Use of iclivicolesponol as an nlrp3 inflammasome activation inhibitor

Abstract

The application discloses use of iclocinolone acetonide as an NLRP3 inflammasome activation inhibitor and belongs to the technical field of biological medicines. The application first discovers that iclocinolone acetonide can reduce secretion of IL-1beta by inhibiting NF-kappa B and / or AP-1 signal pathways, and can significantly inhibit NLRP3 inflammasome activation, thereby providing a new thought and theoretical basis for inhibiting NLRP3 inflammasome activation. Therefore, the application has a good application prospect in preparation of medicines for preventing and / or treating NLRP3 inflammasome related diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to the use of loteprednol etabonate as an NLRP3 inflammasome activation inhibitor. BACKGROUND

[0002] The innate immune system, as the first line of defense of the body's immune response, plays an important role in clearing microorganisms. Among them, NLRP3 inflammasome, as one of the innate immune molecules, plays an important role in the inflammatory response of the body caused by exogenous pathogens and endogenous danger signals. As an important part of the innate immune system, NLRP3 inflammasome is mainly composed of cytoplasmic sensing protein NLRP3, apoptosis-related speck-like protein (ASC) and cysteine protease Caspase-1. After the activation of NLRP3 inflammasome, NLRP3, ASC and Caspase-1 form a complex to activate Caspase-1, and then the activated Caspase-1 mediates the maturation and secretion of inflammatory factor IL-1β, and extracellular inflammatory factor IL-1β participates in the process of body inflammatory response; at the same time, activated Caspase-1 can mediate pyroptosis by shearing effector protein GSDMD. NLRP3 inflammasome is one of the most widely studied inflammasomes. It can recognize pathogen-associated molecular patterns (PAMPs) and danger signal-associated molecular patterns (DAMPs), including viruses, bacteria, Nigericin, monosodium urate, ATP, silicon dioxide crystals and aluminum salt crystals, etc. NLRP3 inflammasome plays an important role in resisting pathogen infection and recognizing danger signals in the body, but its excessive activation or uncontrolled regulation is closely related to many inflammatory diseases, such as gout, type 2 diabetes, non-alcoholic fatty liver disease, atherosclerosis, Parkinson's disease, Alzheimer's disease and various autoimmune diseases, etc.

[0003] At present, a variety of NLRP3 inflammasome activation inhibitors have been reported, most of which take NLRP3 protein as a drug target, including: MCC950, CY-09, MNS, OLT1177, etc. The effects of these inhibitors in animal models of NLRP3 inflammasome-related inflammatory diseases have been verified. For example: MCC950 can reduce the severity of experimental autoimmune encephalomyelitis in mice; oral MCC950 can inhibit NLRP3 inflammasome activation in a mouse model of Parkinson's disease, and also has a neuroprotective effect, which can effectively reduce motor deficits, substantia nigra striatal dopaminergic degeneration and accumulation of alpha-synuclein aggregates; CY-09 has good therapeutic effect in mouse models of cryopyrin-associated autoinflammatory syndrome and type 2 diabetes. Although a variety of NLRP3 inflammasome inhibitors have good effects in animal models, few inhibitors have been approved for marketing at present, and most of the inhibitors are in the clinical trial stage or have stopped at the clinical trial stage. Therefore, it is of great clinical significance to find new and effective drugs for inhibiting the activation of NLRP3 inflammasome.

[0004] Loteprednol etabonate is an anti-inflammatory corticosteroid for ophthalmology, which can be used for treating inflammation of eyelid and bulbar conjunctiva, uveitis, inflammation of cornea and anterior segment of eye, and other inflammation sensitive to corticosteroids. At present, there is no report that loteprednol etabonate can inhibit the activation of NLRP3 inflammasome. SUMMARY

[0005] The purpose of the present application is to provide the use of loteprednol etabonate as an NLRP3 inflammasome activation inhibitor. It is first found in the present application that loteprednol etabonate can significantly inhibit the activation of NLRP3 inflammasome by reducing the secretion of IL-1β, and therefore has a good application prospect in the preparation of drugs for preventing and / or treating NLRP3 inflammasome-related diseases.

[0006] In a first aspect, the present application provides the use of loteprednol etabonate as an NLRP3 inflammasome activation inhibitor.

[0007] In the present application, the inventors have found that loteprednol etabonate can inhibit the activation of NLRP3 inflammasome, and therefore can be used for the preparation of drugs for preventing and / or treating NLRP3 inflammasome-related diseases.

[0008] .

[0009] In the present application, the inventors have found that loteprednol etabonate can inhibit the activation of NLRP3 inflammasome, and therefore can be used for the preparation of drugs for preventing and / or treating NLRP3 inflammasome-related diseases.

[0010] In some embodiments, the icdecloperol inhibits the expression of IL-1β by inhibiting the NF-κB and / or AP-1 signaling pathway, thereby inhibiting the activation of NLRP3 inflammasome.

[0011] In the present application, it is found that the icdecloperol can inhibit the NF-κB and / or AP-1 signaling pathway by detecting the phosphorylation level of p65 protein in the NF-κB signaling pathway and / or c-jun protein in the AP-1 signaling pathway, and the inhibition is dose-dependent, thereby inhibiting the expression of IL-1β, and finally inhibiting the activation of NLRP3 inflammasome.

[0012] In the second aspect, the present application provides the use of icdecloperol in the preparation of a medicament for preventing and / or treating NLRP3 inflammasome-related diseases.

[0013] In some embodiments, the NLRP3 inflammasome-related diseases include at least one of gout, type 2 diabetes, non-alcoholic steatohepatitis, atherosclerosis, Parkinson's disease, Alzheimer's disease, sepsis, and colitis.

[0014] It can be understood that the NLRP3 inflammasome-related diseases also include other common diseases in the prior art, and the NLRP3 inflammasome-related diseases in the present application preferably include at least one of gout, type 2 diabetes, non-alcoholic steatohepatitis, atherosclerosis, Parkinson's disease, Alzheimer's disease, sepsis, and colitis.

[0015] In the third aspect, the present application provides an NLRP3 inflammasome activation inhibitor comprising icdecloperol.

[0016] In the fourth aspect, the present application provides a pharmaceutical composition comprising the above-mentioned NLRP3 inflammasome activation inhibitor.

[0017] In some embodiments, the above-mentioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0018] In the present application, the term "pharmaceutically acceptable carrier" refers to an excipient that is widely employed in the pharmaceutical production field. The excipient is mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method for allowing the active ingredient to be dissolved at a desired rate after the subject receives the administration, or to facilitate the active ingredient to be effectively absorbed after the subject receives the administration of the composition. The pharmaceutical excipient can be an inert filler, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipient can include one or more of the following excipients: binding agents, suspending agents, emulsifying agents, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adherents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavorings and sweeteners.

[0019] The pharmaceutical composition provided by the present application can be prepared according to the disclosed content using any method known to those skilled in the art. For example, including but not limited to conventional mixing, dissolving, granulating, emulsifying, pulverizing, encapsulating, entrapping or lyophilizing processes.

[0020] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of a solid preparation, a semi-solid preparation, a liquid preparation.

[0021] The pharmaceutical composition provided by the present application can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical composition of the present application can also be a controlled or sustained release dosage form (such as liposome or microsphere). Examples of solid oral preparations include but are not limited to powder, capsule, caplet, soft capsule and tablet. Examples of liquid preparations for oral or mucosal administration include but are not limited to suspension, emulsion, elixir and solution. Examples of topical preparations include but are not limited to emulsion, gel, ointment, cream, patch, paste, foam, lotion, drop or serum preparation. Examples of preparations for parenteral administration include but are not limited to injection solution, dry powder preparation that can be dissolved or suspended in a pharmaceutically acceptable carrier, injection suspension and injection emulsion. Examples of other suitable preparations of the pharmaceutical composition include but are not limited to eye drops and other ophthalmic preparations; aerosols such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0022] In a fifth aspect, the present application provides the use of the above-mentioned NLRP3 inflammasome activation inhibitor or any of the above-mentioned pharmaceutical compositions in the preparation of a medicament for preventing and / or treating NLRP3 inflammasome-related diseases.

[0023] In some embodiments, the NLRP3 inflammasome-related disease comprises at least one of gout, type 2 diabetes, non-alcoholic steatohepatitis, atherosclerosis, Parkinson's disease, Alzheimer's disease, sepsis, and colitis.

[0024] The beneficial effects of the present application are: different from the prior art, the present application first discovers that icdeclopotasone can inhibit the expression and secretion of IL-1β by inhibiting the NF-κB and / or AP-1 signaling pathway, and inhibit the activation of NLRP3 inflammasome, thus providing a new idea and theoretical basis for inhibiting the activation of NLRP3 inflammasome; therefore, it has good application prospect in the preparation of drugs for preventing and / or treating NLRP3 inflammasome-related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1A A model result graph of LPS+Nigericin activating NLRP3 inflammasome in Example 1 of the present application;

[0026] Figure 1B A result graph of the content of IL-1β in the supernatant of cells after different drug treatments in Example 1 of the present application;

[0027] Figure 1C A result graph of the content of IL-1β in the supernatant of cells after different drug treatments in Example 1 of the present application;

[0028] Figure 1D A result graph of the content of IL-1β in the supernatant of cells after different drug treatments in Example 1 of the present application;

[0029] Figure 1E A result graph of the content of IL-1β in the supernatant of cells after different drug treatments in Example 1 of the present application;

[0030] Figure 1F A result graph of the content of IL-1β in the supernatant of cells after different drug treatments in Example 1 of the present application;

[0031] Figure 1G A result graph of the content of IL-1β in the supernatant of cells after different drug treatments in Example 1 of the present application;

[0032] Figure 1H A result graph of the content of IL-1β in the supernatant of cells after different drug treatments in Example 1 of the present application;

[0033] Figure 1I A result graph of the content of IL-1β in the supernatant of cells after different drug treatments in Example 1 of the present application;

[0034] Figure 2A A result graph of the expression level of IL-1β mRNA in cells after different drug treatments in Example 1 of the present application;

[0035] Figure 2B Figure for the expression level of IL-1β mRNA in cells after different drug treatments in Example 1 of the present application;

[0036] Figure 2C Figure for the expression level of IL-1β mRNA in cells after different drug treatments in Example 1 of the present application;

[0037] Figure 2D Figure for the expression level of IL-1β mRNA in cells after different drug treatments in Example 1 of the present application;

[0038] Figure 2E Figure for the expression level of IL-1β mRNA in cells after different drug treatments in Example 1 of the present application;

[0039] Figure 2F Figure for the expression level of IL-1β mRNA in cells after different drug treatments in Example 1 of the present application;

[0040] Figure 2G Figure for the expression level of IL-1β mRNA in cells after different drug treatments in Example 1 of the present application;

[0041] Figure 2H Figure for the expression level of IL-1β mRNA in cells after different drug treatments in Example 1 of the present application;

[0042] Figure 3 Figure for the results of Loteprednol etabonate inhibiting NLRP3 inflammasome activation in Example 2 of the present application, wherein (A) is the content of IL-1β in the supernatant of cells after treatment; (B)-(D) are respectively the expression level of IL-1β, NLRP3 and Caspase-1 mRNA in cells after treatment;

[0043] Figure 4 Figure for the results of Western blotting of Loteprednol etabonate inhibiting NF-κB signaling pathway and AP-1 signaling pathway related proteins in cells in Example 2 of the present application, wherein (A) is the results of Western blotting of Loteprednol etabonate inhibiting NF-κB signaling pathway and AP-1 signaling pathway related proteins in THP-1 cells, (B) is the results of Western blotting of Loteprednol etabonate inhibiting NF-κB signaling pathway and AP-1 signaling pathway related proteins in BMDMs cells;

[0044] Figure 5 Figure for the cytotoxicity results of Loteprednol etabonate in Example 3 of the present application;

[0045] Figure 6 Figure for the results of inhibiting NLRP3 inflammasome activation by different concentrations of Loteprednol etabonate in Example 4 of the present application, wherein (A) is the content of IL-1β in the supernatant of the treated cells; (B)-(D) are the expression levels of IL-1β, NLRP3 and Caspase-1 mRNA in the treated cells, respectively;

[0046] Figure 7 Figure for the results of inhibiting NLRP3 inflammasome activation by different concentrations of Loteprednol etabonate in Example 4 of the present application, wherein (A) is the content of IL-1β in the supernatant of the treated cells; (B) is the half effective concentration IC50 results figure of Loteprednol etabonate;

[0047] Figure 8 Figure for the content of IL-1β and TNF-ɑ in the blood of sepsis mice after treatment with Loteprednol etabonate in Example 5 of the present application;

[0048] Figure 9 Figure for the survival rate and body weight of sepsis mice after treatment with Loteprednol etabonate (first intraperitoneal injection of Loteprednol etabonate, 2h later, then injection of LPS) in Example 5 of the present application;

[0049] Figure 10 Figure for the survival rate and body weight of sepsis mice after treatment with Loteprednol etabonate (first intraperitoneal injection of LPS, 1h later, then injection of Loteprednol etabonate) in Example 5 of the present application;

[0050] Figure 11A Figure for the body weight of colitis mice after treatment with Loteprednol etabonate in Example 6 of the present application;

[0051] Figure 11B Figure for the fecal disease index of colitis mice after treatment with Loteprednol etabonate in Example 6 of the present application;

[0052] Figure 11C Figure for the measurement of colon length of colitis mice after treatment with Loteprednol etabonate in Example 6 of the present application;

[0053] Figure 11D Figure 6 is a graph of the colon length statistics of colitis mice treated with Loteprednol etabonate in Example 6 of the present application;

[0054] Figure 11E Figure 6 is a graph of the colon length statistics of colitis mice treated with Loteprednol etabonate in Example 6 of the present application; DETAILED DESCRIPTION

[0055] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present application.

[0056] The experimental methods not specified in the embodiments are usually performed according to the conventional conditions and the conditions described in the manuals, or the conditions suggested by the manufacturers. The general equipment, materials, reagents, etc. used are commercially available, unless otherwise specified.

[0057] Example 1 Screening of NLRP3 inflammasome activation inhibitors

[0058] 1.1 Construction of NLRP3 inflammasome activation model

[0059] THP-1 cells were cultured in 1640 medium containing 10% FBS, and TPA was added at a final concentration of 100 ng / ml, and the culture was continued for 12 h to obtain induced differentiated THP-1 macrophages; then LPS was added to the above induced differentiated THP-1 macrophages at a final concentration of 100 ng / ml, and the culture was continued for 6 h, and then Nigericin was added at a final concentration of 10 μM, and the culture was continued for 1 h, and the cell culture supernatant (LPS and LPS+Nigericin added separately) was collected, and the content of IL-1β in the supernatant was detected, and the supernatant of induced differentiated THP-1 macrophages was used as a control;

[0060] Meanwhile, THP-1 cells were cultured in 1640 medium containing 10% FBS, and TPA was added to a final concentration of 100 ng / ml, and the culture was continued for 12 h to obtain induced differentiated THP-1 macrophages; different concentrations of MCC950 (1, 10, 100 μM) were added to the above induced differentiated THP-1 macrophages, and the culture was continued for 12 h, then LPS was added to a final concentration of 100 ng / ml, and the culture was continued for 6 h, then Nigericin was added to a final concentration of 10 μM, and the culture was continued for 1 h, the cell culture supernatant was collected, and the content of IL-1β in the supernatant was detected, taking the supernatant of cells without the addition of MCC950 as a control, and the results are shown in Figure 1A .

[0061] The content of IL-1β in the supernatant was tested by ELISA method (referring to the method in ELISA MAX TM Deluxe Set Human IL-1β kit), specifically including the following steps:

[0062] 1) 100 μl of caputure antibody (1x) was added to a 96-well plate, sealed, and incubated at 4°C overnight;

[0063] 2) The liquid in the well was poured out, and each well was washed with 300 μl of PBST for 3 times, each time after shaking gently, the liquid was poured out, if there were bubbles on the plate after pouring, the bubbles were patted off on the absorbent paper before adding PBST, and the residual washing liquid was patted dry on the absorbent paper after the last washing;

[0064] 3) 200 μl of Blocking Buffer (1x) was added to each well, sealed, and incubated at room temperature for 1 h;

[0065] 4) After incubation, each well was washed with 300 μl of PBST for 3 times;

[0066] 5) 50 μl of Assay Buffer D was added to each well, and 50 μl of sample diluent or standard was added to the corresponding well, and each sample diluent and standard was repeated in 2 wells; the plate was sealed, and incubated at room temperature for 2 h;

[0067] 6) After incubation, each well was washed with 300 μl of PBST for 3 times;

[0068] 7) 100 μl of Detection Antibody (1x) was added to each well, the plate was sealed, and incubated at room temperature for 1 h;

[0069] 8) After incubation, each well was washed with 300 μl of PBST for 3 times;

[0070] 9. Add 100 μl diluted Avdin-HRP (1x) to each well, seal the plate and incubate at room temperature for 30 min;

[0071] 10) After incubation, wash each well with 300 μl PBST for 4 times;

[0072] 11) Add 100 μl Substrate Solution F to each well, do not seal the plate, and incubate at room temperature for 5 min or so. The positive control wells will turn blue.

[0073] 12) Add 100 μl Stop Solution to each well to terminate the reaction. The positive control wells will turn yellow from blue. Measure the absorbance at 450 nm within 15 min using a microplate reader.

[0074] According to the standard curve prepared from the standard, the content of IL-1β in the sample can be calculated.

[0075] As can be seen from Figure 1A , LPS and Nigericin can activate NLRP3 inflammasome, and MCC950 can effectively inhibit the activation of NLRP3 inflammasome. The results show that the NLRP3 inflammasome activation model is successfully constructed.

[0076] 1.2 Screening of NLRP3 inflammasome activation inhibitors

[0077] THP-1 cells were cultured in 1640 medium containing 10% FBS, and TPA was added to a final concentration of 100 ng / ml, and the culture was continued for 12 h to obtain induced differentiated THP-1 macrophages. The above induced differentiated THP-1 macrophages were added with positive control MCC950 and other drugs (concentration of 10 μM), and the culture was continued for 12 h, then LPS was added to a final concentration of 100 ng / ml, and the culture was continued for 6 h, then Nigericin was added to a final concentration of 10 μM, and the culture was continued for 1 h. The cell culture supernatant was collected, and the content of IL-1β in the supernatant was detected by the method in step 1.1, and the results are shown in Figures 1B-1I .

[0078] As can be seen from Figures 1B-1I , 8 drugs (Loteprednol etabonate (LE), Betamethason, Hydrocortison, Prednisolone, Dexamethaso, Andrographolide, Luteolin, Apigenin (LY08040)) can effectively inhibit the activation of NLRP3 inflammasome.

[0079] Furthermore, THP-1 cells were cultured in 1640 medium containing 10% FBS, and TPA was added to a final concentration of 100 ng / ml. The cells were cultured for another 12 h to obtain induced THP-1 macrophages. Positive control MCC950 and other drugs (both at 10 μM) were added to the induced THP-1 macrophages, and the cells were cultured for another 12 h. Then, LPS was added to a final concentration of 100 ng / ml, and the cells were cultured for another 6 h. Next, Nigericin was added to a final concentration of 10 μM, and the cells were cultured for another 1 h. Cells were then collected, and total RNA was extracted using an RNA extraction kit (Kangwei Century, #CW0581M). The RNA was then analyzed using 5×EasyQuick RT. MasterMix (Kangwei Century, #CW2019M) was reverse transcribed into cDNA. The cDNA template was then used in the following mixture: 10 μl of 2×SYBR Green mixture (Kangwei Century, #CW3008H), 1 μl of cDNA template, 1 μl of specific primers (F and R primers), and 8 μl of ddH2O for RT-PCR. GAPDH was used as an internal control gene to obtain the expression level of IL-1β mRNA in cells. The results are shown below. Figures 2A-2H As shown.

[0080] The specific primers have the following sequences:

[0081] hGAPDH-F: 5′-AAGGCTGTGGGCAAGG-3′ (SEQ ID NO: 1);

[0082] hGAPDH-R: 5′-TGGAGGAGTGGGTGTCG-3′ (SEQ ID NO: 2);

[0083] hIL-1β-F: 5′-CACGATGCACCTGTACGATCA-3′ (SEQ ID NO: 3);

[0084] hIL-1β-R: 5′-GTTGCTCCATATCCTGTCCCT-3′ (SEQ ID NO: 4).

[0085] from Figures 2A-2H As shown, all eight drugs mentioned above can effectively downregulate the expression level of IL-1β mRNA, and loteprednol etabonate has the most significant inhibitory effect. Therefore, this invention screens and obtains the NLRP3 inflammasome activation inhibitor (loteprednol) with the best inhibitory effect.

[0086] Example 2 Mechanism study of Loteprednol etabonate inhibiting NLRP3 inflammasome activation

[0087] THP-1 cells were cultured in 1640 medium containing 10% FBS, and TPA was added at a final concentration of 100 ng / ml, and the culture was continued for 12 h to obtain induced differentiated THP-1 macrophages; the above induced differentiated THP-1 macrophages were added with positive control MCC950 and positive control Luteolin screened in Example 1 (both at a concentration of 10 μM), and the culture was continued for 12 h, then LPS was added at a final concentration of 100 ng / ml, and the culture was continued for 6 h, then Nigericin was added at a final concentration of 10 μM, and the culture was continued for 1 h, and the cells and cell culture supernatant were collected, respectively, and the content of IL-1β in the cell culture supernatant was detected by the method in Example 1, and at the same time, the mRNA expression levels of IL-1β, NLRP3 and Caspase-1 in the cells were detected by the method in Example 1, and the results are shown in Figure 3

[0088] Among them, the specific primers have the following sequences:

[0089] hNLRP3-F: 5'-AAGGGCCATGGACTATTTCC-3' (SEQ ID NO: 5);

[0090] hNLRP3-R: 5'-GACTCCACCCGATGACAGTT-3' (SEQ ID NO: 6);

[0091] hCaspase-1-F: 5'-GCTGAGGTTGACATCACAGGCA-3' (SEQ ID NO: 7);

[0092] hCaspase-1-R: 5'-TGCTGTCAGAGGTCTTGTGCTC-3' (SEQ ID NO: 8).

[0093] As can be seen from Figure 3 , Loteprednol etabonate inhibits NLRP3 inflammasome activation by reducing the secretion of IL-1β and inhibiting the expression level of IL-1β mRNA, in addition, Loteprednol etabonate has little effect on the mRNA transcription level of NLRP3 and Caspase-1.

[0094] ​To further study the inhibitory effect of Loteprednol etabonate on IL-1β mRNA level, THP-1 cells and BMDMs cells were treated with different doses (0.1, 1, 10 μM) of Loteprednol etabonate for 12 h, and then treated with LPS at a final concentration of 100 ng / ml for 6 h. The expression levels of GAPDH, p-c-jun, c-jun, p-p65 and p65 proteins were detected by Western blotting, and the results are shown in Figure 4 .

[0095] As can be seen from Figure 4 , in THP-1 cells and BMDMs, Loteprednol etabonate can inhibit the phosphorylation levels of p65 protein in NF-κB signaling pathway and c-jun protein in AP-1 signaling pathway, proving that Loteprednol etabonate can inhibit the activation of NF-κB signaling pathway and AP-1 signaling pathway, thereby reducing the expression and secretion of IL-1β, and the above inhibition is dose-dependent.

[0096] Example 3 Cell toxicity test of Loteprednol etabonate

[0097] THP-1 cells were cultured in 1640 medium containing 10% FBS, and TPA was added at a final concentration of 100 ng / ml, and the culture was continued for 12 h to obtain induced differentiated THP-1 macrophages. Different concentrations (0, 2.5, 5, 10, 20, 40, 80, 160, 320 μM) of Loteprednol etabonate were added to the above induced differentiated THP-1 macrophages, and the cell toxicity of the drug was detected by CCK8 kit, and the results are shown in Figure 5 .

[0098] As can be seen from Figure 5 , when the concentration of Loteprednol etabonate is below 80 μM, the toxicity to cells is low, and the results show that Loteprednol etabonate has good biological safety.

[0099] Example 4 Concentration-dependent test of Loteprednol etabonate inhibiting NLRP3 inflammasome activation

[0100] THP-1 cells were cultured in 1640 medium containing 10% FBS, and TPA was added to a final concentration of 100 ng / ml, and the cells were cultured for 12 h to obtain induced differentiated THP-1 macrophages; different concentrations of Loteprednol etabonate (0, 0.1, 1, 10 μM) were added to the above induced differentiated THP-1 macrophages, and the cells were cultured for 12 h, then LPS was added to a final concentration of 100 ng / ml, and the cells were cultured for 6 h, then Nigericin was added to a final concentration of 10 μM, and the cells were cultured for 1 h, and the cells and cell culture supernatant were collected, and the content of IL-1β in the cell culture supernatant was detected by the method in Example 1, and at the same time, the mRNA expression levels of IL-1β, NLRP3 and Caspase-1 in the cells were detected by the method in Example 2, and the results are shown in Figure 6 .

[0101] As can be seen from Figure 6 , Loteprednol etabonate inhibits the activation of NLRP3 inflammasome by reducing the secretion of IL-1β and inhibiting the mRNA expression level of IL-1β, and is concentration-dependent; in addition, Loteprednol etabonate has little effect on the mRNA transcription levels of NLRP3 and Caspase-1.

[0102] Further, THP-1 cells were cultured in 1640 medium containing 10% FBS, and TPA was added to a final concentration of 100 ng / ml, and the cells were cultured for 12 h to obtain induced differentiated THP-1 macrophages; different concentrations of Loteprednol etabonate (0, 10 -6 , 10 -4 , 10 -2 μM) were added to the above induced differentiated THP-1 macrophages, and the cells were cultured for 12 h, then LPS was added to a final concentration of 100 ng / ml, and the cells were cultured for 6 h, then Nigericin was added to a final concentration of 10 μM, and the cells were cultured for 1 h, and the cell culture supernatant was collected, and the content of IL-1β in the cell culture supernatant was detected by the method in Example 1, and the results are shown in Figure 7 .

[0103] As can be seen from Figure 7 , the half-effective concentration IC 50 of Loteprednol etabonate is 7.034 x 10 -5 μM.

[0104] Example 5 Loteprednol etabonate effectively alleviates LPS-induced sepsis and improves survival rate in mice

[0105] C57BL / 6 mice were divided into four groups, and each group of mice was treated as follows: the first group was injected intraperitoneally with normal saline (n = 4), the second group was injected intraperitoneally with Loteprednol etabonate (20 mg / kg, n = 4), the third group was injected intraperitoneally with LPS (30 mg / kg, n = 6), and the fourth group was injected intraperitoneally with LPS (30 mg / kg) and Loteprednol etabonate (20 mg / kg, n = 6). In the fourth group, Loteprednol etabonate was injected intraperitoneally first, and then LPS was injected 2 h later. After 4 h, blood was taken from the eyeball to measure inflammatory factors (IL-1β and TNF-ɑ), and the results are shown in Figure 8 .

[0106] As can be seen from Figure 8 , Loteprednol etabonate can effectively inhibit the secretion of IL-1β and TNF-ɑ.

[0107] Further, C57BL / 6 mice were divided into seven groups, and each group of mice was treated as follows: the first group was injected intraperitoneally with normal saline (n = 8), the second group was injected intraperitoneally with Loteprednol etabonate (40 mg / kg, n = 8), the third group was injected intraperitoneally with LPS (20 mg / kg, n = 12), the fourth group was injected intraperitoneally with LPS (20 mg / kg) and Loteprednol etabonate (20 mg / kg, n = 12), the fifth group was injected intraperitoneally with LPS (20 mg / kg) and Loteprednol etabonate (40 mg / kg, n = 12), the sixth group was injected intraperitoneally with MCC950 (20 mg / kg, n = 8), and the seventh group was injected intraperitoneally with LPS (20 mg / kg) and MCC950 (20 mg / kg, n = 12). In the fourth, fifth, and seventh groups, Loteprednol etabonate or MCC950 was injected intraperitoneally first, and then LPS was injected 2 h later. Then, the survival rate and body weight of the mice were observed daily, and the results are shown in Figure 9 .

[0108] The C57BL / 6 mice were divided into seven groups, and each group of mice was treated as follows: the first group was intraperitoneally injected with normal saline (n = 8), the second group was intraperitoneally injected with Loteprednol etabonate (40 mg / kg, n = 8), the third group was intraperitoneally injected with LPS (20 mg / kg, n = 12), the fourth group was intraperitoneally injected with LPS (20 mg / kg) and Loteprednol etabonate (20 mg / kg, n = 12), the fifth group was intraperitoneally injected with LPS (20 mg / kg) and Loteprednol etabonate (40 mg / kg, n = 12), the sixth group was intraperitoneally injected with MCC950 (20 mg / kg, n = 8), and the seventh group was intraperitoneally injected with LPS (20 mg / kg) and MCC950 (20 mg / kg, n = 12). In the fourth, fifth, and seventh groups, LPS was injected intraperitoneally first, and then Loteprednol etabonate or MCC950 was injected intraperitoneally 1 h later. The survival rate and body weight of the mice were observed at regular intervals every day, and the results are shown in Figure 10 .

[0109] As can be seen from Figures 9-10 , whether Loteprednol etabonate or MCC950 was injected intraperitoneally first and LPS was injected intraperitoneally 2 h later, or LPS was injected intraperitoneally first and Loteprednol etabonate or MCC950 was injected intraperitoneally 1 h later, Loteprednol etabonate could effectively inhibit the LPS-induced sepsis model, improve the survival rate of the mice, and alleviate the decrease in body weight.

[0110] Example 6 Loteprednol etabonate effectively alleviates DSS-induced colitis in mice

[0111] The C57BL / 6 mice were divided into five groups, and each group of mice was treated as follows: the first group was intraperitoneally injected with normal saline (n = 12), the second group was intraperitoneally injected with normal saline and orally administered DSS (3%), the third group was intraperitoneally injected with Loteprednol etabonate (20 mg / kg) and orally administered DSS (3%), the fourth group was intraperitoneally injected with Loteprednol etabonate (40 mg / kg) and orally administered DSS (3%), and the fifth group was intraperitoneally injected with MCC950 (20 mg / kg) and orally administered DSS (3%), wherein the mouse colitis model was induced by orally administering DSS (3%). After eight days, each group was fed with normal water, and all the mice were treated on the tenth day. The body weight of the mice was observed and recorded every day, and the results are shown in Figure 11AAs shown; daily observation of mouse fecal changes and disease index scoring were performed, with 0 points indicating normal, 1 point indicating soft feces, 2 points indicating soft feces with bleeding, 3 points indicating loose feces with bleeding, and 4 points indicating massive bleeding. Results are as follows. Figure 11B As shown.

[0112] from Figure 11A As can be seen, Loteprednol etabonate can alleviate the weight loss in mice with colitis. From Figure 11B As can be seen, Loteprednol etabonate can alleviate the symptoms of diarrhea and bloody stools in mice with colitis.

[0113] On the tenth day, colons from each treatment group were photographed and their lengths measured. Loteprednol etabonate was found to alleviate colonic shortening caused by acute colitis. Figure 11C and 11D HE staining pathological analysis was performed on the colons of each treatment group, and the results are as follows: Figure 11E As shown.

[0114] from Figure 11E As can be seen, Loteprednol etabonate can effectively alleviate symptoms such as mucosal edema, goblet cell loss, and inflammatory cell infiltration induced by DSS. The results indicate that Loteprednol etabonate can effectively alleviate DSS-induced colitis in mice.

[0115] In summary, this invention is the first to discover that etacloprofen significantly inhibits NLRP3 inflammasome activation by reducing IL-1β secretion, and therefore can be used to prepare drugs for the prevention and / or treatment of NLRP3 inflammasome-related diseases.

[0116] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0117] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Use of descinolone acetonide for the manufacture of a medicament for the prevention and / or treatment of NLRP3 inflammasome-related diseases, characterized in that, The NLRP3 inflammasome related disease is at least one of sepsis, colitis.

2. Use according to claim 1, characterized in that, The iclocinlotide inhibits the expression of IL-1β by inhibiting the NF-κB and / or AP-1 signaling pathway, thereby inhibiting the activation of the NLRP3 inflammasome.

3. Use of an NLRP3 inflammasome activation inhibitor or a pharmaceutical composition in the manufacture of a medicament for the prevention and / or treatment of an NLRP3 inflammasome-related disease, characterized in that, The NLRP3 inflammasome related disease is at least one of sepsis, colitis. The NLRP3 inflammasome activation inhibitor comprises iclocinlotide. The pharmaceutical composition comprises the NLRP3 inflammasome activation inhibitor.

4. Use according to claim 3, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

5. Use according to claim 3, characterized in that, The dosage form of the pharmaceutical composition comprises at least one of a solid preparation, a semi-solid preparation, and a liquid preparation.

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