Application of tripterygium wilfordii diol in preparation of medicine for preventing and/or treating NLRP3 inflammasome-mediated inflammatory diseases

文档序号：1944104 发布日期：2021-12-10 浏览：12次中文

阅读说明：本技术 雷公藤二醇在制备预防和/或治疗nlrp3炎症小体介导的炎症性疾病药物中的应用 (Application of tripterygium wilfordii diol in preparation of medicine for preventing and/or treating NLRP3 inflammasome-mediated inflammatory diseases ) 是由徐晶王颖宁澄清丁漠雨于 2021-09-30 设计创作，主要内容包括：本发明提供了雷公藤二醇在制备预防和/或治疗NLRP3炎症小体介导的炎症性疾病药物中的应用,雷公藤二醇的结构如下所示。雷公藤二醇能够有效抑制NLRP3炎症小体的组装,进而抑制炎症反应和细胞焦亡,此外,雷公藤二醇活性优、毒性低,为NLRP3炎症小体介导的炎症性疾病药物的开发提供了新的解决方案。(The invention provides application of tripterygium wilfordii diol in preparing a medicament for preventing and/or treating NLRP3 inflammasome-mediated inflammatory diseases, wherein the structure of the tripterygium wilfordii diol is shown as follows. The tripterygium wilfordii diol can effectively inhibit the assembly of NLRP3 inflammasome, further inhibit inflammatory reaction and cell apoptosis, and in addition, the tripterygium wilfordii diol has excellent activity and low toxicity, thereby providing a new solution for the development of medicaments for treating the NLRP3 inflammasome-mediated inflammatory diseases.)

1. The application of tripterygium wilfordii diol in preparing a medicament for preventing and/or treating inflammatory diseases mediated by NLRP3 inflammasome is as follows:

2. the use of triptodiol according to claim 1 for the preparation of a medicament for the prevention and/or treatment of an inflammatory disease mediated by an NLRP3 inflammasome, wherein the prevention and/or treatment is: targeted prevention and/or targeted therapy.

3. The use of triptodiol according to claim 1 for the preparation of a medicament for the prevention and/or treatment of an inflammatory disease mediated by NLRP3 inflammasome, wherein the inflammatory disease is inflammatory bowel disease.

4. The use of triptodiol according to claim 1 for the preparation of a medicament for the prevention and/or treatment of an inflammatory disease mediated by NLRP3 inflammasome, wherein the inflammatory disease is pneumonia.

5. The use of triptodiol according to claim 1 for the preparation of a medicament for the prevention and/or treatment of an inflammatory disease mediated by NLRP3 inflammasome, wherein the inflammatory disease is nonalcoholic steatohepatitis.

6. The use of triptodiol according to claim 1 for the preparation of a medicament for the prevention and/or treatment of NLRP3 inflammasome-mediated inflammatory diseases, wherein the inflammatory diseases are nonalcoholic liver disease, coronary heart disease, heart failure, rheumatoid arthritis, gout, type II diabetes, atherosclerosis, alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, cryopyrine-related periodic syndrome, gastric cancer, colorectal cancer, lung cancer or melanoma.

7. The use of the triptodiol of claim 1 for preparing a medicament for preventing and/or treating an NLRP3 inflammasome-mediated inflammatory disease, wherein the triptodiol is prepared by the steps of:

mixing triptolide with a catalyst, and carrying out a regioselective ring-opening reaction;

the triptolide has the following structure:

8. the use of triptodiol according to claim 7, wherein the catalyst is LiBH in the preparation of a medicament for preventing and/or treating inflammatory diseases mediated by NLRP3 inflammasome₄THF solution with BF₃·Et₂At least one of O solutions.

9. The application of the triptodiol as claimed in any one of claims 1 to 8 in the preparation of the medicine for preventing and/or treating the NLRP3 inflammasome-mediated inflammatory disease, wherein the dosage form of the NLRP3 inflammasome-mediated inflammatory disease medicine is tablets, capsules, granules or pills.

10. The application of the triptodiol of any one of claims 1 to 8 in preparing a medicament for preventing and/or treating the NLRP3 inflammasome-mediated inflammatory disease, wherein the medicament for preventing and/or treating the NLRP3 inflammasome-mediated inflammatory disease is in a dosage form of oral liquid or injection.

Technical Field

The invention relates to the field of medicines, in particular to application of tripterygium glycol in preparation of a medicine for preventing and/or treating an inflammatory disease mediated by NLRP3 inflammasome.

Background

Apoptosis is a programmed cell death mode discovered in recent years and can release cellular contents and proinflammatory mediators, resulting in the occurrence of inflammation. Inflammatory corpuscle formation is one of the factors inducing the occurrence of apoptosis, and the NLRP3(NOD-like receptor protein 3) inflammatory corpuscle is the most deeply studied inflammatory corpuscle at present. The NLRP3 inflammasome is a large protein complex formed intracytoplasmically by NEK7, NLRP3, ASC, procaspase-1 in multimeric form. The NLRP3 protein mainly contains an N-terminal PYD domain, a NACHT domain and a C-terminal LRR domain. Typically, cells stimulated by a cause-related molecular pattern and a risk-related molecular pattern activate NRLP3, which causes a conformational change in its activation exposing the NACHT domain, NLRP3 oligomerizes via homotypic interaction of the exposed NACHT domain with the NEK7 protein, followed by binding of the exposed PYD domain to the adapter protein ASC, which in turn recruits procase-1 in a CARD-CARD homotypic interaction to assemble into NLRP3 inflammasome. The activated small inflammatory bodies crack procaspase-1 to form caspase-1, then the GSDMD is cracked to trigger cell apoptosis, and meanwhile, the activated caspase-1 promotes the maturation and the secretion of proinflammatory cytokines such as IL-1 beta, IL-18 and the like, so that inflammatory response is triggered. Activation of NLRP3 inflammasome can trigger an inflammatory response to eliminate pathogens. Preclinical data show that NLRP3 inflammatory-corpuscle transitional activation is closely associated with the onset and progression of more than twenty diseases, such as acute respiratory distress syndrome, multiple sclerosis, autoimmune encephalomyelitis, peritonitis, Muckle-Wells syndrome, nonalcoholic and nonalcoholic steatohepatitis, pulmonary fibrosis, coronary heart disease, heart failure, enteritis, type II diabetes, rheumatoid arthritis, gout, atherosclerosis, coldness-imidacloprid-related periodic syndrome, neurodegenerative diseases. NLRP3 inflammasome-mediated apoptosis of cells also plays an important role in the development and progression of some tumors. In summary, NLRP3 plays an important role in various diseases such as inflammatory diseases, metabolic diseases, and neurodegenerative diseases.

The traditional NLRP3 inhibitor has low in vivo activity and is in the mg/kg grade, and for example, the MCC950 (also called CP-456,773) and CY-09 have an effective dose of 40mg/kg in a mouse Muckle-Wells syndrome model. In addition, MCC950 can block NLRP3 inflammatory body activation at nanomolar concentrations, and the rate of pfeiffer was first studied in phase II trials of MCC950 on rheumatoid arthritis, but the trials were discontinued due to the induction of elevated liver function-related enzyme activity, i.e., the presence of hepatotoxicity, by MCC 950.

Disclosure of Invention

Based on the above, the main purpose of the present invention is to provide a new application of tripterygium glycol, in particular to an application of tripterygium glycol in preparation of an NLRP3 inflammasome inhibitor, wherein the tripterygium glycol can effectively inhibit assembly of NLRP3 inflammasome, so as to inhibit inflammatory reaction and cell apoptosis, and in addition, the tripterygium glycol has excellent activity and low toxicity.

The invention is realized by the following technical scheme.

The application of tripterygium wilfordii diol in preparing a medicament for preventing and/or treating inflammatory diseases mediated by NLRP3 inflammasome is as follows:

in one embodiment, the prevention and/or treatment is: targeted prevention and/or targeted therapy.

In one embodiment, the inflammatory disease is inflammatory bowel disease.

In one embodiment, the inflammatory disease is pneumonia.

In one embodiment, the inflammatory disease is nonalcoholic steatohepatitis.

In one embodiment, the inflammatory disease is nonalcoholic liver disease, coronary heart disease, heart failure, rheumatoid arthritis, gout, type II diabetes, atherosclerosis, alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, cold imidacloprid-related periodic syndrome, gastric cancer, colorectal cancer, lung cancer, or melanoma.

In one embodiment, the preparation of tripterygium glycol comprises the following steps:

mixing triptolide with a catalyst, and carrying out a regioselective ring-opening reaction;

the triptolide has the following structure:

in one embodiment, the catalyst is selected from LiBH₄THF solution with BF₃·Et₂At least one of O solutions.

In one embodiment, the dosage form of the NLRP3 inflammasome-mediated inflammatory disease drug is tablets, capsules, granules or pills.

In one embodiment, the dosage form of the NLRP3 inflammatory body-mediated inflammatory disease drug is oral liquid or injection.

Compared with the prior art, the application of the tripterygium wilfordii glycol in preparing the medicament for preventing and/or treating the inflammatory disease mediated by the NLRP3 inflammasome has the following beneficial effects:

according to the invention, research shows that tripterygium wilfordii glycol has an obvious inhibition effect on assembly of NLRP3 inflammasome, and the inhibition effect is verified through biological experiments. In addition, the activity of triptodiol is superior to that of the traditional NLRP3 inhibitor MCC950, and the triptodiol has obvious effect of reducing the overall verification level of mice when the in vivo active dose is 200 micrograms/kg, and is obviously lower than the in vivo active dose of MCC950 mg/kg body weight. Meanwhile, no toxic or side effect is observed under the active dose of the tripterygium wilfordii diol, which indicates that the tripterygium wilfordii diol has a wider treatment window. Aiming at the discovery, the application field of tripterygium wilfordii glycol is developed, and a solution is provided for the development of a novel NLRP3 inflammation body mediated inflammatory disease drug.

Drawings

FIG. 1 shows the results of the action of the compounds provided by the present invention on different immune signaling pathway proteins and RNA polymerase II in THP-1 cells;

FIG. 2 is the result of the action of Tripterygium wilfordii glycol and MCC950 on NLRP3 inflammasome in primary macrophage derived from mouse bone marrow provided by the present invention;

FIG. 3 shows the specific binding result of tripterygium glycol provided by the present invention to Cys280 residue in NACHT domain in NLRP3 protein;

FIG. 4 shows the effect of Tripterygium wilfordii glycol on the combination of NLRP3 with NEK7 and ASC;

FIG. 5 is a graph showing the effect of Tripterygium wilfordii diol on the content of proinflammatory cytokines in cell culture fluid after stimulation of mouse bone marrow-derived primary macrophages with Bacillus subtilis flagellin and poly (deoxyadenylate-deoxythymidylate) sodium salt (poly dA: dT);

FIG. 6 shows the protective effect of tripterygium glycol provided by the present invention on acute lung injury induced by LPS;

FIG. 7 shows the lethal protection effect of tripterygium glycol on LPS-induced septic shock.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention provides an application of tripterygium wilfordii diol in preparing a medicament for preventing and/or treating an inflammatory disease mediated by NLRP3 inflammasome, wherein the structure of the tripterygium wilfordii diol is shown as follows:

in one particular example, the prevention and/or treatment is: targeted prevention and/or targeted therapy.

The traditional triptolide and other derivatives inhibit the expression of NLRP3 mRNA by using the action of RNA polymerase inhibitors, namely, the triptolide and other derivatives are not direct and targeted NLRP3 inhibitors. The tripterygium wilfordii diol can be directly combined with NLRP3 to influence the combination of NLRP3 and NEK7 and further influence the formation of NLRP3 inflammasome, and the tripterygium wilfordii diol has no inhibiting effect on RNA polymer.

More specifically, triptodiol inhibits the assembly of NLRP3 inflammasome by binding to the C280 residue of NLRP3 protein.

It is understood that, in the present invention, inflammatory diseases include, but are not limited to: inflammatory bowel disease, pneumonia, nonalcoholic liver disease, nonalcoholic steatohepatitis, coronary heart disease, heart failure, rheumatoid arthritis, gout, type II diabetes, atherosclerosis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, coldimidacloprid-associated periodic syndrome, gastric cancer, colorectal cancer, lung cancer, or melanoma.

In one specific example, the preparation of triptodiol comprises the following steps:

mixing triptolide with a catalyst, and carrying out a regioselective ring-opening reaction;

the triptolide has the following structure:

more specifically, the catalyst is selected from LiBH₄THF solution with BF₃·Et₂At least one of O solutions.

More specifically, the catalyst is LiBH₄THF solution with BF₃·Et₂And (4) O solution.

In a specific example, the dosage form of the NLRP3 inflammasome-mediated inflammatory disease drug is a tablet.

In a specific example, the dosage form of the NLRP3 inflammasome-mediated inflammatory disease drug is a capsule.

In a specific example, the dosage form of the NLRP3 inflammasome-mediated inflammatory disease drug is granules.

In a specific example, the NLRP3 inflammasome-mediated inflammatory disease drug is in the form of a pill.

In a specific example, the dosage form of the NLRP3 inflammasome-mediated inflammatory disease drug is oral liquid.

In a specific example, the formulation of the NLRP3 inflammasome-mediated inflammatory disease drug is an injection.

It is understood that triptodiol can be combined with suitable types of excipients to prepare drugs in different forms (e.g., liquid, semi-solid, solid) and then be formulated into different dosage forms, including but not limited to: tablet, capsule, granule, pill, oral liquid, and injection.

In the above, the embodiment of the present invention is only an example of the kind of "dosage form", and it should be understood that this is not a specific limitation to the "dosage form", and besides the dosage forms of "tablet", "capsule", "granule", "pill", "oral liquid" and "injection" exemplified in the embodiment of the present invention, other suitable pharmaceutically acceptable pharmaceutical dosage forms can be prepared.

The use of triptodiol of the invention in the preparation of NLRP3 inflammasome inhibitors is described in further detail below with reference to specific examples. The starting materials used in the following examples are all commercially available products unless otherwise specified.

Example 1

The embodiment provides a synthesis method of tripterygium wilfordii diol, which comprises the following specific steps:

dissolving triptolide (27mg, 0.07mmol) in anhydrous tetrahydrofuran (4mL), sequentially adding LiBH₄THF solution (2N, 84. mu.L, 0.17mmol) and BF₃·Et₂O solution (45. mu.L, 0.35mmol) was stirred at room temperature for 3h under argon. After the reaction is finished, adding dilute hydrochloric acid to quench the reaction, continuously stirring for 10min, adding dichloromethane to extract for 3 times, combining organic layers, washing with saturated sodium bicarbonate water solution and saturated salt water in sequence, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and performing column chromatography to obtain 7mg of white tripterygium glycol with the yield of 25.9%.

The product was characterized as: 1H NMR (500MHz, CDCl3, Δ): 4.73(m, 2H), 3.85(d, J ═ 1.5Hz, 1H), 3.83(d, J ═ 3.0Hz, 1H), 3.55(d, J ═ 12.0Hz, 1H), 3.44(dd, J ═ 3.0Hz, J ═ 1.0Hz, 1H), 2.53(m, 2H), 2.29(m, 4H), 1.97(m, 1H), 1.63(m, 2H), 1.28(m, 2H), 1.23(s, 3H), 1.03(d, J ═ 7.0Hz, 3H), 0.87(d, J ═ 7.0Hz, 3H), 13C NMR (125MHz, CDCl3, δ): 173.9, 162.1, 124.6, 74.3, 72.5, 70.4, 70.0, 65.7, 57.3, 54.2, 44.1, 37.9, 35.8, 29.5, 28.7, 19.6, 17.9, 17.6, 16.7, 14.1 HRMS (M/z) [ M + H ] + cacld for C20H27O6, 363.1808; found, 363.1797.

Example 2 biological experiments

Method and device

1. Animals and reagents

All animal experiments were ethically reviewed and approved by the university of australia. C57BL/6J mice were provided by the university of Australia pathogen free (SPF) animal center. 1 week prior to the experiment, mice should acclimatize in a 12 hour dark/light cycle, freely available food and water, with temperature controlled between 22.5 ± 0.5 ℃. Tumor necrosis factor (tumor necrosis factor- α, TNF- α) was purchased from PeproTech. Lipopolysaccharide (LPS) and the gram-positive bacterium bacillus subtilis flagellin (FLA-BS) were purchased from InvivoGen. MCC950 was purchased from Cayman Chemical. All other materials and reagents were purchased from Thermo Fisher Scientific and Sigma-Aldrich or otherwise indicated.

2. Cell culture

AD-293 cell lines were purchased from Strategene (La Jolla, CA, USA). AD-293 cells were maintained in DMED high glucose medium supplemented with 10% FBS. AD-293 stable cell lines containing NF-. kappa.B response elements were maintained in the presence of 1. mu.g/ml puromycin. AD-293 cells consistently over-expressed TLR4 plus the NF-. kappa.B response element luciferase reporter in the presence of 1. mu.g/ml puromycin and 40. mu.g/ml hygromycin B gold (InvivoGen).

The THP-1 monocytes were maintained in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS, 1% penicillin and streptomycin and 50. mu.M 2-mercaptoethanol and differentiated into macrophage-like cells 3 days earlier using 10nM phorbol 12-myristate 13-acetate (PMA).

AD-293 cells stably expressing His-procaspase-1 were maintained in high-glucose DMEM medium supplemented with 40. mu.g/ml hygromycin B. All cells were incubated with 5% CO at 37 deg.C₂And culturing in a supermarket environment.

3. Isolation of bone marrow-derived macrophages

Bone marrow-derived macrophages were isolated from 6-8 week-old C57BL/6J mice, differentiated into mature macrophages in DMEM medium plus 10% heat-inactivated FBS and 10% L-929 conditioned medium, 5% CO at 37 ℃₂Culturing in a humid environment.

4. Lactate Dehydrogenase (LDH) cytotoxicity assays

Lactate dehydrogenase released from the cells into the culture medium was measured by a lactate dehydrogenase kit of Shanghai Biyuntian Biotech Co., Ltd.

5. Molecular cloning and transfection

All plasmids were constructed using standard molecular biology methods including PCR, overlap extension PCR, restriction enzyme fragmentation and ligation. The open reading frame of the different genes was amplified and inserted into pcDNA5/TO vector (Thermo Fisher Scientific), NLRP 3C 280A and C280S mutant plasmids were generated by using QuikChange site-directed mutagenesis kit (Stratagene). All plasmids constructed were verified by DNA sequencing.

6. Western blot analysis

Cells were lysed using RIPA lysate, protein-containing lysates were separated by SDS-PAGE after high speed centrifugation, and transferred to PVDF membranes for western blot analysis. The specific information of the antibodies used is as follows: pol II (# sc-56767, Santa Cruz Biotechnology), NLRP3(# ALX-804-819-C100, ENZO Life sciences; # AG-20B-0014, AdipoGen), MYD88(#4283, Cell Signaling Technology), phosphorylated p65(Ser536, #3033, Cell Signaling Technology), p65(#6959, Cell Signaling Technology), RNA polymerase II (Pol II, # sc-56767, Santa Cruz Biotechnology), TH1L (#3088, Cell Signaling Technology), ASC (# 180799, Abcam; # AG-25B-0006, AdipoGen), NEK7(# 133ab 5B 5-13384

14, Abcam), MYC tag (#2276, Cell Signaling Technology), HA tag (# ab9110, Abcam), His tag (# ab9108, Abcam), ASC (# AG-25B-0006, AdipoGen), β -actin (# a5316, Sigma-Aldrich). All antibodies were used for Western blot analysis at a dilution of 1: 1000.

7. Microbead-based serum cytokine level determination

Levels of proinflammatory cytokines and chemokines were determined based on an immune response to legendexplex (tm) microbeads. The specific assay was performed according to the BioLegend instructions.

8. Real-time quantitative PCR (qRT-PCR)

Tissue total RNA was extracted using Trizol reagent (Invitrogen), and the extracted total RNA was reverse-transcribed into cDNA using a cDNA synthesis kit (Bio-Rad). The cDNA diluted 50 times was used as a template for qRT-PCR. According to the kit operation instructions, SYBR Green fluorescent probe is adopted to detect the expression level of mRNA. The primers required for qRT-PCR are shown in Table 1.

TABLE 1

9. Immunofluorescence

After the mouse bone marrow-derived macrophages were treated, they were fixed with 4% paraformaldehyde at room temperature for 30 minutes and then permeabilized with 0.5% TritonX-100 (PBS) for 15 minutes. The cells were washed 3 times with PBS-0.5% Tween 20 for 10 minutes each, and then blocked in PBS-0.5% Tween 20 with 2% bovine serum albumin for 1 hour at room temperature, followed by incubation with 1: 200 diluted antibody at 4 ℃ overnight. Washed 3 times with PBS-0.5% Tween 20 for 10 minutes each, added 1: 500 diluted fluorescent secondary antibody and incubated for 1 hour at room temperature. Washed 3 times with PBS-0.5% Tween 20 for 10 minutes each time, added with Hoechst dye, and then mounted with a discoloration preventing agent, and photographed under a fluorescence confocal microscope.

10. Co-immunoprecipitation

After the mouse bone marrow-derived macrophages were treated, they were treated in NT-2 buffer (50mM Tris-HCl, pH7.4, 150mM NaCl, 1mM MgCl)₂0.05% Nonidet P-40 and a cOmpletetM protease inhibitor cocktail without EDTA (Roche Scientific)) were suspended, freeze-thawed three times repeatedly, and the supernatant was centrifuged at 15000rpm at 4 ℃ at high speed. Adding NEK7 antibody or NLRP3 antibody into cell protein lysate, performing immunoprecipitation by adopting protein G magnetic beads, and analyzing the protein expression level in the co-immunoprecipitation compound by using Western blot.

11. Simulated binding studies

The NACHT domain of NLRP3 is constructed based on NOD2 protein (PDB: 5IRN) template by homologous modeling. Prediction of binding of NLRP3 to compound 3e was obtained by computational analysis of MOE software.

12. Purification of wild-type and C280A mutant NLRP3 NATCH structural domain recombinant protein

The coding regions of the wild-type and C280A mutant NACHT domains used to construct the NLRP3 recombinant protein were ligated to pET28a plasmid by PCR, transfected into E.coli BL21(DE3) strain (Promega), induced with 1mM isopropyl beta-D-thiogalactoside (IPTG), and cultured at 16 ℃ for 16 hours. The bacteria were sonicated in lysis buffer (50mM Tris HCl, pH 8.0, 300mM NaCl, 20mM imidazole, 1% Triton X-100, 1mM DTT and 1mM PMSF). The recombinant protein was recovered from insoluble inclusion bodies using a buffer containing 50mM Tris HCl, pH 8.0, 300mM NaCl, 8M urea and 20mM imidazole and purified using Ni-IDA resin. The purity of the recombinant protein was determined using SDS-PAGE and Coomassie blue staining.

13. Isothermal titration quantitative calorimetry

Isothermal titration quantitative thermal measurements were performed on a MicroCal-ITC microcalorimeter (Malverm Panalytical). The wild-type and C280A mutant NACHT NLRP3 recombinant proteins were dialyzed against PBS containing 10% glycerol. Compound 3e was dissolved in DMSO. All measurements were performed at 25 ℃ in dialysis buffer containing 5% DMSO. Isothermal titration calorimetry used a final concentration of 7. mu.M protein and 500. mu.M 3e in the titration cell. Thermograms were analyzed using GraphPad Prism 6.0 and ORIGIN 2019 software.

14. Determination of stability of drug affinity reaction target

In the assay of stability of target of drug affinity reaction, differentiated mouse bone marrow-derived macrophages were seeded at a density of 5X 105 cells/ml in a 10cm plate. After stimulation of cells with 100ng/ml lipopolysaccharide for 4h, further incubation with different concentrations of compound 3e or MCC950 for 2 h. Cells were lysed in lysis buffer containing 50mM Tris-HCl pH7.4, 150mM NaCl, 2mM EDTA, 1mM ATP and 0.5% Igepal CA-630 in the presence of a complete protease inhibitor cocktail. After centrifugation the clarified lysate was digested with pronase and analyzed by immunoblotting.

15. Lipopolysaccharide nose-dropping induced acute lung injury model of mouse

Mixed sex 6-8 week old C57BL/6J mice the 3e treatment group (100, 200, and 500. mu.g/kg body weight, dissolved in clinically used adjuvantsPEG-15-hydroxystearate, administered twice daily by gavage at 12 hour intervals) for 5 days of pre-protection, after which the same volume of sterile saline will be administered to the sham group by intranasal instillation of lipopolysaccharide (LPS, 10mg/ml LPS solution in sterile saline, 2mg/kg body weight). As a treatment control group, sham groups were given the same volume of Solutol. Dosing was continued for 7 days with daily body weight recordings (figure 3). The administration of LPS was continued for three days after nasal drip. At the end of the experiment, all mice were anesthetized with CO2 and sacrificed. Lung tissue and serum will be collected for further analysis.

16. Lipopolysaccharide intraperitoneal injection mouse septicemia shock

6-8 week old C57BL/6J mice were injected intraperitoneally with lipopolysaccharide (35mg/kg body weight) to induce septic shock. Compound 3e was administered by intraperitoneal injection (200mg/Kg body weight) and the control drug was Dexamethasone (Dexamethasone, 5mg/Kg body weight). The administration was performed twice daily with 12 hour intervals. After the experiment, the death of the mice was closely observed, and a survival curve was drawn. Lipopolysaccharide injection was terminated at 72 hours, and the mice were anesthetized with CO2 and sacrificed.

17. Hematoxylin-eosin staining of lung tissue

Hematoxylin-eosin staining solution was purchased from Nanjing to build the bioengineering institute and stained according to the manufacturer's method.

18. Immunohistochemical and immunofluorescent staining

Mouse lung tissue is fixed, dehydrated, embedded and sliced, then heated and antigen-repaired by 0.05M citric acid buffer (pH 6.0), 1% Triton X-100 is penetrated and sliced for 30min, 10% horse serum and 10% bovine serum albumin are incubated for 1h to block nonspecific sites, and the ratio of the specific sites is determined according to the following steps of 1: the antibodies were diluted at 200 ℃ and incubated overnight at 4 ℃. Then reacting with horseradish peroxidase or a secondary antibody marked by a fluorescent probe, and observing the fluorescence stained tissue section by using DAB peroxidase substrate kit color development or Leica TCS SP8 laser confocal.

19. Statistical analysis

Data from any animal will not be excluded from analysis. All data obtained will be expressed as mean ± SEM. All experiments will be repeated at least two or more times. Differences in measured variables between groups will be analyzed by GraphPad Prism 5.0 software using one-way or two-way ANOVA methods. When p is less than 0.05, the results are considered to be significantly different, and have statistical significance.

Second, result in

In the figure: DMSO is dimethyl sulfoxide; 3a, 3c and 3g are triptolide derivatives, and the specific structural formula is shown as follows; 3e is tripterygium glycol; 1 is triptolide.

1. FIG. 1 shows the effect of Tripterygium wilfordii diol on different immune signaling pathway proteins and RNA polymerase II in THP-1 cells.

FIG. 1(A) shows PMA differentiated THP-1 cells were treated with compounds DMSO, 3a, 3c, 3e, 3g and 1 alone or together with Lipopolysaccharide (LPS) and analyzed for intracellular expression levels of NLRP3, ASC, MYD88, phosphorylated p65(p-p65), p65, beta-actin by immunoblotting.

FIG. 1(B) shows PMA differentiated THP-1 cells treated with compounds DMSO, 3a, 3c, 3e, 3g and 1 alone or in combination with LPS and analyzed by immunoblotting for intracellular Pol II, TH1L and β -actin expression levels.

FIG. 1(C) shows the IL-1. beta. content in the supernatant of cell culture medium of PMA-differentiated THP-1 cells treated with Tripterygium wilfordii glycol alone or together with LPS.

FIG. 1(D) shows the expression level of IL-1. beta. mRNA relative to 18S rRNA in PMA-differentiated THP-1 cells treated with Tripterygium wilfordii glycol alone or in combination with LPS.

FIG. 1(E) shows the expression level of NLRP3 mRNA relative to 18S rRNA in PMA differentiated THP-1 cells treated with Tripterygium wilfordii diol alone or in combination with LPS.

The results show that triptodiol can effectively regulate the daily immune response activated by Toll-like receptor (TLR)4 ligand LPS by inhibiting NLRP3 inflammasome, reduce the LPS-induced NLRP3 and phosphorylated p65 (figure 1A), and has no influence on molecular targets of triptolide, namely RNA polymerase II (pol II) and translation elongation factor TH1L (figure 1B). Tripterygium wilfordii diol significantly induced IL-1 β expression (FIG. 1C) and mRNA levels (FIG. 1D) by LPS, but had no effect on NLRP3 mRNA levels (FIG. 1E).

2. FIG. 2 shows the effect of Tripterygium wilfordii diol on NLRP3 inflammasome in mouse bone marrow derived primary macrophages.

FIG. 2(A) is a graph of the effect of Tripterygium wilfordii diol and MCC950 on LPS and Adenosine Triphosphate (ATP) stimulation of the release of primary macrophage lactate dehydrogenase from mouse bone marrow, respectively.

FIG. 2(B) is a graph of the effect of Tripterygium wilfordii diol and MCC950 on LPS and nigericin (Nig), respectively, to stimulate the release of mouse bone marrow-derived primary macrophage lactate dehydrogenase.

FIG. 2(C) is a graph showing the effect of Tripterygium wilfordii diol and MCC950 on the content of I1-1 β released into cell culture medium after stimulation of primary mouse bone marrow-derived macrophages with LPS and Adenosine Triphosphate (ATP), respectively.

FIG. 2(D) is a graph showing the effect of Tripterygium wilfordii diol and MCC950 on the levels of I1-1 β released into cell culture medium after LPS and nigericin (Nig) stimulate primary mouse bone marrow-derived macrophages, respectively.

FIG. 2(E) is a graph of the effect of Tripterygium wilfordii glycol and MCC950 on the expression levels of pro-caspase1, NLRP3, ASC, NEK7, and β -actin in LPS-and ATP-treated mouse bone marrow-derived primary macrophages.

FIG. 2(F) is a graph of the effect of Tripterygium wilfordii glycol and MCC950 on the expression levels of pro-caspase1, NLRP3, NEK7, ASC, phosphorylated p65(p-p65), p65, and β -actin in LPS and Nig treated mouse bone marrow-derived primary macrophages.

FIG. 2(G) shows co-localization of LPS and APT to mouse bone marrow-derived primary macrophage-activated NLRP3 and ASC by immunofluorescence staining analysis under the action of Tripterygium wilfordii diol and MCC 950.

Tripterygium wilfordii diol alone or in combination with Lipopolysaccharide (LPS) and Adenosine Triphosphate (ATP), or nigericin (Nig), released NLRP3 lactate dehydrogenase (FIGS. 2A-B), Il-1 β released into cell culture (FIGS. 2C-D), pro-caspase1 lytic activation (FIGS. 2E-F), and NLRP3 inflammasome formation (FIG. 2F) were all significantly inhibited in mouse bone marrow-derived primary macrophages.

3. FIG. 3 shows the specific binding of Tripterygium wilfordii glycol to Cys280 residue in the NACHT domain of NLRP3 protein.

FIG. 3(A) is a simulated binding graph of NLRP3 and Tripterygium wilfordii glycol.

FIG. 3(B) shows that in AD-293 cells, wild type, C280S and C280A mutant NLRP3 and ASC and pro-caspase1 are transfected respectively in a homeotropic manner, after 48 hours of transfection, DMSO or triptodiol is added for treatment for 2 hours, and the expression levels of MYC-NLRP3, HA-ASC, His-pro-caspase1 and beta-actin in cells are analyzed by an immunoblotting method.

FIG. 3(C) is a graph of the relative expression level of procaspase 1 in FIG. 3 (B).

FIG. 3(D) is an isothermal titration calorimetry analysis of the direct binding of Tripterygium wilfordii glycol to wild-type NLRP3 NACHT domain protein.

FIG. 3(E) is an isothermal titration calorimetry analysis of the direct binding of Tripterygium wilfordii glycol to the C280A mutant NLRP3 NACHT domain protein.

FIG. 3(F) is the binding constants of Tripterygium wilfordii glycol to wild-type NLRP3 NACHT domain protein and Tripterygium wilfordii glycol to C280A mutant NLRP3 NACHT domain protein.

FIG. 3(G) shows the detection of the binding of Tripterygium wilfordii glycol and MCC950 to NLRP3, NEK7 and beta-actin by the drug affinity reaction target protein stability experiment.

Tripterygium wilfordii glycol was found to bind directly to amino acid residue Cys280 of NLRP3 NACHT domain by molecular docking simulation (FIG. 3A). Wild type, C280S and C280A mutant NLRP3 are transfected and expressed in AD-293 cells along with co-transformation of ASC and pro-caspase1, and immunoblotting shows that triptodiol (3e) can only effectively inhibit the cleavage of pro-caspase1 generated under the expression of wild type NLRP3 (FIG. 3B-C). C280S and C280A mutant NLRP3 did not promote pro-caspase1 cleavage by itself, but instead increased pro-caspase1 cleavage upon addition of Tripterygium wilfordii diol (3e) (FIGS. 3B-C). Isothermal titration calorimetry analysis revealed that Tripterygium wilfordii diol (3e) bound to wild-type NLRP3 (FIGS. 3D-F) much more than C280A mutant NLRP3 (FIGS. 3D-F). Drug affinity reaction target protein stability experiments detect that tripterygium glycol (3e) and MCC950 are combined with NLRP3, NEK7 and beta-actin, and experiments show that the tripterygium glycol (3e) and the MCC950 can effectively protect the degradation of Pronase (Pronase) on NLRP3 but cannot protect the degradation of Pronase (Pronase) on NEK7 and beta-actin (figure 3G).

4. FIG. 4 is a graph showing the effect of Tripterygium wilfordii glycol on the binding of NLRP3 to NEK7 and ASC.

Primary macrophages derived from mouse bone marrow are pretreated by tripterygium wilfordii diol for 1 hour, and then are stimulated by LPS and nigericin (Nig) for 2 hours respectively, and then cells are collected.

FIG. 4(A) shows the analysis of binding of NLRP3 and NEK7 in cells by co-immunoprecipitation using NEK7 antibody.

FIG. 4(B) shows analysis of binding of NLRP3 and ASC in cells by co-immunoprecipitation using NLRP3 antibody.

Tripterygium wilfordii diol (3e) was found to effectively reduce binding of NEK7 to NLRP3 (FIG. 4A) and NLRP3 to NEK7 or ASC (FIG. 4B) in LPS and nigericin (Nig) stimulating mouse bone marrow-derived primary macrophages using antibodies NEK7 and NLRP3, respectively, for co-immunoprecipitation.

5. FIG. 5 shows the effect of Tripterygium wilfordii diol on the level of proinflammatory cytokines in cell culture broth after stimulation of mouse bone marrow-derived primary macrophages by the gram-positive bacteria Bacillus subtilis flagellin (FLA-BS) and poly (deoxyadenosine-deoxythymidylate) sodium salt (poly dA: dT).

The content of (A) Il-1 beta, (B) Tnf-alpha and (C) Il-6 in cell culture fluid under the stimulation of FLA-BS by triptodiol. The content of (D) Il-1 beta, (E) Tnf-alpha, and (F) Il-6 in the cell culture fluid under the stimulation of poly dAdT by triptodiol.

Because the structures and sequences of different nodding head-like receptor proteins are highly similar, in order to verify whether triptediol specifically inhibits NLRP3, gram-positive bacteria Bacillus subtilis flagellin (FLA-BS) and poly (deoxyadenosine-deoxythymidylate) sodium salt (poly dA: dT) are adopted to respectively activate primary macrophages derived from mouse bone marrow to activate the two nodding head-like receptors with similar structures of NLRP3, namely NLRC4 and AIM 2. Tripterygium wilfordii diol had no effect on levels of pro-inflammatory cytokines including Il- β, Tnf- α, Il-6 downstream from activation of NLRC4 and AIM2 (FIG. 5).

6. FIG. 6 shows the protective effect of Tripterygium wilfordii diol on LPS-induced acute lung injury.

Tripterygium wilfordii diol was administered together with LPS five days after pre-protection, i.e. intraperitoneal injection (twice daily), and LPS was administered for four days (0.4 mg/kg, twice daily) by nasal drip.

FIG. 6(A) shows the results of hematoxylin and eosin staining and F4/80 immunohistochemical staining of mouse lung tissues.

FIG. 6(B) shows the results of immunohistochemical staining of mouse lung tissue Nlrp3 and Asc.

FIG. 6(C) shows the mouse lung tissue Nlrp3 and Asc immunofluorescence co-localization, and Hoechest staining as nuclei.

FIG. 6(D) shows Il-1. beta. content in mouse serum.

FIG. 6(E) is the Il-1. beta. relative to 18S rRNA content in mouse lung tissue. (. p, p < 0.05;. p < 0.01).

Acute lung injury was induced in mice by nasal drops of LPS after prior oral administration of tripterygium diol, which was found to reduce LPS-induced lung injury and macrophage infiltration dose-dependently (fig. 6A). NLRP3 and ASC expression (fig. 6B) and binding (fig. 6C) in lung tissue were also significantly dose-dependent under triptodiol action. The Il- β content in mouse serum and lung tissue was also restored to the sham level by the action of triptodiol (fig. 6D-E).

7. FIG. 7 shows the lethal protection effect of triptodiol on LPS-induced septic shock.

Triptodiol was pre-protected for 6 hours, administered intraperitoneally (200 μ g/kg twice daily), dexamethasone (DEXA, 1 mg/kg) was administered for the same time period as triptodiol, and mice were initially scored for time to death after LPS intraperitoneal (35mg/kg, one) LPS injection. The mortality and LPS-induced septic shock to death p-values of mice in the triptodiol and dexamethasone dosing groups are plotted.

A mouse septic shock model is constructed by adopting a large dose of LPS, and triptodiol and Dexamethasone (DEXA) can remarkably protect lipopolysaccharide-induced mouse septic shock from death under the condition of twice daily administration (figure 7).

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments, are within the scope of the appended claims. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.

Sequence listing

<110> Shenzhen Ex Ke Chen Biotech Co., Ltd

University OF MACAU

Application of tripterygium wilfordii glycol in preparation of medicine for preventing and/or treating NLRP3 inflammasome-mediated inflammatory diseases

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA