Application of deoxyrapaquinone in preparing medicine for treating septic shock or colitis

Abstract

The present invention belongs to the technical field of inflammation treatment, and specifically relates to the use of deoxylabaquinone in inhibiting the activation of NLRP3 inflammasome. The technical problem to be solved by the present invention is to provide a new option for the treatment of NLRP3 inflammasome-related diseases. The technical solution of the present invention is the use of deoxylabaquinone in the preparation of NLRP3 inflammasome inhibitors. The present invention studies the interaction between DE and NLRP3 inflammasome, and provides a better strategy for the treatment of NLRP3-related inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of inflammation treatment, and specifically relates to the application of deoxylabaquinone in inhibiting the activation of NLRP3 inflammasome. Background Art

[0002] There are many types of macroalgae, which are widely found in nature. Because of their large size, it is easy to obtain sufficient samples, so they have attracted more and more researchers. Among them, brown algae in macroalgae has obvious anti-tumor effect. Studies have shown that deoxylapacho (hereinafter referred to as DE) isolated from New Zealand brown algae (Landsburgia quercifolia) has anti-tumor effect, but there are relatively few literature reports on the clinical application of deoxylapacho (DE). Therefore, it is of great significance to further develop and utilize its resources effectively.

[0003] Abnormal activation of NLRP3 inflammasomes can lead to a variety of diseases. Various NLRP3 inflammasome inhibitors have shown significant anti-inflammatory effects in animal models, such as MCC950, Tranilast, Rubescensine A, OLT1177, etc. Among them, MCC950 was abandoned due to its hepatotoxicity; Tranilast is used to treat bronchial asthma, and Rubescensine A is derived from Chinese herbal medicine ingredients, and neither has entered clinical trials; OLT1177 is currently the only drug that has entered Phase II clinical trials for the treatment of gouty arthritis. Therefore, there is still a long way to go to find better NLRP3 inflammasome inhibitors.

[0004] The current clinical treatment strategy for NLRP3-related diseases is to target IL-1β, but this method cannot treat diseases caused by IL-18 secretion and cell pyroptosis after NLRP3 inflammasome activation, and there are many limitations and nonspecificity compared with inhibitors targeting NLRP3. Rilonacept is an IL-1 inhibitor and the first drug approved by the FDA for the treatment of CAPS. In 2012, the FDA approved Rilonacept for the treatment of gout. Multiple clinical trials have evaluated the direct inhibitory effect of IL-18 in B-cell non-Hodgkin's lymphoma and inflammatory bowel disease. Since IL-1β is derived from a variety of inflammasomes, treatments that directly target IL-1βR may increase the risk of infection in the body.

[0005] Abnormal activation of NLRP3 inflammasomes leads to the release of a large number of inflammatory factors. Septic shock induced by intraperitoneal injection of LPS is a common acute disease model associated with NLRP3 inflammasomes, mainly caused by bacterial infection and accompanied by severe systemic inflammatory response. Septic shock is an excessive systemic inflammatory response caused by fungal or bacterial infection. Studies have reported that septic shock is accompanied by abnormally active NLRP3 inflammasome activation, which produces a large number of inflammatory cytokines, induces cell pyroptosis, and aggravates systemic inflammation. During the occurrence of septic shock, the secretion levels of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6 in serum and peritoneal fluid increase. LPS-induced septic shock is closely related to abnormal activation of NLRP3 inflammasomes. The model based on intraperitoneal injection of LPS in mice is the most similar to human septic shock and one of the earliest models. The main advantage of this model is that they are highly standardized, the severity of the disease can be adjusted, and it can be applied to study the relevance of specific issues, such as the relationship between proinflammatory cytokines in serum and peritoneal fluid and NLRP3 inflammasome-related septic shock.

[0006] Inflammatory bowel disease is divided into ulcerative colitis and Crohn's disease. At present, the exact pathogenesis of inflammatory bowel disease (IBD) is still not completely clear, but it has been confirmed that NLRP3 inflammasome plays an important role in the occurrence and development of IBD. The severity of IBD disease is positively correlated with the upregulation of NLRP3 inflammasome-related components in the colonic mucosal tissue of patients, as well as the secretion of IL-1β by colonic tissue and macrophages of patients. The secretion of IL-1β by macrophages and colonic tissues of patients with inflammatory bowel disease increases with the severity of the disease, and the expression of NLRP3, ASC and IL-1β in the colonic mucosa of patients with inflammatory bowel disease increases. Studies have shown that IL-18 induced by NLRP3 inflammasomes helps repair the intestinal mucosal barrier; however, different studies have shown that in animal models of DSS-induced colitis, NLRP3 deficiency showed a protective effect on experimental animals. In the early stages of intestinal inflammatory response, NLRP3 inflammasome helps maintain intestinal barrier integrity and promote tissue repair. When intestinal inflammation develops into chronic inflammation, overactivation of NLRP3 inflammasome leads to massive secretion of IL-1β and IL-18, thereby aggravating intestinal inflammation.

[0007] Gout is a chronic inflammatory arthritis characterized by the deposition of MSU crystals in joints or other sites in the presence of increased urate concentrations. MSU crystals are well-known activators of the NLRP3 inflammasome, and the activation of the NLRP3 inflammasome and the release of IL-1β play an important role in acute attacks of gout. Therefore, the proper use of NLRP3 inflammasome inhibitors may be a potential strategy for the treatment of IBD, septic shock, and gout. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a new option for the treatment of NLRP3 inflammasome-related diseases.

[0009] The technical solution of the present invention is the use of deoxylabaquinone in the preparation of NLRP3 inflammasome inhibitors.

[0010] In particular, the inhibitor inhibits the activation or assembly of the NLRP3 inflammasome.

[0011] Furthermore, the NLRP3 inflammasome is induced by an agonist.

[0012] Specifically, the agonist is nigericin, cLPS, adenosine triphosphate (ATP) and / or monosodium urate crystals (MSU).

[0013] The present invention also provides the use of deoxylabaquinone in the preparation of cytokine inhibitors that depend on NLRP3 inflammasomes.

[0014] Specifically, the cytokine is IL-1β or TNF-α.

[0015] The present invention also provides the use of deoxyrapaquinone in preparing a drug for treating NLRP3 inflammasome-related diseases.

[0016] Specifically, the NLRP3 inflammasome-related diseases are systemic inflammation, colitis, septic shock, type II diabetes, metabolic syndrome, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, Behcet's disease, sepsis, gout, arthritis, multiple sclerosis, obesity, viral inflammation such as viral hepatitis and pneumonia, acute and chronic tissue damage caused by infection, atherosclerosis, myocardial infarction, UV-induced skin sunburn, contact hypersensitivity, familial cold autoinflammatory syndrome, chronic infantile neurocutaneous joint syndrome, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, depression, asbestosis, silicosis and silicosis, asthma or acute respiratory distress syndrome, peritonitis, Muller-Weigler syndrome and cryoporphyrin-associated periodic syndrome (CAPS) or cytokine storm caused by tumor treatment (such as CART therapy and immune checkpoint inhibitor therapy), etc.

[0017] The present invention also provides a drug for inhibiting NLRP3 inflammasome, wherein the main component of the drug is deoxylabaquinone.

[0018] Wherein, the medicine also includes pharmaceutically acceptable auxiliary ingredients.

[0019] The present invention also provides a drug for treating NLRP3 inflammasome-related diseases, wherein the main component of the drug is deoxylabaquinone.

[0020] Wherein, the medicine also includes pharmaceutically acceptable auxiliary ingredients.

[0021] Beneficial effects of the present invention: The present invention studies the interaction between DE and NLRP3 inflammasome, and explores the effect of DE on NLRP3 inflammasome activation at the cellular and animal levels, in order to provide safer, more efficient and more therapeutically potential NLRP3 targeted inhibitors for the treatment of NLRP3 inflammasome-related diseases, and provide a better strategy for the treatment of NLRP3-related inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 , deoxylabaquinone inhibits Nigericin-induced NLRP3 inflammasome activation, where Figure (A) is Western blot detection of caspase-1 (p20) protein expression in cell culture supernatant (SN) and Pro-caspase-1 (Pro-casp1) and β-actin protein expression in cell lysate (Input). Figure (B) is ELISA detection of inflammatory factor IL-1β secretion level in cell culture supernatant.

[0023] Figure 2 , deoxylabaquinone inhibited Nigericin-induced NLRP3 inflammasome activation in THP-1 cells. Figure (A) Western blot was used to detect the protein expression of p20 in cell culture supernatant (SN) and the protein expression of Pro-casp1 and β-actin in cell lysate (Input). ELISA was used to detect the secretion levels of inflammatory factors IL-1β (Figure (B)) and TNF-α (Figure (C)) in cell culture supernatant.

[0024] Figure 3 , deoxylabaquinone inhibited the activation of NLRP3 inflammasome induced by other stimulants. Figures (A, B) are Western blot detection of p20 protein expression in cell culture supernatant (SN) and Pro-casp1 and β-actin protein expression in cell lysate (Input) under the action of adenosine triphosphate (ATP) stimulator, and ELISA detection of the secretion level of inflammatory factor IL-1β in cell culture supernatant. Figures (C, D) are Western blot detection of p20 protein expression in cell culture supernatant (SN) and Pro-casp1 and β-actin protein expression in cell lysate (Input) under the stimulation of monosodium urate crystals (MSU), and ELISA detection of the secretion level of inflammatory factor IL-1β in cell culture supernatant.

[0025] Figure 4, DE inhibits the activation of non-classical NLRP3 inflammasome. Figure (A) shows the expression of p20 protein in cell culture supernatant (SN), and Figure (B) shows the secretion of IL-1β in cell lysate (Input).

[0026] Figure 5 , DE does not affect the activation of AIM2 inflammasome induced by Poly(dA:dT). Figure (A) shows the expression of p20 protein in cell culture supernatant (SN), and Figure (B) shows the secretion of IL-1β in cell lysate (Input).

[0027] Figure 6 , DE does not affect the LPS-mediated pre-stimulation signal stage. Figure (A) shows the secretion of IL-6 inflammatory factors, Figure (B) shows the secretion of TNF-α, and Figure (C) Western blot detects the protein expression levels of IL-1β precursor (Pro-IL-1β), NLRP3, ASC, Pro-casp1 and β-actin in cell lysates.

[0028] Figure 7 , DE did not affect Nigericin-induced potassium ion efflux, mitochondrial damage and reactive oxygen species production; among them, Figure (A) shows mitochondrial damage, and Figure (B) shows potassium ion efflux.

[0029] Figure 8 , DE inhibited the interaction between NLRP3 and ASC.

[0030] Fig. 9 , DE specifically binds to NLRP3 protein. Figure (A) Oridonin at different concentrations was used as a positive control. Figure (B) DE at different concentrations was used as an analyte. The interaction affinity between DE and NLRP3 protein was determined by surface plasmon resonance (SPR) technology. KD is the equilibrium dissociation constant between protein and small molecule substances. The smaller the KD value, the stronger the affinity. Compared with Oridinin, DE has a stronger affinity for interaction with NLRP3.

[0031] Fig.10 , DE alleviated LPS-induced septic shock in mice. ELISA was used to detect the secretion of inflammatory factors IL-1β (A) and TNF-α (B) in mouse serum, and the secretion of inflammatory factors IL-1β (C) and TNF-α (D) in peritoneal fluid.

[0032] Fig.11 , DE effectively alleviated DSS-induced weight loss and disease activity index in mice. Figure (A) weighed and recorded the weight of mice every day; Figure (B) observed and scored the disease symptoms of mice every day.

[0033] Fig.12 , DE alleviated DSS-induced colon shortening and colon tissue lesions. Figure (A) and Figure (B) took photos and counted the colon length of each group. Figure (C) took paraffin sections of colon tissue and stained with HE. DETAILED DESCRIPTION

[0034] There are many NLRP3 inflammasome inhibitors, which can be roughly divided into three categories: endogenous small molecules, exogenous synthetic small molecules and plant extracts. In recent years, more and more plant-derived small molecule compounds have been shown to alleviate NLRP3 inflammasome-related diseases. Deoxylapachol isolated from New Zealand brown algae (Landsburgia quercifolia) has anti-tumor effects. In view of the anti-tumor effect of DE and the close relationship between inflammation and tumors, this study explored the interaction between DE and NLRP3 inflammasome for the first time, and planned to explore the effect of DE on NLRP3 inflammasome activation at the cellular and animal levels, in order to provide safer, more efficient and more therapeutically potential NLRP3 targeted inhibitors for the treatment of NLRP3 inflammasome-related diseases, and provide a better strategy for the treatment of NLRP3-related inflammatory diseases.

[0035] In order to improve the research, the applicant constructed the following models: ① LPS-induced septic shock model, detecting the levels of inflammatory cytokines such as IL-1β in serum; ② DSS-induced mouse colitis model, monitoring the weight and disease activity index of mice and analyzing the degree of tissue pathological damage to evaluate the alleviating effect of deoxylabaquinone on NLRP3 inflammasome-related inflammatory diseases. The results showed that DE inhibited NLRP3 inflammasome activation by specifically binding to NLRP3 protein. By blocking the activation of NLRP3 inflammasome in vivo, DE alleviated LPS-induced mouse septic shock and DSS-induced mouse colitis models.

[0036] The sources of the reagents and cells used in the following examples are as follows:

[0037] Table 1 Experimental materials and experimental animals

[0038] name source THP-1 cell line Donated by Wang Hongtao's research group at Bengbu Medical College BMDM cells Extracted from C57BL / 6J mouse leg bones C57BL / 6J mice Purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0039] Table 2 Source of materials

[0040] Material Name source 6-well cell culture plate Guangzhou Jiete Biofiltration Co., Ltd. 12-well cell culture plate Guangzhou Jiete Biofiltration Co., Ltd. 10cm cell culture dish Guangzhou Jiete Biofiltration Co., Ltd. ELISA plate CORNING Cell cryopreservation tube CORNING 15mL centrifuge tube CORNING 50mL centrifuge tube CORNING 10mL pipette ThermoFisher 0.6mm syringe needle Dawn Warriors 1 mL disposable syringe Dawn Warriors 20 mL disposable syringe Dawn Warriors Insulin syringe BD PVDF membrane (0.22μM) Millipore Cell slide (20mm) Biosharp

[0041] Table 3 Antibody sources

[0042]

[0043]

[0044] Table 4 Sources of reagents

[0045]

[0046]

[0047] Deoxylapachol is the main cytotoxic component of New Zealand brown algae Landsburgia quercifolia, which has antifungal and anticancer activities. It is referred to as DE. Its full name is 2-(3-methylbutene-1-yl)naphthalene-1,4-dione, and its molecular formula is: 15 H 14 O2, the molecular structure is as shown below:

[0048]

[0049] Example 1: Deoxylabaquinone can specifically inhibit NLRP3 inflammasome activation

[0050] Experiment 1: Determine whether DE inhibits Nigericin-induced NLRP3 inflammasome activation in BMDM cells

[0051] First, we investigated whether DE affects the activation of NLRP3 inflammasome. ① Mouse BMDM cells were pre-stimulated with LPS (100 ng / mL) for 3 h, and then stimulated with different doses of DE for 30 min, and then stimulated with Nigericin, an activator of NLRP3 inflammasome, for 30 min. The expression of caspase-1 and IL-1β in the supernatant was detected by Western Blot, and the secretion of IL-1β and TNF-α in the cell culture supernatant was detected by ELISA. Figure 1 A and 1B show that DE inhibited the secretion and maturation of IL-1β and caspase-1 in BMDM cells induced by Nigericin.

[0052] Experiment 2: Determine whether DE inhibits Nigericin-induced NLRP3 inflammasome activation in THP-1 cells

[0053] Human THP-1 cells were used to further explore the effect of DE on NLRP3 inflammasome. THP-1 cells with good growth status were selected, centrifuged and resuspended, and then cultured overnight in 12-well cell culture plates. The culture medium in the cell culture plates was replaced with RMPI 1640 culture medium plus LPS (500ng / mL), 500μL liquid per well, and stimulated for 3h; Nigericin was added after the cells were treated with DE (0μM, 0.5μM, 1μM, 2μM). The expression of caspase-1 and IL-1β in the supernatant was detected by Western Blot ( Figure 2 A), ELISA detection of IL-1β in cell culture supernatant ( Figure 2B) TNF-α( Figure 2 C). The results showed that DE inhibited NLRP3 inflammasome activation in THP-1 cells.

[0054] Experiment 3: Does DE inhibit NLRP3 inflammasome activation induced by other agonists?

[0055] The effects of two other classic activators of NLRP3 inflammasome, ATP and MSU, were further explored. BMDM cells were pretreated with LPS (100 ng / mL) for 3 h, and treated with different concentrations of DE (0 μM, 2.5 μM, 5 μM, 10 μM) for 30 min. Then, ATP (12 mM) was added to activate the cells for 30 min, and MSU (120 μg / mL) was added to activate the cells for 3 h. The cell culture supernatant and cell lysate were collected. Western blot was used to detect the protein expression of p20 in the cell culture supernatant (SN) and the protein expression of Pro-casp1 and β-actin in the cell lysate (Input). Figure 3 A, 3C). ELISA was used to detect the secretion level of inflammatory factor IL-1β in cell culture supernatant ( Figure 3 B, 3D). The results showed that DE could broadly inhibit NLRP3 inflammasome activation induced by multiple agonists.

[0056] Experiment 4: DE inhibits non-canonical NLRP3 inflammasome activation in BMDM cells

[0057] BMDM cells were pretreated with Pam3CSK4 (100 ng / mL) for 3 h, and treated with different concentrations of DE (0 μM, 5 μM, 10 μM, 20 μM) for 30 min. LPS was then transfected into the cells. After 16 h of transfection, the cell culture supernatant and cell lysate were collected. Western blot was used to detect the protein expression of p20 in the cell culture supernatant (SN) and the protein expression of Pro-casp1 and β-actin in the cell lysate (Input). ELISA was used to detect the secretion level of inflammatory factor IL-1β in the cell culture supernatant. Figure 4 As shown in A and 4B, DE can also inhibit the non-classical activation pathway of NLRP3 inflammasome.

[0058] Experiment 5: Exploring whether DE affects AIM2 inflammasome activation

[0059] In addition to the NLRP3 inflammasome, there are other classic inflammasomes that respond to exogenous pathogens or endogenous danger signals to trigger inflammatory responses, such as the AIM2 inflammasome. The results of previous experiments have clarified the inhibitory effect of DE on the NLRP3 inflammasome, and further explored whether DE affects the activation of other inflammasomes. After pre-stimulating BMDM cells with LPS for 3 hours, DE was added to treat the cells for 30 minutes, and the AIM2 inflammasome was activated by transfecting poly(dA:dT). Figure 5 A and 5B show that compared with the inhibition of Nigericin activation of NLRP3 inflammasome by DE, the addition of DE did not inhibit the p20 expression and IL-1β secretion induced by transfection of poly(dA:dT), indicating that DE did not affect the activation of AIM2 inflammasome.

[0060] Example 2: Whether DE affects the upstream signaling stage of NLRP3 inflammasome assembly and activation

[0061] Mitochondrial damage and potassium ion outflow are considered to be important upstream signals for NLRP3 inflammasome activation. Previous studies have shown that a variety of plant-derived small molecule drugs can inhibit NLRP3 inflammasome activation by inhibiting mitochondrial damage, such as epigallocatechin-3-glucose (EGCG). EGCG inhibits NLRP3 inflammasome activation in macrophages by blocking mitochondrial DNA synthesis, effectively alleviating acute gout models in mice. Therefore, we explored whether DE affects the upstream signaling stage of NLRP3 inflammasome assembly and activation.

[0062] Experiment 1: Determine whether DE affects LPS-mediated prestimulation

[0063] BMDM cells were pretreated with LPS (100 ng / mL) for 3 h, and different concentrations of DE (0 μM, 2.5 μM, 5 μM, 10 μM) were added to treat the cells for 30 min before LPS treatment, or different concentrations of DE (0 μM, 2.5 μM, 5 μM, 10 μM) were added to treat the cells for 30 min after LPS treatment, and the cell culture supernatant and cell lysate were collected. ELISA was used to detect the inflammatory factor IL-6 ( Figure 6 A) and TNF-α ( Figure 6 Western blot was used to detect the secretion level of IL-1β precursor (Pro-IL-1β), NLRP3, ASC, Pro-casp1 and β-actin in cell lysate ( Figure 6 C). The results showed that DE did not affect the LPS-mediated pre-stimulation process.

[0064] Experiment 2: Determine whether DE affects nigericin-induced mitochondrial damage and potassium ion efflux

[0065] ① After pre-treating BMDM cells with LPS (100 ng / mL) for 3 hours, DE (10 μM) was added to treat the cells for 30 minutes, and then Nigericin (10 μM) was added to treat the cells for 30 minutes. The cells were stained with Mitotracker and DAPI, and observed and photographed with a laser confocal microscope. ② After pre-treating BMDM cells with LPS (100 ng / mL) for 3 hours, different doses of DE (0 μM, 2.5 μM, 5 μM, 10 μM) were added to treat the cells for 30 minutes, and then Nigericin (10 μM) was added to treat the cells for 30 minutes. After the cells were lysed with concentrated nitric acid, the intracellular potassium ion content was detected. Figure 7 As shown in A and 7B, DE did not affect mitochondrial damage and potassium ion efflux.

[0066] Experiment 3: Whether DE affects the assembly of NLRP3 inflammasome complex

[0067] NLRP3 binding to NEK7 and recruiting ASC are important events in the assembly of NLRP3 inflammasome. After pre-treatment of BMDM cells with LPS (100 ng / mL) for 3 hours, DE (10 μM) was added to treat the cells for 30 minutes, and then Nigericin (10 μM) was added to treat the cells for 30 minutes, and then NP-40 solution was added to lyse the cells. NEK7 or ASC antibodies were used for immunoprecipitation, and IgG antibodies were used as negative controls. It is clear whether DE directly inhibits the assembly process of NLRP3 inflammasome. Figure 8 As shown, DE inhibited the interaction between endogenous ASC and NLRP3, thereby suppressing the assembly of NLRP3 inflammasome.

[0068] Experiment 4: Exploring whether DE specifically binds to NLRP3 protein

[0069] DE irreversibly inhibits the activation of NLRP3 inflammasomes, and it is speculated that DE may directly bind to a target protein. The interaction between DE and NLRP3 protein was determined by surface plasmon resonance (SPR) technology. Studies have shown that Oridonin can specifically bind to NLRP3 inflammasomes. Using Oridonin as a positive reference, the experimental results show that as the dose of Oridonin increases, the coupling between Oridonin and NLRP3 protein increases ( Fig. 9 A); Similarly, as the DE concentration increases, more DE binds to NLRP3 protein ( Fig. 9 B), KD is the equilibrium dissociation constant between proteins and small molecules. The smaller the KD value, the stronger the affinity. Compared with Oridonin, DE has a stronger affinity for interaction with NLRP3. Fig. 9As shown in A and 9B, DE has a strong affinity with NLRP3.

[0070] Example 3 Exploring the disease relevance of DE inhibiting NLRP3 inflammasome activation

[0071] Experiment 1: LPS-induced septic shock model, detection of serum IL-1β and other inflammatory cytokine levels

[0072] Mice were intraperitoneally injected with DE (10 mg / kg) or an equal volume of solvent 1 hour later, and then intraperitoneally injected with LPS (20 mg / kg). Mice were killed 4 hours later, and serum and peritoneal fluid were collected. ELISA was used to detect the secretion of inflammatory factors IL-1β and TNF-α in serum and in peritoneal fluid. Fig.10 As shown in A, 10B, 10C, and 10D, DE effectively alleviated LPS-induced septic shock in mice.

[0073] This shows that DE exhibits a highly effective inhibitory effect on the activation of NLRP3 inflammasomes in vitro. Septic shock is an excessive systemic inflammatory response caused by fungal or bacterial infection, accompanied by abnormally active NLRP3 inflammasome activation, which produces a large number of inflammatory cytokines and aggravates systemic inflammation. The study found that 10 mg / kg of DE can effectively inhibit the production of IL-1β in mouse serum and peritoneal lavage fluid, but has no significant effect on the secretion of TNF-α, indicating that DE can effectively improve LPS-induced septic shock in mice. Compared with other NLRP3 inflammasome inhibitors, such as cardamomum, which effectively relieves the mouse septic shock model at 50 mg / kg, the minimum relief concentration of geranyl is 15 mg / kg, and the DE dose is lower.

[0074] Experiment 2: DSS-induced colitis model in mice, monitoring the body weight and disease activity index of mice and analyzing the degree of tissue pathological damage to evaluate the alleviating effect of DE on NLRP3 inflammasome-related inflammatory diseases

[0075] While inducing colitis with 3% DSS, mice were intraperitoneally injected with 10 mg / kg of DE every day, and the weight changes of mice were monitored. The disease symptoms of mice, including erect hair, loose stools, diarrhea, bloody stools, rectal prolapse, etc., were observed and recorded, and the disease activity was scored. The mice were killed 10 days later, the length of the colon was measured, the intestinal epithelial damage and the infiltration of immune cells in the colon tissue were analyzed histologically, the secretion level of IL-1β in mouse serum and colon tissue was detected by ELISA, the level of caspase-1 in mouse colon tissue was detected by Western Blot, and the level of pathogenic Th17 cells in mesenteric lymph nodes (MLN) was detected by flow cytometry. Fig.11As shown in A and 11B, DE effectively alleviated DSS-induced weight loss in mice, delayed the progression of disease activity, and alleviated the occurrence of colitis symptoms. Fig.12 A, 12B, and 12C indicate that DE alleviated DSS-induced colon shortening and colon tissue lesions.

[0076] The DSS-induced acute colitis model in mice can damage epithelial cells, increase colon permeability, and induce intestinal inflammation. This model is often used to evaluate the efficacy of new anti-inflammatory drugs. Various NLRP3 inflammasome inhibitors have been shown to effectively relieve DSS-induced colitis, such as curcumin at 100 mg / kg, Palmatine at 40 mg / kg, and L-Fucose at 250 mg / kg, which are effective in relieving colitis in mice, but the effective concentrations are too high. The present application found that daily injection of 10 mg / kg DE significantly alleviated the weight loss, increased disease activity index, shortened colon, damaged colon tissue, and infiltration of inflammatory cells in mice, and significantly reduced IL-17A in mesenteric lymph nodes. + CD4 + The proportion of T cells in the intestine improves the immune environment of the mouse intestine. Macrophages are the main phagocytes in the intestine. As inflammatory bowel disease worsens, IL-1β produced in macrophages and IL-1β induced by NLRP3 inflammasome activation play an important pro-inflammatory role in the course of colitis. Western blot and ELISA results showed that DE significantly inhibited the expression of mature caspase-1 and the secretion of IL-1β in mouse colon tissue. Therefore, the results showed that DE may inhibit the activation of NLRP3 inflammasomes in vivo and inhibit the secretion of IL-1β, thereby improving the disease progression of DSS-induced colitis in mice. This study found that DE inhibited the activation of NLRP3 inflammasomes induced by multiple agonists, had no inhibitory effect on AIM2 inflammasomes, and had a good inhibitory effect in both mouse and human cells. DE inhibited the activation of NLRP3 inflammasomes by specifically binding to NLRP3 protein. By blocking the activation of NLRP3 inflammasomes in vivo, DE alleviated the LPS-induced mouse septic shock and DSS-induced mouse colitis models. The findings of this study provide an experimental basis for further in-depth research on the specific mechanism of DE in inhibiting NLRP3 inflammasome activation, and also provide new directions and strategies for the treatment of NLRP3 inflammasome-related diseases.

Claims

1. Application of deoxyrapaquinone in the preparation of drugs for the treatment of septic shock.

2. Application of deoxyrapaquinone in the preparation of drugs for treating colitis.

Images