Application of tetracyclic diterpenoid compounds from the genus Isodon in the preparation of NLRP3 inflammasome inhibitors

Abstract

The present invention belongs to the technical field of inflammation treatment, and specifically relates to the application of tetracyclic diterpenoid compounds of the genus Camellia in the preparation of NLRP3 inflammasome inhibitors. 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 scheme of the present invention is the application of tetracyclic diterpenoid compounds of the genus Camellia in the preparation of NLRP3 inflammasome inhibitors, and the tetracyclic diterpenoid compounds of the genus Camellia are Ros B, Bul, Pon, Liang A or Exc A. The present invention screened a variety of tetracyclic diterpenoid compounds of the genus Camellia, and found that 5 diterpenoid compounds can inhibit the activation of NLRP3 inflammasomes. Pharmacophore modification and transformation based on the tetracyclic diterpenoid compounds of the genus Camellia is expected to obtain safer, more efficient and more therapeutically potential NLRP3 targeted inhibitors, providing 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 tetracyclic diterpene compounds of the genus Ilex to the preparation of NLRP3 inflammasome inhibitors. Background Art

[0002] Tetracyclic diterpenoid compounds of the genus Ampelopsis belong to the subfamily of the Lamiaceae family, Ociliaceae. They are produced in many places from southern Africa to tropical Africa. They are distributed throughout my country, with the largest number of species in the southwestern provinces. Ampelopsis plants are widely used herbal medicines in my country, with the effects of clearing heat and detoxifying, antibacterial and anti-inflammatory, relaxing muscles and activating blood circulation, and anti-tumor. At present, there are relatively few literature reports on the clinical application of tetracyclic diterpenoid compounds of the genus Ampelopsis, so it is of great significance to further develop and utilize its resources effectively.

[0003] Current studies have shown that the NLRP3 inflammasome is a multimeric protein complex composed of the pattern recognition receptor NLRP3, the adaptor protein ASC, and the effector protein Pro-caspase-1, which can recognize a variety of pathogen-related molecular patterns, environmental and endogenous risk-related molecular patterns. The activation process of NLRP3 can be divided into two stages, namely the pre-stimulation stage and the activation stage. The pre-stimulation (priming) stage: mainly through the activation of the NF-κB signaling pathway, promotes the expression of inflammasome component proteins NLRP3 and Pro-IL-1β, and prepares for the assembly and activation of the inflammasome; after pre-stimulation, the activator can initiate the activation of the NLRP3 inflammasome. Activation stage: a variety of PAMPs or DAMPs can stimulate cells to cause the assembly of NLRP3 inflammasomes. NLRP3 binds to NEK7 to promote the oligomerization and activation of NLRP3. The PYD domain of the adaptor protein ASC couples the pattern recognition receptor NLRP3. The CARD domain of ASC recruits the effector protein pro-caspase-1 to complete the assembly and activation of the NLRP3 inflammasome, cleaves pro-caspase-1 to make it mature and secreted. The activated caspase-1 mediates the mature secretion of IL-1β and IL-18 and cell pyroptosis, thereby eliminating pathogenic microorganisms and danger signals and maintaining the body's immune homeostasis.

[0004] At present, some NLRP3 inflammasome inhibitors have been discovered, which can be roughly divided into three categories: endogenous small molecules, such as β-hydroxybutyric acid (BHB), ω-fatty acids (ω-3), bile acids and dopamine (DA); exogenous synthetic small molecules, such as MCC950 and CY-09, IAA94 and quercetin; plant extracts, such as cardamom, quercetin, isoliquiritigenin, and oridonin. 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 non-specificity compared with inhibitors targeting NLRP3. Direct or indirect inhibition of NLRP3 protein avoids the risk of infection caused by inhibiting other inflammasomes, and on the other hand, IL-1β, IL-18 and cell pyroptosis produced by NLRP3 inflammasome activation can be effectively inhibited. MCC950 is the most intensively studied NLRP3 small molecule inhibitor. Recently, it has been reported that MCC950 can alleviate cognitive impairment caused by diabetes-induced stroke by inhibiting NLRP3, but there is a risk of chronic infection in rats in the later stage of administration. The sulfonylurea drug Glyburide can inhibit the activation of NLRP3 inflammasomes and treat NLRP3-related mouse disease models such as LPS-induced sepsis and bronchopulmonary dysplasia, but the dose of Glyburide in treating NLRP3-related diseases is high and is prone to off-target effects and adverse reactions. Therefore, it is of great significance to further strive to find inhibitors that target NLRP3 and specifically inhibit the activation of NLRP3 inflammasomes and have therapeutic effects on NLRP3 inflammasome-related diseases. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a new option for the treatment of NLRP3 inflammasome-related diseases.

[0006] The technical solution of the present invention is the use of tetracyclic diterpenoid compounds of the genus Isodon in the preparation of NLRP3 inflammasome inhibitors, and the tetracyclic diterpenoid compounds of the genus Isodon are Ros B, Bul, Pon, Liang A or Exc A.

[0007] Rosthornin B (Ros B, Chinese name: Rosthornin B), molecular structure:

[0008]

[0009] Bulleyanin (Bul, Chinese name: Cangshanxiangchain), molecular structure:

[0010]

[0011] Liangshanin A (Liang A, Chinese name: Liangshan Xiangchain A), molecular structure:

[0012]

[0013] Excisanin A (Exc A, Chinese name: Excisanin A), molecular structure:

[0014]

[0015] Ponicidin (Pon, Chinese name: Oridonium rubescens B), molecular structure:

[0016]

[0017] Furthermore, the inhibitor inhibits the assembly of NLRP3 inflammasome.

[0018] The technical solution of the present invention is the use of tetracyclic diterpenoid compounds of the genus Isodon in the preparation of cytokine inhibitors that depend on NLRP3 inflammasomes, and the tetracyclic diterpenoid compounds of the genus Isodon are Ros B, Bul, Pon, Liang A or ExcA.

[0019] Specifically, the cytokine is IL-1β.

[0020] The present invention also provides the use of a tetracyclic diterpene compound of the genus Isodon in the preparation of a drug for treating NLRP3 inflammasome-related diseases, wherein the tetracyclic diterpene compound of the genus Isodon is Ros B, Bul, Pon, Liang A or Exc A.

[0021] 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.

[0022] The present invention also provides a drug for inhibiting NLRP3 inflammasome, wherein the main component of the drug is a tetracyclic diterpene compound of the genus Isodonis, and the tetracyclic diterpene compound of the genus Isodonis is Ros B, Bul, Pon, Liang A or Exc A.

[0023] Wherein, the medicine also includes pharmaceutically acceptable auxiliary ingredients.

[0024] The present invention also provides a drug for treating NLRP3 inflammasome-related diseases, wherein the drug has a main component of a tetracyclic diterpene compound of the genus Isodonis, and the tetracyclic diterpene compound of the genus Isodonis is Ros B, Bul, Pon, Liang A or Exc A.

[0025] Wherein, the medicine also includes pharmaceutically acceptable auxiliary ingredients.

[0026] Beneficial effects of the present invention: The multiple small molecule inhibitors screened by the present invention through the Chinese herbal medicine library all belong to the tetracyclic diterpenoid compounds of the genus Rosthorne after structural analysis. Through further screening of a variety of tetracyclic diterpenoid compounds of the genus Rosthorne, it was found that 5 diterpenoid compounds can inhibit the activation of NLRP3 inflammasome. This characteristic of the tetracyclic diterpenoid compounds of this genus provides an important experimental basis and target for the development of clinical drugs for NLRP3-related inflammatory diseases. A certain type of natural small molecule compounds with a common structure has not yet been found to specifically inhibit the activation of NLRP3 inflammasome. Among them, the main active ingredient of Rosthorne (also known as wild Patchouli), acetyl Rosthornin B (abbreviated as Ros B) contained in the leaves, has the best inhibitory effect. Experiments have shown that the tetracyclic diterpenoid compounds of the genus Rosthorne, represented by Ros B, specifically target and inhibit the assembly of NLRP3 inflammasomes, which can overcome the limitations of easy off-target and increased risk of infection. By modifying the pharmacophore based on the tetracyclic diterpenoid compounds of the genus Ipomoea screened in the present invention, it is expected to obtain safer, more efficient and more therapeutically potential NLRP3 targeted inhibitors, providing a better strategy for the treatment of NLRP3-related inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1、Ten tetracyclic diterpenoids from the genus Oryza sativa inhibit NLRP3 inflammasome activation induced by Nigericin; Figure A shows the secretion of IL-1β, and Figure B shows the expression of P20 protein. Notes: Ori: Oridonin; Gla A: Glaucocalyxin A; Bul: Bulleyanin; Pon: Ponicidin; Kam: Kamebakaurin; Gla B: Glaucocalyxin B; Liang A: Liangshanin A; Exc A: Excisanin A; Ros B: Rosthornin B; Odo: Odonicin.

[0028] Figure 2 , Ros B inhibits the activation of NLRP3 inflammasome induced by Nigericin; Figure A shows the expression of P20 protein, and Figure B shows the secretion of IL-1β.

[0029] Figure 3 , Ros B inhibits the activation of NLRP3 inflammasome induced by various NLRP3 inflammasome agonists; Figure A shows the expression of P20 protein, and Figure B shows the secretion of IL-1β.

[0030] Figure 4 , Ros B inhibits the activation of non-classical NLRP3 inflammasome induced by intracellular transfection of LPS; Figure A shows the expression of P20 protein, and Figure B shows the secretion of IL-1β.

[0031] Figure 5 , Ros B does not affect the activation of AIM2 inflammasome induced by Poly(dA:dT); Figure A shows the expression of P20 protein, and Figure B shows the secretion of IL-1β.

[0032] Figure 6 , Ros B does not affect Nigericin-induced mitochondrial damage and potassium ion efflux; Figure A shows the mitochondrial damage, and Figure B shows the potassium ion efflux.

[0033] Figure 7 , Ros B inhibited the binding between endogenous NEK7 and NLRP3.

[0034] Figure 8, Ros B alleviates LPS-induced septic shock in mice; Figure A shows the secretion of IL-1β in mouse serum, and Figure B shows the survival curve of mice.

[0035] Fig. 9 , Ros B alleviates DSS-induced colitis in mice; wherein, Figure A shows the changes in mouse body weight, Figure B shows the changes in the disease activity index of mouse colitis, Figure C shows the shortening of mouse colon damage, Figure D shows the measurement statistics of mouse colon length, Figure E shows the expression of P20 protein in mouse intestinal tissue, Figure F shows the secretion of IL-1β in mouse intestinal tissue, and Figure G shows H&E staining of mouse colon sections. DETAILED DESCRIPTION

[0036] At present, it has been reported that a variety of small molecules derived from Chinese herbal medicines can target and inhibit the activation of NLRP3 inflammasomes. Therefore, the multiple small molecule inhibitors screened by the inventors through the Chinese herbal medicine library belong to tetracyclic diterpenoid compounds of the genus Rosthornia after structural analysis. Through further screening of a variety of tetracyclic diterpenoid compounds of the genus Rosthornia, it was found that 5 diterpenoid compounds can inhibit the activation of NLRP3 inflammasomes. Subsequently, Rosthornin B (abbreviated as Ros B) containing acetyl gall flowers in leaves was selected as a representative for subsequent in vitro and in vivo experiments.

[0037] In vitro experiments used classical NLRP3 inflammasome activators (Nigericin, ATP, MSU) and non-classical NLPR3 inflammasome activators (intracellular transfection of LPS) as well as AIM2 inflammasome activators (poly(dA:dT)) to stimulate mouse bone marrow-derived macrophages (BMDMs), and Western blot (WB) and ELISA were used to detect the expression levels of caspase-1 (p20) and IL-1β in the cell supernatant to clarify the effect of Ros B on NLRP3 inflammasome activation; and the effects of Ros B on mitochondrial damage and intracellular potassium ion efflux as well as the effect on inflammasome assembly were detected.

[0038] In vivo experiments, by constructing a mouse inflammation model, found that Ros B has a good therapeutic effect on both systemic inflammation and colitis in mice. Ros B can significantly reduce the expression of IL-1β in the serum of mice with systemic inflammation, but has no significant inhibitory effect on TNF-α that is independent of NLRP3 inflammasomes. In addition, by drawing a 48-hour survival curve analysis, it was found that mice in the LPS group began to die 16 hours after LPS injection, and the mortality rate of mice reached 100% at 31 hours, while mice treated with Ros B did not die until 26 hours, and the mortality rate at 31 hours was only 50%. Ros B can inhibit the release of inflammatory factors in mice with septic shock and delay the death of mice, showing a better therapeutic effect at a lower concentration. Continuous drinking of 3% DSS solution in mice will lead to colitis characterized by diarrhea, bloody stools, weight loss, and inflammatory ulcers. The experimental results showed that Ros B can improve weight loss, disease activity index, shortened colon, mucosal damage, and inflammatory cell infiltration in mice. IL-17A + CD4 + T cells can promote inflammation by producing IL-17, participate in the intestinal immune disorder of DSS-induced colitis mice, and play an important role in the development of colitis. Flow cytometry detection found that Ros B significantly reduced IL-17A in mesenteric lymph nodes. + CD4 + T cell ratio, improve the intestinal inflammatory response of mice. In addition, as the severity of the disease increases, the expression of IL-1β in macrophages and colon tissues of patients with inflammatory bowel disease increases, and excessive release of IL-1β aggravates the intestinal inflammatory response. WB and ELISA results showed that Ros B significantly inhibited the maturation of caspase-1 and the expression of IL-1β in the colon. It shows that Ros B may improve mouse colitis by targeting and reducing IL-1β secretion induced by NLRP3 inflammasome activation. In summary, the various tetracyclic diterpenoid compounds of the genus Camellia screened by the present invention can inhibit NLRP3 inflammasome activation. By inhibiting NLRP3-NEK7 binding, tetracyclic diterpenoid compounds of the genus Camellia can effectively inhibit the assembly and activation of NLRP3 inflammasomes. In addition, tetracyclic diterpenoid compounds of the genus Camellia represented by Ros B have good therapeutic effects on systemic inflammation and colitis, indicating that tetracyclic diterpenoid compounds of the genus Camellia play an important role in the treatment of inflammatory diseases related to NLRP3 inflammasomes. The discovery that tetracyclic diterpenoid compounds from the genus Ilex can inhibit NLRP3 inflammasomes provides a new option for the treatment of NLRP3 inflammasome-related diseases, and also provides new evidence that diterpenoid compounds from the genus Ilex can inhibit NLRP3 inflammasomes.

[0039] The sources of the reagents and cells used in the following examples are as follows:

[0040] Table 1 Cell and mouse information

[0041] name source 293T cell line Purchased from Wuhan Prosai Life Science Co., Ltd. THP-1 cell line Donated by Wang Hongtao's research group at Bengbu Medical College BMDMs Extracted from C57BL / 6J mouse leg bones C57BL / 6J mice Purchased from Jiangsu Jicui Yaokang Biological Co., Ltd.

[0042] Table 2 Antibody information

[0043] Antibody Name Part Number Manufacturer anti-β-actin P30002 Abmart Anti-mouse caspase-1 AG-20B-0042 AdipoGen anti-NLRP3 AG-20B-0014 AdipoGen anti-NEK7 ab133514 Abcam anti-ASC 67824S Cell Signaling Technology anti-Flag F1804 Sigma Anti-VSV V4888 Sigma Anti-GFP M20004M Abmart Anti-Rabbit IgG-HRP 111-035-144 Jackson IR anti-Goat IgG-HRP SA00004-3 Proteintech Group Anti-Mouse IgG-HRP 115-035-146 Jackson IR PB-CD3 100214 BioLegend APC / Cy7-CD19 115530 BioLegend PE-F4 / 80 123110 BioLegend PE / CY7-CD11b 101216 BioLegend FITC-Ly6G 108406 BioLegend BV510-CD4 100553 BioLegend APC-IL-17A 506916 BioLegend

[0044] Table 3 Experimental reagents

[0045]

[0046]

[0047] Example 1 Preliminary screening of NLRP3 inflammasome inhibitors

[0048] Through the screening of the Chinese herbal medicine library, more than 100 diterpenoid compounds were screened. Mouse bone marrow-derived macrophages (BMDM) were pre-stimulated with LPS (50ng / mL) for 3h, and then stimulated with different doses (1μM, 2μM, 10μM) of tetracyclic diterpenoid compounds of the genus Camellia for 30min, and then Nigericin, an activator of the NLRP3 inflammasome, was added for 30min. The secretion of caspase-1 and IL-1β was detected by Western Blot and ELISA, respectively, to screen out efficient NLRP3 inflammasome inhibitors. The obtained multiple small molecule inhibitors all belonged to tetracyclic diterpenoid compounds of the genus Camellia after structural analysis. A total of 10 compounds were obtained, and the inhibitory effect was good.

[0049] Ros B Rosthornin B (Chinese name: Rosthornin B), molecular structure:

[0050]

[0051] Bulleyanin (Chinese name: Cangshanxiangchain), molecular structure:

[0052]

[0053] Liangshanin A (Chinese name: Liangshan Xiangchain A), molecular structure formula:

[0054]

[0055] Excisanin A (Chinese name: Excisanin A), molecular structure:

[0056]

[0057] Ponicidin (Chinese name: Oridonin B), molecular structure:

[0058]

[0059] Example 2 Ros B inhibits the activation of NLRP3 inflammasome in macrophages in vitro

[0060] 1. Determining that various tetracyclic diterpenoids from the genus Isodon can inhibit NLRP3 inflammasome activation

[0061] The inhibitory effects of 10 tetracyclic diterpenoid compounds from Isophyllum plants on Nigericin-induced IL-1β and caspase-1 secretion were detected by ELISA and Western Blot, respectively, and highly effective NLRP3 inflammasome inhibitors were screened out. Their functions were verified through in vivo and in vitro experiments.

[0062] Mouse bone marrow-derived macrophages (BMDM) were pre-stimulated with LPS (50 ng / mL) for 3 h, and then stimulated with tetracyclic diterpenoids of the genus Ilex (1 μM, 2 μM, 10 μM) for 30 min. Nigericin, an activator of NLRP3 inflammasome, was then added to stimulate for 30 min. The secretion of caspase-1 and IL-1β was detected by Western Blot and ELISA, respectively, to screen out highly effective NLRP3 inflammasome inhibitors.

[0063] like Figure 1 As shown, five tetracyclic diterpenoid compounds from the genus Isophyllum inhibited the secretion and maturation of IL-1β and caspase-1 induced by Nigericin; Ros B had the best effect and was selected for subsequent experiments.

[0064] 2. Clarify the specific involvement of Ros B in NLRP3 inflammasome activation

[0065] Ros B, a highly effective NLRP3 inflammasome inhibitor, was screened by ELISA. Western Blot and ELISA were used to detect the inhibitory effect of Ros B on NLRP3 inflammasome activation in mouse BMDM cells and human THP1 cells; and the inhibitory effect of Ros B on the processing and maturation of caspase-1 and IL-1β secretion induced by various NLRP3 inflammasome-specific activators (MSU, ATP, Nigericin, etc.) was explored; the above method was also used to detect whether Ros B had an inhibitory effect on non-classical NLRP3 inflammasome activation and AIM2 inflammasome activation. ELISA was used to detect the effect of Ros B on the secretion of cytokines that are not dependent on NLRP3 inflammasomes, such as TNF-α and IL-6.

[0066] Experiment 1: Determine whether Ros B inhibits Nigericin-induced NLRP3 inflammasome activation: In order to explore the inhibitory effect of Ros B on NLRP3 inflammasome activation in mouse BMDM cells and human THP1 cells, the cells were treated according to the following operating procedures: ① Mouse BMDM cells were pre-stimulated with LPS (50 ng / mL) for 3 h, and different doses (0.5 μm, 1 μm, 2 μm) of Ros B were added for further stimulation for 30 min, and then Nigericin, an activator of NLRP3 inflammasome, was added for stimulation for 30 min. ② Human THP1 cells and PBMCs cells were cultured overnight in 1640 complete medium containing 100 nM PMA. After THP1 cells were pretreated with 200 ng / mL LPS for 3 h, different doses of Ros B were added for 30 min, and then Nigericin was added to stimulate cell activation. 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 2 As shown in A and 2B, Ros B dose-dependently inhibited the maturation and secretion of p20 and IL-1β induced by nigericin.

[0067] Experiment 2: To clarify whether Ros B inhibits NLRP3 inflammasome activation induced by other agonists: BMDM were pre-stimulated with LPS (50 ng / mL) for 3 h, then stimulated with Ros B for 30 min, and then stimulated with ATP, MSU, and Nigericin, respectively. Western Blot was used to analyze the activation of caspase-1 and the secretion of IL-1β, and ELISA was used to quantitatively detect the secretion of IL-1β in the supernatant. Figure 3 As shown in A and 3B, Ros B inhibits NLRP3 inflammasome activation induced by multiple NLRP3 inflammasome agonists.

[0068] Experiment 3: To investigate whether Ros B has an inhibitory effect on the activation of non-classical NLRP3 inflammasomes: BMDM cells were pretreated with 400 ng / mL Pam3CSK4 for 3 h, and then different doses of Ros B (0 μM, 0.5 μM, 1 μM, 2 μM) were added to treat the cells for 30 min, and then transfected with 500 ng / mL LPS to stimulate the cells for 16 h. The activation of caspase-1 and the secretion of IL-1β were analyzed by Western Blot, and the secretion of IL-1β in the supernatant was detected by ELISA. Figure 4 As shown in A and 4B, Ros B dose-dependently inhibited non-classical inflammasome activation.

[0069] Experiment 4: To investigate the effect of Ros B on the activation of AIM2 inflammasomes: BMDM were pre-stimulated with LPS (50 ng / mL) for 3 h, then stimulated with Ros B for 30 min, and then stimulated with Nigericin and poly(dA:dT), respectively. The activation of caspase-1 and the secretion of IL-1β were analyzed by Western Blot, and the secretion of IL-1β in the supernatant was quantitatively detected by ELISA. Figure 5 As shown, Ros B specifically inhibits NLRP3 inflammasome activation and does not affect poly(dA:dT)-activated AIM2 inflammasome.

[0070] 3. Clarify the specific mechanism by which Ros B affects NLRP3 inflammasome activation

[0071] In order to explore how tetracyclic diterpenoids from the genus Ampelopsis play a role in the activation of NLRP3 inflammasomes, we first determined whether tetracyclic diterpenoids from the genus Ampelopsis affect upstream signals of NLRP3 inflammasome activation, such as potassium ion efflux and mitochondrial damage. Secondly, we used immunoprecipitation to explore whether tetracyclic diterpenoids from the genus Ampelopsis affect the assembly of the NLRP3 inflammasome complex.

[0072] Experiment 1: Determine whether the tetracyclic diterpenoid compounds of the genus Ampelopsis affect potassium ion efflux and mitochondrial damage: Mouse bone marrow cells were isolated and induced to differentiate into bone marrow-derived macrophages (BMDM). BMDM cells were pretreated with LPS for 3h: ① Treat the cells with different doses (0.5μm, 1μm, 2μm) of tetracyclic diterpenoid compounds of the genus Ampelopsis for 30min, then stimulate with Nigericin for 30min, collect the cell lysate, and detect the potassium ion content in the cells by plasma atomic chromatography. ② Treat the cells with a high concentration of tetracyclic diterpenoid compounds of the genus Ampelopsis for 30min, then add Mitotracker staining, and use laser confocal microscopy to observe mitochondrial damage. This verifies whether the tetracyclic diterpenoid compounds of the genus Ampelopsis inhibit NLRP3 inflammasome activation by blocking the upstream signals of inflammasome activation. Figure 6 As shown, tetracyclic diterpenoid compounds from the genus Isodon do not affect mitochondrial damage and potassium ion efflux.

[0073] Experiment 2: To clarify whether the tetracyclic diterpenoid compounds of the genus Camellia affect the assembly of the NLRP3 inflammasome complex: After nigericin activated BMDM cells for 30 minutes, Western Blot was used to detect IL-1β and caspase-1 in the supernatant, and the lysate was used to detect the oligomerization of ASC, an important component of the NLRP3 inflammasome. NLRP3 binding to NEK7 and recruiting ASC are important events in the assembly of the NLRP3 inflammasome. After nigericin activated BMDM cells for 30 minutes, NP-40 solution was added to lyse the cells, and NEK7 or ASC antibodies were used for immunoprecipitation, and IgG antibodies were used as negative controls. It was determined whether the tetracyclic diterpenoid compounds of the genus Camellia directly inhibited the assembly process of the NLRP3 inflammasome. Finally, the specific mode of action of Ros B and NLRP3 protein was explored through SPR experiments and target stability experiments of drug affinity reactions. Figure 7 As shown, various tetracyclic diterpenoids from the genus Isophyllum, including RosB, inhibited the interaction between endogenous NEK7 and NLRP3, thereby suppressing the assembly of the NLRP3 inflammasome.

[0074] Example 3 Ros B inhibits LPS-induced septic shock

[0075] (1) Seven-week-old male C57BL / 6J mice were purchased and fed in an SPF mouse house for one week. Mice with similar body weights were selected and randomly divided into groups; the first group, the WT group (n=3), did not receive any treatment; the second group, the LPS group (n=6), was intraperitoneally injected with LPS (20 mg / kg); the third group, the Ros B treatment group (n=6), was intraperitoneally injected with Ros B (10 mg / kg) 50 minutes later and then injected with LPS (20 mg / kg); 4 hours after the intraperitoneal injection of LPS, the eyeballs were removed and blood was collected. The blood was left to stand for 6-8 hours, and the serum was separated. The inflammatory factors in the serum were detected by ELISA;

[0076] (2) Seven-week-old male C57BL / 6J mice were purchased and fed in the SPF mouse house of our laboratory for one week. Mice with similar body weights were selected and randomly divided into groups; the first group, the WT group (n=3), did not receive any treatment; the second group, the LPS group (n=6), was intraperitoneally injected with LPS (20 mg / kg); the third group, the Ros B treatment group (n=6), was intraperitoneally injected with Ros B (10 mg / kg) and then injected with LPS (20 mg / kg) 50 minutes later; Survival curve: within 48 hours after the mice were injected with LPS, the time of death was recorded, and the survival curve was subsequently drawn using statistical software.

[0077] Results: Intraperitoneal injection of LPS is the most common method to establish a mouse systemic inflammation model. Systemic inflammation can cause increased secretion of inflammatory cytokines such as IL-1β and TNF-α in mouse serum. We further verified the role of Ros B at the animal level in a mouse systemic inflammation model. The results showed that compared with the LPS group, the secretion of IL-1β in the serum of mice in the LPS+Ros B group was significantly reduced (P<0.01)( Figure 8 A), while there was no significant difference in TNF-α levels. In addition, the survival status of mice within 48 hours after LPS injection was observed. The survival curve results showed that mice in the LPS group began to die at 16 hours, and the mortality rate of mice reached 100% at 31 hours. However, mice in the LPS+Ros B group began to die at 26 hours, the mortality rate of mice at 31 hours was only 50%, and the mortality rate of mice at 44 hours reached 100% (P<0.05)( Figure 8 B). The above results suggest that Ros B can alleviate inflammation in mice with systemic inflammation and significantly prolong their survival time.

[0078] like Figure 8 As shown, intraperitoneal injection of LPS significantly reduced the secretion of IL-1β in mouse serum and significantly prolonged the survival curve.

[0079] Example 4 Ros B inhibits DSS-induced colitis

[0080] (1) 7-week-old male C57BL / 6J mice were purchased, acclimatized for one week, and randomly divided into groups;

[0081] (2) The first WT group (n = 3) was provided with normal drinking water every day. The second DSS modeling group (n = 8) was provided with 3% DSS solution every day for 6 days, and then changed to normal drinking water on the 7th day. The third Ros B treatment group (n = 8) was intraperitoneally injected with Ros B (10 mg / kg) for 10 consecutive days, and provided with 3% DSS solution for 6 days, and then changed to normal drinking water on the 7th day;

[0082] (3) Record the weight changes of mice and the disease activity of mice (indicators such as blood in stool, diarrhea, and anal prolapse) every day;

[0083] (4) On the 10th day, mesenteric lymph nodes were removed and analyzed for changes in cell subpopulations using flow cytometry. The length of the colon was compared among the groups. The colon was stained with HE.

[0084] like Fig. 9As shown in the results, drinking water containing DSS significantly caused the mice to lose weight, increase the disease activity index of enteritis, shorten the colon, increase the infiltration of colon lymphocytes, and cause severe colon damage. Injection of Ros B significantly inhibited the weight loss of mice, reduced the disease activity index, inhibited the shortening of the colon, inhibited the increase of colon immune cell infiltration, and improved colon damage. This shows that RosB can effectively inhibit DSS-induced colitis.

[0085] It can be seen from the above two groups of modeling that the tetracyclic diterpene compounds of the genus Ilex provided by the present invention, represented by Ros B, have a specific alleviating effect on NLRP3 inflammasomes and a series of inflammatory diseases induced by them.

Claims

1. Use of tetracyclic diterpenoid compounds of the genus Isodonis in the preparation of drugs for treating systemic inflammation, characterized in that: The tetracyclic diterpene compound of the genus Isodon is Ros B.

2. The use of tetracyclic diterpenoid compounds of the genus Isodonis in the preparation of a drug for treating colitis, characterized in that: The tetracyclic diterpene compound of the genus Isodon is Ros B.

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