Application of eupatolide in preparation of medicine for preventing and treating inflammatory bowel disease

Abstract

The invention discloses application of eupatolide in preparation of a medicine for preventing and treating inflammatory bowel disease, application of eupatolide in preparation of a medicine for preventing and treating inflammatory bowel disease and application of a medicine preparation containing eupatolide in preparation of a medicine for preventing and treating inflammatory bowel disease, and researches that eupatolide (EPT) can be used for preparing the medicine for preventing and treating inflammatory bowel disease through targeting pyruvate kinase isoenzyme 2 (PKM2, Gene ID: 5135). The inflammatory response, glycolysis metabolism and mitochondrial homeostasis are synergistically regulated and controlled, so that the inhibition effect on the inflammatory bowel disease is exerted. Meanwhile, EPT has the advantages of being low in dosage, feasible in oral administration, free of obvious toxic and side effects and the like, and a solid experimental basis is provided for further developing EPT into candidate drugs for preventing and treating inflammatory bowel diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of zebulin in the preparation of drugs for the prevention and treatment of inflammatory bowel disease. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic, relapsing, nonspecific inflammatory bowel diseases with incompletely understood etiologies. It primarily includes ulcerative colitis (UC) and Crohn's disease (CD). Inflammation in IBD typically affects the colonic mucosa. It is a recurrent, immune-mediated gastrointestinal disease, and its pathogenesis is believed to be caused by a combination of host genetic susceptibility and environmental triggers. The global prevalence of IBD is rising at an extremely rapid rate.

[0003] Currently, traditional treatments such as salicylates, glucocorticoids, and immunosuppressants remain fundamental. However, biologics and small molecule targeted drugs, such as anti-tumor necrosis factor-α, anti-interleukin (IL)-12 / 23 (anti-IL-12 / 23), and Janus kinase inhibitors, have become core treatments for patients with moderate to severe IBD, achieving remission rates of 50%–70%. Finding new targets for treating inflammatory bowel disease is of great significance for the development of drugs for its prevention and treatment.

[0004] Eutoprololide (EPT, CAS No.: 6750-25-0) is a natural compound isolated from costus root. Currently, there are no reports of its application in the preparation of drugs for the prevention and treatment of inflammatory bowel disease. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of zebulin in the preparation of drugs for the prevention and treatment of inflammatory bowel disease.

[0006] A second objective of this invention is to provide the use of pharmaceutical preparations containing zebulin in the preparation of drugs for the prevention and treatment of inflammatory bowel disease.

[0007] The technical solution of this invention is summarized as follows: Application of zebuline in the preparation of drugs for the prevention and treatment of inflammatory bowel disease.

[0008] Application of pharmaceutical preparations containing zebulin in the preparation of drugs for the prevention and treatment of inflammatory bowel disease.

[0009] Beneficial effects This invention investigates how eupatolide (EPT) exerts its inhibitory effect on inflammatory bowel disease by targeting pyruvate kinase isoenzyme 2 (PKM2, Gene ID: 5135) and synergistically regulating inflammatory responses, glycolytic metabolism, and mitochondrial homeostasis. Furthermore, EPT possesses advantages such as low dosage, feasibility of oral administration, and no observed significant toxic side effects, providing solid experimental evidence for its further development as a candidate drug for the prevention and treatment of inflammatory bowel disease. Attached Figure Description

[0010] Figure 1 EPT can alleviate DSS-induced inflammatory bowel disease and improve intestinal barrier function in vivo, including: The structure of Eupatolide (EPT); B mice body weight, rectal bleeding, and DAI score (n = 8); Colon length and analysis in C mice (n = 6); Representative images of H&E staining and Occludin immunohistochemical staining of the colon of D mice.

[0011] Figure 2 EPT modulates immune function by balancing Th17 and Treg immune cells in DSS-mediated IBD mice, where: Flow cytometry atlas of Th17 cells (CD4+IL-17A+) and Treg cells (CD4+CD25+Foxp3+); Quantitative results of B Th17 cells and Treg cells (n=3); C. Expression levels of RORc and Foxp3 mRNA in spleen (n=3); Effects of DEPT on the expression of RORγt and Foxp3 proteins in the spleen and colon.

[0012] Figure 3 EPT blocked the NF-κB and NLRP3 signaling pathways, where: Effects of EPT on the expression of iNOS, p-p65, IL-1β, pro-IL-1β, Cleaved Caspase 1, ASC and NLRP3 proteins in the colon; Representative images of immunofluorescence of B RORγt and Foxp3, COX-2, ASC and NLRP3.

[0013] Figure 4EPT can reduce LPS-induced glycolysis in RAW264.7 cells and inhibit the activation of the NLRP3 inflammasome and NF-κB signaling pathways, thereby suppressing inflammation and restoring mitochondrial homeostasis. A EPT inhibited the LPS-induced release of inflammatory cytokines NO, IL-6, and TNF-α in RAW264.7 cells (n=3); B EPT inhibits LPS-induced lactate release in RAW264.7 cells; Effects of CEPT on LPS-induced expression of LDHA and GLUT1 proteins in RAW264.7 cells.

[0014] mRNA expression levels of iNOS, COX-2, IL-1α, IL-6, CCL5, Mfn1, Mfn2, and Opa1 in D RAW264.7 cells (n=3); Effects of EPT on LPS-induced expression of Mfn1, Mfn2, Opa1, Fis1, COX-2, iNOS and p-p65 proteins in RAW264.7 cells; Representative images of F p65 and COX-2 immunofluorescence.

[0015] Figure 5 EPT inhibits pyroptosis by suppressing NLRP3 inflammasome activation, wherein: A flow cytometry atlas of IL-1β and NLRP3 (n=3); B EPT inhibited LPS-induced IL-1β mRNA expression in RAW264.7 cells (n=3); Effects of CEPT on LPS-induced expression of Cleaved Caspase 1, IL-1β, pro-IL-1β, ASC and NLRP3 proteins in RAW264.7 cells.

[0016] Figure 6 PKM2 is a potential target for EPT, where: A. The pull-down technique combined with LC-MS / MS analysis was used to identify the cellular targets of AR; B. Results of DARTS and CETSA experiments on binding proteins and EPT and PKM2 were obtained by Western blot analysis. Immunofluorescence co-localization assay of PKM2 and EPT; D Microscale thermoelectrophoresis (MST) was used to detect the binding affinity of EPT to PKM2.

[0017] Figure 7 Knockdown of PKM2 negated the in vitro protective effect of EPT, where: A Western blot and PCR confirmed the knockdown of PKM2 in RAW264.7 cells (n=3); B PKM2 knockout eliminated the effect of EPT on the levels of IL-1β, LDHA, HK2, GLUT1, Opa1, Drp1, IL-1α and IL-6 mRNA in LPS-induced RAW264.7 cells (n=3); PKM2 knockout eliminated the effects of EPT on the NF-κB and NLRP3 signaling pathways and mitochondrial fusion-fission-related proteins (n=3).

[0018] Representative immunofluorescence images of D p65. Detailed Implementation

[0019] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The embodiments of the present invention are merely some examples and not all embodiments. Based on the embodiments of the present invention, any other embodiments made by those skilled in the art without inventive effort should fall within the protection scope of the present invention.

[0020] For any experimental steps or conditions not explicitly specified in the examples, refer to the standard experimental procedures or conditions described in conventional literature in this field. If the manufacturers of the reagents or instruments used are not specified, they are all commercially available conventional reagent products, and appropriate suppliers can be selected as needed.

[0021] Example 1 Animal level: EPT significantly alleviated the typical inflammatory bowel disease phenotype induced by DSS in mice, including weight loss, rectal bleeding, and shortened colon length. Histological and molecular analyses showed that EPT reduced inflammatory cytokine levels, restored the expression of the intestinal tight junction protein occludin, improved intestinal crypt structure disruption, and regulated Treg / Th17 cell immune balance, thereby improving the overall intestinal inflammatory microenvironment.

[0022] (1) Experimental materials: Eutopolide (EPT, Shanghai Yuanye Biotechnology Co., Ltd., CAS No.: 6750-25-0), see Figure 1A.

[0023] Preparation of EPT gavage solution: Based on the experimentally determined dosage (5, 10, 20 mg / kg), combined with the average weight of the experimental animals (21 g / animal), the number of animals (10 animals), and the dosing period (14 days), calculate the total mass of drug required for each dosage group. The formula is: .

[0024] According to the formula, the 5, 10, and 20 mg / kg dosage groups require 14.7 mg, 29.4 mg, and 58.8 mg of EPT powder, respectively. For each dosage group, 2.94 g of hydroxypropyl-β-cyclodextrin was added as a solubilizer, followed by 29.4 mL of deionized water. The powder was then sonicated to completely dissolve, yielding the corresponding concentration of the oral administration solution. (If the final concentration of the solution (e.g., mg / mL) needs to be specified, it can be calculated as follows: for the 20 mg / kg group, 58.8 mg of EPT powder dissolved in 29.4 mL of water results in a final concentration of 2 mg / mL; similarly, the 10 mg / kg group requires 1 mg / mL, and the 5 mg / kg group requires 0.5 mg / mL).

[0025] Dextran sulfate sodium salt (DSS, Shanghai Haohong Biomedical Technology Co., Ltd., CAS No. 9011-18-1).

[0026] Preparation of 3% DSS solution: Weigh 30 g of sodium dextran sulfate (DSS) powder, add deionized water to 1 L, mix and dissolve.

[0027] Specific pathogen-free grade male C57BL / 6 mice (22-25 g, 8 weeks old), commercially available. Room temperature 22-24℃, relative humidity 50%-65%, good ventilation, artificial day / night cycle (12 h / 12 ​​h), acclimatization feeding before the start of the experiment, free access to food and water during the experiment.

[0028] Primary antibodies used in immunoblotting include RORγt (Proteintech, 29910-1-AP, 1:1000), Foxp3 (Proteintech, 22228-1-AP, 1:1000), iNOS (Proteintech, 22226-1-AP, 1:500), p-p65 (ABclonal, AB-AP1294, 1:1000), p65 (Proteintech, 10745-1-AP, 1:1000), IL-1β / proIL-1β (Proteintech, 26048-1-AP, 1:1000), Cleaveed-caspase 1 / caspase 1 (Proteintech, 22915-1-AP, 1:2000), and ASC (Proteintech, 10500-1-AP, 1:5000). NLRP3 (Proteintech, 68102-1-AP, 1:2000) and GAPDH (Proteintech, 60004-1-Ig, 1:4000); secondary antibodies include goat anti-rabbit recombinant secondary antibody (Proteintech, RGAR001, 1:4000) and goat anti-mouse recombinant secondary antibody (Proteintech, RGAM001, 1:4000).

[0029] Primary antibodies used in immunofluorescence applications include RORγt (Proteintech, 29910-1-AP, 1:50), Foxp3 (Proteintech, 22228-1-AP, 1:200), COX-2 (Proteintech, 66351-1-AP, 1:200), ASC (Proteintech, 10500-1-AP, 1:200), and NLRP3 (Proteintech, 68102-1-AP, 1:200); secondary antibodies include Alexa Fluor™ 488 GAR (Invitrogen, R37116, 1:200) and Alexa Fluor™ 594 GAM (Invitrogen, R37120, 1:200).

[0030] The primary antibody used in immunohistochemistry includes Occludin (Proteintech, 27260-1-AP, 1:1000).

[0031] The antibodies used in flow cytometry include FITC-CD4 (100405, BioLegend, 0.5 μl / 100 μl), PE-CD25 (102007, BioLegend, 5 μl / 100 μl), AF700-FOXP3 (126421, BioLegend, 0.12 μl / 100 μl), and APC-IL-17A (50915, BioLegend, 1.25 μl / 100 μl).

[0032] (2) Experimental method: 1) Experimental procedure: After the acclimatization period, the mice were randomly divided into 6 groups of 10 mice each. The groupings are as follows: Blank control group (Ctrl), blank administration group (EPT (20 mg / kg), model group (DSS), model + low-dose administration group (DSS + EPT (5 mg / kg), model + medium-dose administration group (DSS + EPT (10 mg / kg)) and model + high-dose administration group (DSS + EPT (20 mg / kg)).

[0033] The experiment lasted for two weeks. During the first week, all mice had free access to water, and their weight was recorded daily. The EPT (20 mg / kg), DSS+EPT (5 mg / kg), DSS+EPT (10 mg / kg), and DSS+EPT (20 mg / kg) groups were administered the drug by gavage (200 μl / mouse / day), while the other groups were given the same volume of physiological saline.

[0034] In the second week, mice in the Ctrl and EPT (20 mg / kg) groups had free access to water.

[0035] Mice in the DSS, DSS+EPT (5 mg / kg), DSS+EPT (10 mg / kg), and DSS+EPT (20 mg / kg) groups were given free access to 3% DSS water to establish a mouse model of inflammatory bowel disease.

[0036] The EPT (20 mg / kg), DSS+EPT (5 mg / kg), DSS+EPT (10 mg / kg), and DSS+EPT (20 mg / kg) groups were administered the drugs by gavage (200 μl / mouse / day) for 14 days, while the other groups were given the same volume of physiological saline. The body weight of all mice was recorded daily, rectal bleeding was observed, and DAI (colonic inflammation index) scores were subsequently calculated.

[0037] On day 15, tissue samples were collected from all mice, including colons and spleens. First, two spleens from each group were collected for flow cytometry analysis. Colonoscopy was performed on two mice from each group. The length of all colons was recorded and photographed uniformly. A small section of each colon was cut from both mice in each group and fixed at room temperature in 4% paraformaldehyde to maintain its structure and morphology. The solution was changed after 24 hours, and the next step was performed after 48 hours.

[0038] 2) Mouse body weight, rectal bleeding, and DAI score: After mice were given free access to 3% DSS in the second week, their body weight, rectal bleeding, and DAI scores were recorded daily. (See Figure 1B). Seven days after DSS administration, mice showed significant weight loss, rectal bleeding, and a marked increase in DAI scores. EPT treatment reversed these pathological changes in a dose-dependent manner.

[0039] 3) Measurement of colon length in mice: The colon tissue of mice was sampled and its length was measured (see Figure 1C). It can be observed that DSS significantly shortened the colon length, while EPT treatment greatly improved this effect.

[0040] 4) H&E staining: Colonic sections were stained using a hematoxylin and eosin (HE) staining kit (Beijing Solarbio Science & Technology Co., Ltd.). See Figure 1D. The H&E staining results show that EPT treatment alleviated DSS-mediated inflammatory cell aggregation and crypt atrophy in IBD mice in a dose-dependent manner.

[0041] 5) Immunohistochemistry: Mouse colon tissue sections were dewaxed with xylene and rehydrated with gradient ethanol. Antigen retrieval was performed using citrate buffer (pH=6.0) in a microwave oven on high for 3 min, followed by low for 10 min. After retrieval, the tissue was incubated with 3% H2O2 aqueous solution at room temperature in the dark for 10 min to block endogenous peroxidase. After thorough washing with PBS, 5% BSA was added for blocking for 1 h. The serum was discarded, and diluted immunohistochemical primary antibody was added. The tissue was incubated overnight at 4°C. The next day, after multiple washes with PBS, the tissue was incubated with high-sensitivity enzyme-labeled secondary antibody from a two-step universal kit (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) at room temperature for 1 h. After washing again, the tissue was subjected to DAB... The chromogenic reaction was performed using a chromogenic reagent kit (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.). The chromogenic time was controlled under a microscope. After chromogenic development, the reaction was terminated with distilled water. Cell nuclei were counterstained with hematoxylin, differentiated with differentiation solution, and then blued with tap water. Gradient ethanol dehydration and xylene clearing were then performed sequentially. Finally, neutral resin was added and the slides were mounted with coverslips. After the mounting resin dried, the location and expression intensity of positive signals could be observed under an optical microscope. (See Figure 1D). Occludin staining results showed that EPT treatment alleviated DSS-mediated intestinal barrier function in IBD mice in a dose-dependent manner.

[0042] 6) Flow cytometry: Fresh mouse spleen tissue was collected and ground in pre-chilled PBS to prepare a single-cell suspension. The suspension was filtered through a 200-mesh sieve to remove tissue debris. Cells were collected by centrifugation at 2000 rpm for 5 min at 4°C. Red blood cell lysis buffer (Solepro Science & Technology Co., Ltd.) was added, and the suspension was incubated on ice for 15 min. DMEM culture medium was then added and mixed thoroughly. The suspension was centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS and passed through a 200-mesh sieve. Cell counts were performed, and the cell concentration was adjusted to 2 × 10⁶ cells / mL. 6Cells were stained at a density of 1 / mL in two groups: One group was stimulated with ionomycin and phorbol ester (PMA) for 1 h, followed by the addition of a protein transport inhibitor (BD GolgiStop™) and cultured in a cell culture incubator for another 5 h. Cells were then collected, blocked with 5% BSA, and incubated with FITC-CD4 at 4°C in the dark for 30 min. After washing with PBS, cells were fixed and perforated with pre-chilled methanol at 4°C in the dark for 10 min, followed by incubation with APC-IL-17A at 4°C in the dark for 30 min (TH17 cell staining). The other group was blocked with 5% BSA, then incubated with FITC-CD4 and PE-CD25 fluorescent antibodies at 4°C in the dark for 30 min. After washing with PBS, cells were fixed and perforated with pre-chilled methanol at 4°C in the dark for 30 min, followed by incubation with AF700-Foxp3 fluorescent antibody at 4°C in the dark for 30 min (Treg cell staining). Both groups were washed with PBS after staining and resuspended in an appropriate amount of PBS. In the middle stage, after passing through a 200-mesh sieve, the cells were analyzed by flow cytometry to determine the proportion and expression levels of TH17 and Treg cells. See [link to relevant documentation]. Figure 2 A, 2B. Using the above method, Th17 and Treg cells were analyzed using CD4, CD25, IL-17A and Foxp3 markers. The results showed that the proportion of CD4+IL-17A+ Th17 cells in the spleen increased after DSS treatment, while the proportion of CD4+CD25+Foxp3+ Treg cells decreased. EPT reversed these changes in a dose-dependent manner.

[0043] 7) Real-time quantitative PCR analysis: RNA was extracted from mouse spleen tissue using TRIzol reagent, and its quantity and purity were assessed using a NanoPhotometer® N50 spectrophotometer. Reverse transcription was performed using a reverse transcription system. The level of target mRNA was quantified by real-time fluorescence PCR using an Applied Biosystems QuantStudio 1, with GAPDH as an internal control gene. (See Figure 2C) This demonstrates that EPT downregulates Th17 cell-associated genes (RORc) and upregulates Treg-associated genes (Foxp3) expression in the spleen.

[0044] 8) Immunoblotting: Colon and spleen tissue samples were collected from mice, proteins were extracted and their concentrations were measured, and proteins were separated by SDS-PAGE gel electrophoresis. The proteins were then wet-transferred to PVDF membranes, blocked, incubated with the primary and secondary antibodies used in the immunoblotting process, and subjected to chemiluminescence imaging to complete the immunoblotting detection of proteins. (See Figure 2D) It can be seen that the DSS-induced changes in RORγt and Foxp3 protein levels in the colon and spleen were reversed by EPT. (See Figure 3A) This indicates that EPT can block the activation of the NLRP3 inflammasome and NF-κB signaling pathway induced by DSS, thereby alleviating DSS-induced intestinal inflammation.

[0045] 9) Immunofluorescence: Mouse colon tissue sections were dewaxed with xylene, rehydrated with graded ethanol, and then subjected to antigen retrieval in a microwave oven on high for 3 min and on low for 10 min using citrate buffer (pH=6.0). After natural cooling, 0.2% Triton solution was added and incubated at room temperature for 10 min. The sections were washed three times with PBS, and 3% H2O2 aqueous solution was added to block endogenous peroxidase. The sections were incubated at room temperature for 10 min, washed with PBS, and then blocked with 5% BSA blocking solution at room temperature for 1.5 h. After discarding the blocking solution, diluted primary antibody for immunofluorescence was added and incubated overnight at 4°C. The next day, the sections were thoroughly washed with PBS, and then incubated with secondary antibody for immunofluorescence at room temperature in the dark for 1.5 h. After washing with PBS, the sections were stained with DAPI at room temperature in the dark for 10 min, washed again with PBS, and then mounted with an anti-fluorescence quencher. The sections were then observed and images were acquired under a Leica DM6B fluorescence microscope. As shown in Figure 3B, the results show that DSS treatment led to enhanced fluorescence of RORγt, COX-2, ASC, and NLRP3, and decreased fluorescence of Foxp3, while EPT administration reversed these effects, consistent with the above results.

[0046] 10) Statistical analysis: Data were analyzed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). Data are presented as mean ± standard error of the mean. Statistical analysis was performed using one-way ANOVA, with p < 0.05 considered statistically significant.

[0047] (3) Experimental results: These results indicate that EPT can maintain intestinal immune homeostasis by regulating Th17 and Treg cells in DSS-mediated IBD mice, and alleviate inflammatory responses and DSS-induced inflammatory bowel disease by blocking the NF-κB and NLRP3 signaling pathways.

[0048] Example 2 At the cellular level: EPT significantly inhibited lipopolysaccharide (LPS)-induced overactivation of RAW264.7 cells, manifested as a reduction in the release of inflammatory factors such as cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6). Simultaneously, EPT restored mitochondrial fusion-division dynamics and downregulated the expression of multiple glycolysis-related genes, thereby alleviating inflammation-related metabolic disorders and cellular stress responses.

[0049] (1) Experimental materials: Mouse mononuclear macrophage leukemia cells (RAW 264.7, Wuhan Pronosai Life Science Technology Co., Ltd.).

[0050] Bio-EPT is biotin-conjugated eupatolide (EPT).

[0051] The primary antibodies used in the immunoblotting experiments included LDHA (Proteintech, 19987-1-AP, 1:2000), GLUT1 (Proteintech, 21829-1-AP, 1:1000), Mfn1 (Proteintech, 13798-1-AP, 1:2000), Mfn2 (Proteintech, 12186-1-AP, 1:5000), Opa1 (Proteintech, 27733-1-AP, 1:5000), Fis1 (Proteintech, 10956-1-AP, 1:2000), and COX-2 (Proteintech, 66351-1-AP, 1:1000).

[0052] Primary antibodies used in immunofluorescence include p65 (Proteintech, 10745-1-AP, 1:200).

[0053] Primary antibodies used in flow cytometry include IL-1β (Proteintech, 26048-1-AP, 1:50) and NLRP3 (Proteintech, 68102-1-AP, 1:200); secondary antibodies include Alexa Fluor™ 488 GAR (Invitrogen, R37116, 1:200) and Alexa Fluor™ 594 GAM (Invitrogen, R37120, 1:200).

[0054] For the remaining materials, please refer to step (1) of Example 2.

[0055] (2) Experimental method: 1) Cell culture and treatment: RAW 264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C and 5% CO2.

[0056] Cells were divided into 5 groups: blank group, LPS group, 0.5 μM EPT+LPS group, 1 μM EPT+LPS group, and 2 μM EPT+LPS group.

[0057] After RAW 264.7 cells were fully adhered, they were pretreated with the appropriate concentration of EPT for 1 h. Then, except for the control group, 500 ng / ml LPS was added for stimulation, and the cells were cultured in an incubator for another 24 h before being collected for subsequent analysis. When studying the NLRP3 inflammasome, after 4 h of LPS stimulation, except for the control group, 10 μM NIG (nigrain) was added for further culture for 6 h. The EPT concentration used in the immunofluorescence experiment was 2 μM.

[0058] 2) NO detection: The nitric oxide (NO) content in RAW264.7 cells was detected using the Griess reagent method. RAW264.7 cells were cultured in 96-well plates for 24 h until complete cell adhesion. After 24 h of treatment with EPT and LPS, the cell supernatant was collected and thoroughly mixed with the corresponding detection reagents. The absorbance of the sample was measured at a specific wavelength using a microplate reader. Finally, the nitric oxide (NO) content was calculated using a NO standard curve. (See Figure 4A). EPT significantly inhibited NO release, indicating that EPT has anti-inflammatory activity.

[0059] 3) Enzyme-linked immunosorbent assay (ELISA): The levels of TNF-α and IL-6 in the supernatant of RAW 264.7 cells were detected according to the instructions of the QuantiCyto® ELISA kit from Xinbosheng Biotechnology Co., Ltd. RAW 264.7 cells were cultured in 96-well plates, protected with drug administration, and stimulated. The supernatant was collected. Standards and samples were diluted as required and added to the wells of the ELISA plate, incubated at 37°C in the dark. After washing, biotinylated antibody working solution and enzyme conjugate working solution were added sequentially, with washing after each incubation. The chromogenic substrate was added, and the OD value was measured after the reaction was terminated. A standard curve was plotted using the OD values ​​of the standards, and the levels of TNF-α and IL-6 in the RAW 264.7 cell supernatant were calculated by substituting the OD values ​​of the samples. (See Figure 4A). The results show that EPT can inhibit the release of inflammatory factors TNF-α and IL-6 at gradient concentrations, reducing inflammation levels.

[0060] 4) Lactate level testing: The experiment was conducted according to the instructions of the L-lactic acid (LAC) test kit (Nanjing Jiancheng Bioengineering Institute). The detected lactic acid content is shown in Figure 4B. It can be seen that EPT gradient administration can reverse LPS-mediated upregulation of lactic acid content, and its effect is closely related to the anti-inflammatory effect of EPT.

[0061] 5) Immunoblotting: RAW 264.7 cell samples were collected from each group. After protein extraction and concentration determination, proteins were separated by SDS-PAGE gel electrophoresis and wet-blown onto PVDF membranes. The membranes were then blocked, incubated with primary antibodies (the primary antibodies used in the immunoblotting experiments disclosed in this embodiment and those disclosed in Example 1, such as iNOS, p-p65, p65, Cleaveed-caspase 1 / caspase 1, IL-1β / proIL-1β, ASC, NLRP3) and secondary antibodies (the secondary antibodies used in the immunoblotting experiments disclosed in Example 1, such as goat anti-rabbit recombinant secondary antibody and goat anti-mouse recombinant secondary antibody), and chemiluminescent imaging to complete the protein immunoblotting detection. See Figures 4C, 4E, and 5C. The results show that EPT can reduce the glycolysis level of immune cells and inhibit LPS-induced activation of the NLRP3 inflammasome and NF-κB signaling pathways, thereby suppressing inflammation and restoring mitochondrial homeostasis.

[0062] 6) Real-time quantitative PCR analysis: RAW 264.7 cells were collected and thoroughly lysed using Trizol lysis buffer. RNA-assisted extraction reagent was added for separation. After centrifugation, the supernatant was collected, and isopropanol was added. After standing, centrifugation was performed to obtain the precipitate. The precipitate was washed with 80% ethanol, centrifuged again, and the supernatant was discarded. The precipitate was then air-dried to obtain the RNA precipitate. The nucleic acid concentration and purity of the samples were measured using a NanoPhotometer® N50 ultra-micro spectrophotometer. cDNA was synthesized using a reverse transcription system, diluted, and the level of the target mRNA (GAPDH was used as an internal reference gene) was assessed by real-time quantitative PCR. As shown in Figures 4D and 5B, EPT downregulated the mRNA levels of iNOS, COX-2, IL-1α, IL-1β, IL-6, and CCL5 in a dose-dependent manner, while upregulating the mRNA levels of Mfn1, Mfn2, and Opa1. This indicates that EPT reduces the expression of related inflammatory factors by inhibiting the activation of the NF-κB inflammatory signaling pathway. At the same time, EPT can also improve mitochondrial fusion function and restore mitochondrial homeostasis.

[0063] 7) Immunofluorescence: Add 1 ml of RAW 264.7 cell suspension of appropriate density to a 12-well plate with cell spreaders, culture in a cell culture incubator until adherent, add EPT for 1 h, then add LPS, and continue culturing for 24 h. Discard the culture medium, wash twice with PBS, add pre-cooled methanol fixative and fix at 4°C for 10 min, then block with 5% BSA blocking solution at 4°C for 30 min (wash with PBS 3 times, 10 min each time between each step). Afterwards, add the corresponding primary antibody (the primary antibody used in the immunofluorescence experiment disclosed in this example and the primary antibody used in the immunofluorescence experiment disclosed in Example 1, such as COX-2) and incubate overnight at 4°C. The next day, use the working solution of the corresponding species' fluorescent secondary antibody (the secondary antibody used in the immunofluorescence experiment disclosed in Example 1, such as Alexa Fluor™ 488 GAR, Alexa Fluor™ 594 GAM) and incubate at room temperature in the dark. After 1 hour, stain the nuclei with DAPI staining solution, incubate at room temperature in the dark for 15 min, wash three times with PBS, then add anti-fluorescence quencher and mount. Observe the fixed slides using a Leica DM6B microscope (Leica GmbH, Germany). See Figure 4 F. The results showed that EPT reversed LPS-induced p65 nuclear ectopic and COX-2 expression, indicating that EPT inhibited the activation of the NF-κB signaling pathway and suppressed the inflammatory response.

[0064] 8) Flow cytometry: RAW 264.7 cells were seeded in 6-well plates. One hour after drug administration, LPS was added and the cells were cultured for another 3 hours. NIG and a protein transport inhibitor (BD GolgiStop™) were added for further stimulation for 5 hours. Cells were then collected, washed with PBS, and fixed with cold methanol fixative for 10 minutes. After fixation, the cells were blocked with 5% BSA blocking solution for 30 minutes. After blocking, the cells were incubated with the primary antibody used for flow cytometry for 2 hours. After washing with PBS and centrifuging, the cells were incubated with the corresponding species' fluorescent secondary antibody for flow cytometry for 1 hour in the dark. After each incubation, the cells were washed with PBS, centrifuged, and the supernatant was discarded. Finally, the cells were resuspended in an appropriate amount of PBS and filtered through a 200-mesh filter to prepare a single-cell suspension. The positive cell population was analyzed using a CytoFlex flow cytometer (Beckman, USA). As shown in Figure 5A, EPT can reverse the LPS+NIG-induced increase in the number of NLRP3 pathway IL-1β and NLRP3 positive cells, indicating that EPT can reduce the maturation and secretion of downstream pro-inflammatory factor IL-1β by inhibiting NLRP3 inflammasome activation.

[0065] 9) Statistical analysis: Data were analyzed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). Data are presented as mean ± standard error of the mean. Statistical analysis was performed using one-way ANOVA, with p < 0.05 considered statistically significant.

[0066] (3) Experimental results: The above results indicate that eupatolide (EPT) exerts its anti-inflammatory effect in vitro by inhibiting LPS-induced activation of the NLRP3 inflammasome and NF-κB signaling pathway, thereby suppressing pyroptosis.

[0067] Example 3 Mechanism study: EPT exerts its effects by targeting PKM2. Changes in PKM2 expression levels significantly affect the anti-inflammatory, anti-pyroptosis, and mitochondrial protective effects of EPT in in vitro models.

[0068] (1) Experimental materials: The primary antibodies used in the immunoblotting assay include PKM2 (Proteintech, 60268-1-AP, 1:5000) and Drp1 (Proteintech, 12957-1-AP, 1:2000).

[0069] For the remaining materials, please refer to step (1) of Example 1 and step (1) of Example 2.

[0070] (2) Experimental methods.

[0071] 1) Cell culture and treatment: RAW cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C and 5% CO2. Before stimulation with lipopolysaccharide (LPS, 500 ng / mL), cells were pretreated with different concentrations of EPT as needed. After 24 h of stimulation, cells were centrifuged, and the cell pellet was collected, lysed, and the supernatant was used for subsequent analysis.

[0072] 2) Bio-EPT fishing target identification: After determining the protein concentration using the BCA method, equal volumes of protein from cell lysates were divided into three groups and brought to a final volume of 500 μL with PBST. The competitive binding group was pre-incubated overnight with 20 μM EPT, while the experimental group received an equal volume of DMSO as a control. Subsequently, 2 μM Bio-EPT was added to each of the three groups, and the mixture was incubated at room temperature for 3 h. Then, 30 μL of pretreated streptavidin magnetic beads (Beyotime Biotechnology Co., Ltd.) were added, and the mixture was incubated at room temperature for another 3 h to enrich the binding protein. After incubation, the magnetic bead-bound protein was eluted with 2× protein loading buffer (Beijing Lanjieke Technology Co., Ltd.), separated by SDS-PAGE, and the differentially expressed protein bands were stained using a silver staining kit (Beyotime Biotechnology Co., Ltd.). The differentially expressed bands were excised and identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Combined with protein / peptide database comparison analysis, the results showed that the differentially expressed protein was PKM2 (…). Figure 6 A). Further pull-down experiments showed that Bio-EPT effectively captured PKM2 in cell lysates, and this interaction was significantly blocked after the addition of excess EPT for competitive binding. Figure 6 B).

[0073] 3) Drug affinity response and target stability: After determining the protein concentration of cell lysates using the BCA method, equal amounts of protein were divided into 5 groups and brought to a final volume of 500 μL with PBST. The experimental groups were incubated with different concentrations of EPT (25, 50, and 100 μM) at room temperature for 1 h, followed by protease digestion with 1 μg / mL Pronase E (Solepro Biotechnology Co., Ltd.). The blank group received an equal volume of DMSO and Pronase E dilution, while the control group received an equal volume of DMSO and 1 μg / mL Pronase E. The mixtures were incubated at 37 °C for 5 min, centrifuged at 20,000 g, and the supernatant was collected and analyzed using PKM2 antibody via Western blotting. The binding of EPT significantly enhanced the resistance of PKM2 to protease digestion, indicating that EPT can improve the structural stability of PKM2. Figure 6 B).

[0074] 4) Cell thermal migration analysis: After quantification of cell lysates using the BCA method, equal volumes of protein were divided into two groups and brought to a final volume of 500 μL with PBST. The experimental group was incubated with 20 μM EPT at room temperature for 1 h, followed by heating at 46, 50, 54, 58, and 62 °C for 5 min, respectively. The control group received an equal volume of DMSO and was treated under the same conditions. After heating, the cells were centrifuged at 20,000 g, and the supernatant was used for Western blotting analysis of PKM2. As shown in Figure 6B, the CETSA results further demonstrate that EPT binding to PKM2 significantly enhances its thermal stability.

[0075] 5) Immunofluorescence: RAW264.7 cells were seeded in 12-well plates in DMEM medium containing 10% fetal bovine serum. siPKM2 and siCtrl were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the PKM2 overexpression plasmid was provided by Ubisoft Biotechnology. Transfection was performed according to the transfection reagent instructions. 36 h after transfection, cells were treated with 2 μM EPT for 1 h, followed by stimulation with LPS (500 ng / mL) for 4 h. Cells were fixed with pre-cooled methanol for 10 min, washed three times with PBS, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% BSA at room temperature for 90 min. Primary antibodies (such as p65, used in the immunofluorescence experiments disclosed in Example 2) were then added, and the cells were incubated overnight at 4 °C. The following day, after washing with PBS, the cells were incubated at room temperature with fluorescently labeled secondary antibody (the secondary antibody used in the immunofluorescence experiments disclosed in Example 1, such as Alexa Fluor™ 488 GAR) and stained with DAPI. The cells were then mounted using anti-fluorescence quenching mounting medium. Images were acquired using a Leica DM6B microscope. Figure 6C shows significant overlap between the red fluorescence of EPT and the green fluorescence of PKM2 protein within the cells; as shown in Figure 7D, in PKM2 knockdown RAW264.7 cells, EPT did not significantly affect LPS-induced p65 expression.

[0076] 6) Microscale thermophoresis: PKM2 protein was fluorescently labeled using a Monolith NT kit. Different concentrations of EPT were added to buffer containing 200 nM labeled PKM2 protein, and after incubation for 15 min, binding affinity was detected and calculated using a Monolith NT.115 instrument (NanoTemper Technologies, Munich, Germany). As shown in Figure 6D, MST results indicated a high binding affinity between EPT and PKM2.

[0077] 7) Immunoblotting: After determining the protein concentration of cell lysates using the BCA method, an equal mass of protein was separated by 7.5%–15% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 h, and the primary antibody (the primary antibody used in the immunoblotting experiments disclosed in this example and the primary antibodies used in the immunoblotting experiments disclosed in Examples 1 and 2, such as iNOS, Mfn1, NLRP3) was diluted with 5% BSA and incubated overnight at 4 °C. After washing three times with TBST containing 0.1% Tween-20, the membrane was incubated with the corresponding secondary antibody (the secondary antibody used in the immunoblotting experiments disclosed in Example 1, such as goat anti-rabbit recombinant secondary antibody and goat anti-mouse recombinant secondary antibody) at room temperature for 2 h, washed again, and developed with enhanced chemiluminescence (ECL). The results showed that the protein level of PKM2 decreased significantly after transfection with siRNA (Figure 7A). Meanwhile, in PKM2 knockdown RAW264.7 cells, the regulatory effects of LPS-induced inflammatory factors iNOS, mitochondrial-associated proteins Drp1 and Mfn1, as well as the levels of inflammasome-associated proteins, were improved, thereby eliminating the therapeutic effect of EPT (Figure 7C).

[0078] 8) Real-time quantitative PCR analysis: Total RNA was extracted from cells using TRIzol reagent, and the concentration and purity of RNA were assessed using a NanoPhotometer® N50 spectrophotometer. After RNA was reverse transcribed into cDNA, the mRNA expression levels of the target genes were detected using an Applied Biosystems QuantStudio 1 real-time fluorescence PCR system, with GAPDH as an internal control gene. PKM2 expression levels were regulated by siRNA or plasmid transfection. Results showed that PKM2 mRNA expression significantly decreased after siRNA transfection (Figure 7A). Simultaneously, LPS stimulation significantly decreased the mRNA expression levels of upregulated IL-6, IL-1α, IL-1β, and genes related to mitochondria and glycolysis, such as OPA1, DRP1, HK2, GLUT, and LDHA. Notably, under conditions of PKM2 knockdown, EPT failed to further reduce the expression levels of these genes (Figure 7B).

[0079] 9) Statistical analysis: Data were analyzed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). Data are presented as mean ± standard error of the mean. Statistical analysis was performed using one-way ANOVA, with p < 0.05 considered statistically significant.

[0080] (3) Experimental results: In summary, EPT directly binds to and regulates the functional state of PKM2, and inhibits inflammatory amplification and cell damage processes by reducing PKM2-mediated inflammatory signaling and metabolic reprogramming.

[0081] Obviously, the above embodiments are merely illustrative examples and do not limit the implementation methods. Those skilled in the art can make other variations or modifications based on the above description. Such variations and modifications are still within the scope of protection of this invention, and it is not necessary to exhaustively describe all embodiments. Obvious variations or modifications derived therefrom should also be considered part of this invention.

[0082] This invention can also be used to prepare common pharmaceutical formulations, such as tablets, capsules, and granules, by combining EPT with pharmaceutically acceptable excipients using conventional techniques. Experiments have shown that EPT-containing pharmaceutical formulations are effective in preventing and treating inflammatory bowel disease and can be used in the preparation of drugs for the prevention and treatment of inflammatory bowel disease.

Claims

1. The use of oridonin in the preparation of a drug for preventing and treating inflammatory bowel disease.

2. The use of a pharmaceutical preparation containing oridonin in the preparation of a drug for preventing and treating inflammatory bowel disease.

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