Thus, evaluate the therapeutic effect of uridine on acute experimental colitis in mice. Use the immunohistochemistry method to detect the expression of SIRT1 protein in mouse colon tissues. Use the enzyme activity detection kit method to detect SIRT1 enzyme activity. Use the Western blot detection method to detect the protein expression level of SIRT1, the total protein expression level and phosphorylated protein expression level of NF - κB, and the protein expression level of IL - 1β. By comparing the ratio of the total protein amount of NF - κB to the phosphorylated protein amount of NF - κB, examine the activation of NF - κB.

[0043] Thus, evaluate the pharmacological action mechanism of uridine.

[0044] Furthermore, in step S1, the total number of male SPF - level C57BL / 6J mice is 36, which are divided into a control group (CON group), a model group (MOD group), a sulfasalazine group (SASP group), a low - dose uridine group (LU group), a medium - dose uridine group (MU group), and a high - dose uridine group (HU group). Each group has 6 male SPF - level C57BL / 6J mice.

[0045] Furthermore, in step S3, the disease activity index score is the DAI score. In step S4, the enzyme - linked immunosorbent assay method is the ELISA method, the Western blot method is the WB method, the reverse transcription quantitative polymerase chain reaction method is the RT - qPCR method, and the immunohistochemistry method is the IHC method.

[0046] It also includes the following steps:

[0047] S11. Uniformly plate mouse macrophages RAW264.7. Divide them into a blank control group, an LPS model group, a low-dose uridine group, a medium-dose uridine group, and a high-dose uridine group.

[0048] S22. Establish an inflammation model by stimulating with LPS for 24 h.

[0049] S33. Use the RT-qPCR method to detect the mRNA levels of pro-inflammatory factors IL-1β, IL-6, TNF-α, and COX-2 in mouse macrophages RAW264.7.

[0050] S44. Use the WB method to detect the protein expression level of SIRT1, the total protein expression level and phosphorylated protein expression level of NF-κB, and the protein expression level of IL-1β in mouse macrophages RAW264.7.

[0051] S55. Use the immunofluorescence method to detect the protein expression level of SIRT1 in mouse macrophages RAW264.7.

[0052] Furthermore, in step S11, the cells are evenly seeded in each well of a 6-well plate at a density of 2×10 6 cells / well. Divide them into a blank control group, an LPS model group, a low-dose uridine group, a medium-dose uridine group, and a high-dose uridine group.

[0053] Furthermore, the immunofluorescence method described in step S55 is the IF method.

[0054] Example 2

[0055] In the experiment of the present invention, the natural nucleoside small molecule uridine (purchased from Merck KGaA, Germany, purity ≥99%) is used as the research object to preliminarily explore the therapeutic effect and mechanism of uridine on dextran sulfate sodium (DSS)-induced acute experimental colitis. Under the guidance of theory and practice, the experiment consists of two parts.

[0056] The first part of the experiment: Thirty-six male SPF-grade C57BL / 6J mice were randomly divided into a control (CON) group, a model (MOD) group, a sulfasalazine (SASP) group, a low-dose uridine (LU) group, a medium-dose uridine (MU) group, and a high-dose uridine (HU) group, with 6 mice in each group. Except for the CON group, acute experimental colitis mouse models were successfully established in the other 5 groups by DSS; disease activity index (DAI) scores, tissue injury index scores and other indicators were evaluated to monitor the therapeutic effect of uridine on experimental colitis mice. The mRNA expression levels of IL-1β, IL-6, TNF-α, Claudin-5, ZO-1, and Occludin in colon tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The protein expression levels of ZO-1, Occludin, SIRT1, NF-κB, p-NF-κB, and IL-1β in colon tissues were detected by Western blotting (WB). The protein expression of SIRT1 in colon tissues was detected by immunohistochemistry (IHC), and the SIRT1 enzyme activity in colon tissues was detected using an enzyme activity kit; the contents of IL-1β, IL-6, and TNF-α in serum were detected by ELISA. To explore the therapeutic effect of uridine on DSS-induced acute experimental colitis in mice and preliminarily explore the possible mechanism.

[0057] The second part of the experiment: Mouse macrophages RAW264.7 were divided into a control group, a lipopolysaccharide (LPS) model group, a low-dose uridine group, a medium-dose uridine group, and a high-dose uridine group. An LPS inflammation model was established. The mRNA expression levels of IL-1β, IL-6, TNF-α, and cyclooxygenase-2 (COX-2) in colon tissues were detected by RT-qPCR. The protein secretion levels of IL-1β and IL-6 in cell supernatants were detected by ELISA. The protein expression levels of SIRT1, NF-κB, p-NF-κB, and IL-1β in cells were detected by WB. The protein expression level of SIRT1 in cells was detected by immunofluorescence (IF).

[0058] Example 3

[0059] Study on the Therapeutic Effect and Mechanism of Uridine on DSS-Induced Acute Experimental Colitis in Mice:

[0060] 1.1 Experimental Materials

[0061] 1.1.1 Experimental Animals

[0062] SPF - level male C57BL / 6J mice, 6 - 8 weeks old, weighing 20 ± 2 g, were purchased from Suzhou Speifo Biotechnology Co., Ltd. The animal experiment was approved by the Experimental Animal Research Ethics Committee of Nanjing First Hospital (approval number: DWSY - 23087391), and the experiment was conducted in the Animal Experiment Center of Nanjing First Hospital. The whole experiment complied with the ARRIVE UPDATE 2.0 guidelines for reporting experiments on laboratory animals.

[0063] 1.2 Experimental methods

[0064] 1.2.1 Preparation of experimental reagents

[0065] Preparation of 3% DSS solution: Prepared by dissolving 3 g of DSS powder in 100 mL of sterile water; uridine and sulfasalazine were dissolved in the corresponding volume of physiological saline.

[0066] 1.2.2 Experimental grouping and model establishment

[0067] The mice were raised in a SPF - level conventional experimental environment (temperature 26 ± 2°C, humidity 50 ± 10%). Thirty - six C57BL / 6J mice were randomly divided into a normal control (Control, CON) group, a model (Model, MOD) group, a sulfasalazine (Salicylazosulfapyridine, SASP) group, a low - dose uridine group (Low Dose Uridine, LU), a medium - dose uridine group (Medium Dose Uridine, MU), and a high - dose uridine group (High Dose Uridine, HU), with 6 mice in each group. The experimental period was 18 days. From day 1 to day 7, the mice drank sterile water. From day 8 to day 14, the MOD, SASP, LU, MU, and HU groups drank 3% DSS solution to establish the model. From day 15 to day 17, they resumed drinking sterile water. During the experimental period, the mental state, activity ability, food and water intake, and fecal characteristics of the mice were recorded in detail every day, and the body weight was accurately measured using an electronic balance (accuracy 0.1 g). Due to the different administration methods of uridine and sulfasalazine, to ensure that all mice were affected equally by the external environment, the following administration plan was designed: The uridine intervention groups (LU / MU / HU) were intraperitoneally injected with 60 / 120 / 240 mg / kg of uridine daily and gavaged with an equal volume of phosphate - buffered saline (Phosphate Buffered Saline, PBS); SASP was gavaged with 200 mg / kg of sulfasalazine daily and intraperitoneally injected with an equal volume of PBS; both the CON and MOD groups were gavaged and intraperitoneally injected with an equal volume of PBS daily. On the 18th day of the experiment, the mice were sacrificed, and colon tissues and serum samples were collected for later use.

[0068] 1.2.3 Scoring criteria for disease activity index and histological injury score

[0069] The disease activity index scoring criteria are shown in Table 1. The histological injury scoring criteria are as follows: Severity of inflammation: 0 points = none; 1 point = mild; 2 points = moderate; 3 points = severe; Depth of lesion: 0 points = none; 1 point = mucosal layer; 2 points = submucosal layer; 3 points = muscular layer; 4 points = transmural; Crypt damage: 0 points = none; 1 point = 1 / 3 of the basal breakage; 2 points = 2 / 3 of the basal breakage; 3 points = only the surface epithelium intact; 4 points = entire crypt and epithelium lost; Extent of lesion: 1 point = 1% - 25%; 2 points = 26% - 50%; 3 points = 51% - 75%; 4 points = 76% - 100%.

[0070] Table 1 DAI Scoring Criteria

[0071]

[0072] 1.2.4 Hematoxylin-Eosin Staining

[0073] The steps of Hematoxylin-Eosin (HE) staining are as follows: After obtaining a fresh colon tissue sample, quickly cut a 1-cm colorectal tissue above the anus. Immediately place the cut tissue into an EP tube containing 1 mL of universal tissue fixative, ensuring that the tissue is completely immersed. Fix for 24 h. The fixed tissue is then dehydrated. According to the method of gradient alcohol dehydration, sequentially place the tissue into 70%, 80%, 90%, and 95% alcohol for 1 - 2 h each, and then place it into absolute ethanol for 2 treatments, 1 - 2 h each time, to thoroughly remove the water in the tissue. Then perform clearing treatment. Place the dehydrated tissue into xylene to further clear the tissue and facilitate the infiltration of paraffin. The tissue is treated in xylene 2 - 3 times, 15 - 30 min each time. Finally, perform wax infiltration. Place the cleared tissue into melted paraffin. First, infiltrate the tissue in paraffin with a lower melting point for 2 - 3 h, and then place it into paraffin with a higher melting point for 2 - 3 h to allow the paraffin to fully infiltrate the tissue.

[0074] Place the wax-infiltrated tissue into an embedding mold, add melted paraffin, quickly place the tissue in the required direction, and wait for the paraffin to solidify to make a paraffin block containing the tissue. Use a microtome to cut the paraffin block into thin slices with a thickness of 4 - 6 μm. During the sectioning process, pay attention to maintaining the continuity and integrity of the sections. The cut thin slices float in warm water at 40 - 45 °C to flatten the sections. Use a glass slide to pick up the sections, let the sections adhere flatly to the glass slide, and then place the glass slide into an oven at 60 °C and bake for 1 - 2 h to firmly attach the sections to the glass slide.

[0075] Put the baked slices into xylene for dewaxing 2 - 3 times, 5 - 10 minutes each time, and then hydrate them through gradient alcohol, successively putting them into 100%, 95%, 90%, 80%, and 70% alcohol for 2 - 3 minutes each. Then perform hematoxylin staining. Put the slices into hematoxylin staining solution for 5 - 10 minutes, and then rinse with tap water for 10 - 15 minutes to fully differentiate and blue the hematoxylin. After that, differentiate with 1% hydrochloric acid alcohol for several seconds, and then rinse with tap water for 10 - 15 minutes. Then perform eosin staining. Put the slices into eosin staining solution for 3 minutes. The stained slices are successively dehydrated through 70%, 80%, 90%, and 95% alcohol, 2 - 3 minutes each, and then dehydrated in absolute ethanol 2 times, 2 - 3 minutes each time. Finally, put them into xylene for clearing 2 - 3 times, 2 - 3 minutes each time. Seal the slices with neutral gum, cover with a coverslip to isolate the slices from the outside for easy observation under the microscope.

[0076] 1.2.5 Protein immunoblotting detection

[0077] First, prepare the colon tissue samples and place them in pre - cooled EP tubes. Add an appropriate amount of RIPA lysis buffer (containing protease inhibitor, phosphatase inhibitor, EDTA) according to the ratio of 1 mL RIPA lysis buffer per 100 mg tissue, shake well to make the lysis buffer fully contact with the tissue. Then add 2 - 3 grinding beads to the EP tube and grind in a tissue grinder. After grinding, place the centrifuge tube on ice for lysis for 30 minutes. After lysis, centrifuge the centrifuge tube at 12000 - 14000 rpm for 20 minutes at 4°C, and transfer the supernatant to a new centrifuge tube. This supernatant is the crude extract containing proteins. Use a BCA protein quantification kit to measure the protein concentration of the crude extract for subsequent adjustment of the loading amount.

[0078] Add 5× loading buffer according to the ratio of crude extract to loading buffer of 4:1, and vortex thoroughly to mix them evenly. Place the centrifuge tube in a metal bath or boiling water bath at 100°C for 10 minutes. After heating, quickly transfer the centrifuge tube to ice for cooling for 2 - 3 minutes. Then use the prepared protein sample for electrophoresis or store it in a - 80°C refrigerator.

[0079] According to the molecular weight of the target protein, select an appropriate concentration of separating gel and use a one - step gel kit to prepare the gel. After the gel solidifies, load the prepared protein samples in sequence. Put the gel into the electrophoresis tank, add electrophoresis buffer, and first perform electrophoresis at a voltage of 100 V. When the protein sample enters the separating gel, raise the voltage to 150 V and continue electrophoresis until the bromophenol blue front is close to the bottom of the gel, then stop electrophoresis.

[0080] After the electrophoresis is completed, take out the gel and stack it in the transfer cassette in the order of gel, polyvinylidene fluoride (PVDF) membrane (activated with methanol in advance), and filter paper, paying attention to removing the air bubbles between each layer. Place the transfer cassette into the transfer tank, add the transfer buffer, and transfer the membrane at a current of 300 mA for 90–120 min at 4 °C (adjust the specific time according to the molecular weight of the target protein). After the transfer is completed, take out the PVDF membrane and block it with a 5% skim milk solution prepared with 1× Tris-buffered saline with Tween (TBST) on a shaker at room temperature for 1–2 h to block the non-specific binding sites on the membrane.

[0081] After the blocking is completed, wash the PVDF membrane 3 times with 1× TBST buffer, 5–10 min each time. Then place the membrane into a 1× TBST buffer containing the primary antibody (select the appropriate primary antibody according to the target protein) and incubate it overnight at 4 °C. The next day, take out the membrane from the primary antibody solution and wash it 3 times with 1× TBST buffer, 10 min each time, to remove the unbound primary antibody. Then place the membrane into a 1× TBST buffer containing the secondary antibody (a secondary antibody species-matched to the primary antibody) and incubate it at room temperature for 1–2 h. After the incubation is completed, wash it 3 times with 1× TBST buffer by shaking, 10 min each time, to thoroughly remove the unbound antibody. After the washing is completed, evenly cover the surface of the membrane with the ECL chemiluminescent agent, and perform exposure using an imaging system to obtain a chemiluminescent image. The gray value of the target protein band is semi-quantitatively analyzed using ImageJ software, with GAPDH as the internal reference protein to calculate the relative expression level of the target protein.

[0082] 1.2.6 Reverse transcription quantitative polymerase chain reaction detection

[0083] First, prepare the colon tissue sample and place it in an enzyme-free EP tube. Use the FreeZol Reagent kit to extract RNA. In the first step, add FreeZol Reagent lysis buffer to the enzyme-free EP tube (add 500 μL of lysis buffer for every 50 mg of tissue), and lyse at room temperature for 5 min. In the second step, add Dilution Buffer, mix well and let it stand at room temperature for 5 min. After 5 min, centrifuge at 12000 rpm for 15 min at room temperature to precipitate impurities. In the third step, pipette the RNA-containing supernatant in the tube, transfer it to a new enzyme-free EP tube, then add an equal volume of isopropanol to the supernatant, mix well and let it stand for 20 min. After the standing time, centrifuge at 12000 rpm for 10 min at room temperature. In the fourth step, discard the supernatant after centrifugation and retain the precipitate, add 1 mL of 75% ethanol solution prepared with diethylpyrocarbonate (DEPC) water and absolute ethanol to wash the precipitate, and centrifuge at 8000 rpm for 5 min at room temperature. Repeat the washing 2 times to obtain the RNA precipitate. Dissolve the precipitate with DEPC water, and use a ultra-micro spectrophotometer to measure the ratios of A260 / A280 and A260 / A230 to evaluate the purity of RNA, and at the same time determine its concentration to ensure the accuracy of subsequent experiments.

[0084] Use the extracted high-quality RNA for reverse transcription to synthesize cDNA. In the reverse transcription reaction system, sequentially add appropriate amounts of RNA template, DEPC water, and 5× gDNA Wiper Mix according to the instructions. React in a PCR instrument at 42 °C for 2 min to remove residual genomic DNA. Then add an appropriate amount of 4× HisyGo qRT Red SuperMix and react in the PCR instrument according to the program of 37 °C for 15 min and 85 °C for 5 s to synthesize cDNA. The synthesized cDNA can be immediately used for subsequent experiments or stored in a -20 °C refrigerator for later use.

[0085] Prepare the qPCR reaction system, which usually includes cDNA template, specific primers (primer sequences are shown in Table 2), fluorescent dye (SYBR Green), etc. Set up corresponding reaction tubes according to the target gene and reference gene. Set a suitable reaction program on the qPCR instrument, which generally includes three stages: pre-denaturation, cyclic amplification, and melting curve analysis. In the pre-denaturation stage, it is usually heated at 95°C for 3 - 5 min to fully denature the cDNA template; in the cyclic amplification stage, generally 40 cycles are carried out, and each cycle includes denaturation (95°C, 10 - 15 s), annealing (the temperature is determined according to the primer Tm value, generally at 55 - 65°C, 15 - 30 s), and extension (72°C, 20 - 30 s); in the melting curve analysis stage, it is slowly heated from 60°C to 95°C to detect the specificity of the PCR product. After the reaction is completed, according to the change of the fluorescence signal recorded by the qPCR instrument, analyze the CT values of the target gene and the reference gene, and calculate the expression level of the target gene by the method of 2 (-△△CT ) relative quantification.

[0086] Table 2 Primer sequences

[0087]

[0088] 1.2.7 Enzyme-linked immunosorbent assay detection

[0089] Obtain the blood of mice by orbital blood collection method. Place the collected blood in an EP tube and let it stand at room temperature for 30 - 60 min to allow the blood to clot fully. Then centrifuge the centrifuge tube at a speed of 3000 rpm for 20 min. After centrifugation, the blood will be stratified, and the clear liquid on the upper layer is the serum. Carefully aspirate the serum into a new centrifuge tube, avoiding sucking the blood cells in the lower layer. If the detection cannot be carried out immediately, the serum samples can be aliquoted and stored at -20°C or -80°C. The subsequent steps are carried out according to the instruction manual process of the ELISA kit. After the experiment, use a multifunctional microplate reader for detection.

[0090] 1.2.8 Immunohistochemistry detection

[0091] Use a microtome to cut the embedded mouse colon paraffin block into thin slices with a thickness of 4 - 6 μm. Float the cut thin slices in warm water at 40 - 45°C to make the slices unfold flat. Pick up the slices with a glass slide, let the slices adhere flatly to the glass slide, and then place the glass slide in an oven at 60°C and bake for 1 - 2 h to make the slices firmly adhere to the glass slide.

[0092] The baked sections were successively dewaxed in xylene 2-3 times, 5-10 min each time, and then hydrated through gradient alcohol, successively placed in 100%, 95%, 90%, 80%, 70% alcohol for 2-3 min each, and finally rinsed with distilled water. To enhance the exposure of antigens, the sections were placed in antigen retrieval solution, and antigen retrieval was performed by methods such as microwave retrieval or high-pressure retrieval. After retrieval, let it cool naturally, and rinse with PBS buffer 3 times, 5 min each time. Dropwise add 3% hydrogen peroxide solution and incubate at room temperature for 10–15 min to block the activity of endogenous peroxidase, and then rinse with PBS buffer 3 times, 5 min each time. Dropwise add normal goat serum blocking solution and incubate at room temperature for 30 min to reduce non-specific binding. Discard the blocking solution without rinsing, and directly dropwise add the primary antibody SIRT1 (diluted with antibody diluent according to the dilution ratio recommended in the instruction manual), and incubate overnight at 4°C. The next day, take out the sections from the refrigerator, restore to room temperature, and rinse with PBS buffer 3 times, 5 min each time. Dropwise add biotin-labeled secondary antibody and incubate at room temperature for 30-60 min, and then rinse with PBS buffer 3 times, 5 min each time. Dropwise add streptavidin working solution labeled with horseradish peroxidase and incubate at room temperature for 30 min, and then rinse with PBS buffer 3 times, 5 min each time. Dropwise add DAB chromogenic solution, observe the chromogenic situation under the microscope, terminate chromogenesis in a timely manner, and rinse with distilled water. Counterstain the cell nuclei with hematoxylin for 1-2 min, then rinse with tap water, differentiate with hydrochloric acid alcohol for a few seconds, and then rinse with tap water to turn blue.

[0093] The stained sections were successively dehydrated through 70%, 80%, 90%, 95% alcohol, 2-3 min each, then dehydrated in absolute ethanol 2 times, 2-3 min each time. Then place it in xylene for clearing 2-3 times, 2-3 min each time. Finally, seal the slices with neutral balsam and cover with a coverslip. Place the sealed slices under the microscope and observe, and judge the expression of SIRT1 in mouse colon tissue according to the staining situation.

[0094] 1.2.9 Detection of SIRT1 activity

[0095] First, add the prepared colon tissue into an EP tube, add physiological saline according to the ratio of tissue sample mass (mg): physiological saline (0.9% NaCl) volume (mL) = 1:9, and homogenize in a tissue grinder. Then centrifuge at 4°C, 10000 g for 10 min, and take the supernatant. Take part of the supernatant and use a BCA detection kit to measure the protein concentration. The remaining supernatant is used for SIRT1 activity detection, and the detection steps are carried out strictly according to the procedure of the SIRT1 activity detection kit instruction manual.

[0096] 1.2.10 Data statistics

[0097] All data were analyzed using GraphPad Prism 8 software. Data values were presented as mean ± standard error of the mean (SEM). If the data met the homogeneity of variance or normal distribution, one-way ANOVA combined with Tukey multiple comparison test was used for statistical analysis. If the data did not meet the homogeneity of variance or normal distribution, the non-parametric Kruskal-Wallis test was selected, and multiple corrections between groups were performed by Dunn's method. When the p-value was less than 0.05 (*), less than 0.01 (**), or less than 0.001 (***), it was considered statistically significant.

[0098] 1.3 Experimental results

[0099] 1.3.1 General situation

[0100] The mice in the CON group were in good mental state, with smooth and shiny hair, normal spontaneous activities, food and water intake, and clean perianal area without abnormalities; the mice in the MOD group showed typical disease characteristics, manifested as mental depression, curled up and less active, dry and messy hair, reduced food and water intake, fresh bleeding points and bloody secretions visible around the anus, and feces adhered to form dirty scabs (as Figure 1 shown); the state of the mice in the LU group was similar to that of the MOD group, but the degree of perianal bleeding was slightly less; the improvement in the SASP group, MU group, and HU group was more significant, with a significant recovery in mental state, improved hair texture, increased food and water intake compared with the MOD group, and significantly less perianal bleeding than the MOD group (as Figure 1 shown).

[0101] 1.3.2 Body weight and body weight percentage

[0102] Weight loss is one of the symptoms of DSS-induced colitis. After 3-5 days of free access to DSS solution for mice, the body weight of mice will show a sharp decline. In the results of this experiment (as Figure 2As shown, the body weight of mice that drank the DSS solution decreased severely 3 days after drinking the DSS solution. However, the decrease in body weight in the SASP group, MU group, and HU group was significantly less than that in the MOD group. By comparing the daily body weight of mice with their initial body weight (the body weight when starting to drink the DSS solution), the percentage of the daily body weight of mice relative to the initial body weight can be obtained. Analyzing the percentage of the body weight of mice on the last day, the results showed that the body weight percentage of mice in the MOD group was significantly lower than that in the CON group (p<0.001), while the body weight percentages in the SASP group (p<0.001), MU group (p<0.05), and HU group (p<0.001) were significantly higher than that in the MOD group. The above results indicate that the positive drug sulfasalazine and medium and high doses of uridine can significantly improve the symptom of weight loss caused by DSS-induced colitis.

[0103] Figure 2 In (A), the daily body weight of mice; (B), the percentage of mouse body weight. n = 6, the results are expressed as Mean±SEM. Compared with the CON group, # p<0.05, ## p<0.01, ### p<0.001; compared with the MOD group, * p<0.05, ** p<0.01, *** p<0.001.

[0104] 1.3.3 Colon length and colon weight / colon length ratio

[0105] After euthanizing the mice and obtaining the colon, the colon length was immediately measured (as shown in Figure 3 A). The colon length of the MOD group was 5.4±0.160 cm, and the colon length of the CON group was 6.9±0.094 cm. The colon of mice in the MOD group was significantly shorter than that in the CON group (p<0.001). Among the drug-administered groups, the colon length of the SASP group was 6.0±0.140 cm; the colon length of the LU group was 5.6±0.130 cm; the colon length of the MU group was 6.1±0.190 cm; the colon length of the HU group was 6.1±0.110 cm. Among them, there were significant differences in the colon lengths between the SASP group (p<0.05), MU group (p<0.05), and HU group (p<0.05) and the colon length of the MOD group (as shown in Figure 3 B). Although the colon length of the LU group was slightly longer than that of the MOD group, there was no statistical difference between them.

[0106] Since colitis can cause colon edema and thickening of the intestinal wall, the colon weight / colon length ratio (colon weight / colon length) of colitis mice is higher than that of normal mice. In the results of this experiment (as shown in Figure 3As shown in C, the intestinal weight / length ratio in the MOD group was higher than that in the CON group (p < 0.001). The intestinal weight / length ratio in the MOD group was 52 ± 2.50 mg / cm, while that in the CON group was 36 ± 2.10 mg / cm. The intestinal weight / length ratios in the SASP group, LU group, MU group, and HU group were 41 ± 0.86 mg / cm, 49 ± 2.60 mg / cm, 42 ± 0.95 mg / cm, and 40 ± 1.20 mg / cm, respectively. Compared with the MOD group, there were significant decreases in the intestinal weight / length ratios in the SASP group (p < 0.001), MU group (p < 0.01), and HU group (p < 0.001).

[0107] Figure 3 Among them, (A) picture of mouse colon measurement; (B) results of mouse colon length; (C) mouse intestinal weight / length ratio, n = 6, results are expressed as Mean ± SEM, compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group, * p < 0.05 ，** p < 0.01, *** p < 0.001.

[0108] Table 3 Colon weight (mg), colon length (cm), and intestinal weight / length (mg / cm)

[0109]

[0110] Note: n = 6, results are expressed as Mean ± SEM, compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0111] 1.3.4 Spleen coefficient

[0112] As the largest immune organ in the body, the spleen often shows enlargement in mouse models of immune diseases such as colitis. Compared with the MOD group, the spleen weight index in the SASP group and the medium- and high-dose uridine intervention groups (MU group, HU group) was significantly decreased (p < 0.01) (as Figure 4 shown), suggesting that uridine may effectively inhibit spleen enlargement and maintain the normal volume of the spleen by regulating immune responses.

[0113] Figure 4Spleen coefficients and spleen pictures of mice: Among them, (A) spleen coefficients of mice; (B) spleen pictures of mice.

[0114] n = 6, the results are expressed as Mean±SEM. Compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0115] Table 4 Spleen weights (g) of mice, body weights (g) of mice at the time of sacrifice, and spleen coefficients of mice

[0116]

[0117] Note: n = 6, the results are expressed as Mean±SEM. Compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0118] 1.3.5 Disease Activity Index (DAI) scoring

[0119] The DAI score is a core indicator for evaluating the severity of colitis. By establishing a DAI scoring system, after starting to use DSS solution to construct a mouse colitis model, the fecal characteristics, occult blood conditions, and body weight changes of mice were recorded daily, and the severity of colitis in each group was comprehensively evaluated. On the 11th day of the experiment (the 4th day of modeling), the DAI scores of all mice that drank DSS solution showed a significant increase. And on the last day of the experiment, the DAI scores of the SASP group, MU group, and HU group were significantly lower than those of the MOD group (as Figure 5 shown).

[0120] Figure 5 DAI scores of mice. n = 6, the results are expressed as Mean±SEM. Compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0121] 1.3.6 Histopathological scoring of colon tissue

[0122] In the CON group, the intestinal structure was normal. The epithelial cells in the mucosal layer were arranged tightly, without degeneration or exfoliation. There were relatively many crypts, and the goblet cells were abundant, as indicated by the yellow arrows; occasional inflammatory cell infiltration was seen, as indicated by the red arrows; in the MOD group, the intestinal damage was obvious. Almost all of the mucosal layer was ulcerated, and the epithelial cells were eroded and exfoliated, as indicated by the black arrows; the crypts and goblet cells disappeared extensively. At the same time, a large number of inflammatory cells and fibrous tissue proliferation were visible, as indicated by the red arrows; in some areas, the inflammation had penetrated the intestinal wall, as indicated by the blue arrows; in the SASP group, the treatment effect was obvious. The degree of intestinal damage was significantly improved compared with the model group. In a small part of the mucosal area, the epithelial cells were eroded and exfoliated, the crypts disappeared, and a small amount of inflammatory cells and fibrous tissue proliferation were visible; in the uridine intervention group, the degree of damage was improved compared with the MOD group. Among them, the LU group had the worst treatment effect. A large area of ulcer was still visible in the mucosal layer, and a large number of inflammatory cells and fibrous tissue proliferation were present; in the MU group and the HU group, the treatment effects were comparable to those of the SASP group. In a small part of the mucosal layer, there was damage, and the inflammatory cells and fibrous tissue were significantly reduced compared with the model group, and the number of goblet cells increased (as Figure 6 shown in A). According to the pathological scoring standard of the colon tissue, the scores of the SASP group, the MU group, and the HU group were significantly lower than those of the MOD group (p < 0.001), indicating that the colon damage in the SASP group, the MU group, and the HU group was lower than that in the MOD group (as Figure 6 shown in B).

[0123] Figure 6 HE staining of mouse colon tissue. Among them, (A) HE staining pictures of mouse colon tissue (40× and 200×); (B) pathological scoring of mouse colon tissue. The yellow arrows indicate normal crypts and goblet cells; the red arrows indicate inflammatory cell infiltration; the black arrows indicate epithelial cell damage; the blue arrows indicate transmural intestinal wall damage, n = 4, and the results are expressed as Mean ± SEM. Compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0124] Table 5 Histopathological scoring

[0125]

[0126] Note: n = 4, and the results are expressed as Mean ± SEM. Compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group,* p < 0.05, ** p < 0.01, *** p < 0.001.

[0127] 1.3.7 mRNA expression levels of inflammatory factors in colon tissue

[0128] The expression of mRNA of inflammatory factors in mouse colon tissue was detected by RT-qPCR (as Figure 7 shown). The mRNA expression level of IL-1β in the MOD group was 11 ± 0.74 times that of the CON group (p < 0.001). In the SASP group, LU group, MU group and HU group, the mRNA expressions of IL-1β were 2.0 ± 0.36, 7.4 ± 1.10, 2.6 ± 0.55 and 1.8 ± 0.14 times that of the CON group, respectively. Therefore, in terms of the mRNA expression of IL-1β, there were statistical differences between the SASP group (p < 0.001), LU group (p < 0.05), MU group (p < 0.001) and HU group (p < 0.001) and the MOD group. The expression trend of IL-6 mRNA was similar to that of IL-1β, but there was no significant difference in the IL-6 mRNA expression level between the LU group and the MOD group. However, there were no statistical differences in the mRNA expression level of TNF-α among all groups.

[0129] Figure 7 mRNA expression levels of inflammatory factors in mouse colon tissue. Among them, (A) IL-1β mRNA expression level in mouse colon tissue (relative to the CON group); (B) IL-6 mRNA expression level in mouse colon tissue (relative to the CON group); (C) TNF-α mRNA expression level in mouse colon tissue (relative to the CON group), n = 3, and the results are expressed as Mean ± SEM. Compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0130] Table 6 mRNA levels of IL-1β, IL-6 and TNF-α

[0131]

[0132] Note: n = 3, and the results are expressed as Mean ± SEM. Compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group,* p < 0.05, ** p < 0.01, *** p < 0.001.

[0133] 1.3.8 Serum inflammatory factor levels

[0134] The concentrations of IL-1β, IL-6, and TNF-α in the sera of mice were detected using an ELISA kit (as Figure 8 shown). The results showed that the serum levels of IL-1β in the six groups were 6.4 ± 0.67, 21 ± 2.20, 9.9 ± 1.10, 17.0 ± 2.80, 9.6 ± 1.20, and 10.0 ± 0.55, respectively. Compared with the MOD group, the serum IL-1β level in the SASP group of mice was significantly lower (p < 0.001). Additionally, the serum IL-1β levels in the MU and HU groups were also significantly lower than those in the MOD group (p < 0.001). The trends of the serum levels of IL-6 and TNF-α in each group were consistent with those of IL-1β, that is, the serum IL-6 levels in the SASP, MU, and HU groups were all significantly lower than those in the MOD group (p < 0.01). At the same time, the serum IL-6 levels in the SASP, MU, and HU groups were all significantly lower than those in the MOD group.

[0135] Figure 8 For the serum inflammatory factor levels in mice. Among them, (A) Serum IL-1β level in mice; (B) Serum IL-6 level in mice; (C) Serum TNF-α level in mice, n = 6, results are expressed as Mean ± SEM, compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0136] Table 7 Serum inflammatory factor levels

[0137]

[0138] Note: n = 6, compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0139] 1.3.9 mRNA and protein expression levels of tight junction proteins in colon tissues

[0140] In addition to the reduction in inflammatory levels, the intestinal barrier function in the SASP, MU, and HU groups was also restored compared with the MOD group. This restoration was mainly reflected in the significantly higher mRNA levels of the tight junction proteins Occludin and ZO-1 in the SASP, MU, and HU groups compared with the MOD group (p < 0.05) (as shown in Figure 9 Figures B and C). However, only the ZO-1 protein levels in the SASP and HU groups were significantly higher than those in the MOD group (p < 0.05) (as shown in Figures E and G), while the Occludin protein levels in the SASP, MU, and HU groups were all increased relative to the MOD group (p < 0.05) (as shown in Figure 9 Figures D and F). For another tight junction protein, Claudin-5, only the mRNA levels in the SASP and HU groups were significantly higher than those in the MOD group among the drug treatment groups (p < 0.05) (as shown in Figure 9 Figure A).

[0141] Figure 9 are the tight junction proteins and mRNA levels in mouse colon tissues. Among them, (A) Claudin-5 mRNA expression level in mouse colon tissues (relative to the CON group); (B) ZO-1 mRNA expression level in mouse colon tissues (relative to the CON group); (C) Occludin mRNA expression level in mouse colon tissues (relative to the CON group); (D) Occludin WB band in mouse colon tissues; (E) ZO-1 WB band in mouse colon tissues; (F) Occludin protein expression level in mouse colon tissues (relative to the CON group); (G) ZO-1 protein expression level in mouse colon tissues (relative to the CON group), n = 3, the results are expressed as Mean ± SEM, compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0142] Table 8 mRNA levels of tight junction proteins

[0143]

[0144] Note: n = 3, the results are expressed as Mean ± SEM compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group, * p < 0.05,** p < 0.01, *** p < 0.001.

[0145] 1.3.10 Expression level and activity of SIRT1 protein in colon tissue

[0146] The results of WB analysis showed that uridine intervention significantly upregulated the expression of SIRT1 protein in mouse colon tissue. Specifically, the ratio of SIRT1 / GAPDH gray value in the MU group (p < 0.05) and the HU group (p < 0.001) was significantly higher than that in the MOD group, while there was no statistical difference between the SASP group and the MOD group (as shown in Figure 10 Figures A and B). This phenomenon may be related to the mechanism of action of sulfasalazine. This drug mainly blocks prostaglandin synthesis by inhibiting the activity of cyclooxygenase (COX) and synergistically inhibits the production of inflammatory mediators such as leukotrienes. Its anti-inflammatory effect does not depend on the rapid recovery of SIRT1 protein. Therefore, even when the inflammation is controlled, the expression level of SIRT1 remains at a level similar to that of the MOD group. The results of immunohistochemistry and activity detection of SIRT1 were consistent with the Western blot results, further indicating that uridine can upregulate the expression and activity of SIRT1 protein in mouse colon tissue (as shown in Figure 10 Figures C, D, and E).

[0147] Figure 10 Expression level and activity of SIRT1 protein in mouse colon tissue. Among them, (A) Expression level of SIRT1 protein in mouse colon tissue (relative to the CON group); (B) SIRT1 WB band in mouse colon tissue; (C) Immunohistochemical results of SIRT1 protein in mouse colon tissue; (D) SIRT1 enzyme activity in mouse colon tissue; (E) Immunohistochemical picture of SIRT1 protein in mouse colon tissue (200×), n = 3, the results are expressed as Mean ± SEM. Compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0148] Table 9 SIRT1 enzyme activity level

[0149]

[0150] Note: n = 3, the results are expressed as Mean ± SEM. Compared with the CON group, # p < 0.05, ## p < 0.01, ###p < 0.001; compared with the MOD group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0151] 1.3.11 Phosphorylation of NF-κB in colon tissue

[0152] The phosphorylation of NF-κB in the MU group and HU group was significantly inhibited compared with the MOD group (p < 0.05) (as Figure 11 shown in A and B). In addition, the phosphorylation of NF-κB in the SASP group was also significantly decreased compared with the MOD group (p < 0.05). Overactivation of the NF-κB pathway leads to an increase in the production of IL-1β. The WB results showed that the protein expression of IL-1β in the colon tissue of the MOD group was significantly increased compared with the CON group (p < 0.001), while the protein expression of IL-1β in the colon tissue of all drug intervention groups, including the SASP group (p < 0.001), LU (p < 0.05) group, MU group (p < 0.01), and HU group (p < 0.001), was significantly decreased compared with the MOD group (as Figure 11 shown in A and C).

[0153] Figure 11 is the phosphorylation of NF-κB in mouse colon tissue. Among them, (A) WB bands of NF-κB, phosphorylation(p)-NF-κB, and IL-1β in mouse colon tissue; (B) Results of NF-κB phosphorylation in mouse colon tissue (compared with the CON group); (C) Expression level of IL-1β in mouse colon tissue (compared with the CON group), n = 3, compared with the CON group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the MOD group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0154] Example 4

[0155] Effect of uridine on inflammatory indexes of LPS-induced mouse macrophages RAW264.7:

[0156] 2.1 Experimental materials

[0157] 2.1.1 Cell source

[0158] Mouse macrophages RAW264.7 were purchased from Wuhan Shang'en Biotechnology Co., Ltd.

[0159] 2.2 Experimental methods

[0160] 2.2.1 Inflammatory model construction

[0161] RAW264.7 cells were subcultured using high-glucose DMEM medium containing 10% fetal bovine serum. When the cells were in the logarithmic growth phase and the confluence reached 70%-80%, they were seeded in the culture plate at a density of 2×10 6 cells / well (6-well plate) or 1×10 4 cells / well (24-well plate). After the cells adhered, the drug intervention groups were respectively added with uridine medium with a final concentration of 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL, and pre-incubated for 2 h under the conditions of 37℃ and 5% CO2. Subsequently, except for the blank control group, LPS was added to the remaining groups to a final concentration of 100 ng / mL, and the culture was continued for 24 h to complete the model construction.

[0162] 2.2.2 Protein immunoblotting detection

[0163] After discarding the medium in the 6-well plate, the cells were washed twice with pre-cooled PBS and transferred to an EP tube. 150 μL of RIPA lysis buffer containing protease inhibitor, phosphatase inhibitor, and EDTA was added. After sonication with an ultrasonic crusher, the cells were lysed on ice for 30 min. After the lysis was completed, the mixture was centrifuged at 12000 rpm for 15 min at 4℃. The supernatant was aspirated and transferred to a new EP tube. The subsequent steps were the same as those in Section 1.2.5 of the second part.

[0164] 2.2.3 Reverse transcription quantitative polymerase chain reaction detection

[0165] After discarding the medium in the 6-well plate, the cells were washed twice with pre-cooled PBS and transferred to a nuclease-free EP tube. The subsequent steps were the same as those in Section 1.2.6 of the second part. The primer sequences are shown in Table 10 (the repeated primer sequences are not listed again).

[0166] Table 10 Primer sequences

[0167]

[0168] 2.2.4 Enzyme-linked immunosorbent assay detection

[0169] The cell culture medium in the 24-well plate was aspirated and centrifuged at 3000 rpm for 10 min at room temperature. The supernatant was aspirated and transferred to a new EP tube. If the detection could not be carried out immediately, the supernatant samples could be aliquoted and stored at -20℃ or -80℃. The subsequent steps were carried out according to the instructions of the ELISA kit. After the experiment, a multifunctional microplate reader was used for detection.

[0170] 2.2.5 Immunofluorescence

[0171] Cells were cultured in 24-well plates. After the model was constructed, the culture medium was removed, and the cells were washed 2-3 times with PBS. Then, they were fixed with 4% paraformaldehyde for 15-20 min at room temperature and washed again with PBS. If the target protein was an intracellular protein, 0.1%-0.5% Triton X-100 was added to the PBS and incubated for 5-10 min, followed by washing with PBS. Nonspecific binding sites were blocked with goat serum for 1 h at room temperature. The primary antibody was diluted at a ratio of 1:100 and added to the cells, and then incubated for 1-2 h at room temperature or overnight at 4°C. Subsequently, the cells were washed 3 times with PBS. After that, the fluorescently labeled secondary antibody was diluted and added to the cells, and incubated for 1 h at room temperature in the dark. After incubation with the secondary antibody, the cells were washed 3 times with PBS. When nuclear counterstaining was required, DAPI was added to the cells and incubated for 5 min at room temperature in the dark, followed by washing with PBS. Finally, the cells were observed and images were taken by fluorescence microscopy.

[0172] 2.2.6 Data statistics

[0173] All data were analyzed using GraphPad Prism 8 software. Data values were presented as mean ± SEM and were derived from three different biological samples. If the data met the homogeneity of variance or normal distribution, one-way ANOVA combined with Tukey multiple comparison test was used for statistical analysis. If the data did not meet the homogeneity of variance or normal distribution, the non-parametric Kruskal-Wallis test was selected, and multiple corrections between groups were performed by Dunn's method. Statistical significance was considered when the p-value was less than 0.05 (*), less than 0.01 (**), and less than 0.001 (***).

[0174] 2.3 Experimental results

[0175] 2.3.1 mRNA expression levels of inflammatory factors in RAW264.7 cells

[0176] The mRNA expression levels of IL-1β, IL-6, TNF-α, and COX-2 genes in RAW264.7 cells after LPS stimulation were detected (as Figure 11(as shown). The mRNA expression levels of IL-1β, IL-6, TNF-α, and COX-2 in RAW264.7 cells stimulated only by LPS without drug intervention were 171.0 ± 13.0 (p < 0.001), 3726.0 ± 257.0 (p < 0.001), 15 ± 0.95 (p < 0.01), and 727 ± 84 (p < 0.001) times those of the blank control group, respectively, showing a statistically significant difference between the two groups. In the drug intervention group, the mRNA expression levels of IL-1β (p < 0.001), IL-6 (p < 0.001), and COX2 (p < 0.01, p < 0.001) at medium and high doses were significantly lower than those in the LPS model group. However, there was no significant difference in the mRNA expression level of TNF-α between all drug intervention groups and the LPS model group.

[0177] Figure 12 are the mRNA expression levels of inflammatory factors IL-1β, IL-6, TNF-α, and COX-2 in RAW264.7 cells. Among them, (A) IL-1β mRNA expression level in RAW264.7 cells (relative to the blank control group); (B) IL-6 mRNA expression level in RAW264.7 cells (relative to the blank control group); (C) TNF-α mRNA expression level in RAW264.7 cells (relative to the blank control group); (D) COX-2 mRNA expression level in RAW264.7 cells (relative to the blank control group), n = 3, and the results are expressed as Mean ± SEM. Compared with the blank control group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the LPS model group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0178] Table 11 mRNA expression levels of inflammatory factors IL-1β, IL-6, TNF-α, and COX-2 in RAW264.7 cells

[0179]

[0180] Note: n = 3, and the results are expressed as Mean ± SEM. Compared with the blank control group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the LPS model group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0181] 2.3.2 Inflammatory factor content in the supernatant of RAW264.7 cell culture medium

[0182] The IL-1β contents in the cell supernatants of the blank control group, LPS model group, and high, medium, and low-dose drug intervention groups were 0.81±0.51, 63±3.9, 42±4.8, 38±1.1, and 36±3.1 pg / mL, respectively. Among them, the IL-1β contents in the low, medium, and high-dose drug intervention groups were significantly lower than those in the LPS model group (p<0.01, p<0.01, p<0.001) (as Figure 12 shown in A). The IL-6 contents in the cell supernatants of the 5 groups were 2.8±2.0, 611±78, 458±53, 392±16, and 335±25 pg / mL, respectively. Among them, the IL-6 contents in the cell supernatants of the medium and high-dose drug intervention groups were significantly lower than those in the LPS model group (p<0.05, p<0.01) (as Figure 13 shown in B).

[0183] Figure 13 They are the inflammatory factor IL-1β and IL-6 contents in the supernatant of RAW264.7 cell culture medium. Among them, (A) IL-1β content in RAW264.7 cell supernatant; (B) IL-6 content in RAW264.7 cell supernatant, n = 3, and the results are expressed as Mean±SEM. Compared with the blank control group, # p<0.05, ## p<0.01, ### p<0.001; compared with the LPS model group, * p<0.05, ** p<0.01, *** p<0.001.

[0184] Table 12 Inflammatory factor IL-1β and IL-6 contents in the supernatant of RAW264.7 cell culture medium

[0185]

[0186] Note: n = 3, and the results are expressed as Mean±SEM. Compared with the blank control group, # p<0.05, ## p<0.01, ### p<0.001; compared with the LPS model group, * p<0.05, ** p<0.01, *** p<0.001.

[0187] 2.3.3 Expression of SIRT1 protein in RAW264.7 cells

[0188] It can be found from the experimental results of observing WB that the expression level of SIRT1 protein in the medium- and high-dose drug intervention groups was significantly higher than that in the LPS model group (p<0.05, p<0.01) (as Figures 2-3 shown in A and B). The results of immunofluorescence were consistent with those of WB, and the expression level of SIRT1 protein in the medium- and high-dose drug intervention groups was also significantly higher than that in the LPS model group (p<0.05) (as Figure 14 shown in C and D).

[0189] Figure 14 It was the expression level of SIRT1 protein in RAW264.7 cells. Among them, (A) WB band of SIRT1 in RAW264.7 cells; (B) Expression level of SIRT1 protein in RAW264.7 cells (relative to the blank control group); (C) Immunofluorescence intensity of SIRT1 protein in RAW264.7 cells; (D) Immunofluorescence picture of RAW264.7 cells (200×), n = 3, the results were expressed as Mean±SEM. Compared with the blank control group, # p<0.05, ## p<0.01, ### p<0.001; compared with the LPS model group, *p<0.05, **p<0.01, ***p<0.001.

[0190] 2.3.4 Phosphorylation of NF-κB in RAW264.7 cells

[0191] With the increase in the expression of SIRT1 protein, the phosphorylation of NF-κB in the high-dose uridine intervention group was significantly lower than that in the LPS model group (p<0.05) (as Figure 15 shown in A and B). And the protein level of IL-1β in the medium- and high-dose uridine intervention groups was significantly lower than that in the LPS model group (p<0.01, p<0.001) (as Figures 2-4 shown in A and C). The results were similar to those of the in vivo experiment.

[0192] Figure 15 It was the phosphorylation of NF-κB in RAW264.7 cells. Among them, (A) WB bands of NF-κB, p-NF-κB, and IL-1β in RAW264.7 cells; (B) Phosphorylation results of NF-κB in RAW264.7 cells; (C) Expression level of IL-1β protein in RAW264.7 cells (relative to the blank control group), n = 3, the results were expressed as Mean±SEM. Compared with the blank control group, # p<0.05, ## p<0.01, ### p<0.001; compared with the LPS model group, * p<0.05,** p < 0.01, *** p < 0.001.

[0193] Results:

[0194] In the in vivo experimental results, the symptoms of colitis were alleviated. Uridine intervention significantly improved the mental state of mice, reduced weight loss (SASP group: p < 0.001; MU group: p < 0.05; HU group: p < 0.001), prolonged the colon length (SASP group: p < 0.05; MU / HU groups: p < 0.05), decreased the intestinal weight / intestinal length ratio (SASP group: p < 0.001; MU group: p < 0.01; HU group: p < 0.001) and spleen coefficient (SASP group: p < 0.01; MU / HU groups: p < 0.01). The DAI score showed that the SASP group, MU group, and HU group were significantly lower than the MOD group (p < 0.05), indicating that uridine effectively alleviated the severity of colitis. In terms of anti-inflammatory effects, uridine significantly inhibited the mRNA expression of IL-1β (SASP group: p < 0.001; LU group: p < 0.05; MU / HU groups: p < 0.001) and IL-6 (SASP group: p < 0.01; MU group: p < 0.01; HU group: p < 0.001) in colon tissues, and had no significant effect on the mRNA level of TNF-α. However, in serum, uridine significantly decreased the protein contents of IL-1β (SASP group: p < 0.001; MU / HU groups: p < 0.001), IL-6 (SASP group: p < 0.01; MU / HU groups: p < 0.01) and TNF-α (SASP group: p < 0.01; MU / HU groups: p < 0.05). In terms of intestinal barrier protection, the results of RT-qPCR and Western blot showed that uridine up-regulated the mRNA and protein expressions of ZO-1 and Occludin in colon tissues (p < 0.05), but had a weak effect on the mRNA level of Claudin-5 (SASP group: p < 0.05; HU group: p < 0.05). HE staining showed that the mucosal damage of the colon in the uridine intervention group was alleviated, the crypt structure was repaired, and the infiltration of inflammatory cells was reduced. The protein expression (p < 0.05) and enzyme activity (p < 0.001) of SIRT1 in the colon tissues of the MU group and HU group were significantly higher than those of the MOD group, and at the same time, the phosphorylation of NF-κB was significantly inhibited (p < 0.05). It is suggested that uridine may play an anti-inflammatory role by enhancing the activity and expression of SIRT1.

[0195] In in vitro experiments, in terms of anti-inflammatory effects, uridine significantly reduced the mRNA expression of IL-1β, IL-6, and COX-2 in LPS-induced RAW264.7 cells (medium and high dose groups: p < 0.01) and the protein secretion of IL-1β and IL-6 (medium and high dose groups: p < 0.01), but had no significant effect on the TNF-α mRNA level. Medium and high doses of uridine significantly upregulated the expression of SIRT1 protein (p < 0.05), while the phosphorylation of NF-κB was significantly inhibited (p < 0.05), and the secretion and intracellular expression of IL-1β were reduced (p < 0.01).

[0196] Conclusion:

[0197] Uridine can improve the symptoms of mice with dextran sulfate sodium-induced acute experimental colitis model and has good therapeutic effects. The experimental results suggest that a satisfactory therapeutic effect can be obtained by using a medium dose of uridine. Its mechanism is related to uridine inhibiting the phosphorylation activation of NF-κB by upregulating the expression and activity of SIRT1 protein in the local colon mucosa, and regulating tight junction proteins and inflammatory indicators. At the same time, the in vitro experimental results are consistent with the in vivo experiments, indicating the scientific nature of the results. However, the more specific mechanism remains to be further explored.

[0198] The present invention provides an idea and method for the application of uridine in the preparation of drugs for preventing and treating ulcerative colitis. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. The application of uridine in the preparation of drugs for preventing and treating ulcerative colitis.

2. The use according to claim 1, characterized in that: The ulcerative colitis is in the acute stage.

3. The use according to claim 1, characterized in that: The ulcerative colitis is in the remission stage.

4. The use according to claim 1, characterized in that: The uridine can improve pathological damage of colon tissue.

5. The use according to claim 1, characterized in that: The uridine can inhibit the secretion of colon pro-inflammatory factors.

6. The use according to claim 1, characterized in that: The colon pro-inflammatory factors are IL-1β and / or IL-6.

7. The use according to claim 1, characterized in that: The uridine can restore the colon mucosal barrier.

8. The use according to claim 1, characterized in that: The uridine can promote the expression and activity of colon SIRT1 protein.

9. The use according to claim 1, characterized in that: The uridine can inhibit the phosphorylation activation of the NF-κB signaling pathway.

10. The use according to claim 1, characterized in that: The dosage form of the drug is selected from any one of tablets, capsules, injections or enteric-coated preparations.

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