Use of 12-O-deacetyl-fungosanoid A in the preparation of drugs for treating ulcerative colitis and intestinal sepsis

Abstract

The application discloses application of 12-O-deacetyl-finenoxanthone A in preparation of a drug for treating ulcerative colitis and sepsis, and relates to the technical field of biological medicines. Experiments prove that 12-O-deacetyl-finenoxanthone A has a remarkable effect on treating ulcerative colitis and sepsis, is a potential drug for preparing ulcerative colitis and sepsis in clinic, and can significantly inhibit P2X7R-mediated Ca 2+ signals, and can be used as a P2X7R inhibitor drug. The drug developed by the application provides a safe and effective new treatment option for two diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of 12-O-deacetylated phenanthrone A in the preparation of a therapeutic drug for ulcerative colitis and intestinal sepsis. Background Technology

[0002] Ulcerative colitis (UC) is an important subtype of inflammatory bowel disease (IBD), characterized by persistent inflammation and damage to the colonic mucosa. Its etiology involves multiple mechanisms, including intestinal barrier dysfunction, dysbiosis, and immune abnormalities. Clinically, it often manifests as recurrent abdominal pain and bloody, mucous stools. Disease progression can affect the entire colon, leading to life-threatening complications such as intestinal perforation and intestinal obstruction. In recent years, the incidence of UC has been steadily increasing, and the patient population is trending younger, making it a significant chronic disease threatening intestinal health.

[0003] Sepsis is a systemic inflammatory response syndrome triggered by infection, characterized by severe and rapidly progressing illness that easily leads to multiple organ failure. Its occurrence and development are closely related to systemic immune dysregulation and excessive inflammatory response. Notably, there is a key pathological link between sepsis and ulcerative colitis (UC): the disruption of the intestinal mucosal barrier in UC increases the risk of enterogenic infections, while intestinal flora translocation often occurs during sepsis, further exacerbating the systemic inflammatory response and forming a bidirectional worsening cycle of "intestinal damage - systemic inflammation."

[0004] Although UC and Sepsis differ in clinical manifestations and site of onset, they share a core pathological pathway: "P2X7R receptor activation - Ca²⁺ signaling disorder - NLRP3 inflammasome activation - pyroptosis." Extracellular ATP can specifically bind to and activate P2X7R receptors, triggering a large influx of extracellular Ca²⁺. Abnormally elevated intracellular Ca²⁺ further induces NLRP3 inflammasome assembly and Caspase-1 activation. Activated Caspase-1 cleaves IL-1β and IL-18 precursors to generate active pro-inflammatory factors and forms pores on the cell membrane to promote the explosive release of inflammatory factors, ultimately leading to macrophage pyroptosis. This creates a vicious cycle of "intensified inflammation - barrier damage - more pyroptosis," becoming a key driver of the progression of both diseases.

[0005] While various intervention options exist for the clinical treatment of ulcerative colitis (UC) and sepsis, their limitations are significant: existing UC therapies often suffer from severe side effects or insufficient efficacy, making long-term disease control difficult; sepsis, on the other hand, suffers from rapid onset, complex mechanisms, a lack of specific clinical treatments, and a persistently high mortality rate. Crucially, existing drugs primarily focus on "suppressing existing inflammatory responses," failing to address the core pathological process shared by UC and sepsis—"Ca²⁺ signaling disorder-pyroptosis"—thus failing to halt disease progression at its source. This results in poor clinical treatment outcomes, high relapse rates, and falls far short of clinical needs.

[0006] 12-O-deacetylated phenanthrone A (12-ODPXA) is a natural dianthrone compound isolated from a co-culture of the fungus Diaporthegoulteri L17 and Alternaria sp. X112. Current research has confirmed that 12-ODPXA possesses significant antitumor activity, effectively inhibiting the proliferation and metastasis of malignant tumor cells such as ovarian cancer, gastric cancer, and melanoma. Its good biocompatibility and potential as an active molecular compound have been preliminarily verified.

[0007] However, there are currently no studies on the anti-inflammatory activity of 12-ODPXA, nor on its use in the preparation of drugs for the treatment of ulcerative colitis and intestinal sepsis. These are the technical problems that urgently need to be solved by those skilled in the art.

[0008] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides the application of 12-O-deacetylated phenanthrone A in the preparation of drugs for the treatment of ulcerative colitis and intestinal sepsis, thereby resolving the issues raised in the background section.

[0010] Application of 12-O-deacetylated phenanthrone A in the preparation of a drug for treating ulcerative colitis.

[0011] Preferably, 12-O-deacetylated phenanthrone A is used to inhibit the expression of TNF-α, IL-6, IL-1β and IL-18.

[0012] Preferably, 12-O-deacetylated phenanthrone A is used to improve the weight of patients with ulcerative colitis; 12-O-deacetylated phenanthrone A is used to improve the stool condition of patients with ulcerative colitis; 12-O-deacetylated phenanthrone A is used to maintain the colon length of patients with ulcerative colitis.

[0013] Application of 12-O-deacetylated phenanthrone A in the preparation of drugs for treating sepsis.

[0014] Preferably, 12-O-deacetylated phenanthrone A is used to improve the survival rate of patients with sepsis.

[0015] Preferably, 12-O-deacetylated phenanthrone A is used to treat inflammation by inhibiting macrophage pyroptosis.

[0016] Preferably, 12-O-deacetylated phenanthrone A inhibits NLRP3 protein expression by suppressing the NF-κB signaling pathway.

[0017] Preferably, 12-O-deacetylated phenanthrone A is used to treat inflammation by inhibiting NLRP3 inflammasome activation to block macrophage pyroptosis.

[0018] Application of 12-O-deacetylated phenanthrone A in the preparation of P2X7R inhibitor drugs.

[0019] Preferably, 12-O-deacetylated phenanthrone A is used to inhibit P2X7R-mediated calcium signaling in macrophages.

[0020] The application of 12-O-deacetylated phenanthrone A provided in this invention in the preparation of drugs for the treatment of ulcerative colitis and intestinal sepsis has the following beneficial effects: This invention has experimentally demonstrated that 12-ODPXA can effectively treat ulcerative colitis and intestinal sepsis, and the effect is significant. The preparation of the drug of this invention provides a safe and effective new treatment option for these two diseases. Attached Figure Description

[0021] Figure 1 The results of experiments on the effects of 12-ODPXA on DSS-induced ulcerative colitis in mice and the expression of inflammatory factors in colonic tissue;

[0022] in, Figure 1 A represents the effect of 12-ODPXA on the body weight of mice with DSS-induced ulcerative colitis;

[0023] Figure 1 B represents the effect of 12-ODPXA on fecal scores in mice with DSS-induced ulcerative colitis;

[0024] Figure 1 C represents the effect of 12-ODPXA on colon length in DSS-induced ulcerative colitis mice;

[0025] Figure 1D represents the effect of 12-ODPXA on the expression level of TNF-α mRNA in the colon tissue of mice with DSS-induced ulcerative colitis;

[0026] Figure 1 E represents the effect of 12-ODPXA on the expression level of IL-6 mRNA in the colon tissue of mice with DSS-induced ulcerative colitis;

[0027] Figure 1 F represents the effect of 12-ODPXA on the expression level of IL-1β mRNA in the colon tissue of mice with DSS-induced ulcerative colitis;

[0028] Figure 1 G represents the effect of 12-ODPXA on the expression level of IL-18 mRNA in the colon tissue of mice with DSS-induced ulcerative colitis.

[0029] Figure 2 The results of the experiment on the preventive effect of 12-ODPXA on sepsis in mice;

[0030] Figure 3 The experimental results show the effect of 12-ODPXA on LPS / ATP-induced macrophage pyroptosis.

[0031] in, Figure 3 A represents the effect of 12-ODPXA on the viability of iBMDMs cells stimulated by LPS / ATP.

[0032] Figure 3 B represents the effect of 12-ODPXA on the viability of PMs cells stimulated by LPS / ATP.

[0033] Figure 3 C represents the effect of 12-ODPXA on improving LPS / ATP-induced pyroptosis in iBMDMs cells;

[0034] Figure 3 D represents the improvement effect of 12-ODPXA on LPS / ATP-induced pyroptosis in PMs cells;

[0035] Figure 3 E represents the effect of 12-ODPXA on LDH levels in iBMDMs cells stimulated by LPS / ATP.

[0036] Figure 3 F represents the effect of 12-ODPXA on LDH levels in PMs cells stimulated by LPS / ATP.

[0037] Figure 4 The experimental results show the effect of 12-ODPXA on the expression of GSDMD-N, a pyroptosis-related protein in macrophages induced by LPS / ATP.

[0038] in, Figure 4 A represents the effect of 12-ODPXA on LPS / ATP-induced GSDMD-N protein expression in macrophages;

[0039] Figure 4 B is a graph showing the quantitative analysis of the relative expression levels of GSDMD-N protein in LPS / ATP-induced iBMDMs by 12-ODPXA and A438079;

[0040] Figure 4 C is a graph representing the quantitative analysis of the relative expression levels of GSDMD-N protein in LPS / ATP-induced PMs by 12-ODPXA and A438079.

[0041] Figure 5 The experimental results show the effect of 12-ODPXA on the expression of LPS / ATP-induced NLRP3 inflammasome-related molecules mRNA in macrophages.

[0042] in, Figure 5 AD represents the effects of 12-ODPXA and A438079 on the expression levels of IL-18 mRNA and IL-1β mRNA in LPS / ATP-induced macrophages;

[0043] Figure 5 EH represents the effects of 12-ODPXA and A438079 on the expression levels of NLRP3 mRNA and Caspase-1 mRNA in LPS / ATP-induced macrophages.

[0044] Figure 6 Results of the effect of 12-ODPXA on LPS-induced NF-κB-dependent NLRP3 protein expression in macrophages

[0045] in, Figure 6 A represents the effect of 12-ODPXA on LPS-induced NF-κB phosphorylation in iBMDMs;

[0046] Figure 6 B is a quantitative analysis diagram of the effect of 12-ODPXA on LPS-induced NF-κB phosphorylation in iBMDMs;

[0047] Figure 6 C represents the effect of 12-ODPXA on LPS-induced NLRP3 protein expression in iBMDMs;

[0048] Figure 6 D is a graph representing the quantitative analysis of the effect of 12-ODPXA on LPS-induced NLRP3 protein expression in iBMDMs.

[0049] Figure 7The experimental results show the effect of 12-ODPXA on LPS / ATP-induced P2X7R expression in macrophages;

[0050] in, Figure 7 A is an RT-qPCR quantitative analysis of the effect of 12-ODPXA and A438079 on the relative expression level of P2X7R mRNA in LPS / ATP-induced iBMDMs;

[0051] Figure 7 B is a graph showing the effect of 12-ODPXA and A438079 on the relative expression level of P2X7R mRNA in LPS / ATP-induced PMs by RT-qPCR.

[0052] Figure 7 C represents the effect of 12-ODPXA on LPS / ATP-induced P2X7R protein expression in macrophages;

[0053] Figure 7 D is Figure 6 Quantitative statistical results of the relative expression level of P2X7R protein in C.

[0054] Figure 8 The experimental results show the effect of 12-ODPXA on P2X7R-mediated calcium signaling in macrophages;

[0055] in, Figure 8 A represents the effect of 12-ODPXA on the increase in intracellular calcium signaling induced by the P2X7R agonist ATP in iBMDM cells.

[0056] Figure 8 B represents the effect of 12-ODPXA on the increase in intracellular calcium signaling induced by the P2X7R-specific agonist BzATP in iBMDM cells. Detailed Implementation

[0057] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0058] To address the aforementioned technical problems, this invention provides the application of 12-O-deacetylated phenanthrone A in the preparation of drugs for the treatment of ulcerative colitis and intestinal sepsis, thereby resolving the issues raised in the background section.

[0059] Example 1: This example is an experiment on the effect of 12-ODPXA on ulcerative colitis in mice.

[0060] 1. Experimental materials:

[0061] Sodium dextran sulfate (DSS) was purchased from MPbio, USA; the preparation method for 3.5% DSS solution is as follows: dissolve 3.5 g of DSS in 100 mL of deionized water.

[0062] 2. Experimental methods:

[0063] (1) Six-week-old Kunming mice were randomly divided into a control group, a model group, and an experimental group. The ulcerative colitis model was induced by allowing Kunming mice to drink 3.5% DSS (mass percentage concentration) solution freely for 10 days. While drinking DSS solution freely, mice in each group were treated with drugs at the corresponding doses. The control group mice were given free access to water and food, and were also given saline by gavage. The model group mice were given free access to a solution containing 3.5% DSS, and were also given saline by gavage. The mice in experimental group 1 were given free access to 3.5% DSS solution, and were given 12-ODPXA (0.5 mg / kg / day) by gavage 3 days before the administration of DSS. The mice in experimental group 2 were given free access to 3.5% DSS solution, and were given 12-ODPXA (1.5 mg / kg / day) by gavage 3 days before the administration of DSS.

[0064] (2) The groups were: i: Control group; ii: DSS group; iii: DSS+12-ODPXA 0.5 mg / kg; iv: DSS+12-ODPXA 1.5 mg / kg

[0065] (3) At the end of the experiment, mice were euthanized by cervical dislocation, and their abdomens were dissected to measure the length of the colon; colon RNA was extracted and subjected to RT-qPCR experiments, and the results are as follows. Figure 1 As shown in the above statistical analysis chart, significant differences are indicated using... * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates that ns represents no statistical difference.

[0066] 3. Analyze the data:

[0067] Statistical results are as follows Figure 1 As shown. From Figure 1 As shown in Figure A, the weight loss in experimental groups 1 and 2 was significantly less than that in the model group, indicating that 12-ODPXA can improve the weight of mice with ulcerative colitis. Figure 1 As shown in Figure B, the fecal scores of experimental groups 1 and 2 were significantly lower than those of the model group, indicating that 12-ODPXA can improve the fecal condition of mice with ulcerative colitis. Figure 1 As shown in Figure C, the colon length of the experimental group mice was longer than that of the model group mice and close to that of the control group, indicating that 12-ODPXA can restore the colon length of mice with ulcerative colitis; this suggests that 12-ODPXA can improve ulcerative colitis. In the subsequent RT-qPCR experiment, in... Figure 1 DG showed that under DSS stimulation, the expression of TNF-α, IL-6, IL-1β and IL-18 increased, while 12-ODPXA in the experimental group significantly inhibited the increase in inflammatory factor expression.

[0068] Example 2: This example demonstrates the preventive effect of 12-ODPXA on sepsis in mice.

[0069] 1. Laboratory animals:

[0070] Thirty C57 mice weighing 21–25 g were randomly divided into three groups: a sham-operated group, a CLP group, and a 0.5 mg / kg 12-ODPXA + CLP group (treated with 1.5 mg / kg / d of 12-ODPXA three hours after CLP modeling), with 10 mice in each group.

[0071] 2. Experimental methods:

[0072] (1) The drug treatment group was given 12-ODPXA by gavage 3 hours after CLP modeling, at a dose of 1.5 mg / kg; the model group and the sham operation group were given an equal volume of physiological saline by gavage.

[0073] (2) CLP was used to construct sepsis models in the model group and the drug pretreatment group. The specific process is as follows:

[0074] a. Mice were fasted for 12 hours before the experiment;

[0075] b. After anesthesia, the animal is placed supine and fixed on the surgical board. The abdominal surgical area is routinely disinfected and the hair is removed. Under aseptic conditions, a 2 cm long incision is made in the abdominal wall with a scalpel. The abdomen is then accessed through the incision, and the cecum is separated distal to the ileocecal valve and ligated with No. 3 silk thread for 1 / 3 of the cecum.

[0076] c. Use an 18-gauge injection needle to puncture the ligation end and squeeze out a small amount of feces, trying to avoid damaging blood vessels. Then, use No. 4 silk sutures to intermittently suture the peritoneum and skin. At the same time, immediately inject 50 mL / kg body weight of normal saline subcutaneously to combat shock. In the sham surgery group, only the abdominal cavity is opened, but the cecum is not ligated or punctured.

[0077] (3) Mice mortality was observed at 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h post-surgery, and survival curves were plotted. The results are as follows: Figure 2 As shown, significant differences are indicated using... *P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates that ns represents no statistical difference.

[0078] 3. Analyze the data:

[0079] from Figure 2 As can be seen, the survival rate of mice in the 12-ODPXA pretreatment group was significantly improved compared to the model group. This indicates that 12-ODPXA can prevent sepsis in mice and improve the survival rate of septic mice.

[0080] Example 3: This example is an experiment on the effect of 12-ODPXA on macrophage pyroptosis.

[0081] 1. Experimental materials:

[0082] Lipopolysaccharide (LPS), ATP, and A438079 were purchased from MCE (USA); the CCK-8 kit was purchased from MCE (USA), and the LDH kit was purchased from Beyotime (China); LPS / ATP was used to induce pyroptosis; A438079 is a known P2X7 receptor antagonist.

[0083] 2. Experimental methods:

[0084] (1) Cell culture medium: by volume percentage, the culture medium contains 89% cell culture medium, 10% fetal bovine serum, and 1% streptomycin / penicillin;

[0085] (2) Cytotoxicity against iBMDMs and PMs was assessed using the MedChemExpress CCK-8 assay kit. The specific method included: seeding cells in 96-well plates overnight, then treating with different concentrations of sample and stimulating with or without LPS / ATP. After 24 hours, the medium was replaced with fresh medium supplemented with 10% CCK-8 solution and cultured for an additional 1 hour. OD450 values ​​were measured using a microplate reader.

[0086] (3) The amount of LDH produced by iBMDM and PMs cells under LPS / ATP stimulation was assessed using an LDH detection kit. The specific method included: seeding cells into 96-well plates overnight, then treating them with different concentrations of sample, and stimulating them with or without LPS / ATP. LDH was then measured according to the reagent manufacturer's instructions. The OD520 value was measured using a microplate reader.

[0087] (4) The groups were: i: Control group; ii: LPS 1 μg / mL stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours; iii: LPS+ATP+A438079 group: A438079 pretreatment for 2 hours followed by LPS stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours; iv: LPS+ATP+12-ODPXA 0.5 μM group: 12-ODPXA 0.5 μM pretreatment for 2 hours followed by LPS stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours; v: LPS+ATP+12-ODPXA 1 μM group: 12-ODPXA 1 μM pretreatment for 2 hours followed by LPS stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours. (4) At the end of the experiment, the statistical analysis graphs above showed significant differences using * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates that ns represents no statistical difference.

[0088] 3. Analyze the data:

[0089] Statistical results are as follows Figure 3 As shown. From Figure 3 A and B show the effect of 12-ODPXA on cell viability; it can be seen that 0.5 and 1 μM have no effect on cell viability. From... Figure 3 As can be seen in C and 3D, 12-ODPXA reversed LPS / ATP-induced pyroptosis. From Figure 3 As shown in E and 3F, 12-ODPXA reversed the LPS / ATP-induced increase in LDH levels. This indicates that 12-ODPXA can inhibit macrophage pyroptosis.

[0090] Example 4: This example demonstrates the effect of 12-ODPXA on the macrophage pyroptosis protein GSDMD-N.

[0091] 1. Experimental materials:

[0092] Lipopolysaccharide (LPS), ATP, and A438079 were purchased from MCE Pharmaceuticals, Inc. (USA); GSDMD-N antibody was purchased from CST Pharmaceuticals, Inc. (USA).

[0093] 3. Experimental methods:

[0094] (1) The groups were: i: Control group; ii: LPS 1 μg / mL stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours; iii: LPS+ATP+A438079 group: A438079 pretreatment for 2 hours followed by LPS stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours; iv: LPS+ATP+12-ODPXA 0.5 μM group: 12-ODPXA 0.5 μM pretreatment for 2 hours followed by LPS stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours; v: LPS+ATP+12-ODPXA 1 μM group: 12-ODPXA 1 μM pretreatment for 2 hours followed by LPS stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours. The Western blot experimental procedure was as follows:

[0095] (2) Cell culture medium: by volume percentage, the culture medium contains 89% cell culture medium, 10% fetal bovine serum, and 1% streptomycin / penicillin;

[0096] (3) After the cells were seeded into six-well plates, the drug was added for stimulation 24 hours later. Stimulation was performed with or without LPS / ATP.

[0097] (4) Add RIPA lysis buffer and lyse on ice for 30 minutes.

[0098] (5) Centrifuge at 14,000 rpm for 15 minutes.

[0099] (6) Determine protein concentration using the BCA method and adjust all samples to the same concentration.

[0100] (7) Add 5× loading buffer and boil for 5 minutes to denature the protein.

[0101] (8) Constant voltage 80V (stacking gel) → 120V (separating gel) until bromophenol blue runs out from the bottom of the gel.

[0102] (9) Activate the PVDF membrane with methanol for 1 minute, and assemble the "sandwich" in the order of "negative electrode-sponge-filter paper-gel-membrane-filter paper-sponge-positive electrode". Transfer the membrane at a constant current of 200-300 mA for 1-2 hours (4℃ ice bath).

[0103] (10) Immerse the membrane in 5% skim milk (prepared with TBST) and block by shaking at room temperature for 1 hour. Primary antibody incubation: Dilute the primary antibody with blocking buffer and incubate overnight at 4°C (or 2 hours at room temperature). Wash the membrane: Wash the membrane 3 times with TBST, 10 minutes each time. Secondary antibody incubation: Dilute the HRP-labeled secondary antibody with blocking buffer and incubate at room temperature for 1 hour. Wash the membrane: Wash the membrane 3 times with TBST, 10 minutes each time.

[0104] (11) Mix ECL A / B solutions at a 1:1 ratio and drop them evenly onto the membrane. Imaging: Expose in a dark room (chemiluminescence analyzer), adjusting the time according to the signal. At the end of the experiment, in the above statistical analysis graph, significant differences are represented by... * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates that ns represents no statistical difference.

[0105] 3. Analyze the data:

[0106] Statistical results are as follows Figure 4 As shown. From Figure 4 As can be seen from A, 12-ODPXA and A438079 can inhibit the LPS / ATP-induced increase in GSDMD-N expression in macrophages. Figure 4 BC is the quantitative plot of Figure A. It shows that 12-ODPXA can inhibit LPS / ATP-induced macrophage pyroptosis.

[0107] Example 5: This example illustrates the effect of 12-ODPXA on the assembly of macrophage inflammasomes.

[0108] 1. Experimental materials:

[0109] Lipopolysaccharide (LPS), ATP, and A438079 (positive control drug) were purchased from MCE Pharmaceuticals, Inc., USA; primer sequences were designed and obtained by China Sangon Biotech Co., Ltd.; reverse transcription kit and RNA extraction kit were purchased from Beyotime Biotech Ltd., China; and SYBRY was purchased from YEASEN Biotech Ltd., China.

[0110] 2. Experimental methods:

[0111] (1) The cells were divided into groups: i: Control group; ii: LPS 1 μg / mL stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours; iii: LPS+ATP+A438079 group: A438079 pretreatment for 2 hours followed by LPS stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours; iv: LPS+ATP+12-ODPXA 0.5 μM group: 12-ODPXA 0.5 μM pretreatment for 2 hours followed by LPS stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours; v: LPS+ATP+12-ODPXA 1 μM group: 12-ODPXA 1 μM pretreatment for 2 hours followed by LPS stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours. Cell culture medium: by volume percentage, the culture medium contained 89% cell culture medium, 10% fetal bovine serum, and 1% streptomycin / penicillin.

[0112] (2) After the cells were seeded into six-well plates, the drug was added for stimulation 24 hours later. Stimulation was performed with or without LPS / ATP.

[0113] (3) RNA extraction: Lyse the sample according to the reagent supplier's instructions and extract RNA. Determine the concentration and purity. Reverse transcription (RT): Take 1 μg of RNA, add reverse transcriptase, primers (Oligo dT / random hexamer) and dNTPs, and react at 37℃ for 15 minutes to synthesize cDNA. qPCR amplification: Add cDNA template, SYBR Green premix, and primers to the reaction wells, and set the program: 95℃ pre-denaturation for 30 seconds; 40 cycles (95℃ for 5 seconds → 60℃ for 30 seconds, collecting fluorescence); finally, run the melting curve (gradually increasing the temperature from 65℃ to 95℃). Data analysis: Calculate the relative expression level of the target gene using the ΔΔCt method (with GAPDH as an internal reference) based on the Ct value. At the end of the experiment, in the above statistical analysis graph, significant differences are represented by... * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates that ns represents no statistical difference.

[0114] 3. Analyze the data:

[0115] Statistical results are as follows Figure 5 As shown. From Figure 5 AD showed that 12-ODPXA and A438079 could inhibit the increase in expression of LPS / ATP-induced pyroptosis markers IL-18 and IL-1β in macrophages. Figure 5 EH showed that 12-ODPXA and A438079 could inhibit the LPS / ATP-induced increase in NLRP3 and Caspase-1 expression in macrophages. These data indicate that 12-ODPXA inhibits NLRP3 assembly. This suggests that 12-ODPXA can block macrophage pyroptosis and inflammatory responses by inhibiting the assembly of the NLRP3 inflammasome.

[0116] Example 6: This example involves an experiment on the effect of 12-ODPXA on the NF-κB pathway in vitro.

[0117] 1. Experimental materials:

[0118] Lipopolysaccharide (LPS) was purchased from MCE Pharmaceuticals, Inc. (USA); iNOS, P-NF-κB, and COX-2 antibody were purchased from CST Pharmaceuticals, Inc. (USA).

[0119] 2. Experimental methods:

[0120] (1) The groups were: i: Control group; ii: LPS 1 μg / mL stimulation for 12 hours; iii: LPS + 12-ODPXA 0.5 μM group: pretreated with 12-ODPXA 0.5 μM for 2 hours and then stimulated with LPS for 12 hours; iv: LPS + ATP + 12-ODPXA 1 μM group: pretreated with 12-ODPXA 1 μM for 2 hours and then stimulated with LPS for 12 hours. Cell culture medium: by volume percentage, the culture medium contained 89% cell culture medium, 10% fetal bovine serum, and 1% streptomycin / penicillin;

[0121] (2) Cells were seeded into six-well plates, and stimulation was performed 24 hours later with or without LPS / ATP.

[0122] (3) The Western blot experimental procedure is as described in Example 4. At the end of the experiment, the statistical analysis graphs above use significant differences. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates that ns represents no statistical difference.

[0123] 3. Analyze the data:

[0124] Statistical results are as follows Figure 8 As shown. From Figure 6 As can be seen from the WB experiment, 12-ODPXA inhibited the LPS-induced increase in P-NF-κB expression in macrophages at the protein level in a concentration-dependent manner. Figure 6 B is Figure 6 A statistical chart. From Figure 6 As can be seen from C, 12-ODPXA inhibited the expression of NLRP3. Figure 6 D is Figure 6 The statistical graph of C. The above results indicate that 12-ODPXA can inhibit NLRP3 expression by suppressing the NF-κB signaling pathway.

[0125] Example 7: This example is an experiment on the effect of 12-ODPXA on P2X7R.

[0126] 1. Experimental materials:

[0127] Lipopolysaccharide (LPS), ATP, and A438079 were purchased from MCE Pharmaceuticals, USA; primer sequences were designed and obtained by China Sangon Biotech, Inc.; reverse transcription and RNA extraction kits were purchased from Beyotime Biotech, Inc., China; SYBRY was purchased from YEASEN, China. P2X7R antibody was purchased from CST Biotech, USA.

[0128] 2. Experimental methods:

[0129] (1) The cells were divided into groups: i: Control group; ii: LPS 1 μg / mL stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours; iii: LPS+ATP+A438079 group: A438079 pretreatment for 2 hours followed by LPS stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours; iv: LPS+ATP+12-ODPXA 0.5 μM group: 12-ODPXA 0.5 μM pretreatment for 2 hours followed by LPS stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours; v: LPS+ATP+12-ODPXA 1 μM group: 12-ODPXA 1 μM pretreatment for 2 hours followed by LPS stimulation for 4 hours followed by ATP 3 mM stimulation for 0.5 hours. Cell culture medium: by volume percentage, the culture medium contained 89% cell culture medium, 10% fetal bovine serum, and 1% streptomycin / penicillin.

[0130] (2) After the cells were seeded into six-well plates, the drug was added for stimulation 24 hours later. Stimulation was performed with or without LPS / ATP.

[0131] (3) The RT-qPCR experimental procedure is as described in Example 5.

[0132] (4) The Western blot experimental procedure is as described in Example 4. At the end of the experiment, the statistical analysis graphs above use significant differences. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates that ns represents no statistical difference.

[0133] 3. Analyze the data:

[0134] Statistical results are as follows Figure 7 As shown. From Figure 7 As shown in AB, RT-qPCR experiments verified that 12-ODPXA and A438079 can inhibit LPS / ATP-induced increase in P2X7R expression in macrophages at the mRNA level. Figure 6 CD results show that 12-ODPXA and A438079 can inhibit LPS / ATP-induced increase in P2X7R expression in macrophages at the protein level. These data indicate that 12-ODPXA inhibits P2X7R expression. P2X7R is a common target for anti-inflammatory drugs.

[0135] Example 8: This example involves an experiment on the effect of 12-ODPXA on P2X7R-mediated calcium signaling in macrophages.

[0136] 1. Experimental materials:

[0137] 12-ODPXA was synthesized in-house; dimethyl sulfoxide (DMSO) was purchased from SIGMA; calcium fluorescence detection reagent Fura-2 AM was purchased from Solarbio; ATP, BzATP, and A438079 were purchased from MCE.

[0138] 2. The experimental reagents are prepared as follows:

[0139] (1) Cell culture medium: by volume percentage, the culture medium contains 89% cell culture medium, 10% fetal bovine serum, and 1% streptomycin / penicillin;

[0140] (2) Phosphate buffer (PSS): Dissolve 0.818 g NaCl, 0.037 g KCl, 0.022 g CaCl2, 0.238 g HEPES, and 0.18 g glucose in 90 mL of double-distilled water, adjust the pH to 7.4, and bring the volume to 100 mL for later use.

[0141] (3) Preparation of the stock solution of calcium ion dye Fura-2, AM fluorescent dye (5 mM): Add 6 μL DMSO and 4 μL Pluronic F127 (20% in DMSO) to 50 μg / vial, and sonicate for 30 s to fully dissolve. Preparation of working solution (5 μM): Take 1 μL of Fura-2, AM (5 mM), dissolve it in 999 μL PSS, mix thoroughly, and prepare fresh before use.

[0142] 3. Experimental methods:

[0143] (1) Culture iBMDM and PMs on a 15 mm diameter circular transparent cell spreader in a culture medium until the cell density reaches about 80% before proceeding to the next step;

[0144] (2) After washing the cells twice with PSS, add Fura-2 and AM dye and incubate at 37°C for 1 h;

[0145] (3) Transfer the cells to a new cell culture dish, wash once with PSS, and incubate for 30 min in PSS containing 12-ODPXA or A438079 or without the drug.

[0146] (4) Imaging was performed under a fluorescence microscope, using blue excitation light and green receiving light; the imaging interval was 3 s. After loading the microscope, ATP was added to achieve a final concentration of 100 μM. The intensity of green fluorescence in the cells was recorded, and the rate of change in calcium fluorescence was statistically analyzed. The results are as follows: Figure 1 As shown. Fura-2, a commonly used calcium fluorescent probe in cell biology, can specifically bind to Ca.2+ (The binding ratio is 1:1), and it can also emit fluorescence, binding Ca. 2+ The maximum excitation wavelength after binding decreased from 380 nm to 340 nm (Ca 2+ (At saturation) shift, its emission fluorescence intensity is related to the binding Ca 2+ There is a quantitative relationship between the concentration and the concentration of calcium ions. Therefore, Fura-2 cells are generally excited at wavelengths of 340 nm and 380 nm, and the intracellular calcium ion concentration is calculated by using the fluorescence intensity ratio corresponding to the two excitations. The results are expressed as mean ± standard error.

[0147] 4. Analyze the data:

[0148] The relationship between the rate of change of calcium fluorescence signal and changes in time and concentration was statistically analyzed, and the results are as follows: Figure 8 As shown in Figure A, 100 μM ATP induces extracellular Ca2+. 2+ Upon entering the cell, A438079 10 μM and 12-ODPXA 1 μM inhibited the ATP-induced increase in intracellular calcium signaling. Figure 8 B indicates that 10 μM BzATP induces extracellular Ca2+. 2+ Upon entering the cells, A438079 10 μM and 12-ODPXA 1 μM inhibited the increase in intracellular calcium signaling induced by BzATP. The inhibitory effect of 12-ODPXA on P2X7R was verified at the cellular level.

[0149] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of 12-O-deacetylated phenanthrone A in the preparation of a drug for treating ulcerative colitis.

2. The use of 12-O-deacetylated phenanthrone A according to claim 1 in the preparation of a medicament for treating ulcerative colitis, characterized in that, 12-O-deacetylated phenanthrone A is used to inhibit the expression of TNF-α, IL-6, IL-1β and IL-18.

3. The use of 12-O-deacetylated phenanthrone A according to claim 1 in the preparation of a medicament for treating ulcerative colitis, characterized in that, 12-O-deacetylated phenanthrone A is used to improve the weight of patients with ulcerative colitis; 12-O-deacetylated phenanthrone A is used to improve the stool condition of patients with ulcerative colitis; 12-O-deacetylated phenanthrone A is used to maintain the colon length of patients with ulcerative colitis.

4. The application of 12-O-deacetylated phenanthrone A in the preparation of drugs for treating sepsis.

5. The use of 12-O-deacetylated phenanthrone A according to claim 4 in the preparation of a drug for treating sepsis, characterized in that, 12-O-deacetylated phenanthrone A is used to improve the survival rate of patients with sepsis.

6. The use of 12-O-deacetylated phenanthrone A according to claim 4 in the preparation of a drug for treating sepsis, characterized in that, 12-O-deacetylated phenanthrone A is used to treat inflammation by inhibiting macrophage pyroptosis.

7. The use of 12-O-deacetylated phenanthrone A according to claim 4 in the preparation of a drug for treating sepsis, characterized in that, 12-O-deacetylated phenanthrone A inhibits NLRP3 protein expression by suppressing the NF-κB signaling pathway.

8. The use of 12-O-deacetylated phenanthrone A according to claim 4 in the preparation of a drug for treating sepsis, characterized in that, 12-O-deacetylated phenanthrone A is used to treat inflammation by inhibiting NLRP3 inflammasome activation to block macrophage pyroptosis.

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