CN116808016A

CN116808016A - Use of eplerenone for inhibiting activation of inflammatory bodies

Info

Publication number: CN116808016A
Application number: CN202310817372.2A
Authority: CN
Inventors: 陈赟; 林微微; 宁旦旦; 史月华
Original assignee: Zhejiang Chinese Medicine University ZCMU
Current assignee: Zhejiang Chinese Medicine University ZCMU
Priority date: 2022-01-13
Filing date: 2022-01-13
Publication date: 2023-09-29
Also published as: CN114931573A

Abstract

The application discloses an application of epstein in inhibiting activation of inflammatory bodies. The application discovers that the epstein can effectively inhibit AIM2 inflammatory corpuscle activation, caspase-1 protein activation and inflammatory cytokine IL-1 beta secretion. The application also discovers that the epstein can also protect mitochondria of macrophages, reduce the reduction of mitochondrial membrane potential, maintain mitochondrial morphology, inhibit VDAC1 protein expression, and particularly inhibit lysosomal rupture caused by aluminum adjuvants. Thus, can be used as a potential drug for preventing and/or treating AIM2 inflammatory corpuscles.

Description

Use of eplerenone for inhibiting activation of inflammatory bodies

The application is a divisional application of application number 202210035588.9 and application date 2022.01.13, and the application name is 'the application of epstein in inhibiting the activation of NLRP3 inflammatory bodies'.

Technical Field

The application relates to the field of biology and medicine, in particular to an application of eplerenone (Ipriflavone) in inhibiting AIM2 inflammation small body activation at a non-treatment destination, an application of eplerenone in inhibiting Caspase-1 protein activation and IL-1 beta secretion at a non-treatment destination, an application of eplerenone in preparing a medicament for treating AIM2 inflammation small body activation related diseases and/or improving related inflammation stages, an application of eplerenone in protecting mitochondria of macrophages at a non-treatment destination, reducing reduction of mitochondrial membrane potential and maintaining mitochondrial morphology, and an application of eplerenone in inhibiting lysosome rupture caused by aluminum adjuvants.

Background

Innate immunity is the first barrier of the body to infection by pathogenic microorganisms and is also one of the important factors in maintaining the immune balance of the body. Deregulation of innate immunity can not only cause infectious diseases, but can lead to susceptibility to autoimmune diseases and a variety of allergic reactions. Macrophages, which act as effector cells of the innate immune system, play an important role in the removal of aging and tissue remodeling following damage to infected cells, the body. Inflammatory corpuscles, which are important components of natural immunity, play a vital role in the recognition of pathogen-associated molecular patterns and danger-associated molecular patterns. Inflammatory cells are activated primarily in myeloid cells (e.g., macrophages) and induce the production of dependent cytokines to protect the host from microbial infection. The inflammation corpuscle is a protein complex composed of receptor protein (mainly NOD-like receptor), linker protein ASC and Caspase-1, when the organism senses infection of external pathogenic microorganism or self 'danger signal', NOD-like receptor or AIM2 (absent in melanoma 2) molecule can be activated, and assembled into a huge protein complex, namely inflammation corpuscle, so as to recruit and activate pro-Caspase-1, cut precursor forms of inflammatory factors such as IL-1 beta, IL-18 and the like to enable the precursor forms to be matured and secreted outside cells, and cause inflammation, and meanwhile, the activation of the inflammation corpuscle can cause a special apoptosis: cell apoptosis (pyrosis) induces cell death under inflammatory and stress-bearing pathological conditions. Many inflammatory corpuscles currently studied include NLRP1b, NLRP3, NLCR4, AIM2, and the like. Each inflammatory body is defined by a unique pattern recognition receptor.

Melanoma lacks factor 2 (AIM 2) as an intracellular DNA receptor, one of the important pattern recognition receptors that make up the inflammatory body. AIM2 protein consists of N-terminal thermal protein domain (PYD) and hemopoietic interferon inducible nuclear protein (HIN 200) domain with 200 amino acid repetitive sequence at C terminal, and is mainly localized in cytoplasm, belonging to PYHIN protein family. Its C-terminal HIN domain has double-stranded deoxyribonucleotide (dsDNA) recognition capability and comprises 2 oligonucleotide/Oligosaccharide Binding (OB) folded subdomains with high affinity for DNA, 70-150 amino acids in length, for binding to a single strand of DNA. The N-terminal PYD domain is a folding structure with 6 alpha helices, belongs to the death domain superfamily, and binds to other proteins through highly specific PYD-PYD interactions. AIM2 inflammatory minibodies consist of AIM2, ASC and pro-caspase-1. The classical activation pathway of AIM2 inflammatory bodies differs from NLRP3 inflammatory bodies in that the stimulus that it directly recognizes and specifically binds to is double stranded DNA within the cytoplasm. Although activation of AIM2 inflammation bodies can play an important role in protecting organisms when the organisms are infected by DNA viruses or bacteria, the overactivated inflammation bodies can cause damage to normal cells, so that finding specific regulatory molecular targets is becoming a new weapon for overcoming related diseases and improving prognosis.

Activation of NLRP3 inflammatory bodies requires two steps: the first step requires transcription and translation of the pro-IL-1β and NLRP3 by NF-. Kappa.B by the initiation signal (primer signal), and the second step activation signal (second driving signal) includes extracellular ATP, nigericin and crystalline material. NLRP3 inflammatory body assembly causes pro-caspase-1 self-cleavage activation, and its main function is to induce processing and cleavage of non-bioactive IL-1β and IL-18 precursors into bioactive IL-1β and IL-18, and promote its secretion, thereby triggering inflammatory response. At present, studies have shown that abnormal activation of NLRP3 inflammatory bodies is closely related to the occurrence of a variety of complex diseases, such as sepsis, type two diabetes, atherosclerosis, obesity, gout, alzheimer's disease, and the like. In addition, activation of NLRP3 inflammatory bodies also occurs at certain physiological stages, such as damage to surrounding tissue cells during implantation of biological materials, thereby releasing danger signals to activate the NLRP3 inflammatory bodies, and released pro-inflammatory mediators will lead to recruitment, differentiation and maturation of osteoclast precursors, which can disrupt osseointegration when bone resorption exceeds osteogenesis, resulting in failure of biological material implantation. Therefore, the research on the metabolic mechanism of NLRP3 inflammation corpuscles in the disease and physiological process searches for specific regulation molecular targets, and is becoming a new weapon for overcoming related diseases and improving prognosis.

Epstein, which belongs to isoflavone derivatives, is a kind of phytoestrogen synthesized from natural isoflavone, and can increase the effect of estrogen after entering human body to treat osteoporosis. At present, the specific action mechanism of the eplerenone in the aspect of AIM2 inflammatory body regulation, the immune metabolism target point and the regulation effect of the eplerenone in the early bone healing process of biomaterial implantation have not been reported. Therefore, the research of the effect of the eplerenone on AIM2 inflammatory minibodies and the effect of the eplerenone on AIM2 inflammatory minibodies related diseases and inflammatory physiological phases is of great significance.

Disclosure of Invention

To address the problems and needs in the background art, in a first aspect, the present application provides the use of epstein in inhibiting activation of AIM2 inflammatory bodies at non-therapeutic destinations. The application discovers that the epstein can effectively inhibit AIM2 inflammation small body activation specifically induced by Poly (dA: dT). And pharmaceutical applications under the above applications.

In a second aspect, the application also provides the use of epstein for inhibiting the activation of Caspase-1 proteins in non-therapeutic destinations, in particular for inhibiting the activation of Caspase-1 proteins, inhibiting the activation of GSDMD and maturation and secretion of IL-1 beta in vivo and/or in vitro. The application discovers that the epstein can inhibit the activation of Caspase-1 protein, reduce the recognition and cleavage of GSDMD and the maturation and secretion of inflammatory cytokines IL-1 beta. And pharmaceutical applications under the above applications.

In a third aspect, the present application also provides the use of epstein in the manufacture of a medicament for the prevention and/or treatment of diseases associated with the activation of the AIM2 inflammatory body and/or for the amelioration of the associated inflammatory stages.

The AIM2 inflammation-related disorder is at least one of multiple sclerosis, colon cancer, liver cancer, prostate cancer, cervical cancer, atopic dermatitis, psoriasis and systemic lupus erythematosus.

The AIM2 inflammatory body activation-related inflammatory phase comprises an immune response caused by injury after implantation of a biomaterial.

In a fourth aspect, the application also provides a product comprising epstein.

The product is a medicament or an agent, and the medicament also comprises a pharmaceutically acceptable auxiliary material or carrier.

In a fifth aspect, the application also provides the use of said product for inhibiting the activation of AIM2 inflammatory bodies for non-therapeutic purposes.

In a sixth aspect, the application also provides the use of the epstein in protecting the mitochondria of macrophages, reducing the decrease in mitochondrial membrane potential, maintaining mitochondrial morphology, inhibiting expression of VDAC1 protein in an in vitro non-therapeutic destination.

In a seventh aspect, the application also provides the use of said epstein in inhibiting lysosomal disruption caused by an aluminium adjuvant at a non-therapeutic destination in vitro.

In an eighth aspect, the application provides that the epstein inhibits lysosomal rupture for non-therapeutic purposes in vitro, thereby inhibiting activation of NLRP3 inflammatory bodies.

The beneficial effects of the application are as follows:

according to the application, the eplerenone can inhibit AIM2 inflammation corpuscle activation specifically induced by Poly (dA: dT), and simultaneously can inhibit activation of NLRP3 inflammation corpuscles by agonists with different properties such as nigericin (niger), ATP, aluminum adjuvant (Alum) and the like, but does not influence the activation of NLRC4 inflammation corpuscles; also, the assembly of AIM2 inflammatory bodies can be inhibited, including inhibition of activation of Caspase-1 protein downstream of inflammatory body activation and secretion of IL-1β. It also protects the mitochondria of macrophages, reduces the decrease in mitochondrial membrane potential, maintains mitochondrial morphology, and inhibits lysosomal rupture by aluminum adjuvants (Alum). Epstein can be used as a potential drug for treating AIM2 inflammatory corpuscles.

Drawings

FIG. 1 shows the inhibition of IL-1β secretion by different concentrations of epstein of LPS+ATP-induced activation of PMs NLRP3 inflammatory bodies in example 1 according to the present application. * P.ltoreq.0.05, p.ltoreq.0.01, p.ltoreq.0.001;

FIG. 2 shows the effect of different concentrations of epstein on TNFα secretion in LPS+ATP-induced activation of PMs NLRP3 inflammatory bodies in example 1 according to the present application;

FIG. 3 shows that epstein at various concentrations inhibits PMs Caspase-1 activation and secretion of inflammatory cytokine IL-1. Beta. In example 1 of the present application;

FIG. 4 shows the inhibition of PMs IL-1. Beta. Secretion by epstein stimulated by various NLRP3 inflammatory small agonists in example 2 of the present application. * P is less than or equal to 0.05;

FIG. 5 is a graph showing the effect of epstein on PMs TNFα secretion upon stimulation of various NLRP3 inflammatory small agonists in example 2 of the present application;

FIG. 6 is a graph showing that epstein inhibits PMs Caspase-1 activation and secretion of inflammatory cytokine IL-1. Beta. Under stimulation of various NLRP3 inflammatory small body agonists in example 2 of the present application;

FIG. 7 shows the inhibition of IL-1β secretion by epstein treatment for various times under LPS+ATP-induced PMs NLRP3 inflammatory body activation conditions in example 3 according to the present application. * P is less than or equal to 0.05, P is less than or equal to 0.01;

FIG. 8 shows the effect of epstein treatment on TNFα secretion at various times under LPS+ATP-induced PMs NLRP3 inflammatory body activation conditions in example 3 according to the present application;

FIG. 9 shows inhibition of Caspase-1 activation and inflammatory cytokine IL-1 beta secretion by epstein treatment for various times under LPS+ATP-induced PMs NLRP3 inflammatory body activation conditions in example 3 according to the present application;

FIG. 10 shows the inhibition of IL-1β secretion by epstein in example 4 according to the present application in various inflammatory body activations. * P is less than or equal to 0.05;

FIG. 11 is a graph showing the effect of epstein on Caspase-1 activation and secretion of inflammatory cytokine IL-1. Beta. In example 4 of the present application in various inflammatory body activations;

FIG. 12 shows the inhibition of BMDMs Caspase-1 activation, GSDMD activation and IL-1β secretion by epstein in example 5 of the present application;

FIG. 13 shows the inhibition of IL-1β secretion by epstein under LPS+ATP-induced inflammatory body activation conditions of BMDMs NLRP3 in example 5 according to the present application. * P is less than or equal to 0.05;

FIG. 14 shows the inhibition of THP-1Caspase-1 activation, GSDMD activation and IL-1β secretion by epstein in example 5 of the present application;

FIG. 15 shows the inhibition of IL-1β secretion by epstein under LPS+ATP-induced THP-1NLRP3 inflammatory body activation conditions in example 5 according to the present application. * P is less than or equal to 0.05;

FIG. 16 shows the inhibition of oligomerization of ASC protein during NLRP3 inflammatory body activation by epstein in example 6 according to the present application;

FIG. 17 shows the inhibition of NLRP3 protein oligomerization during NLRP3 inflammatory body activation by epstein in example 6 of the present application;

FIG. 18 is a photograph showing immunofluorescence assay of the inhibition of ASC oligomerization (spotting) by epstein in example 6 of the present application;

FIG. 19 shows the statistics of immunofluorescence assay of the inhibition of ASC oligomerization (spotting) by epstein in example 6 of the present application. * P is less than or equal to 0.01;

FIG. 20 shows the inhibition of Caspase-1 activation, GSDMD activation and IL-1β secretion by epstein under LPS+ATP-induced NLRP3 inflammatory body activation conditions in example 7 of the present application;

FIG. 21 shows LPS+K-free in example 7 of the present application ⁺ Under the culture medium induced NLRP3 inflammatory body activation condition, the effect of the epstein on the activation of Caspase-1, GSDMD activation and IL-1 beta secretion;

FIG. 22 shows the LPS+ATP-induced NLRP3 inflammatory body activation by epstein and LPS+K-free in example 7 of the present application ⁺ Culture medium induced NLRP3 inflammationResults of effects of IL-1 beta secretion in activation of the symptomatic individuals.

***P≤0.001；

FIG. 23 shows the LPS+ATP-induced NLRP3 inflammatory body activation by epstein and LPS+K-free in example 7 of the present application ⁺ Influence of tnfα secretion in culture medium-induced NLRP3 inflammatory body activation;

FIG. 24 is an immunofluorescence photograph showing the effect of epstein on mitochondrial morphology in example 7 of the present application;

FIG. 25 is a graph showing the effect of different doses of epstein on membrane potential using JC-1 for monitoring mitochondrial membrane potential in example 7 of the present application;

FIG. 26 is a comparison of ROS production under conditions of LPS+ATP-induced NLRP3 inflammatory body activation by epstein at various concentrations in example 7 of the present application. * P is less than or equal to 0.05;

FIG. 27 shows the effect of epstein on IL-1β concentration in serum of mice after LPS-induced sepsis in example 8 according to the present application. * P is less than or equal to 0.05;

FIG. 28 shows the effect of epstein on TNFα concentration in mouse serum after LPS-induced sepsis in mice in example 8 of the present application;

FIG. 29 shows the effect of epstein on IL-1β concentration in bone tissue at the site of implantation in example 9 according to the present application. * P.ltoreq.0.01, p.ltoreq.0.001;

FIG. 30 shows the effect of epstein on TNFα concentration in bone tissue at the site of implantation in example 9 of the present application;

FIG. 31 is a graph showing the effects of epstein on Caspase-1 activation, GSDMD activation and IL-1β secretion at the planting site and semi-quantitative statistical results of Caspase-1 and GSDMD 3 days after implantation in example 9 of the present application;

FIG. 32 is a graph showing the effects of epstein on Caspase-1 activation, GSDMD activation and IL-1β secretion at the site of implantation and semi-quantitative statistical results of Caspase-1, GSDMD 7 days after implantation in example 9 of the present application;

FIG. 33 is a statistical result of evaluation of the effect of epstein on activation of NLRP3 inflammatory bodies at implantation site cells, by FLICA-labeled NLRP3 inflammatory bodies 3 days after implantation in example 9 of the present application;

FIG. 34 is a statistical result of evaluation of the effect of epstein on activation of NLRP3 inflammatory bodies at the site of implantation by FLICA-labeled NLRP3 inflammatory bodies 7 days after implantation in example 9 of the present application;

FIG. 35 shows the results of HE (hematoxylin-eosin) staining of peri-implant tissues 3, 7 and 14 days after implantation in example 9 of the present application;

FIG. 36 shows Masson staining of peri-implant tissue 14 days after implantation in example 9 of the present application;

FIG. 37 shows TRAP staining results of peri-implant tissue 14 days after implantation in example 9 of the present application;

fig. 38 shows Micro-CT scan, three-dimensional reconstruction, and BV/TV statistics of tibia with implant at 14 days after implantation in example 9 of the present application, P < 0.001.

FIG. 39 is a graph showing the effect of varying concentrations of epstein on the translocation of NLRP3 to mitochondria in example 7 of the present application.

Detailed Description

The conception and the technical effects produced by the present application will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present application. The endpoints of the ranges and any values disclosed in the application are not limited to the precise range or value, and the range or value should be understood to include values close to the range or value. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

Materials and methods:

(1) In the present application, the epstein has a structure as shown in formula (I):

(2) Isolated culture of macrophages

Macrophages in particular embodiments of the application relate to Peritoneal Macrophages (PMs), bone marrow-derived macrophages (BMDMs) and the RAW264.7 stably transformed cell line expressing ASC-GFP protein (RAW-ASC-GFP).

(1) Abdominal macrophage (PMs) isolation culture: the mice were injected intraperitoneally with 3% sodium thioglycolate for 4 days at 2 mL/day each, after the completion of the stimulation, dislocation was sacrificed, 75% ethanol was sterilized, 10mL PBS buffer was injected into the abdominal cavity of the mice by a syringe, the needle was left in the abdominal cavity, the large bellies of the mice were gently warmed, PBS buffer in the abdominal cavity was recovered after 4 minutes, the recovered cell suspension was centrifuged at 1000rpm for 5min, washed once with physiological saline, resuspended with DMEM containing 10% FBS, and inoculated on a cell culture plate at 37℃at 5% CO ₂ Culturing for 3h. After 3h, the cell culture plate was removed, the DMEM medium was washed twice, the supernatant was discarded, the non-adherent cells were removed, and DMEM containing 10% fbs was added for further culture.

(2) Bone Marrow Derived Macrophages (BMDMs) isolation induction: mice were sacrificed, disinfected, and pulled bilaterally back until a crisp sound was heard (suggesting dislocation of the femur from the hip bone). The skin was cut off along one thigh ring using an ophthalmic scissors and an ophthalmic forceps, and peeled off in the direction of the paw. The muscles were dissected with forceps and the bones below the knee were cut with an ophthalmic scissors. The physiological saline is sucked by a needle head of the injector, inserted into the marrow cavity, repeatedly washed for a plurality of times, and the washing liquid is collected. The crushed bones and other tissue particles in the flushing liquid are removed by adopting a cell filter, and the cell suspension is collected into another centrifuge tube, centrifuged at 1000rpm for 5min. The supernatant was discarded, DMEM medium containing 10% FBS was added, 1% penicillin/streptomycin, 10ng/mL macrophage colony stimulating factor (M-CSF) was added, and the mixture was placed at 37℃and 5% CO ₂ Culturing. After 3 days DMEM washed the cells three times, the culture medium was changed and continued to be cultured for 3 days.

(3) Construction of RAW264.7 stable transgenic cell line expressing ASC-GFP protein (RAW-ASC-GFP): the 293T cells were plated in 100mm diameter cell culture dishes the day prior to transfection. On day 2, cells grow to 50-70%, plasmid is transfected, and the amount of cells per 100mm of culture plate is: 60. Mu.L PEI, 3. Mu.g pMD2.G, 7.5. Mu.g pSPAX2, 10. Mu.g PLVX-ASC-GFP. The DNA-PEI mixture was added dropwise to 293T cells, the dish was gently shaken to disperse the pellet evenly, the culture was performed normally, and fresh medium was changed after 6h of transfection. Collecting virus supernatant after 48h, centrifugingCell debris was removed and transferred to a new 15mL centrifuge tube. Then polybrene and 10mM HEPES were added at a final concentration of 8. Mu.g/mL and mixed well for use. The specific steps of lentivirus infection of RAW264.7 cells are as follows: RAW264.7 cells were first prepared, using 24 well plates as an example, 1X 10 wells were added to each well ⁶ Individual cells. Then, 2mL of the virus supernatant prepared in the above step was added, the culture plate was sealed with a sealing film, and the mixture was centrifuged at 1500rpm for 12min at 32 ℃. After centrifugation, the plates were allowed to stand in an incubator at 32℃for 4 hours. Finally, the supernatant was replaced with fresh medium and transferred to a 6-well plate for culture in an incubator at 37 ℃. After 48h of cell culture, RAW264.7 cells were subjected to screening culture using DMEM complete medium containing puromycin (final concentration of 2. Mu.g/mL). After 2 days, non-resistant RAW264.7 cells were killed and virus-infected cells grew normally.

Example 1

This example demonstrates that different concentrations of eplerenone significantly inhibit activation of PMs NLRP3 inflammatory bodies, caspase-1 activation and IL-1β release.

The implementation method comprises the following steps: PMs were seeded in 2.5X105 cells in 24-well plates, DMEM medium+10% FBS was cultured overnight, then 500ng/mL lipopolysaccharide (LPS, sigma Co., L2630) was used to stimulate PMs for 4h, then different concentrations of Ipriflavone (Ipriflavone, 0. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M; MCE Co., HY-N0094) were added for 2h, then 2mM ATP (Simga Co., A7699) was used to stimulate IL-1. Beta. In the supernatant for 30min, and ELSIA detection kit (Thermo Fisher Co., 88-7013-77) and TNF. Alpha. Was used as shown in FIGS. 1, 2. The supernatant was concentrated using a 10kD ultrafiltration tube, intracellular proteins were lysed using a 2 Xloading buffer, and Western Blot was performed to detect the expression of IL-1β, caspase-1 (p 20) and pro-IL-1β, pro-Caspase-1 and NLRP3 in cell culture supernatants, as shown in FIG. 3.

The results of the attached figures 1-3 show that the eplerenone can inhibit the activation of PMs NLRP3 inflammatory corpuscles in a gradient way, the maturation of pro-IL-1 beta and pro-Caspase-1, the enzyme activity of Caspase-1 and the release of IL-1 beta with high efficiency.

Example 2

This example demonstrates that epstein significantly inhibits PMs NLRP3 inflammatory body activation and Caspase-1 activation and IL-1β release, and inhibits NLRP3 inflammatory body activation due to lysosomal rupture, all under stimulation with different NLRP3 specific agonists.

Method of implementation

(1) PMs inoculation 2.5X10 ⁵ The cells were grown in 24 well plates with DMEM medium+10% FBS overnight, then 500ng/mL lipopolysaccharide (LPS, sigma Co., L2630) stimulated PMs for 4h, followed by 50. Mu.M epstein (Ipriflavone, MCE Co., HY-N0094) treatment for 2h, control group added equivalent DMSO, 10. Mu.M Nigericin (nigericin, invivogen Co., tlrl-nig) stimulated with other NLRP3 inflammatory minibody agonists respectively for 30min, 300mg/mL aluminum adjuvant (Alum, thermo Fisher Co., 77161) stimulated for 6h. IL-1β in the supernatant was detected using an ELSIA assay kit (Thermo Fisher Co., 88-7013-77), and TNFα was detected using an ELSIA assay kit (Thermo Fisher Co., 88-7324-22), and the results are shown in FIGS. 4 and 5.

(2) PMs inoculation 2.5X10 ⁵ The cells were grown in 24 well plates overnight with DMEM medium+10% FBS, then 500ng/mL lipopolysaccharide (LPS, sigma Co., L2630) stimulated PMs for 4h, followed by 50. Mu.M epstein (Ipriflavone, MCE Co., HY-N0094) treatment for 2h, 10. Mu.M nigericin (nigericin, invivogen Co., tlrl-nig) stimulated with the other NLRP3 inflammatory body agonists respectively for 30min, 300mg/mL aluminum adjuvant (Alum, thermo Fisher Co., 77161) stimulated for 6h, the supernatant was concentrated with a 10kD ultrafiltration tube, intracellular proteins were lysed with a 2 Xloading buffer, and the expression of IL-1β, caspase-1 (p 20) and pro-IL-1 and NLRP3 in the cell lysates was detected as shown in FIG. 6.

The results of FIGS. 4-6 show that under the stimulation of different NLRP3 specific agonists, the eplerenone can inhibit the activation of PMs NLRP3 inflammatory bodies, the maturation of pro-IL-1 beta and pro-Caspase-1, the enzyme activity of Caspase-1 and the secretion of IL-1 beta.

When particles or crystals such as aluminum adjuvants are phagocytized by cells, the lysosome structure is destroyed, thereby releasing proteases in the lysosome, especially cathepsin B, which ultimately results in activation of NLRP3 inflammatory corpuscles. Also, whether or not aluminum adjuvants are phagocytosed by cells is often used to verify whether lysosomes are ruptured. While epstein can inhibit activation of PMs NLRP3 inflammatory bodies under the stimulation of different NLRP3 specific agonists, thus epstein can inhibit activation of NLRP3 inflammatory bodies caused by lysosomal rupture.

Example 3

This example demonstrates that different epstein treatment times can inhibit activation of PMs NLRP3 inflammatory bodies, but do not affect the LPS initiation signal activation of NLRP3 inflammatory body stage.

The implementation method comprises the following steps: PMs NLRP3 inflammatory bodies were activated in the same manner as in example 1 and PMs were vaccinated 2.5X10 ⁵ The cells were grown in 24-well plates with DMEM medium+10% FBS overnight, then 500ng/mL lipopolysaccharide (LPS, sigma Co., L2630) was used to stimulate PMs for 4h, 50. Mu.M epstein (Ipriflavone, MCE Co., HY-N0094) was added for 0h,2h,4h, respectively, followed by 2mM ATP (Simga Co., A7699) for 30min. IL-1β in the supernatant was detected using an ELSIA assay kit (Thermo Fisher Co., 88-7013-77), and TNFα was detected using an ELSIA assay kit (Thermo Fisher Co., 88-7324-22), and the results are shown in FIGS. 7 and 8. The supernatant was concentrated using a 10kD ultrafiltration tube, intracellular proteins were lysed using a 2 Xloading buffer, and Western Blot was performed to detect the expression of IL-1β, caspase-1 (p 20) and pro-IL-1β, pro-Caspase-1 and NLRP3 in cell culture supernatants, as shown in FIG. 9.

The results of FIGS. 7-9 show that IL-1β secretion gradually decreases with increasing epstein treatment time, and that PMs NLRP3 inflammatory body activation and Caspase-1 inhibition effect gradually increases. Taken together with the ELISA results of tnfα in this example and examples 1 and 2, it was shown that the concentration of eplerenone and the treatment time had no significant effect on the release of tnfα, and therefore, the inhibition of activation of NLRP3 inflammatory bodies by eplerenone did not affect the initiation phase of inflammatory body activation (LPS stimulation phase) but directly the assembly phase of inflammatory body activation process (ATP stimulation phase).

Example 4

This example is intended to demonstrate that epstein inhibits activation of PMs AIM2 inflammatory bodies but has no effect on NLRC4 inflammatory bodies.

Method of implementation

(1) PMs inoculation 2.5X10 ⁵ Cells were plated in 24-well plates, DMEM medium+10% FBS overnight, then 500ng/mL lipopolysaccharide (LPS, sigma Co., L2630) stimulated PMs for 4h, the experimental group was treated with 50. Mu.M epstein (Ipriflavone, MCE Co., HY-N0094) for 2h, the control group was added with an equal amount of DMSO, followed by the addition of different inflammatory body activators. IL-1β in the supernatant was detected using an ELSIA assay kit (Thermo Fisher Co., 88-7013-77), and the results are shown in FIG. 10.

NLRP3 inflammatory body: 2mM ATP (Simga Co., A7699) was stimulated for 30min.

AIM2 inflammatory body: 1. Mu.g/mL Poly- (dA: dT) (Invivogen, tlrl-patn) was stimulated for 2h.

NLRC4 inflammatory body: inoculation of 2X 10 ⁹ CFU/mL Salmonella in PMs was stimulated for 2h.

(2) PMs were inoculated 2.5X10 according to the above-described method of activation of different inflammatory corpuscles ⁵ Cells were plated in 24-well plates, DMEM medium+10% FBS overnight, then 500ng/mL lipopolysaccharide (LPS, sigma Co., L2630) stimulated PMs for 4h, the experimental group was treated with 50. Mu.M epstein (Ipriflavone, MCE Co., HY-N0094) for 2h, the control group was added with an equal amount of DMSO, followed by the addition of different inflammatory body activators. The supernatant was concentrated using a 10kD ultrafiltration tube, and intracellular proteins were lysed using a 2 Xloading buffer, and Western Blot was used to detect the expression of IL-1β, caspase-1 (p 20) and pro-IL-1β and pro-Caspase-1 in cell culture supernatants and cell lysates, as shown in FIG. 11.

As can be seen from FIGS. 10, 11 and examples 1,2 and 3, epstein can inhibit activation of PMs NLRP3 inflammatory bodies, activation of Caspase-1 and secretion of IL-1β, and activation of AIM2 inflammatory bodies, caspase-1 and secretion of IL-1β. But does not inhibit activation of the NLRC4 inflammatory small Caspase-1 and secretion of IL-1β.

Example 5

This example illustrates that epstein can also inhibit Caspase-1 enzyme activity and its activation-induced GSDMD activation, IL-1β secretion in other macrophage NLRP3 inflammatory bodies.

Method of implementation

(1) BMDMs vaccinated 2.5X10 ⁵ In individual cells to 24 well plates, RPMI1640 medium+10% FBS was cultured overnight, then 500ng/mL lipopolysaccharide (LPS, sigma Co., L2630) was used to stimulate BMDMs for 4h, 50. Mu.M epstein (Ipriflavone, MCE Co., HY-N0094) was added for 2h, followed by 10. Mu.M Nigericin (Invivogen Co., tlrl-nig) for 30min. The supernatant was concentrated using a 10kD ultrafiltration tube, intracellular proteins were lysed using a 2 Xloading buffer, and Western Blot was used to detect expression of IL-1β, caspase-1 (p 20) in cell culture supernatants and GSDMD, pro-IL-1β and pro-Caspase-1 in cell lysates, as shown in FIG. 12. IL-1β in the supernatant was detected using an ELSIA assay kit (Thermo Fisher Co., 88-7013-77), and the results are shown in FIG. 13.

(2) Human monocyte cell line (THP-1) at 2.5X10 ⁵ Each was seeded in 24-well plates. THP-1 cells were induced to differentiate with 50nM phorbol 12-tetradecanoate 13-acetate (PMA) for 3h prior to NLRP3 inflammatory body stimulation. After 3h, the culture supernatant in the plates was discarded, and each well was replaced with 500. Mu.L of RPMI1640 medium+10% FBS containing 250ng/mL LPS for 4h, treated with 50. Mu.M epstein (Ipriflavone, MCE Co., HY-N0094) for 2h, followed by 10. Mu.M Nigericin (Invivogen Co., tlrl-nig) for 30min. The supernatant was concentrated using a 10kD ultrafiltration tube, intracellular proteins were lysed using a 2 Xloading buffer, and Western Blot was used to detect expression of IL-1β, caspase-1 (p 20) in cell culture supernatants and GSDMD, pro-IL-1β and pro-Caspase-1 in cell lysates, as shown in FIG. 14. IL-1β in the supernatant was detected using an ELSIA assay kit (Thermo Fisher Co., 88-7013-77), and the results are shown in FIG. 15.

Studies have demonstrated that Caspase-1 activation can cause cleavage of GSDMD, inducing apoptosis of the cell coke. FIGS. 12-15 show that epstein also inhibits Caspase-1 enzyme activity in BMDMs and THP-1 cells NLRP3 inflammatory bodies and activation thereof, GMDM activation and apoptosis, IL-1 beta secretion.

Example 6

This example is intended to illustrate that epstein inhibits activation of NLRP3 inflammatory bodies and inhibits oligomerization of ASC proteins.

Method of implementation

(1) Chemical cross-linking method for detecting oligomerization: RAW-ASC-GFP was spread on 6-well plates 2X 10 per well one day in advance ⁶ Individual cells. The following day, cells were stimulated for NLRP3 inflammatory body activation, 500ng/mL lipopolysaccharide (LPS, sigma Co., L2630), treated with 50. Mu.M epstein (Ipriflavone, MCE Co., HY-N0094) for 2h, followed by stimulation with 2mM ATP (Simga Co., A7699) for 30min, the supernatant was discarded, the cells were rinsed with pre-chilled PBS buffer, and 500. Mu.L lysate (50 mM Tris-HCl, pH7.6,0.5% Triton X-100,0.1mM PMSF and protease inhibitor cocktail) was added. The cells were then scraped and transferred to a 1.5mL centrifuge tube and blown 10 times with a needle. 6000g, centrifuged at 4℃for 10min, the pellet was washed 2 times with PBS and resuspended. Adding a cross-linking agent DSS with the final concentration of 2mM, and shaking at room temperature for 30min; then centrifuged and 30. Mu.L of 2 XSDS/SDD loading buffer was added. Finally, the mixture was boiled at 100℃for 10min, and ASC monomers and oligomers and NLRP3 monomers and oligomers were detected by using a Westem Blot, and the results are shown in FIGS. 16 and 17.

(2) For ASC spot formation experiments, immunofluorescent staining was mainly performed, and the specific steps were as follows: a75% ethanol sterilized coverslip was placed on a 24-well plate, followed by inoculation with RAW-ASC-GFP 5X 10 ⁵ After RPMI1640 medium plus 10% serum was incubated overnight without MCSF, then 500ng/mL lipopolysaccharide (LPS, sigma, L2630) was stimulated for 4h, 50 μm epstein (Ipriflavone, MCE, HY-N0094) was added for 2h, then 2mM ATP (Simga, a 7699) was used to stimulate 30min,4% paraformaldehyde was used to fix the cells, and the immunofluorescence method examined ASC spot formation, the results of which are shown in fig. 18, 19, wherein part of the ASC spots formed in fig. 18 were circled.

As can be seen from fig. 16-19, the result of cellular immunofluorescence in RAW-ASC-GFP showed that many spots, ASC spots, appeared around the nucleus under the stimulation of the second signal ATP (fig. 18), which is a phenomenon of oligomerization of ASC protein, and ASC spots were significantly reduced after epstein treatment (fig. 19). Detection of ASC monomers and oligomers by Western Blot also found that epstein significantly inhibited ASC oligomerization (fig. 16). In addition, the oligomerization of NLRP3 was detected by semi-denaturing gel (SDD-PAGE), and the treatment with epstein showed that the oligomerization of NLRP3 protein was inhibited (FIG. 17), which indicates that the activation of the NLRP3 inflammatory corpuscles by epstein was occurred in the upstream event and the assembly of NLRP3 inflammatory corpuscles was inhibited.

Example 7

The embodiment is used for explaining that the action target of the epstein for inhibiting the activation of NLRP3 inflammatory bodies improves the mitochondrial function and reduces the ROS release; and mitochondrial protection, reducing the decrease in mitochondrial membrane potential, maintaining mitochondrial morphology, and inhibiting expression of mitochondrial voltage dependent anion channel 1 (VDAC 1).

Method of implementation

(1) Epstein pair K ⁺ Influence of the discharge: BMDMs vaccinated 2.5X10 ⁵ Cells were plated into 24-well plates, RPMI1640 medium+10% FBS overnight, and BMDMs were stimulated with 500ng/mL lipopolysaccharide (LPS, sigma Co., L2630) for 4h. Treatment with 50. Mu.M eplerenone (Ipriflavone, MCE company, HY-N0094) for 2h followed by stimulation with 2mM ATP (Simga company, A7699) for 30min, respectively, and addition of K-free ⁺ Supplement Na ⁺ The medium was stimulated for 30min. The supernatant was concentrated using a 10kD ultrafiltration tube, intracellular proteins were lysed using a 2 Xloading buffer, and Western Blot was performed to detect expression of IL-1β, caspase-1 (p 20) and GSDMD, pro-IL-1β and pro-Caspase-1 in cell culture supernatants and cell lysates, as shown in FIGS. 20 and 21. IL-1. Beta. And TNF. Alpha. In the supernatant were detected by ELSIA, and the results are shown in FIGS. 22 and 23.

(2) Mitochondrial MitoTraker Deep Red staining and ultra-high resolution AriyScan reconstruction: BMDMs approximately 5X 10 in sheet form ⁵ Every well (24-well plate) of cells, the culture supernatant from the plate was discarded the next day, and every well was replaced with 500. Mu.L of Opti-MEM medium containing 200ng/mL LPS, and stimulated for 4 hours. Adding 50 μm epstein, allowing to act for 2 hr, washing twice with preheated PBS, adding 300 μl PBS (containing 5000× MitoTraker Deep Red), and staining in incubator for 30min, washing again with preheated PBS, adding NLRP3 inflammatory corpuscle secondary signal ATP for 30min. After the immunofluorescent staining step is carried out to prepare the flakes, the flakes are observed with ultra-high resolution by using an AriyScan technology through a fluorescent confocal microscope, and rendering reconstruction is carried out, and the result is shown in figure 24.

(3) Determination of mitochondrial membrane potential and mitochondrial ROS: BMDMs approximately 5X 10 in sheet form ⁵ Each cell per well (12 well plate). The next day the culture supernatant from the plates was discarded, each well was replaced with 500. Mu.L of opti-MEM medium containing 200ng/ml LPS, and after 4h of stimulation, treated with Ipriflavone (25. Mu.M, 50. Mu.M) for 2h and NLRP3 inflammatory corpuscle secondary signal ATP was added for 30min. Wash 2 times with pre-warmed PBS, add 200 μl PBS per well, gently scrape to 1.5mL centrifuge tube with a cell scraper, and gently mix the cells with a pipette. JC-1 (final concentration 5000X) or Mitosox dye (final concentration 1000X) was added respectively according to the measurement of mitochondrial membrane potential or ROS, and stained in an incubator for 30min. After staining, the cells were washed 2 times with ice PBS, and then were filtered through a 300 mesh cell filter, and the results were shown in FIG. 25.

(4) Detection of ROS: fluorescent probe MitoSox (Thermo Fisher Co., M36008) detects intracellular ROS formation. BMDMs cells at 5X 10 per well ⁵ Is seeded in 12-well plates. Following the next day of NLRP3 inflammatory body activation stimulation, cells were incubated with DCFH-DA at a final concentration of 10. Mu.M/L for 30min at 37 ℃. Finally, the cells were washed with PBS, resuspended and then examined on a flow cytometer, the results of which are shown in FIG. 26.

(5) VDAC1 protein is a porous protein with extremely rich content on the outer membrane of mitochondria, forms a common path for metabolite exchange between mitochondria and cytoplasm, controls the entry and exit of mitochondrial metabolites, and plays a vital role in regulating the metabolism and energy functions of mitochondria. Activation of BMDMs NLRP3 inflammasome as in fig. 39, detection of VDAC1 protein by Western Blot, showed that epstein inhibited VDAC1 protein expression.

Potassium (K) ⁺ ) Efflux and mitochondrial dysfunction are two upstream events of NLRP3 inflammatory body assembly. Thus, explore epperWhether the flavone affects K ⁺ The outer row, as shown in FIGS. 20-23, uses a K-free ⁺ Supplement Na ⁺ Culture medium, stimulating K ⁺ Efflux as second signal for NLRP3 inflammation small body activation obviously compared with ATP as second activation signal for NLRP3 inflammation small body, the epstein has no effect on Caspase-1 activation, GSDMD activation and IL-1β secretion, i.e. has no effect on K ⁺ Efflux regulates activation of NLRP3 inflammatory bodies. The effect of epstein on mitochondrial function was also investigated, as shown in figures 24 and 25. The results of the ultra-high resolution laser confocal microscopy showed that the mitochondrial morphology disintegrated from "linear" to "punctiform" and the cell morphology shrunken under the second signal ATP stimulation, whereas the mitochondria recovered "linear" after epstein treatment (fig. 24). Injury to mitochondria is often manifested by rupture of mitochondria and ROS production. Mitochondria function, mitochondrial membrane potential was monitored with JC-1, at which JC-1 was present as a green fluorescent monomer, and at which JC-1 was present as an orange fluorescent aggregate, wherein the orange fluorescent aggregate was located within each black ellipse in fig. 25, and the green fluorescent monomer was located in a light region outside each black ellipse in fig. 25. ATP-treated cells showed increased monomer levels, mitochondrial damage, membrane potential depolarization, while epstein rescued membrane potential in a dose-dependent manner (fig. 25). Detection of ROS production indicated that epstein reduced ROS production in a dose-dependent manner (fig. 26). Therefore, the action target of the epstein for inhibiting the activation of NLRP3 inflammatory bodies can improve mitochondrial function and reduce ROS release without depending on K ⁺ And (5) discharging.

The results of the ultra-high resolution laser confocal microscope showed that the mitochondria morphology disintegrated from "linear" to "punctiform" and the cell morphology shrunken under the second signal ATP stimulation, and the mitochondria recovered "linear" again after the epstein treatment (fig. 24). And mitochondria functionally, monitoring mitochondrial membrane potential with JC-1, at which JC-1 exists as a green fluorescent monomer, and at which JC-1 exists as an orange fluorescent aggregate, wherein the orange fluorescent aggregate is located within each black ellipse in fig. 25, and the green fluorescent monomer is located in a light region outside each black ellipse in fig. 25. ATP-treated cells showed increased monomer levels, mitochondrial damage, membrane potential depolarization, while epstein rescued membrane potential in a dose-dependent manner (fig. 25). It is known that epstein reduces the decrease in mitochondrial membrane potential and maintains mitochondrial morphology during inhibition of NLRP3 inflammatory body activation. As can be seen from a comparison of lanes 4 and 3 of FIG. 39, in LPS-induced peritoneal macrophages, epstein inhibited activation of the VDAC1 protein, thereby inhibiting translocation of NLRP3 protein to mitochondria. Thus, there is also mitochondrial metabolism protection during the inhibition of NLRP3 inflammatory body activation by epstein.

Example 8

This example is used to demonstrate that epstein promotes the resistance of mice against LPS-induced sepsis while validating the inhibition of NLRP3 inflammatory body activation by epstein in vivo.

The implementation method comprises the following steps: c57BL/6 mice were intraperitoneally injected with LPS, a sepsis model was established, 30mg/kg of epstein was intraperitoneally injected, 25mg/kg of LPS was injected after 1h, blood was collected after 4h, and the concentrations of cytokines such as serum IL-1. Beta. And TNF-alpha were detected, and the results are shown in FIGS. 27 and 28, respectively.

LPS treatment can trigger a storm of systemic inflammatory factors in mice, leading to sepsis, ultimately leading to death in mice. While epstein greatly reduces the serum IL-1 beta cytokine level in mice (FIG. 27), thereby promoting the onset of sepsis in mice. The level of tnfα was not affected by epstein (fig. 28), which is consistent with the results in examples 1-3 and example 7, and indirectly suggests that epstein is achieved by inhibiting the second signaling stage of NLRP3 inflammatory bodies. The above results indicate that epstein can promote the resistance of mice against LPS-induced sepsis.

Example 9

This example is intended to demonstrate that epstein improves early stages of immune response in vivo when biomaterials are implanted and promotes early osseointegration.

Method of implementation

(1) Modeling: the titanium nails (diameter 2.2mm, length 6 mm) were blasted and ultraviolet sterilized before being implanted into 3 month old male C57BL/6 mice tibia. In implantation, a 3mm longitudinal incision was made in the tibia, the soft tissue was carefully removed with a periosteum stripper, and a drill of 2.2mm diameter was used to penetrate the bone marrow cavity. And meanwhile, physiological saline is sprayed on the operation area, so that the damage to tissues is reduced. The titanium nail is directly rotated into the hole until the bottom of the implant reaches the cortical bone at the other side. Two screws (one titanium nail per tibia) were inserted per animal. Mice were randomly divided into 2 groups (Ipriflavone group and Mock group). Ipriflavone group mice were intraperitoneally injected with epstein (30 mg/kg body weight) and Mock group mice were intraperitoneally injected with an equivalent amount of DMSO as a control. The following corresponding tests were performed on days 3, 7 and 14, respectively.

(2) Detecting the index: mice were sacrificed 3 days, 7 days, and 14 days after implantation, and after tibial extraction, peri-implant cells were flushed out with a PBS-loaded needle and syringe, centrifuged, and IL-1β and tnfα in the supernatant were detected by ELSIA, and the results are shown in fig. 29 and 30. The supernatant from the 3-day and 7-day samples was concentrated using a 10kD ultrafiltration tube, the intracellular proteins were lysed using a 2 Xloading buffer, and the Western Blot was assayed for expression of IL-1β, caspase-1 (p 20) and GSDMD, pro-IL-1β and pro-Caspase-1 in the supernatant, as shown in FIGS. 31 and 32, respectively, wherein FIGS. 31 (a), (b) and (c) were the Western Blot assay results of Caspase 1 activation, GSDMD activation and IL-1β secretion at the 3-day implant site, the semi-quantitative statistical results of Caspase 1 and GSDMD, FIGS. 32 (a), (b) and (c) were the Western Blot assay results of Caspase 1 activation, GSDMD activation and IL-1β secretion at the 7-day implant site, the semi-quantitative statistical results of Caspase 1 and GSDMD, respectively. After the activation of the NLRP3 inflammatory bodies in the samples of 3 days and 7 days is dyed by FLICA, flow cytometry is carried out, and the results are shown in figures 33 and 34 respectively. Histological examination the tibia was decalcified with EDTA and then HE (hematoxylin-eosin) stained (fig. 35), masson stained (fig. 36), TRAP stained (fig. 37). The results of Micro-CT examination of the tibia on 14 days are shown in FIG. 38, wherein FIGS. 38 (a) and (c) are respectively three-dimensional reconstruction diagrams of the tibia and the titanium nail with the implant and the new bone on 14 days after implantation of the control group, FIGS. 38 (b) and (d) are respectively three-dimensional reconstruction diagrams of the tibia and the titanium nail with the implant and the new bone on 14 days after implantation of the epstein, and FIG. 38 (e) is a statistical result of BV/TV in the tibial bone tissue with the implant on 14 days after implantation.

During the implantation of biological materials into the body, the wound may initiate an immune response phase at the beginning of implantation, typically for one week, followed by a shift to tissue healing. The effect of epstein use on immune response and promotion of the prognosis at the initial stage of biomaterial implantation were studied. IL-1. Beta. Inflammatory factors were significantly reduced in the Ipriflavone group at 3 and 7 days of biomaterial implantation, and TNFα results were consistent with those in examples 1-3 and 7,8 (FIGS. 29, 30). At the same time, the wound also causes activation of NLRP3 inflammation bodies, and the result of Western Blot detection shows that the use of the epstein can inhibit the enzymatic activity of Caspase-1 and the GMDM cleavage and cell apoptosis caused by the activation of the epstein, and the maturation and secretion of IL-1 beta (figures 31 and 32) and inhibit the activation of NLRP3 inflammation bodies (figures 33 and 34). The results of HE staining showed that at 3 and 7 days post-implantation, both groups had inflammatory infiltrates, but the inflammatory infiltrates were lower for Ipriflavone group than for the control group, and no inflammatory infiltrates were seen for 14 days (fig. 35). Masson staining showed that bone tissue fibers were remodelled around the existing material for 14 days (fig. 36), TRAP was used to label osteoclasts, and the TRAP stained group had more TRAP positive cells than the control group (fig. 37). IL-1β is thought to promote osteoclast differentiation, leading to bone resorption. Micro-CT measurements were performed 14 days after implantation and early osteogenesis (BV/TV) was measured, and the results showed that the epstein group had better early bone remodeling than the control group (fig. 38). Thus, epstein can improve early immune response phase of biological material implanted in vivo and promote early osseointegration.

From the results of examples 1-9 above, it can be seen that epstein can inhibit activation of NLRP3 inflammatory bodies and AIM2 inflammatory bodies by agonists of different properties such as ATP, nigericin, alu, but does not affect activation of NLRC4 inflammatory bodies; epstein inhibits activation of NLRP3 inflammatory bodies by inhibiting oligomerization of ASC proteins, and specific targets of action are improvement of mitochondrial function and reduction of ROS production. Epstein also inhibits Caspase-1 enzyme activity and GSDMD activation, and IL-1β secretion. Meanwhile, the epstein can also inhibit septicemia induced by LPS and promote early osseointegration of biological materials by regulating immune response. It can be seen that eplerenone can be used as a potential drug for the prevention and/or treatment of diseases associated with small inflammatory bodies of NLRP3 and/or for the amelioration of the physiological phase of inflammation.

The preferred embodiments of the present application have been described in detail above, but the present application is not limited thereto. Within the scope of the technical idea of the application, a number of simple variants of the technical solution of the application are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the application, all falling within the scope of protection of the application.

Claims

1. Use of eplerenone for inhibiting AIM2 inflammatory body activation at a non-therapeutic destination, including use in inhibiting AIM2 inflammatory body activation in vivo and/or in vitro.

2. Use of eplerenone for inhibiting activation of Caspase-1 protein during activation of AIM2 inflammatory bodies at a non-therapeutic destination, in particular for inhibiting activation of Caspase-1 protein and maturation and secretion of IL-1 beta in vivo and/or in vitro.

3. Use of epstein for the manufacture of a medicament for the prevention and/or treatment of diseases associated with activation of the AIM2 inflammatory body and/or for the amelioration of the associated inflammatory phases.

4. The use according to claim 3, wherein the disease associated with AIM2 inflammatory body activation is at least one of multiple sclerosis, colon cancer, liver cancer, prostate cancer, cervical cancer, atopic dermatitis, psoriasis, systemic lupus erythematosus.

5. A product comprising epstein.

6. The product of claim 5, wherein the product is a medicament or agent, the medicament further comprising a pharmaceutically acceptable adjuvant or carrier.

7. Use of a product according to claim 5 or 6 for inhibiting AIM2 inflammatory body activation for non-therapeutic purposes.

8. The application of epstein in protecting macrophage mitochondria, reducing the reduction of mitochondrial membrane potential, maintaining mitochondrial morphology and inhibiting VDAC1 protein expression in non-therapeutic destination in vitro.

9. Use of epstein for inhibiting lysosomal disruption caused by an aluminum adjuvant at a non-therapeutic in vitro destination.