Use of garciniaflavone c

Abstract

The application relates to the technical field of medicines, in particular to the application of gambogeflavone C. The application of gambogeflavone C in the preparation of an NLRP3 inflammasome inhibitor. The application of gambogeflavone C in the preparation of a medicine for treating infectious shock. The application of gambogeflavone C in the preparation of a medicine for treating acute liver injury. The application of gambogeflavone C in the preparation of a medicine for treating acute peritonitis. The application provides a new choice for medicines for preventing or treating NLRP3 inflammasome related diseases, and also provides a new medicine with treatment potential for infectious shock, acute liver injury and acute peritonitis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of gambogeine C. Background Technology

[0002] Natural compounds have received increasing attention as potential sources of anticancer agents. Flavonoids are oxygen-containing heterocyclic compounds widely distributed in various plants and microorganisms. Among them, Garcinone C (Gar C) is an xanthone natural compound isolated from the popular Southeast Asian fruit, purple mangosteen (Garcinia mangostana). Its unique xanthone tricyclic core structure and specific functional groups endow it with a variety of significant biological functions. The most remarkable feature of Gar C is its broad-spectrum and highly effective antitumor activity. Recent studies have shown that it exerts multi-target antitumor effects in various tumor models by targeting key signaling pathways and regulating cell cycle progression. Its mechanism of action varies depending on the cancer type, exhibiting high specificity: In colorectal cancer, Gar C directly inhibits the key transcription factor Gli1 in the non-canonical Hedgehog signaling pathway, blocking its nuclear translocation and transcriptional activity, thereby downregulating downstream cell cycle regulatory proteins such as cyclin D1 and CDK6, and inducing the production of p21; in addition, it can also inhibit AKT phosphorylation, and through these dual effects, it induces G0 / G1 phase cell cycle arrest, thus effectively inhibiting tumor growth. In nasopharyngeal carcinoma, Gar C significantly inhibits cancer cell viability in a time- and dose-dependent manner and induces S phase cell cycle arrest; it upregulates the expression of ATR and 4E-BP1, while inhibiting the expression of cyclins and related kinases and Stat3, ultimately inducing necrotizing cell death.

[0003] Although Gar C demonstrates clear multi-target potential in directly inhibiting tumor proliferation, its role in inflammation and related signaling pathways remains unexplored. The NLRP3 inflammasome, as a core hub connecting chronic inflammation and the tumor microenvironment, plays a crucial role in their pathological processes. Therefore, in-depth investigation into whether Gar C can modulate the tumor immune microenvironment and related inflammatory responses by intervening in the activation of the NLRP3 inflammasome is of significant value for comprehensively elucidating its pharmacological mechanism and expanding the application prospects of this natural compound as a multi-target anti-tumor and anti-inflammatory therapeutic strategy.

[0004] The NLRP3 inflammasome, a multi-protein complex in the innate immune system, is a crucial component of the body's defense system, composed of three core proteins: NLRP3, ASC, and pro-caspase-1. It plays a central role in immune surveillance and inflammatory responses by recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Activation of the NLRP3 inflammasome promotes caspase-1-mediated maturation of IL-1β and IL-18 and induces Gasdermin D (GSDMD)-mediated pyroptosis. While this mechanism maintains immune homeostasis, its overactivation is also closely associated with the development of various chronic diseases, including type 2 diabetes, Alzheimer's disease, atherosclerosis, and cancer.

[0005] Current treatment strategies targeting the NLRP3 inflammasome mainly include small molecule inhibitors such as MCC950 and CY-09, as well as anti-IL-1β antibodies (Canakinumab). However, existing inhibitors generally have shortcomings in terms of specificity, bioavailability, and safety. Furthermore, some compounds may interfere with normal immune defense functions, posing potential safety risks. This is likely one of the main reasons why no drugs directly targeting the NLRP3 inflammasome have been approved for clinical use to date. Therefore, developing novel, highly effective, and safe NLRP3 inhibitors is of significant clinical importance. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide the application of garcinolone C as a new option for the prevention or treatment of NLRP3 inflammasome-related diseases, and also as a new drug with therapeutic potential for septic shock, acute liver injury and acute peritonitis.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This technical solution focuses on Garcinia cambogia C (Gar C), a natural flavonoid compound derived from plants of the Garcinia genus, and systematically studies its inhibitory effect on the NLRP3 inflammasome and its therapeutic potential in inflammatory diseases.

[0009] Application of Garcinia Cambogia C, specifically its use in the preparation of NLRP3 inflammasome inhibitors.

[0010] Garcinone C is a natural flavonoid compound derived from plants of the genus Garcinia, also known as Garcinone C, with the molecular formula: C 23 H 26 O7 has the following molecular structure:

[0011] .

[0012] The inventors of this case have determined through relevant experiments that Garcinia cambogia C can inhibit Nigericin-induced NLRP3 inflammasome activation in THP-1 cells and PBMCs (peripheral blood mononuclear cells) of healthy volunteers, thus proving that Garcinia cambogia C can be used as an NLRP3 inflammasome inhibitor, providing a new drug option for the prevention or treatment of NLRP3 inflammasome-related diseases.

[0013] Preferably, the dosage of gamboge ketone C is 20 mg / kg.

[0014] Experimental studies have shown that garcinolone C of the present invention exhibits significant inhibition of the release of IL-1β and caspase-1 p20 subunits at low doses (20 mg / kg), thus demonstrating that garcinolone C may have good bioavailability and strong anti-inflammatory activity, providing important experimental evidence for the development of novel NLRP3 inflammasome inhibitors.

[0015] Preferably, the NLRP3 inflammasome inhibitor is prepared by adding pharmaceutical excipients with Garcinia cambogia C as the active ingredient;

[0016] Preferably, the dosage form of the NLRP3 inflammasome inhibitor is at least one of tablets, capsules, granules, powders, patches, suspensions, oral liquids, injections, and suppositories.

[0017] Preferably, the use of garcinolone C as an inhibitor of Nigericin-induced NLRP3 inflammasome activation in BMDM cells.

[0018] This invention also provides the use of gamboge ketone C in the preparation of medicaments for treating septic shock.

[0019] Preferably, the septic shock is lipopolysaccharide (LPS)-induced septic shock.

[0020] Preferably, the drug for treating septic shock is prepared by adding pharmaceutical excipients to Garcinia cambogia C as the active ingredient.

[0021] Preferably, in the drug for treating septic shock, the dosage of gamboge ketone C is 20 mg / kg.

[0022] Experimental studies have shown that garcinolone C of this invention exhibits significant therapeutic effects at low doses (20 mg / kg). This demonstrates that garcinolone C may possess high bioavailability and strong anti-inflammatory activity, providing important clues for the development of novel anti-septic shock drugs.

[0023] Preferably, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, syrups, oral liquids, injections, and suppositories.

[0024] Preferably, when the garcinolone C is used as a drug for treating septic shock, it can significantly reduce the content of IL-1β in serum and peritoneal lavage fluid.

[0025] This invention also provides the use of gamboge ketone C in the preparation of a medicament for treating acute liver injury.

[0026] Preferably, the acute liver injury is concanavalin A-induced acute liver injury.

[0027] Preferably, the drug for treating acute liver injury is prepared by adding pharmaceutical excipients, with Garcinia cambogia C as the active ingredient.

[0028] Preferably, in the medicament for treating acute liver injury, the dosage of gamboge ketone C is 20 mg / kg.

[0029] Experimental studies have shown that garcinolone C of this invention exhibits good therapeutic effects even at low doses (20 mg / kg), with an effective dose significantly lower than the 60 mg / kg reported for celecoxib in the literature. This demonstrates that garcinolone C may possess high bioavailability and high anti-inflammatory activity, providing important clues for the development of novel drugs for the treatment of acute liver injury.

[0030] Preferably, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, syrups, oral liquids, injections, and suppositories.

[0031] Preferably, when the gamboge ketone C is used as a drug for treating acute liver injury, it can inhibit the production of caspase-1p20 and the release of IL-1β in liver tissue.

[0032] This invention also provides the use of gamboge ketone C in the preparation of a medicament for treating peritonitis.

[0033] Preferably, the peritonitis is acute peritonitis.

[0034] Preferably, the peritonitis is acute peritonitis induced by potassium aluminum sulfate (Alum).

[0035] In experimental studies, alum (potassium aluminum sulfate), a classic inorganic salt adjuvant, is widely used to establish acute peritonitis models due to its stable crystal structure and well-defined immunostimulatory properties. After intraperitoneal injection of alum, its positively charged aluminum salt crystals can be taken up by innate immune cells such as peritoneal macrophages and dendritic cells through phagocytosis. This process alters lysosomal membrane permeability, causing intracellular potassium ion efflux and inducing mitochondrial dysfunction, leading to a surge of reactive oxygen species (ROS). These cellular stress responses activate multiple inflammatory signaling pathways, induce inflammatory cell death, and release large amounts of damage-associated molecular patterns (DAMPs). These inflammatory mediators infiltrate the peritoneal cavity through chemotaxis of immune cells such as neutrophils and monocytes, activating signaling pathways such as TLR4 / MyD88, ultimately forming a "cytokine storm" and resulting in the typical pathological changes of acute peritonitis.

[0036] Preferably, the drug for treating acute peritonitis is prepared by adding pharmaceutical excipients, with Garcinia cambogia C as the active ingredient.

[0037] Preferably, in the medicament for treating acute peritonitis, the dosage of gamboge ketone C is 20 mg / kg.

[0038] Experimental studies have shown that garcinolone C of this invention exhibits significant therapeutic effects at low doses (20 mg / kg). This demonstrates that garcinolone C may possess high bioavailability and strong anti-inflammatory activity, providing important clues for the development of novel anti-peritonitis drugs.

[0039] Preferably, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, syrups, oral liquids, injections, and suppositories.

[0040] Preferably, when the garcinolone C is used as a drug for treating acute peritonitis, it can inhibit the release of IL-1β in peritoneal lavage fluid and reduce the amount of Ly6G in peritoneal lavage fluid. + CD11b + The proportion and absolute count of neutrophils.

[0041] Preferably, the use of the garcinolone C in a medicament for reducing the content of IL-1β in serum and peritoneal lavage fluid.

[0042] Preferably, when the garcinol C acts as an inhibitor of Nigericin-induced NLRP3 inflammasome activation in BMDM cells, it can reduce the content of IL-1β in serum and peritoneal lavage fluid.

[0043] The beneficial effects of this invention are:

[0044] Garcinia galanga C of this invention exhibited inhibitory effects on the NLRP3 inflammasome in various cell models. Under induction by different agonists such as Nigericin, ATP, and MSU, Garcinia galanga C significantly inhibited the release of IL-1β and caspase-1 p20 subunits in mouse BMDM cells, human THP-1 cells, and healthy human PBMCs, showing a clear dose-dependent effect without affecting the expression levels of Pro-IL-1β and Pro-caspase-1, indicating that its inhibitory effect occurs during the inflammasome activation stage. Simultaneously, Garcinia galanga C effectively inhibited the caspase-11-mediated non-classical inflammasome activation pathway, demonstrating a comprehensive inhibitory ability on the NLRP3 pathway. Furthermore, activation of the AIM2 inflammasome via Poly(A:T) revealed no significant inhibitory effect from Garcinia galanga C treatment, confirming its highly specific inhibitory effect on the NLRP3 inflammasome. Mechanistic studies showed that Garcinia galanga C directly interferes with the upstream assembly process of the inflammasome by blocking the protein-protein interactions between NEK7-NLRP3 and ASC-NLRP3, thereby inhibiting its activation at its source. These findings not only reveal the unique mechanism of action of Gar C, but also lay a solid experimental foundation for its subsequent development into a highly effective and specific NLRP3 inflammasome inhibitor.

[0045] This invention systematically validated the therapeutic efficacy of Gar C using three inflammatory models (septic shock, acute liver injury, and acute peritonitis) with different inducing factors and pathological manifestations. In the LPS-induced septic shock model, this study confirmed that Gar C exerts its anti-inflammatory effect by specifically reducing IL-1β levels. ELISA analysis showed that while significantly reducing IL-1β levels in serum and peritoneal lavage fluid, the compound did not significantly affect inflammatory factors such as IL-6 and TNF-α, indicating that its anti-inflammatory effect has high targeting specificity. In the ConA-induced acute liver injury model, Gar C showed significant therapeutic effects. Compared with the model group, the Gar C treatment group showed significant inhibition of caspase-1 p20 production and IL-1β release in liver tissue, while liver injury markers (ALT / AST) levels and histopathological damage were also simultaneously improved. Moreover, Gar C showed significant efficacy at a dose of 20 mg / kg, which is lower than the 60 mg / kg required by celecoxib to achieve similar efficacy levels reported in the literature. In an Alum-induced acute peritonitis model, Gar C also demonstrated a good intervention effect. Flow cytometry analysis showed that after Gar C intervention, Ly6G levels in the peritoneal lavage fluid of mice increased. + CD11b +The proportion and absolute count of neutrophils were significantly decreased. Further ELISA analysis confirmed that the release of IL-1β in the peritoneal lavage fluid of this group was also significantly inhibited. The results indicate that it can exert significant anti-inflammatory and protective effects in different disease scenarios by targeting the NLRP3 inflammasome, demonstrating broad clinical application prospects. It provides a breakthrough solution for the treatment of septic shock, acute liver injury, and acute peritonitis, with significant clinical value and socioeconomic benefits. It also has application value in the field of biomedical materials technology. Attached Figure Description

[0046] Figure 1 This illustrates the effect of Gar C in inhibiting NLRP3 inflammasome activation induced by Nigericin (name: Nigerian styracin; stimulates the activation of the NLRP3 inflammasome, thereby stimulating the secretion of IL-1β cytokine) in BMDM cells, as described in Example 1. Specifically, 1A shows the protein expression of IL-1β and p20 in the cell culture supernatant (SN), 1B shows the secretion level of the inflammatory factor IL-1β in the cell culture supernatant, 1C shows the secretion level of the inflammatory factor TNF-α in the cell culture supernatant, and 1D shows the secretion level of the inflammatory factor IL-6 in the cell culture supernatant.

[0047] Figure 2 This illustrates the effect of Gar C in inhibiting Nigericin-induced NLRP3 inflammasome activation in human THP-1 cells and PBMCs, as shown in Example 1. Specifically, 2A shows the protein expression of p20 in the THP-1 cell culture supernatant (SN), 2B shows the secretion level of the inflammatory factor IL-1β in the THP-1 cell culture supernatant, 2C shows the secretion level of the inflammatory factor IL-1β in the PBMC cell culture supernatant, and 2D shows the secretion level of the inflammatory factor TNF-α in the PBMC cell culture supernatant.

[0048] Figure 3 This illustrates the role of Gar C in inhibiting ATP and MSU-induced NLRP3 inflammasome activation in Example 1; wherein, 3A and 3C show the protein expression of IL-1β and p20 in cell culture supernatant (SN), and 3B and 3D show the secretion level of the pro-inflammatory cytokine IL-1β in cell culture supernatant;

[0049] Figure 4 This illustrates the effect of Gar C in inhibiting cLPS-induced non-classical NLRP3 inflammasome activation in Example 1; wherein, 4A shows the protein expression of IL-1β and p20 in the cell culture supernatant (SN), and 4B shows the secretion level of the pro-inflammatory cytokine IL-1β in the cell culture supernatant;

[0050] Figure 5In Example 1, Gar C had no effect on poly(A:T)-induced activation of the AIM2 inflammasome; 5A shows the protein expression of IL-1β and p20 in the cell culture supernatant (SN), and 5B shows the secretion level of the pro-inflammatory cytokine IL-1β in the cell culture supernatant;

[0051] Figure 6 In Example 2, Gar C inhibited the binding of endogenous NEK7 to NLRP3 and NLRP3 to ASC; wherein, 6A shows the levels of NLRP3 and NEK7 proteins in cell immunoprecipitation (IP) and cell lysate (Input), and 6B shows the levels of NLRP3 and ASC proteins in cell immunoprecipitation (IP) and cell lysate (Input);

[0052] Figure 7 This illustrates the effect of Gar C in alleviating LPS-induced septic shock in mice in Example 3; wherein, 7A, 7B, and 7C show the secretion levels of inflammatory factors IL-1β, IL-6, and TNF-α in the peritoneal fluid of mice, and 7D, 7E, and 7F show the secretion levels of inflammatory factors IL-1β, IL-6, and TNF-α in the serum of mice;

[0053] Figure 8 This is to illustrate the role of Gar C in alleviating ConA-induced acute liver injury model in Example 3; wherein, 8A shows the appearance of mouse liver, 8B shows the IL-1β secretion level in mouse ocular blood, 8C shows HE-stained liver tissue, 8D and 8E show the ALT and AST levels in mouse ocular blood, and 8F shows the p20 expression in mouse liver homogenate.

[0054] Figure 9 In Example 3, Gar C effectively alleviated Alum-induced peritonitis in mice; 9A showed the presence of CD11b in the peritoneal fluid of mice in each group. + Ly6G + Flow cytometry plots of neutrophils: 9B shows the percentage of neutrophils in the peritoneal fluid of mice in each group; 9C shows the number of neutrophils in the peritoneal fluid of mice in each group; 9D shows the IL-1β secretion level in the peritoneal fluid of mice in each group; and 9E shows the TNF-α secretion level in the peritoneal fluid of mice in each group. Detailed Implementation

[0055] The following description, with reference to preferred embodiments, illustrates the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are merely illustrative of the present invention and not intended to limit the scope of protection of the present invention.

[0056] Experimental reagents and materials

[0057] The following experiment was conducted using Garcinone C (Gar C) as an inhibitor of the NLRP3 inflammasome. Garcinone C, also known as Garcinone C, has the molecular formula: C 23 H 26 O7 has the following molecular structure:

[0058]

[0059] Table 1: Cells and mice used in the experiment

[0060]

[0061] Table 2: Consumables Involved in the Experiment

[0062]

[0063] Table 3: Antibody Information

[0064]

[0065] Table 4: Reagents Used in the Experiment

[0066]

[0067]

[0068] To address the shortcomings of existing technologies, this application provides a specific NLRP3 inflammasome inhibitor and its use in the preparation of drugs for the prevention or treatment of NLRP3 inflammasome-related diseases.

[0069] Inflammation is a core defense response of the body to harmful stimuli, and its precise regulation is crucial for maintaining homeostasis. The NLRP3 inflammasome, an important intracellular multiprotein complex, plays a key role in the initiation and amplification of inflammatory responses, and its aberrant activation is a common pathway in various inflammatory diseases. Therefore, targeted regulation of the NLRP3 inflammasome has become an important strategy for treating inflammation-related diseases. Currently, research on the natural compound Gar C mainly focuses on its anti-tumor effects, while its anti-inflammatory potential and molecular mechanisms in directly regulating the NLRP3 inflammasome and related signaling pathways remain to be elucidated. Given its promising anti-inflammatory prospects, Gar C is expected to be developed into a novel NLRP3-targeted anti-inflammatory drug. This study systematically evaluated its inhibitory effects on the assembly and activation of the NLRP3 inflammasome using in vitro cell models and in vivo animal disease models. Specific examples are as follows.

[0070] Example 1

[0071] Gar C inhibits the activation of the NLRP3 inflammasome in macrophages in vitro.

[0072] Experiment 1: Mouse BMDM (bone marrow-derived macrophages) were pre-stimulated with LPS (100 ng / ml) for 3 h, then treated with different doses of Gar C for 30 min, followed by stimulation with the activator Nigericin for 30 min to induce NLRP3 inflammasome activation. The secretion levels of IL-1β, TNF-α, and IL-6 in the cell culture supernatant were detected by ELISA; the protein expression of caspase-1 (P20) and IL-1β in the cell culture supernatant (SN), and the protein expression of Pro-casp1, Pro-IL-1β, and β-actin in the cell lysate (Input) were detected by Western blotting. Results are as follows: Figure 1 As shown.

[0073] from Figure 1 Analysis showed that Western blot results indicated that Gar could dose-dependently inhibit the protein expression of mature IL-1β and caspase-1 cleavage product p20 in mouse BMDM cells, while having no significant effect on the expression levels of pro-IL-1β and pro-caspase-1. Figure 1 A). ELISA results further showed that this compound significantly reduced the secretion of IL-1β in cell supernatant in a concentration-dependent manner, but had no significant effect on the secretion of TNF-α and IL-6. Figure 1 This demonstrates that Gar C can inhibit Nigericin-induced activation of the classical NLRP3 inflammasome in BMDM cells.

[0074] Experiment 2: Well-grown THP-1 cells and PBMCs isolated from peripheral blood of healthy individuals were centrifuged and resuspended, then seeded into 12-well plates and cultured overnight. The next day, the medium was replaced with RPMI 1640 medium containing LPS (500 ng / mL) (500 μL per well) for pre-stimulation for 3 hours. Subsequently, different doses of Gardner C were added for 30 minutes, and finally, Nigericin was added to induce NLRP3 inflammasome activation. The secretion levels of IL-1β and TNF-α in the cell supernatants of both groups were detected by ELISA; Western blotting was used to detect the protein expression of caspase-1 (p20) in the THP-1 cell culture supernatant (SN) and the protein expression of Pro-casp1 and β-actin in the cell lysate (Input). Results are as follows: Figure 2 As shown.

[0075] from Figure 2 Analysis showed that in THP-1 cells, Western blot analysis revealed that Gard C could dose-dependently inhibit the caspase-1 activation fragment p20, while having no significant effect on the basal expression of pro-caspase-1. Figure 2 A). ELISA results further confirmed that this compound significantly reduced IL-1β secretion in THP-1 cell supernatant in a concentration-dependent manner. Figure 2 B). Furthermore, Gar C also exhibited similar inhibitory effects in human PBMCs, dose-dependently reducing IL-1β release without affecting TNF-α secretion levels. Figure 2 (C~2D). This demonstrates that Gar C can inhibit the activation of NLRP3 inflammasomes in THP-1 cells and human PBMCs.

[0076] Experiment 3: Two other classic NLRP3 inflammasome agonists (ATP and MSU) were selected to verify whether Gar C could also inhibit the inflammasome activation induced by them.

[0077] BMDM cells were pretreated with LPS (100 ng / ml) for 3 h, and then treated with different concentrations of Gardner C for 30 min. Cells were then activated with ATP (12 mM) for 30 min or MSU (120 μg / ml) for 3 h. Cell culture supernatant and cell lysate were collected. The secretion level of the inflammatory factor IL-1β in the cell culture supernatant was detected by ELISA. Western blotting was used to detect the protein expression of caspase-1 (p20) and IL-1β in the cell culture supernatant (SN), and the protein expression of Pro-casp1, Pro-IL-1β, and β-actin in the cell lysate (Input). Results are as follows: Figure 3 As shown.

[0078] from Figure 3 Analysis showed that, according to Western blot analysis, in ATP-activated NLRP3 inflammasomes, Gar C inhibited the generation of mature IL-1β and its caspase-1 cleavage fragment p20 in a dose-dependent manner, but did not affect the expression of pro-IL-1β and pro-caspase-1 precursor proteins. Figure 3 A); Further ELISA assays showed that Gar C was able to dose-dependently inhibit ATP-triggered IL-1β secretion (A). Figure 3 B). Similarly, under MSU-induced inflammasome activation conditions, this compound also significantly inhibited the production of p20 and mature IL-1β in a dose-dependent manner. Figure 3 C), IL-1β secretion is also inhibited to a similar degree in a concentration-dependent manner ( Figure 3 D). This demonstrates that Gar C can broadly inhibit NLRP3 inflammasome activation induced by multiple agonists.

[0079] Experiment 4: BMDM cells were pretreated with Pam3CSK4 (palmitoyl triacyl lipopeptide) (100 ng / ml) for 3 h, and then treated with different concentrations of Gar C for 30 min. LPS was then transfected into the cells. After 16 h, cell culture supernatant and cell lysate were collected. The secretion level of the inflammatory factor IL-1β in the cell culture supernatant was detected by ELISA. Western blotting was used to detect the protein expression of caspase-1 (p20) and IL-1β in the cell culture supernatant (SN), and the protein expression of Pro-casp1, Pro-IL-1β, and β-actin in the cell lysate (Input). Results are as follows: Figure 4 As shown.

[0080] from Figure 4 Analysis showed that Western blot analysis revealed that Gar C dose-dependently reduced the secretion levels of IL-1β and its caspase-1 cleavage fragment p20 after activation of the non-classical NLRP3 inflammasome, while having no significant effect on the expression of pro-IL-1β and pro-caspase-1 precursor proteins. Figure 4 A); ELISA experiments further showed that this compound was able to inhibit the secretion of IL-1β in cells in a dose-dependent manner ( Figure 4 B). This demonstrates that Gar C can also inhibit the non-classical activation pathway of the NLRP3 inflammasome.

[0081] Experiment 5: To investigate the specificity of Gar C inhibition, the study also evaluated its potential impact on the AIM2 (melanoma deletion gene 2) inflammasome. The AIM2 inflammasome can respond to dsDNA and mediate defense responses.

[0082] To activate the AIM2 inflammasome, we first pretreated BMDM cells with LPS, followed by Gard C intervention, and finally transfected them with poly(A:T) for 3 hours to induce AIM2 inflammasome activation. The secretion level of the inflammatory factor IL-1β in the cell culture supernatant was detected by ELISA; the protein expression of caspase-1 (p20) and IL-1β in the cell culture supernatant (SN), and the protein expression of Pro-casp1, Pro-IL-1β, and β-actin in the cell lysate (Input) were detected by Western blotting. Results are as follows: Figure 5 As shown.

[0083] from Figure 5 Analysis showed that, unlike the significant inhibition of the NLRP3 pathway, GarC treatment did not cause significant changes in the expression of key component proteins after AIM2 inflammasome activation. Figure 5 A). Consistent with this, ELISA results showed that while the compound significantly inhibited IL-1β secretion triggered by Nigericin, it had no significant effect on IL-1β release mediated by the Poly A:T-activated AIM2 pathway. Figure 5 B). This demonstrates that Gar C has no effect on AIM2 inflammasome activation.

[0084] Example 2

[0085] Research on the mechanism by which Gar C inhibits NLRP3 inflammasome activation

[0086] Experiment 1: The interactions between NEK7 and NLRP3, and between NLRP3 and ASC, play a central role in the activation, assembly, and functional regulation of the NLRP3 inflammasome. Specifically, NEK7, as a key pivot protein, directly binds to NLRP3, which is a prerequisite for initiating NLRP3 oligomerization, promoting ASC spot formation, and successfully activating caspase-1 under specific conditions (such as potassium ion efflux).

[0087] BMDM cells were pretreated with LPS (100 ng / ml) for 3 hours, followed by Gard C treatment for 30 minutes, and then activated with Nigericin (10 μM). Cell supernatant and lysis buffer were collected. Western blotting was used to analyze the protein interactions and expression levels of NLRP3, NEK7, and ASC in immunoprecipitation (IP) and lysis buffer (Input). Results are as follows. Figure 6 As shown.

[0088] from Figure 6 Analysis showed that the immunoprecipitation experiment indicated that Gar C treatment significantly weakened the interaction between NLRP3 and NEK7. Figure 6 A), and also blocked the binding of NLRP3 and ASC. Figure 6 B). This indicates that Gar C inhibits the binding of NEK7 to NLRP3 and NLRP3 to ASC, thus proving that Gar C can block the interaction between endogenous NEK7 and NLRP3, and NLRP3 and ASC.

[0089] Example 3

[0090] In vivo study of Gar C inhibiting NLRP3 inflammasome in mice

[0091] Experiment 1: LPS (lipopolysaccharide)-induced septic shock in mice

[0092] An LPS-induced septic shock model was constructed, and the levels of inflammatory cytokines such as IL-1β in serum and peritoneal lavage fluid were detected to comprehensively evaluate the alleviating effect of Gar C on NLRP3 inflammasome-related inflammatory diseases.

[0093] In the lipopolysaccharide (LPS)-induced septic shock model, activation of the NLRP3 inflammasome is a core driver of the pathological process. LPS induces the expression of pro-inflammatory factors (TNF-α, IL-6) and inflammasome-related components (pro-IL-1β, NLRP3) through the TLR4 / MyD88 / NF-κB signaling pathway, thus completing the "pre-initiation" of the inflammatory response. Subsequently, signals such as K⁺ efflux and ROS generation directly or indirectly induced by LPS trigger the assembly of the NLRP3 inflammasome, activating caspase-1. Activated caspase-1 catalyzes the maturation and release of pro-IL-1β / pro-IL-18 and mediates pyroptosis by cleaving GSDMD, forming a "cytokine storm" dominated by NLRP3. This explosive inflammatory response ultimately leads to microcirculatory disturbances, vasodilatory shock, and multiple organ dysfunction, accurately mimicking the typical characteristics of clinical septic shock and establishing the importance of NLRP3 as a key therapeutic target.

[0094] The specific steps are as follows:

[0095] Seven-week-old male C57BL / 6J mice of similar weight were randomly divided into three groups of six each: a blank control group (Control group), a septic shock model group (LPS group), and a Gar C treatment group (LPS+Gar C group). Mice were separated into different cages the night before. The following morning, the LPS+Gar C group mice were given an intraperitoneal injection of Gar C (20 mg / kg), while the Control group mice were injected with an equal volume of PBS. Fifty minutes later, except for the blank control group mice, the other groups were injected with LPS solution (20 mg / kg) to induce septic shock. Four hours later, ocular blood was collected by enucleation, followed by cervical dislocation to sacrifice the mice and collect peritoneal lavage fluid. The blood sample was allowed to stand at room temperature for 3-4 hours, then centrifuged at 8500 rpm for 5 minutes at 4 °C. The supernatant serum was transferred to a new EP tube, taking care to avoid contact with the cell layer. The collected mouse peritoneal lavage fluid was centrifuged, and the supernatant was then transferred to a new tube. The secretion levels of IL-1β and other related inflammatory factors in mouse serum and peritoneal lavage fluid were measured using ELISA. Results are as follows: Figure 7 As shown.

[0096] from Figure 7 Analysis showed that in the LPS-induced septic shock model, Gar C intervention significantly reduced the concentration of IL-1β in the peritoneal lavage fluid of mice. Figure 7 A), but had no significant effect on IL-6 and TNF-α levels (A), while it did not have a significant effect on IL-6 and TNF-α levels. Figure 7 B, 7C). Simultaneously, this treatment also effectively inhibited the release of IL-1β from ocular blood (B, 7C). Figure 7 D), but it did not have a significant effect on the levels of IL-6 and TNF-α in the same sample ( Figure 7 E, 7F). This demonstrates that Gar C can effectively alleviate LPS-induced septic shock in mice.

[0097] Experiment 2: ConA-induced acute liver injury

[0098] An acute liver injury model induced by concanavalin A (ConA) was constructed, and serum liver function indicators such as ALT and AST were detected. Simultaneously, combined with HE staining of liver tissue sections and detection of key inflammatory factors such as caspase-1 (p20) in tissue homogenates by Western blotting, the alleviating effect of Gar C on NLRP3 inflammasome-related inflammatory diseases was comprehensively evaluated from the histopathological and molecular levels.

[0099] In ConA-induced acute liver injury (as a model of autoimmune hepatitis), the NLRP3 inflammasome and its downstream product IL-1β play a central pathogenic role. Studies have shown that ConA attack significantly activates the NLRP3 inflammasome in the liver, leading to caspase-1 cleavage and the massive release of mature IL-1β, inducing pyroptosis. Genetic evidence confirms that mice lacking the NLRP3 or caspase-1 genes are protected from ConA-induced hepatitis damage. The mechanism lies in the fact that NLRP3 activation is critically driven by reactive oxygen species (ROS), and intervention with an IL-1 receptor antagonist (rhIL-1Ra) not only effectively inhibits liver inflammation and cell infiltration but also, in turn, weakens the activation of the NLRP3 inflammasome, forming a positive feedback loop. These findings collectively demonstrate that the ROS / NLRP3 / IL-1β signaling axis is a key mechanism in ConA-induced liver injury, providing an important theoretical basis for targeting this pathway to treat autoimmune hepatitis.

[0100] The specific steps are as follows:

[0101] Male C57BL / 6 mice aged 8-10 weeks with similar weights were randomly divided into three groups of six mice each: a blank control group (Control group), an acute liver injury model group (ConA group), and a Gar C treatment group (ConA+Gar C group). Mice were separated into different cages the night before. The next day, before modeling, mice in the ConA+Gar C group were pre-injected intraperitoneally with Gar C (20 mg / kg), while mice in the Control group were injected with an equal volume of PBS. Thirty minutes later, except for mice in the Control group, mice in the ConA and ConA+Gar C groups were injected intravenously with ConA solution (20 mg / kg) via the tail vein to establish the acute liver injury model. Sample collection and indicator detection: 1. Liver function assessment: 24 hours after ConA injection, mice were sacrificed by cervical dislocation, and ocular blood was collected. After centrifugation, serum was separated, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using an ELISA kit. 2. Histological analysis: Mouse liver tissue was analyzed as follows: Western Blot: to detect the protein expression level of caspase-1 in liver tissue; ELISA: to detect the IL-1β content in liver tissue homogenate; HE staining: to stain liver tissue sections and observe pathological morphological changes. Results are as follows: Figure 8 As shown.

[0102] from Figure 8 Analysis showed that, upon visual inspection, the livers of mice in the ConA model group were dark red, brittle, and had diffuse hemorrhages on the surface; however, after treatment with Gar C (20 mg / kg), the appearance of the livers was significantly improved, the color was close to normal, and the number of hemorrhages was significantly reduced. Figure 8 A). Histological analysis further showed that this compound could significantly alleviate ConA-induced inflammatory cell infiltration and hepatocyte necrosis in liver tissue ( Figure 8 C), accompanied by a decrease in IL-1β levels in liver tissue ( Figure 8 B). Furthermore, Gar C treatment significantly reduced serum ALT and AST levels (B). Figure 8 D, 8E), Western blot results further showed that it could inhibit caspase-1 activation, manifested as a reduction in p20 fragment generation ( D, 8E). Figure 8 F). This demonstrates that Gar C can effectively alleviate ConA-induced acute liver injury.

[0103] Experiment 3: Alum-induced acute peritonitis

[0104] An Alum-induced mouse model of acute peritonitis was established. The proportion of neutrophils in peritoneal lavage fluid was detected by flow cytometry, and inflammatory factors in peritoneal fluid were detected by ELISA to comprehensively evaluate the alleviating effect of Gar C on NLRP3 inflammasome-related inflammatory diseases.

[0105] In the classic Alum (aluminum hydroxide adjuvant)-induced acute peritonitis model, activation of the NLRP3 inflammasome is a key step in initiating and amplifying the neutrophil-driven acute inflammatory response. When Alum crystals are phagocytosed by innate immune cells such as macrophages in the peritoneal cavity, a series of upstream signaling events are triggered, the most critical of which include lysosomal rupture and intracellular potassium efflux. These danger signals are recognized by the intracellular sensor NLRP3, prompting NLRP3 to assemble with the adaptor protein ASC and the protease caspase-1 into an active inflammasome complex. Activated caspase-1 then cleaves its downstream substrates: on the one hand, it processes and matures pro-IL-1β and pro-IL-18, releasing these potent pro-inflammatory factors that recruit large numbers of neutrophils to the peritoneal cavity.

[0106] The specific steps are as follows:

[0107] Eight-week-old male C57BL / 6J mice of similar weight were randomly divided into three groups of six mice each: a blank control group (Control group), an Alum-induced acute peritonitis model group (Alum group), and a Gar C treatment group (Gar C + Alum group). Mice were separated into different cages the night before. The next day, before modeling, the Gar C treatment group mice were intraperitoneally injected with Gar C (20 mg / kg), while the Control group was injected with an equal volume of PBS. One hour later, the Alum group and the Gar C + Alum group mice were intraperitoneally injected with Alum (1 mg / mouse) to establish the acute peritonitis model. Twelve hours after modeling, ocular blood was collected from the mice, followed by cervical dislocation, and peritoneal lavage fluid was collected. Evaluation was performed using the following methods: ① Flow cytometry to detect the proportion of neutrophils in the peritoneal lavage fluid. ② ELISA to detect the secretion of inflammatory factors (IL-1β, TNF-α) in ocular blood and peritoneal fluid. Results are as follows: Figure 9 As shown.

[0108] from Figure 9 Analysis of the data, including flow cytometry analysis, showed that after Gard C intervention, CD11b levels in the peritoneal fluid of mice decreased. + Ly6G + The proportion of neutrophils decreased significantly. Figure 9 A), its absolute quantity and relative percentage also decreased simultaneously ( Figure 9 B, 9C). Further ELISA analysis revealed that Gar C treatment significantly inhibited the secretion level of IL-1β in peritoneal fluid, but had no significant effect on the release of TNF-α. Figure 9 (D, 9E). This demonstrates that Gar C can effectively alleviate Alum-induced peritonitis in mice.

[0109] The above experimental studies fully demonstrate that the natural compound Gar C can specifically inhibit the activation of the NLRP3 inflammasome, while having no significant effect on other inflammasomes (such as AIM2). At the molecular level, Gar C effectively blocks the assembly process of the NLRP3 inflammasome by interfering with the interaction between NLRP3 and the key adaptor protein NEK7. This inhibitory effect was verified in bone marrow-derived macrophages (BMDM) stimulated by various activators (such as ATP, nigrain, and Alum), and showed consistent inhibitory effects in both murine and human cells. At the animal model level, Gar C effectively alleviated pathological symptoms and improved disease progression in various NLRP3-related disease models, including LPS-induced septic shock, ConA-induced acute liver injury, and Alum-induced acute peritonitis, fully demonstrating its broad-spectrum anti-inflammatory effect. This study not only elucidated the mechanism by which Gar C specifically targets the NLRP3 / NEK7 interaction at the molecular level, but also further solidified the key pathological role of NLRP3 in diseases such as infection, autoimmunity, and tumors, providing important experimental evidence and candidate molecules for the development of novel anti-inflammatory drugs targeting NLRP3.

[0110] At the cellular level, this study systematically evaluated the inhibitory effect of Gar C on the NLRP3 inflammasome in various cell models. Under induction by different agonists such as Nigericin, ATP, and MSU, Gar C significantly inhibited the release of IL-1β and caspase-1 p20 subunits in mouse BMDM cells, human THP-1 cells, and healthy human PBMCs, exhibiting a clear dose-dependent effect without affecting the expression levels of Pro-IL-1β and Pro-caspase-1, indicating that its inhibitory effect occurs during the inflammasome activation stage. Notably, Gar C also effectively inhibited the caspase-11-mediated non-classical inflammasome activation pathway, demonstrating a comprehensive inhibitory capacity on the NLRP3 pathway. To explore its specificity, this study further activated the AIM2 inflammasome using Poly(A:T), and found that Gar C treatment did not produce a significant inhibitory effect, confirming its highly specific inhibitory effect on the NLRP3 inflammasome. Mechanistic studies have shown that Gar C directly interferes with the upstream assembly process of the inflammasome by blocking the protein-protein interaction between NEK7-NLRP3 and ASC-NLRP3, thereby inhibiting its activation at its source. These findings not only reveal the unique mechanism of action of Gar C, but also lay a solid experimental foundation for its subsequent development into a highly effective and specific NLRP3 inflammasome inhibitor.

[0111] In animal model studies, the therapeutic effect of Gar C was systematically validated using three inflammatory models (septic shock, acute liver injury, and acute peritonitis) with different inducing factors and pathological manifestations. In the LPS-induced septic shock model, this study confirmed that Gar C can exert its anti-inflammatory effect by specifically reducing IL-1β levels. ELISA analysis showed that while significantly reducing IL-1β levels in serum and peritoneal lavage fluid, this compound did not significantly affect inflammatory factors such as IL-6 and TNF-α, indicating that its anti-inflammatory effect has high targeting specificity. In the ConA-induced acute liver injury model, Gar C showed significant therapeutic effects. Compared with the model group, the Gar C treatment group showed significant inhibition of caspase-1 p20 production and IL-1β release in liver tissue, while liver injury markers (ALT / AST) levels and histopathological damage were also improved simultaneously. Notably, Gar C demonstrated significant efficacy at a dose of 20 mg / kg, lower than the 60 mg / kg required for celecoxib to achieve similar efficacy as reported in the literature. Gar C also showed good intervention effects in an Alum-induced acute peritonitis model. Flow cytometry analysis revealed increased levels of Ly6G in the peritoneal lavage fluid of mice after Gar C intervention. + CD11b + The proportion and absolute count of neutrophils were both significantly decreased. Further ELISA analysis confirmed that the release of IL-1β in the peritoneal lavage fluid of this group was also significantly inhibited. The results indicate that it can exert significant anti-inflammatory and protective effects in different disease scenarios by targeting the NLRP3 inflammasome, demonstrating broad prospects for clinical application.

[0112] The above studies confirmed that Gar C effectively inhibited the activation of the NLRP3 inflammasome in BMDMs and various human cell lines. Its mechanism of action lies in blocking the interactions between NLRP3 and NEK7, as well as between NLRP3 and ASC, thereby interfering with the assembly process of the NLRP3 inflammasome. Furthermore, this study systematically evaluated the anti-inflammatory effects of Gar C by constructing a series of mouse models, including septic shock, ConA-induced acute liver injury, and acute peritonitis. The results showed that Gar C effectively alleviated disease symptoms in three different inflammation models, consistently demonstrating its therapeutic potential as a dual inhibitor of the NLRP3 inflammasome with both broad spectrum and high specificity.

[0113] In summary, the Gar C provided in this application exhibits specific inhibitory effects on the NLRP3 inflammasome and a series of inflammatory diseases it induces. It offers a novel, potentially therapeutic drug for the treatment of septic shock, acute liver injury, and acute peritonitis, demonstrating significant clinical value and socioeconomic benefits. Furthermore, it has potential for widespread application in the field of biomedical materials technology.

[0114] The above embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. The application of gamboge ketone C, characterized in that, The application of gamboge ketone C in the preparation of a drug for treating acute peritonitis.

2. The application according to claim 1, characterized in that, In medications for treating acute peritonitis, the dosage of garcinolone C is 20 mg / kg.

3. The application according to claim 1, characterized in that, The drug for treating acute peritonitis is made with gamboge ketone C as the active ingredient and pharmaceutical excipients.

4. The application according to claim 1, characterized in that, The dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, oral liquids, injections, and suppositories.

Images