Use of glabridin in the preparation of a drug for preventing or / and treating ulcerative colitis

Abstract

The application provides a use of glabridin in preparation of a drug for preventing or / and treating ulcerative colitis, relates to the technical field of biological medicine, and can improve the symptoms of a 3% DSS induced acute experimental colitis model mouse, has excellent therapeutic effect, and increases the body weight of the mouse. The experimental results show that the anti-colitis effect of the glabridin has concentration dependence, and the glabridin can effectively treat ulcerative colitis. The glabridin has no damage to the functional organs of the mouse, such as heart, liver, spleen, lung and kidney. The application provides a theoretical basis for developing a safe and effective new ulcerative colitis treatment drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to an application of glabriden in preparation of a drug for preventing or / and treating ulcerative colitis. BACKGROUND

[0002] Ulcerative colitis (UC) is a chronic, non-specific inflammatory bowel disease (IBD) mainly involving the mucosal layer of the colon and rectum, which is characterized by recurrent diarrhea, mucopurulent bloody stool, abdominal pain and tenesmus. The inflammation is localized in the colon and is continuously distributed, and severe cases can lead to intestinal wall ulceration, perforation and even cancer. The etiology of UC is not fully understood, and it is currently believed to be the result of the combined effects of genetics, immune dysfunction, intestinal flora disorder and environmental factors.

[0003] The current treatment goal of UC is to induce and maintain clinical remission, promote mucosal healing and improve the quality of life. The main treatment methods include: traditional drugs such as 5-aminosalicylic acid (5-ASA) for mild to moderate patients, glucocorticoids for acute phase control of inflammation, and immunosuppressants (such as azathioprine) for maintenance of remission. Biological agents such as anti-TNF-α (infliximab, adalimumab, anti-integrin (vedolizumab) significantly improve the prognosis of patients with moderate to severe UC. Small molecule drugs such as JAK inhibitors (tofacitinib, upadacitinib) and S1P receptor modulators (ozanimod) have become emerging treatment options due to their oral convenience and strong targeting. Despite the continuous progress in treatment methods, UC management still faces many challenges, such as large individual differences in efficacy, non-response (primary resistance) or late failure (secondary resistance) of some patients to biological agents or small molecule drugs; long-term safety issues, such as the potential for JAK inhibitors to increase the risk of infection and thrombosis, and the potential for biological agents to induce immune suppression-related complications; low histological healing rate, even if clinical remission, some patients still have microscopic inflammation, which increases the risk of recurrence and cancer; heavy economic burden: biological agents are expensive, and long-term treatment costs are high. Small molecule drugs have become a hot research direction for UC drug development due to their high oral bioavailability, low production cost and ability to target specific signaling pathways.

[0004] Therefore, the present application is provided. SUMMARY

[0005] One of the purposes of the present application is to provide an application of glabriden in preparation of a drug for preventing or / and treating ulcerative colitis, so as to solve the technical problem that there is no safe and effective small molecule drug for ulcerative colitis in the prior art.

[0006] The second purpose of the present application is to provide an application of glabriden in preparation of a drug for repairing intestinal barrier damage.

[0007] A third objective of this invention is to provide the application of glycyrrhizin in the preparation of drugs for preventing the carcinogenesis of ulcerative colitis.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] In a first aspect, the present invention provides the use of glycyrrhizin in the preparation of medicaments for the prevention and / or treatment of ulcerative colitis.

[0010] Furthermore, the prevention and / or treatment of ulcerative colitis includes downregulating the expression of inflammatory factors and / or inhibiting the activation of the inflammatory signaling pathway NF-κB.

[0011] Furthermore, the inflammatory factors include at least one of TNF-α, IL-6, CCL2, or CXCL2.

[0012] Furthermore, the ulcerative colitis mentioned is acute ulcerative colitis.

[0013] Secondly, this invention provides the application of glycyrrhizin in the preparation of drugs for repairing intestinal barrier damage.

[0014] Furthermore, the repair of intestinal barrier damage includes at least one of repairing the intestinal mucosal layer, restoring the number of goblet cells, and promoting the expression of barrier proteins.

[0015] Furthermore, the barrier protein includes at least one of ZO-1, Occludin-1, and CDH1.

[0016] Thirdly, the present invention provides the use of glycyrrhizin in the preparation of drugs for preventing carcinogenesis of ulcerative colitis.

[0017] Furthermore, the prevention of ulcerative colitis from becoming cancerous includes inhibiting the activation of the cancer signaling pathway PI3K-Akt, reducing p-PI3K expression, or reducing p-Akt expression at least one of these.

[0018] Furthermore, the drug includes glycyrrhizin and / or its derivatives.

[0019] This invention provides the application of glycyrrhizin in the preparation of drugs for the prevention and / or treatment of ulcerative colitis. Glycyrrhizin can improve the symptoms of mice with 3% DSS-induced acute experimental colitis, exhibiting excellent therapeutic effects and increasing the body weight of the mice. Experimental results show that the anti-colitis effect of glycyrrhizin is concentration-dependent and can effectively treat ulcerative colitis; moreover, glycyrrhizin does not damage the functional organs of mice, including the heart, liver, spleen, lungs, and kidneys. This provides a theoretical basis for the development of safe and effective novel therapeutic drugs for ulcerative colitis. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 The pharmacological results of the prevention of ulcerative colitis by glycyrrhizin provided in Example 1 of the present invention are shown in the figure. In the figure, A is the mouse body weight, B is the DAI score, C is the colon length, D is the colon tissue appearance, and E is the colon histological staining.

[0022] Figure 2 The results of glaucamine inhibiting the release of inflammatory factors in UC mice provided in Example 2 of this invention;

[0023] Figure 3 The results of barrier protein expression in the intestinal barrier repair effect of glycyrrhizin provided in Example 2 of the present invention are shown in Figure 2. In Figure 2, A represents the expression of barrier protein at the mRNA level and B represents the expression of barrier protein at the protein level.

[0024] Figure 4 The mucosal layer observation results of the intestinal barrier repair effect of glycyrrhizin provided in Example 2 of the present invention are shown in Figure a, where a is PAS staining and b is immunohistochemistry of ZO-1 and Occludin-1.

[0025] Figure 5 The results of the inflammatory inhibition of NCM460 cells after LPS stimulation by glycyrrhizin provided in Example 3 of the present invention;

[0026] Figure 6 The results of Example 3 of this invention show the effect of glycyrrhizin on tight junctions between cells in NCM460 cells stimulated by LPS.

[0027] Figure 7 The results of Example 4 of this invention show the effect of glycyrrhizin on the mRNA expression levels of inflammatory factors IL-6, IL-17α and TNFα in RAW264.7 cells;

[0028] Figure 8 The results of glycyrrhizin inhibiting the activation of the PI3K-Akt signaling pathway provided in Example 5 of the present invention are shown in Figure A, which is a protein expression detection graph of p-PI3K and p-Akt, and Figure B is a statistical graph of the relative protein expression levels of p-PI3K and p-Akt.

[0029] Figure 9Results of glycyrrhizin inhibiting NF-κB signaling pathway activation, where A is the protein expression detection graph of P65 and p-P65, and B is the statistical graph of the relative protein expression of p-P65 / P65. Detailed Implementation

[0030] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0031] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] This invention provides, in one aspect, the use of glycyrrhizin in the preparation of drugs for the prevention and / or treatment of ulcerative colitis.

[0034] Glycyrrhizin effectively improved the symptoms of a 3% DSS-induced acute experimental colitis model in mice, demonstrating excellent therapeutic effects and increasing mouse body weight. Experimental results showed that the anti-colitis effect of glabridin was concentration-dependent and could effectively treat ulcerative colitis. Furthermore, glabridin did not damage the functional organs of mice—heart, liver, spleen, lungs, and kidneys. This provides a theoretical basis for developing safe and effective novel therapeutic drugs for ulcerative colitis.

[0035] This study reveals for the first time the effects and mechanisms of glabridin in preventing and treating ulcerative colitis. In vitro and in vivo experiments validated its function of regulating the inflammatory response and inhibiting NF-κB phosphorylation activation through the PI3K-Akt signaling pathway. In some specific embodiments, the prevention and / or treatment of ulcerative colitis includes downregulating the expression of inflammatory factors and / or inhibiting the activation of the NF-κB signaling pathway to reduce the flare-up of inflammation and / or downregulating p-65 levels. In some specific embodiments, the inflammatory factors include at least one of TNF-α, IL-6, CCL2, or CXCL2. In some specific embodiments, the ulcerative colitis is acute ulcerative colitis.

[0036] According to another aspect of the present invention, the application of glycyrrhizin in the preparation of drugs for repairing intestinal barrier damage is also provided. Experimental results show that glycyrrhizin can regulate the release of inflammatory factors and the expression of tight junction proteins between cells, promote the recovery of colonic goblet cells, and repair the intestinal mucosal barrier while having anti-inflammatory effects.

[0037] In some specific embodiments, the repair of intestinal barrier damage includes at least one of repairing the intestinal mucosal layer, restoring the number of goblet cells, and promoting the expression of barrier proteins. In some specific embodiments, the barrier protein includes at least one of ZO-1, Occludin-1, and CDH1.

[0038] Glycyrrhizin can inhibit the activation of the cancer signaling pathway PI3K-Akt, reducing the expression of p-PI3K and p-Akt, and has the potential to prevent the transformation of ulcerative colitis into colorectal cancer. According to another aspect of the present invention, the use of glycyrrhizin in the preparation of a medicament for preventing the carcinogenesis of ulcerative colitis is also provided.

[0039] In some specific embodiments, the prevention of ulcerative colitis from becoming cancerous includes inhibiting the activation of the cancer signaling pathway PI3K-Akt, reducing p-PI3K expression, or reducing p-Akt expression at least one of these.

[0040] In some specific embodiments, the drug includes glycyrrhizin and / or its derivatives.

[0041] The dosage form of the drug is any one of tablets, capsules, injections, or enteric-coated preparations. Derivatives include pharmaceutically acceptable salts, solvent compounds, tautomers, or mixtures thereof.

[0042] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0043] The glycyrrhizin used in the following examples was purchased from Chengdu EFA Biotechnology Co., Ltd.

[0044] Example 1: Treatment of Acute Experimental Colitis in Mice with Glycyrrhizin

[0045] This embodiment evaluates the therapeutic effect of glycyrrhizin on acute experimental colitis in mice by detecting mouse body weight, fecal characteristics, comparing disease activity index scores, colon length, and colon histological staining.

[0046] 1. Experimental Animals: A total of 48 male SPF-grade C57BL / 6J mice were used, and they were divided into a control group, a model group, a mesalazine group, a low-dose glycyrrhizin group, a medium-dose glycyrrhizin group, and a high-dose glycyrrhizin group. Each group contained 8 mice. The control group was the control group, the model group was the 3% DSS group, the mesalazine group was the 5-ASA group, the low-dose glycyrrhizin group was the Glabrone-L group, the medium-dose glycyrrhizin group was the Glabrone-M group, and the high-dose glycyrrhizin group was the Glabrone-H group.

[0047] 2. To establish an acute experimental colitis model in mice, the mice were given drinking water containing 3% sodium dextran sulfate for 7 days to establish the colitis model.

[0048] 3. Drug administration: The control group was given normal drinking water, while the model group was given drinking water containing 3% sodium dextran sulfate. The low-dose glycyrrhizin group, medium-dose glycyrrhizin group, and high-dose glycyrrhizin group were given intraperitoneal injections of 12.5 mg / kg, 25 mg / kg, and 50 mg / kg glycyrrhizin, respectively, once a day, with the corresponding administration volume calculated according to the mouse's body weight.

[0049] 4. The Disease Activity Index score is the DAI score.

[0050] The DAI scoring criteria are shown in Table 1. Scoring is conducted and recorded daily.

[0051] Table 1 Disease Activity Index (DAI) Scoring Criteria

[0052]

[0053] 5. Colon length analysis

[0054] At the end of the experiment, the colon of the mice was removed, from above the anus to below the cecum, and the changes in colon length in each group of mice were measured.

[0055] 6. Hematoxylin-eosin staining (HE staining):

[0056] At the end of the experiment, mouse colon tissue was taken, fixed with 4% (w / v) paraformaldehyde for 48 h, treated with 80% (v / v) ethanol for 5 h, 90% (v / v) ethanol for 5 h, and left overnight with 95% (v / v) ethanol. It was then treated with anhydrous ethanol I, II and III for 30 min each, xylene I, II and III for 30 min each, paraffin I and II for 30 min each, and paraffin III for 1 h. After dehydration, paraffin infiltration, embedding and sectioning were completed to obtain paraffin sections.

[0057] Paraffin sections were stained with hematoxylin and eosin (HE) according to the steps in Table 2, and mounted with neutral resin. All percentages in Table 2 are volume concentrations.

[0058] Table 2 HE staining steps

[0059]

[0060] 7. Experimental Results

[0061] like Figure 1 As shown in Figure A, compared with the 3% DSS model group, the weight of each drug intervention group increased to varying degrees, and this increase was concentration-dependent. Figure 1 As shown in Figure B, the DAI score results indicate that the disease activity index of mice in the drug treatment group was significantly reduced, and the symptoms of loose stools and bloody stools in the mice were alleviated. Figure 1 As shown in C and D, measurements of colon length in mice revealed that glycyrrhizin significantly reduced the degree of colonic shortening caused by inflammation, and high-dose glycyrrhizin restored colonic damage in a manner consistent with the control group. Figure 1 Figure E shows the HE staining results, which indicate that 3% DSS caused severe damage to the mouse colon, even the disappearance of the mucosal layer, and significant inflammatory infiltration. Glycyrrhizin repaired this damage, and the colonic damage and inflammatory infiltration in mice were reduced after administration.

[0062] Example 2: Glycyrrhizin repairs intestinal barrier damage

[0063] In this embodiment, RT-qPCR was used to detect the mRNA expression levels of IL-6 and IFN-γ, as well as intestinal barrier factors ZO-1, Occludin-1, and CDH1 in mouse colon tissue. PAS staining was used to detect the number of goblet cells in the intestine. Western blot was used to detect the protein expression of ZO-1, Occludin-1, and E-cadherin-1. Immunohistochemistry was used to detect the expression of ZO-1 and Occludin-1 to evaluate the anti-inflammatory and barrier repair effects of glycyrrhizin.

[0064] 1. Experimental Animals: A total of 48 male SPF-grade C57BL / 6J mice were used, and they were divided into a control group, a model group, a mesalazine group, a low-dose glycyrrhizin group, a medium-dose glycyrrhizin group, and a high-dose glycyrrhizin group. Each group contained 8 mice. The control group was the control group, the model group was the 3% DSS group, the mesalazine group was the 5-ASA group, the low-dose glycyrrhizin group was the Glabrone-L group, the medium-dose glycyrrhizin group was the Glabrone-M group, and the high-dose glycyrrhizin group was the Glabrone-H group.

[0065] 2. To establish an acute experimental colitis model in mice, the mice were given drinking water containing 3% sodium dextran sulfate for 7 days to establish the colitis model.

[0066] 3. Drug administration: The control group was given normal drinking water, while the model group was given drinking water containing 3% sodium dextran sulfate. The low-dose glycyrrhizin group, medium-dose glycyrrhizin group, and high-dose glycyrrhizin group were given intraperitoneal injections of 12.5 mg / kg, 25 mg / kg, and 50 mg / kg glycyrrhizin, respectively, once a day, with the corresponding administration volume calculated according to the mouse's body weight.

[0067] 4. Testing

[0068] Periodic acid Schiff staining (PAS staining): Dewaxing of paraffin sections: Dewaxing paraffin sections in xylene twice, 15 min each time, to obtain dewaxed sections.

[0069] Hydration: The dewaxed sections were hydrated by sequentially passing them through a gradient of alcohols. The specific steps were: 5 min of anhydrous ethanol, 3 min of 90% ethanol, 3 min of 80% ethanol, 3 min of 70% ethanol; 3 min of distilled water, repeated 3 times; and 3 min of PBS buffer, repeated 3 times, to obtain hydrated sections.

[0070] Oxidation: The hydrated sections were placed in a 1% (w / v) periodic acid solution and oxidized for 6-20 min to obtain oxidized sections.

[0071] Washing: Rinse the oxidized sections thoroughly with distilled water to remove excess periodic acid solution for 10 min to obtain washed oxidized sections.

[0072] Staining: Place the washed oxidized sections into fuchsin-sulfurite reagent (Schiff reagent) and stain for 10-20 minutes. The staining time can be adjusted according to the room temperature. In summer, when the room temperature is high, the staining time can be reduced, while in winter, when the room temperature is low, the staining time can be extended to 20 minutes to obtain Schiff-stained sections.

[0073] Rinsing: Rinse the Schiff stained sections with running water until the water color changes from red to colorless for 10 minutes to obtain the rinsed Schiff stained sections.

[0074] Counterstaining: The rinsed Schiff stained sections are counterstained with hematoxylin to make the cell nuclei appear blue. This usually takes 2 to 4 minutes to obtain counterstained sections.

[0075] Differentiation: If the cell nuclei are stained too darkly during the counterstaining process, hydrochloric acid alcohol can be used to differentiate the counterstained sections for about 2 to 5 seconds to obtain differentiated sections.

[0076] Blueing: Use Scott's Bluing Reagent to blue the counterstained or differentiated sections for 1-2 minutes, then rinse with running water for 3 minutes to obtain the blue-rebounded sections.

[0077] Dehydration: The blue-returned sections were sequentially dehydrated by gradient alcohol treatment. The specific steps were: 70% (v / v) ethanol for 1 min, 80% (v / v) ethanol for 1 min, 95% (v / v) ethanol for 2 min, and 100% (v / v) ethanol for 4 min to obtain dehydrated sections.

[0078] Clearing: The dehydrated sections are cleared with xylene, usually twice, for 15 minutes each time, to obtain clear sections.

[0079] Mounting: Dry the transparent slide, add a drop of neutral resin to mount the slide, and cover with a coverslip.

[0080] Immunohistochemical (IHC) assay:

[0081] The hydrated sections were obtained by following the above steps. The hydrated sections were then immersed in a 3% (v / v) hydrogen peroxide solution for 10 minutes to block endogenous peroxidase activity. After rinsing with tap water, the hydrogen peroxide-treated sections were obtained.

[0082] Hydrogen peroxide-treated sections were placed in citrate buffer (pH 6.0) for heat-induced antigen retrieval, and then microwaved for 20 min to obtain retrieval sections.

[0083] The repaired sections were allowed to cool naturally to room temperature, washed with PBS buffer, covered with bovine serum albumin (BSA), and incubated at room temperature for 10 min to reduce non-specific binding, resulting in BSA-treated sections.

[0084] Add 3 drops of ZO-1 and Occludin-1 antibody reagent to each BSA-treated slide (preferably to completely cover the slide tissue), incubate at room temperature for 30 min, wash with PBS buffer for 5 min each time, repeat 3 times to obtain Ki67-treated slides.

[0085] Secondary antibody incubation: Add approximately 100 μL of enzyme-labeled goat anti-mouse / rabbit IgG polymer to each treated slide (preferably to completely cover the slide tissue), incubate at room temperature for 30 min, wash with PBS buffer for 5 min each time, repeat 3 times to obtain secondary antibody-incubated slides.

[0086] The secondary antibody-incubated sections were stained with freshly prepared DAB (diaminobenzidine) chromogenic solution. Approximately 100 μL of DAB chromogenic solution was added to each secondary antibody-incubated section, and the chromogenic time was 1–5 min (staining was controlled under a microscope). The chromogenic reaction was terminated by rinsing with tap water to obtain DAB-treated sections.

[0087] The DAB-treated sections were counterstained, dehydrated, cleared, and mounted according to the above method.

[0088] 5. Experimental Results

[0089] like Figure 2 As shown, by detecting the mRNA levels of inflammatory factors after drug administration, it was found that DSS caused a large release of inflammatory factors in the colon of mice. After intervention with glycyrrhizin, the release of inflammatory factors in UC mice was significantly inhibited, including IFN-γ, IL-6, and chemokines CCL2 and CXCL2.

[0090] like Figure 3 As shown in Figures A and B, DSS stimulation, while causing an outbreak of colonic inflammation, also led to severe damage to the colonic mucosa in mice, with reduced expression of intercellular barrier proteins at both the mRNA and protein levels, including Occludin-1, CDH1, and ZO-1. After treatment with glycyrrhizin, the expression of colonic barrier proteins in mice showed an increasing trend at both the mRNA and protein levels, and the recovery effect became more pronounced with increasing drug concentration. Figure 4 As shown, PAS staining revealed mucosal damage and significant loss of goblet cells in the model group mice. Treatment with glycyrrhizin significantly reduced this damage and restored the number of colonic goblet cells. Immunohistochemistry of ZO-1 and Occludin-1 also demonstrated the barrier repair effect of glycyrrhizin.

[0091] Example 3: Effects of glycyrrhizin on LPS-induced inflammation and barrier markers in human colonic epithelial cells NCM46

[0092] 1. Preparation

[0093] Human colonic epithelial cells NCM460 were purchased from Wuhan Pronosei Biotechnology Co., Ltd. NCM460 cells were cultured and passaged in high-glucose DMEM medium containing 10% fetal bovine serum. When the cells reached 70%–80% confluence in the logarithmic growth phase, they were sputtered at a concentration of 3 × 10⁻⁶ g / mL. 5Cells were seeded into 6-well plates at a density of 10 cells per well. After cell attachment, NCM460 cells were stimulated with 20 μg / mL LPS for 24 h under serum-free conditions to establish the cell model. Then, drug intervention was performed by adding glycyrrhizin medium at final concentrations of 2.5 µmol / L, 5 µmol / L, 10 µmol / L, and 20 µmol / L, respectively, and incubating at 37°C and 5% CO2 for 24 h.

[0094] 2. Quantitative reverse transcription polymerase chain reaction (PCR) detection

[0095] Cell samples after drug treatment were collected into enzyme-free EP tubes using a cell scraper. Cells were lysed using Trizol reagent. 500 µL of lysis buffer was added to each tube, and lysis was performed on ice for 10 min. 100 µL of chloroform was added, the mixture was thoroughly mixed, allowed to stand for 15 min, and then centrifuged at 12000 rpm / min, 4°C for 15 min. The supernatant was collected, and an equal volume of isopropanol was added for precipitation for 10 min. The mixture was then centrifuged at 12000 rpm / min, 4°C for 10 min. The supernatant was discarded, and the cells were washed with 500 µL of pre-chilled anhydrous ethanol. The cells were centrifuged at 7500 rpm / min, 4°C for 5 min, and this process was repeated twice. The cells were air-dried at room temperature for 7 min, and the precipitate was dissolved in DEPC water. RNA purity was assessed and its concentration determined using an ultra-micro spectrophotometer by measuring the A260 / A280 and A260 / A230 ratios to ensure the accuracy of subsequent experiments.

[0096] The extracted high-quality RNA was used for reverse transcription to synthesize cDNA. In the reverse transcription reaction system, appropriate amounts of RNA template, DEPC water, and 5×PrimeScript RT Master Mix (Perfect Real Time) were added sequentially according to the manufacturer's instructions. The reaction was then performed in a PCR instrument at 37°C for 15 minutes and 85°C for 5 seconds to synthesize cDNA. The synthesized cDNA can be used immediately for subsequent experiments or stored at -20°C for later use.

[0097] Prepare the qPCR reaction system, including cDNA template, specific primers (primer sequences are shown in Table 3), and fluorescent dye (SYBR Green). Set up appropriate reaction tubes according to the target gene and internal reference gene. Set up a suitable reaction program on the qPCR instrument, including three stages: pre-denaturation, cyclic amplification, and melting curve analysis. In the pre-denaturation stage, heat at 95℃ for 3–5 min to fully denature the cDNA template; perform 40 cycles, each cycle including denaturation (95℃, 10–15 s), annealing (temperature determined according to primer Tm value, generally 55–65℃, 15–30 s), and extension (72℃, 20–30 s). In the melting curve analysis stage, slowly increase the temperature from 60℃ to 95℃ to detect the specificity of the PCR product. After the reaction, analyze the CT values ​​of the target gene and internal reference gene based on the changes in fluorescence signal recorded by the qPCR instrument, and calculate the expression level of the target gene relatively quantitatively using the 2(-ΔΔCT) method.

[0098] Table 3 Primer Sequences

[0099]

[0100] 3. Experimental Results

[0101] like Figure 5 As shown, the mRNA expression levels of inflammatory factors IL6, TNFα, and IL-1β in NCM460 cells stimulated with LPS were detected. LPS stimulation significantly increased the expression of IL6, TNFα, and IL-1β in NCM460 cells, even increasing them fourfold compared to the control group. After treatment with glycyrrhizin, inflammatory factors decreased to varying degrees, showing significant differences compared to the model group, indicating that glycyrrhizin has an anti-inflammatory effect on LPS-induced inflammation in NCM460 cells.

[0102] like Figure 6 As shown, LPS stimulation leads to impaired intercellular tight junctions, manifested as decreased mRNA expression of ZO-1, Claudin-1, and Occludin-1 genes. Under the intervention of glycyrrhizin, intercellular tight junctions are restored, and the expression of related tight junction factors is increased with significant differences, indicating that the barrier repair effect of glycyrrhizin is basically consistent with animal experiments.

[0103] Example 4: Effects of glycyrrhizin on LPS-induced inflammation in mouse macrophages RAW264.7

[0104] 1. Preparation

[0105] Mouse macrophages RAW264.7 were purchased from Wuhan Pronosei Biotechnology Co., Ltd. RAW264.7 cells were cultured and passaged in high-glucose DMEM medium containing 10% fetal bovine serum. When the cells reached 70%–80% confluence in the logarithmic growth phase, they were sputtered at a concentration of 3 × 10⁻⁶ g / mL. 5 Cells were seeded into 6-well plates at a concentration of 100 μg / mL. After cell attachment, drug intervention was performed by adding glycyrrhizin medium at final concentrations of 2.5 µmol / L, 5 µmol / L, 10 µmol / L, and 20 µmol / L, respectively, and incubating at 37°C and 5% CO2 for 24 h. Four h before the end of drug treatment, RAW264.7 cells were stimulated with 8 μg / mL LPS for 4 h to complete the model construction.

[0106] 2. Quantitative reverse transcription polymerase chain reaction (PCR) detection

[0107] After discarding the culture medium in the 6-well plate, wash twice with pre-cooled PBS. Use a cell scraper to transfer the cells to enzyme-free EP tubes. Subsequent steps are the same as in Example 3. Primer sequences are shown in Table 4.

[0108] Table 4 Primer Sequences

[0109]

[0110] 3. Experimental Results

[0111] like Figure 7 As shown, LPS induced an inflammatory outburst in mouse macrophages RAW264.7, with a sharp increase in the expression of IL-6, IL-17α, and TNFα genes, even more than 10 times that of the control group. Glycyrrhizin administration for 24 hours reduced the expression of inflammatory factors to some extent, although it did not reach the calm levels seen in the untreated control group. The degree of decrease in inflammatory factors increased with increasing drug concentration, indicating that the anti-inflammatory effect of glabridin was concentration-dependent.

[0112] Example 5: Application of glycyrrhizin in the preparation of drugs for the prevention and treatment of ulcerative colitis

[0113] In this embodiment, the expression of proteins related to the PI3K / Akt signaling pathway was detected by Western blot, and the regulatory effect of glycyrrhizin on this signaling pathway was evaluated.

[0114] 1. Experimental Animals: A total of 48 male SPF-grade C57BL / 6J mice were used, and they were divided into a control group, a model group, a low-dose glycyrrhizin group, a medium-dose glycyrrhizin group, and a high-dose glycyrrhizin group. Each group contained 8 mice. The control group was the Control group, the model group was the 3% DSS group, the low-dose glycyrrhizin group was the Glabrone-L group, the medium-dose glycyrrhizin group was the Glabrone-M group, and the high-dose glycyrrhizin group was the Glabrone-H group.

[0115] 2. To establish an acute experimental colitis model in mice, the mice were given drinking water containing 3% sodium dextran sulfate for 7 days to establish the colitis model.

[0116] 3. Drug administration: The control group was given normal drinking water, while the model group was given drinking water containing 3% sodium dextran sulfate. The low-dose glycyrrhizin group, medium-dose glycyrrhizin group, and high-dose glycyrrhizin group were given intraperitoneal injections of 12.5 mg / kg, 25 mg / kg, and 50 mg / kg glycyrrhizin, respectively, once a day, with the corresponding administration volume calculated according to the mouse's body weight.

[0117] 4. Western blot is an immunoblotting method.

[0118] First, prepare a colon tissue sample and place it in a pre-chilled EP tube. Add an appropriate amount of RIPA lysis buffer (containing protease inhibitor, phosphatase inhibitor, and EDTA) at a ratio of 1 mL per 100 mg of tissue, and vortex thoroughly to ensure adequate contact between the lysis buffer and the tissue. Then, add 2-3 grinding beads to the EP tube and grind the tissue using a tissue homogenizer. After homogenization, place the centrifuge tube on ice for 30 min to lyse. After lysis, centrifuge the tube at 12000-14000 rpm for 20 min at 4°C. Transfer the supernatant to a new centrifuge tube; this supernatant is the crude protein extract and should be quantified.

[0119] Add 5× loading buffer to the crude extract at a ratio of 4:1, and vortex thoroughly to ensure homogeneity. Heat the centrifuge tube in a 100°C metal bath or boiling water bath for 10 min. After heating, quickly transfer the centrifuge tube to ice to cool for 2–3 min. Then, use the prepared protein sample for electrophoresis or store it at -80°C.

[0120] Based on the molecular weight of the target protein, a separating gel concentration of 10% was selected, and a one-step gel preparation kit was used to prepare the gel. After the gel solidified, the prepared protein samples were loaded sequentially. The gel was placed in an electrophoresis tank, and electrophoresis buffer was added. Electrophoresis was initially performed at 100 V. Once the protein sample entered the separating gel, the voltage was increased to 150 V, and electrophoresis was continued until the bromophenol blue front was close to the bottom of the gel, at which point electrophoresis was stopped.

[0121] After electrophoresis, remove the gel and stack it in the transfer holder in the following order: gel, polyvinylidene fluoride (PVDF) membrane (pre-activated with methanol), and filter paper, taking care to remove air bubbles between the layers. Place the transfer holder into the transfer tank, add transfer buffer, and transfer the membrane at 300 mA for 90–120 min at 4°C (adjust the time according to the molecular weight of the target protein). After transfer, remove the PVDF membrane and block it on a shaker at room temperature for 1–2 h using a 5% skim milk powder solution prepared with 1× Tween Tris buffer (TBST) to block non-specific binding sites on the membrane.

[0122] After blocking, the PVDF membrane was washed three times with 1×TBST buffer, 5–10 min each time. The membrane was then placed in 1×TBST buffer containing primary antibody (selected according to the target protein) and incubated overnight at 4°C. The next day, the membrane was removed from the primary antibody solution and washed three times with 1×TBST buffer, 10 min each time, to remove unbound primary antibody. The membrane was then placed in 1×TBST buffer containing secondary antibody (species matching the primary antibody) and incubated at room temperature for 1–2 h. After incubation, the membrane was washed three times with 1×TBST buffer, 10 min each time, to thoroughly remove unbound antibody. After washing, the membrane surface was uniformly coated with ECL chemiluminescent agent, and the membrane was exposed using an imaging system to acquire chemiluminescent images. The grayscale values ​​of the target protein bands were semi-quantitatively analyzed using ImageJ software, with GAPDH as an internal control protein, to calculate the relative expression level of the target protein.

[0123] 5. Experimental Results

[0124] The PI3K-Akt signaling pathway is an important intracellular regulatory network involved in cell proliferation, survival, metabolism, and inflammatory responses. In colitis, aberrant activation of the PI3K-Akt pathway can promote the release of NF-κB-mediated inflammatory factors (TNF-α, IL-6), exacerbating intestinal mucosal damage. Simultaneously, it affects intestinal barrier function by regulating tight junction proteins (such as occludin), and Akt promotes inflammatory responses in macrophages and T cells by activating the IKK / NF-κB pathway. Therefore, targeting this pathway may alleviate inflammation and repair mucosal damage. Western blot results are as follows... Figure 8As shown, glycyrrhizin effectively inhibited the phosphorylation activation of the PI3K-Akt signaling pathway, reduced the protein expression of p-PI3K and p-Akt, and decreased their ratio to total protein, with statistically significant differences. This indicates that glycyrrhizin may inhibit the overactivation of the PI3K-Akt signaling pathway by targeting it. Figure 9 As shown, glycyrrhizin can effectively inhibit the activation of the NF-κB signaling pathway and downregulate p-65 levels, thereby treating ulcerative colitis, reducing inflammation in mice, and repairing intestinal mucosal damage.

[0125] This invention is the first to investigate the therapeutic effect of glycyrrhizin on ulcerative colitis, studying its anti-inflammatory and barrier repair effects in vitro and in vivo, and preliminarily elucidating its pharmacological mechanism of action. It provides options for the development of small-molecule drugs for the clinical treatment of ulcerative colitis.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of glycyrrhizin in the preparation of drugs for the prevention and / or treatment of ulcerative colitis; The ulcerative colitis mentioned is acute ulcerative colitis; Glycyrrhizin is the only active ingredient.

2. The application according to claim 1, characterized in that, The prevention and / or treatment of ulcerative colitis includes downregulating the expression of inflammatory factors and / or inhibiting the activation of the NF-κB inflammatory signaling pathway.

3. The application according to claim 2, characterized in that, The inflammatory factors include at least one of TNF-α, IL-6, CCL2, or CXCL2.

4. Application of glycyrrhizin in the preparation of drugs for preventing carcinogenesis in ulcerative colitis; The prevention of ulcerative colitis from becoming cancerous includes inhibiting the activation of the cancer signaling pathway PI3K-Akt, reducing p-PI3K expression, or reducing p-Akt expression at least one of these methods. Glycyrrhizin is the only active ingredient.

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