CN112716928B

CN112716928B - Application of capsaicin in inhibiting activation of NLRP3 inflammatory corpuscle

Info

Publication number: CN112716928B
Application number: CN202110196432.4A
Authority: CN
Inventors: 张云娇; 邓育辉; 温龙平
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2021-02-22
Filing date: 2021-02-22
Publication date: 2022-05-24
Anticipated expiration: 2041-02-22
Also published as: CN112716928A

Abstract

The invention discloses application of capsaicin in inhibiting activation of NLRP3 inflammatory bodies. According to the invention, through research, capsaicin can inhibit the activation of various agonists such as nigericin, microcrystalline sodium urate and alum on NLRP3 inflammasome, but does not affect the activation of NLRC4, AIM2 and other inflammasome, and can also inhibit the assembly of NLRP3 inflammasome, including the inhibition of the combination of NLRP3 and ASC protein and the formation of ASC spots. In addition, the invention also discovers that the capsaicin can inhibit the lipopolysaccharide-induced septicemia. Therefore, the capsaicin can be used as an inhibitor for activation of NLRP3 inflammasome and a potential medicament for preventing or treating diseases related to abnormal activation of NLRP3 inflammasome.

Description

Application of capsaicin in inhibiting activation of NLRP3 inflammatory corpuscle

Technical Field

The invention belongs to the field of biology and medicine, and particularly relates to application of capsaicin in inhibition of activation of NLRP3 inflammasome.

Background

Inflammasome is a cytoplasmic polyprotein complex composed of members of the Nod-like receptor (NLR) family and members of the pyhin (pyrin and HIN domain) family, which can be activated by a variety of pathogen-or injury-associated molecular patterns. The function of the inflammasome is to activate Caspase 1(Caspase-1), which in turn causes the maturation and secretion of the proinflammatory cytokines Interleukin (IL) -1 beta and IL-18, and induces apoptosis. The NLR family protein 3(NLRP3) inflammasome is a macromolecular polyprotein complex composed of NLRP3, linker protein ASC and effector protein Caspase-1. Unlike other inflammasomes, NLRP3 inflammasomes can be activated by a variety of stimuli, including microbial components and endogenous molecules.

When the body is stimulated by foreign microbial infections (PAMPs) or injury signals from the body (DAMPs), the innate immune system is activated to activate inflammatory responses through pattern recognition receptors or induction systems on the surface of the body, and release of type I interferons (interferon-alpha and interferon-beta) or inflammatory factors IL-1 beta, IL-18, IL-33 and the like is promoted.

Abnormal activation of nucleotide-binding oligomerization domain-like receptor family 3(NOD-like receptor family, pyrin domain-binding 3, NL-RP3) inflammasome is closely related to occurrence and development of various inflammatory diseases of human body, and researches indicate that abnormal activation of NLRP3 inflammasome is closely related to central nervous system diseases, cardiovascular system diseases, respiratory system diseases, urinary system diseases, digestive system diseases and the like. Therefore, specific inhibitors of the NLRP3 inflammasome were found to be of great interest for the treatment of NLRP 3-related diseases.

Capsaicin (hereinafter abbreviated as CAP) is an active ingredient of capsicum and is clinically used for analgesia, but specific studies on inflammatory mechanisms have not been reported so far.

Disclosure of Invention

The primary object of the present invention is to overcome the disadvantages and drawbacks of the prior art and to provide the use of capsaicin for inhibiting the activation of NLRP3 inflammatory bodies.

Another object of the present invention is to provide the use of capsaicin for the preparation of a medicament for inhibiting the activation of NLRP3 inflammasome.

Still another object of the present invention is to provide the use of capsaicin for the preparation of an inhibitor of NLRP3 inflammasome activation.

The purpose of the invention is realized by the following technical scheme:

use of capsaicin for inhibiting activation of NLRP3 inflammasome; the environment of the application is an in vitro environment.

Use of capsaicin for the manufacture of a medicament for inhibiting the activation of NLRP3 inflammasome.

Use of capsaicin in preparation of NLRP3 inflammation body activation inhibitor.

The activation of NLRP3 inflammasome comprises NLRP3 inflammasome activation induced by NLRP3 inflammasome activation activator and activation of NLRP3 inflammasome induced by Lipopolysaccharide (LPS); preferably activation of NLRP3 inflamed bodies induced by at least one of Nigericin (Nigericin), sodium urate (MSU), Alum (Alum) and Lipopolysaccharide (LPS).

The sodium urate is preferably Microcrystalline Sodium Urate (MSU).

The application of capsaicin in preparing a medicament for preventing and/or treating NLRP3 inflammatory-corpuscle abnormal activation related diseases, wherein the NLRP3 inflammatory-corpuscle abnormal activation related diseases are at least one of peritonitis, type II diabetes atherosclerosis, gout, rheumatoid arthritis, Parkinson multiple sclerosis, Alzheimer disease and septicemia.

The capsaicin can inhibit NLRP3 inflammatory corpuscle from activating oligomerization of downstream ASC and inhibit formation of ASC oligomers; can inhibit self oligomerization of NLRP3 protein and inhibit outflow of K ions, thereby achieving the effect of inhibiting NLRP3 inflammatory pathway; capsaicin can also reduce the level of IL-1 beta in serum and ascites, and can also improve the infiltration condition of neutrophils in the abdominal cavity, thereby promoting the body to resist septicemia.

The medicament can also contain one or at least two pharmaceutically acceptable carriers.

The carrier is preferably a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an absorption enhancer, an adsorption carrier, a surfactant, a lubricant and the like.

The medicine can be prepared into various dosage forms by adopting a conventional method in the field, including decoction, tablets, pills, capsules, injection (such as powder injection), granules (such as granules), oral liquid and syrup, or the medicine can be prepared into tablets, capsules, injection, powder injection, granules and the like by adopting a micro-nano technology.

Compared with the prior art, the invention has the following advantages and effects:

(1) during research, the inventor of the invention finds that capsaicin can inhibit the activation of different agonists such as Alum, Nigericin, MSU and the like on NLRP3 inflammasome, but does not influence the activation of other inflammasome, such as NLRC4 and AIM2 inflammasome (after adding NLRC4 inflammasome agonist Salmonella, the capsaicin cannot influence the release of IL-1 beta caused by the activation), and can also inhibit the assembly of NLRP3 inflammasome, including the inhibition of the combination of NLRP3 and ASC protein and the formation of ASC spots; capsaicin can inhibit Lipopolysaccharide (LPS) induced septicemia, and can be used as an inhibitor for activation of NLRP3 inflammatory bodies and a potential medicament for preventing or treating diseases related to abnormal activation of NLRP3 inflammatory bodies.

(2) The invention initially researches the action and mechanism of capsaicin on NLRP3 inflammatory corpuscle activation through methods such as biology, chemistry, zoology and the like, and the capsaicin belongs to natural plant components and has higher biological safety as a mature analgesic drug, and the research provides a new strategy for the capsaicin to be used for treating inflammation-related diseases. The invention researches the influence of capsaicin on the activation of inflammatory corpuscles, and particularly has great research significance on the action and the mechanism of NLRP3 inflammatory corpuscles.

Drawings

FIG. 1 is a graph showing the inhibitory effect of capsaicin on IL1- β release after LPS + Nigericin-induced activation of NLRP3 inflammasome in example 1 of the present invention.

FIG. 2 is a graph showing the inhibitory effect of capsaicin on IL1- β release after LPS + Nigericin-induced activation of NLRP3 inflammasome in example 1 of the present invention.

FIG. 3 is a graph showing the inhibitory effect of capsaicin in example 1 of the present invention on IL1- β release after activation of NLRP3 inflammasome induced by LPS + MSU.

FIG. 4 is a graph showing the inhibitory effect of capsaicin in example 1 on the release of IL 1-beta after LPS + Alum-induced activation of NLRP3 inflammasome.

FIG. 5 is a graph showing the effect of capsaicin on IL1- β release after activation of NLRC4 inflammasome induced by LPS + Salmonella bacterial suspension in example 1 of the present invention.

FIG. 6 is a graph of the effect of capsaicin on ROS expression in bone marrow-derived macrophages after stimulation with LPS + Nigericin in example 2 of the present invention.

FIG. 7 is a graph of the effect of capsaicin on the oligomerization of bone marrow-derived macrophages after stimulation with LPS + Nigericin in example 3 of the present invention.

FIG. 8 is a graph showing the effect of capsaicin on the formation of bone marrow-derived macrophage ASC spots after LPS + Nigericin stimulation in example 4 of the present invention.

FIG. 9 is a graph of the effect of capsaicin on the oligomerization of bone marrow-derived macrophages NLRP3 following LPS + Nigericin stimulation in example 5 of the invention.

FIG. 10 shows intracellular K after stimulation of LPS + Nigericin by capsaicin in example 6 of the present invention⁺Influence of outflow.

FIG. 11 shows intracellular Cl after stimulation of LPS + Nigericin by capsaicin in example 7 of the present invention^–Graph of the effect of outflow.

FIG. 12 is a graph showing the effect of capsaicin in example 8 on neutrophil infiltration in the abdominal cavity of mice after LPS-induced sepsis in the mice.

FIG. 13 is a graph showing the effect of capsaicin on the expression of IL-1. beta. in mouse serum after LPS-induced sepsis in mice in example 8 of the present invention.

FIG. 14 is a graph showing the effect of capsaicin in example 8 on the expression of IL-1. beta. in the abdominal cavity of mice after LPS-induced sepsis in the mice.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.

The present invention provides the use of Capsaicin (CAP) for inhibiting the activation of NLRP3 inflammasome.

In the present invention, the capsaicin has a structure represented by formula (I):

in the invention, the term "activation of NLRP3 inflammasome" refers to the combination of NLRP3, ASC and Pro-Caspase1 to assemble a multifunctional protein complex under the action of activator lipopolysaccharide and Nigericin (LPS + Nigericin), wherein Pro-Caspase1 is self-sheared into Caspase1 and participates in the subsequent inflammation generation and development process; activated caspase1 cleaves Pro-IL-1. beta. and Pro-IL-18 precursors as active factors and releases them out of the cell.

In the present invention, the capsaicin specifically inhibits the activation of NLRP3 inflammasome without affecting the activation of NLRC4 inflammasome.

In the present invention, the capsaicin does not affect the transcriptional expression of NLRP3 inflammatory-body key proteins and inflammatory factors during the activation of the inflammatory-body; the key proteins are NLRP3, ASC, Pro-Caspase1 and Pro-IL-1 beta; the inflammatory factor is IL-1 beta; and the inhibition effect of the capsaicin on NLRP3 inflammasome is to inhibit the combination of NLRP3 and ASC protein in the activation process of the inflammasome.

In the present invention, the administration mode is in vivo administration and in vitro administration; wherein, the in vivo administration mode of the capsaicin is particularly intraperitoneal injection.

In the present invention, there is also provided a method for preventing and treating diseases associated with abnormal activation of NLRP3 inflammasome, which refers to: capsaicin is administered to a patient suffering from, or likely to suffer from, a disease associated with abnormal activation of NLRP3 inflammasome.

In the invention, the NLRP3 inflammatory body abnormal activation related diseases are septicemia and peritonitis.

In the present invention, the formulation of the capsaicin to be administered is not particularly limited, and a general formulation is an injection.

The present invention is described in detail below with reference to examples.

Example 1

This example illustrates the inhibitory effect of capsaicin on the maturation and release of IL-1 β processing downstream of the NLRP3 inflammasome pathway.

(1) Acquisition of mouse bone marrow-derived macrophages (BMDM): bone marrow cells of C57 mice (purchased from Shanghai, Sjogren-Biotech, SPF grade 8 week old male mice) were harvested at 1X 10⁶Each/ml was inoculated into a large dish, and cultured for 5 days in a DMEM medium containing 20% (v/v) of L929 cell supernatant (L929 cells purchased from Shanghai Korea, Ltd., product No. CBP60878, cell culture in laboratory L929 to obtain a medium containing secretion) and 10% (v/v) of fetal bovine serum (purchased from Shanghai Microbiol., product No. S1580-500), and the DMEM complete medium containing L929 cell supernatant was changed every 3 days.

(2) In 24-well plates, according to 5X 10⁵Each cell/well was inoculated with BMDM obtained in step (1), incubated overnight, treated with 100ng/ml lipopolysaccharide (LPS, from Sigma-Aldrich, cat # L2880) for 3h, then treated with various concentrations (0, 10, 20, 40. mu.M) of Capsaicin (Capsaicin, from Cayman, cat # 92350) for 1h, the supernatant removed, and 5 addedmu.M Nigericin (Nigericin, from Sigma, cat # 481990) was treated for 30min, the supernatant was collected and IL-1. beta. levels were detected using immunoblotting techniques and the Elisa method. The results are shown in FIGS. 1 and 2 (Mock: negative control; capsaicin concentration in FIG. 2: 40. mu.M).

(3) In 24-well plates, according to 5X 10⁵Each cell/well was inoculated with BMDM obtained in step (1), cultured overnight, treated with 100ng/ml lipopolysaccharide for 3h, then treated with capsaicin at various concentrations (0, 10, 20, 40. mu.M) for 1h, the supernatant was removed, treated with 400ug/ml microcrystalline sodium urate (MSU, available from Sigma, cat. No. U2875) for 3h, the supernatant was collected, and IL-1. beta. level was detected by immunoblotting technique. The results are shown in FIG. 3.

(4) In 24-well plates, according to 5X 10⁵Each cell/well was inoculated with BMDM obtained in step (1), cultured overnight, treated with 100ng/ml lipopolysaccharide for 3 hours, then treated with capsaicin at various concentrations (0, 10, 20, 40. mu.M) for 1 hour, the supernatant was removed, treated with 50ug/ml Alum (Alum; Imject Alum Adjuvant, available from Thermo Fisher, cat. No. 77161) for 3 hours, the supernatant was collected, and IL-1. beta. level was determined by immunoblotting technique. The results are shown in FIG. 4.

(5) Putting 6ml of sterile LB culture medium into a 15ml centrifuge tube, adding 10ul of Salmonella (Salmonella) ATCC 14028 (bacterial strain purchased from microbiology, Cat. 0363) bacterial liquid, then putting the mixture on a shaking table at 37 ℃ and shaking at 180rpm for 8-12 hours, and stopping culturing when the culture solution is slightly turbid; taking 1ml of cultured bacteria culture solution, setting the rotating speed at 4000rpm, and centrifuging for 5 min; taking the bacterial precipitate, adding 1ml of PBS buffer solution for re-suspending for later use;

② in 24-hole plate, according to 5 × 10⁵Inoculating BMDM obtained in the step (1) into each cell/hole, culturing overnight, replacing the original culture medium with Opti-MEM containing 100ng/ml lipopolysaccharide for 3h, then adding capsaicin with different concentrations (0, 10, 20 and 40 mu M) for 1h, removing supernatant, adding 10ul of the heavy suspension liquid for treating for 30min, replacing the original Opti-MEM culture medium with Opti-MEM containing gentamicin (with a final concentration of 50 mu g/ml) to inhibit continuous propagation of the Salmonella extracellular (Salmonella), collecting supernatant after culturing for 3h, and detecting the level of IL-1 beta by using an immunoblotting technology. The results are shown in the figure5, respectively.

As can be seen from the results of fig. 1, 2, 3 and 4, under the action of NLRP3 inflammation corpuscle specific agonists Alum (Alum), Nigericin (Nigericin), sodium urate (MSU) and the like, BMDM cells release a large amount of IL-1 β, and capsaicin can inhibit the effect; as shown in FIG. 5, capsaicin did not have inhibitory effect on IL-1 β release caused by Salmonella (Salmonella), an inflammasome-specific agonist of NLRC4 inflammasome, and therefore capsaicin specifically inhibited IL-1 β processing maturation and release downstream of NLRP3 inflammasome activation.

Example 2

This example is presented to illustrate the effect of capsaicin on ROS production during activation of NLRP3 inflammasome.

(1) Acquisition of BMDM cells: same as in step (1) of example 1.

(2) In a six-well plate, according to 2X 10⁶The density of individual cells/well was inoculated with BMDM overnight culture. Adding LPS with the final concentration of 100ng/ml for treatment for 3h the next day, then adding capsaicin with the final concentration of 40 mu M for treatment for 1h, adding Nigericin (Nigericin) with the final concentration of 5 mu M for treatment for 30min, removing supernatant, digesting and dissociating cells to obtain cell suspension, adding PBS buffer solution containing ROS fluorescent probe DCFH-DA (purchased from Thermo Fisher, product number C2938), incubating for 15min in the dark, and detecting the ROS level in the cells by using flow cytometry. The results are shown in FIG. 6 (LPS + Capsaicin: the treatment group to which only LPS and Capsaicin were added; Nigericin + Capsaicin: the treatment group to which LPS, Nigericin and Capsaicin were added; Medium: the untreated negative control group).

As can be seen from fig. 6, compared to the agonist Nigericin group, the addition of capsaicin can significantly inhibit the right shift of the curve, which indicates that capsaicin can significantly inhibit the generation of ROS, and ROS participates in a series of inflammation-related reactions of the body as an important intermediate in the activation process of NLRP3 inflammatory bodies, which indicates that capsaicin has an inhibitory effect on the generation of inflammation.

Example 3

This example serves to illustrate the inhibitory effect of capsaicin on the ASC oligomerization event during NLRP3 inflammasome activation.

(1) Acquisition of BMDM: same as in step (1) of example 1.

(2) In a six-well plate, according to 2X 10⁶The density of individual cells/well was inoculated with BMDM overnight culture. The following day, LPS was added to a final concentration of 100ng/ml for 3h, then Capsaicin (CAP) was added to a final concentration of 40. mu.M for 1h, Nigericin (Nigericin) was added to a final concentration of 5. mu.M for 30min, the supernatant was removed, NP-40 lysate containing protease inhibitor was added to lyse the cells, and the cells were incubated with shaking on ice for 30 min. Then, a part of cell lysate was taken as an internal reference for the next immunoprecipitation experiment, and the remaining lysate was centrifuged at 6000 Xg for 10min at 4 ℃. The pellet was washed 3 times with PBS buffer, DSS (Disuccinimidyl suberate, Disuccinimidyl suberate, Disuccinimidyl suberate from Thermo Fisher Scientific, cat # 21555) was added as a protein cross-linker, the reaction was shaken for 30min, the reaction product was centrifuged at 6000 Xg for 10min, and the pellet was subjected to immunoblotting (LPS alone, and LPS + Nigericin as controls). The results are shown in FIG. 7.

As can be seen from FIG. 7, capsaicin inhibited the production of ASC oligomers during activation of NLRP3 inflammasome, which are an essential component of the NLRP3-ASC-Pro caspase1 complex whose assembly is also a "central event" during activation of NLRP3 inflammasome. Thus, it can be concluded that capsaicin can inhibit the formation of ASC oligomers under treatment with the Nigericin, an inflammasome agonist.

Example 4

This example illustrates on the other hand the inhibitory effect of capsaicin on the ASC oligomerization downstream of NLRP3 inflammasome activation.

(1) Acquisition of BMDM: same as in step (1) of example 1.

(2) In 24-well plates, according to 5X 10⁵BMDM is inoculated at the density of each cell/well for overnight culture, LPS with the concentration of 100ng/ml is added for 3h on the next day, then capsaicin with the final concentration of 40 mu M is added for treatment for 1h, nigericin with the concentration of 5 mu M is added for treatment for 30min, and the formation of ASC spots is detected by an immunofluorescence method. The results are shown in FIG. 8.

As can be seen in fig. 8, capsaicin reduced the formation of ASC spots under treatment with the NLRP3 inflammasome agonist, Nigericin, indicating that capsaicin inhibited the oligomerization of ASCs downstream of NLRP3 inflammasome activation.

Example 5

This example serves to illustrate the inhibitory effect of capsaicin on NLRP3 oligomerization in the NLRP3 inflammatory pathway.

(1) 6ml of sterile LB medium was placed in a 15ml centrifuge tube, 10ul of Escherichia coli solution (Escherichia coli K12 strain, purchased from BioMed, Cat. No. BC208-02) carrying Flag-NLRP3 (purchased from Shanghai boundary Ming Biotech, Cat. No. MZB391) and m-Cherry tagged plasmid (purchased from Youbao, Cat. No. VT1431) were added, and the mixture was placed on a 37 ℃ shaker at 180rpm and cultured for 8-12 h with shaking, and the culture was stopped when the culture solution was slightly turbid. Plasmid DNA containing Flag-NLRP3, m-Cherry tag was extracted according to the procedure of Plasmid DNA extraction Kit (OMEGA Kit, Plasmid Mini Kit II D6945, cat # D6945-02).

(2) 293T cells (HEK 293T cells, purchased from Shanghai Baibo, Inc., cat # CBP60439) were seeded at a cell density of 80% in the field of view, LPS was added to a final concentration of 100ng/ml for 3 hours, capsaicin was added to a final concentration of 40. mu.M for 1 hour, Nigericin was added to a final concentration of 5. mu.M for 30 minutes, and plasmids were transfected into 293T cells using a Lipofectamine 3000 kit (purchased from Thermo Fisher, cat # L3000150) (Flag-NLRP 3, M-Cherry-tagged plasmids were simultaneously transferred into 293T cells, and Flag-NLRP3 and M-Cherry-tagged plasmids were separately transferred as controls, while 293T cells not transferred with plasmids were used as blanks). 293T cells were cultured for 2 days after transfection, and cell lysates were taken for the next experiment.

(3) Immunoblotting experiments were performed using antibodies DYKDDDDK Tag Recombinant Antibody (Binds to FLAG, available from Proteintech, cat No. 80010-1-RR) and MCherry Polyclonal Antibody (available from Proteintech, cat No. 26765-1-AP) corresponding to Flag-NLRP3 and mherry-NLRP 3, respectively, to examine the effect of capsaicin on NLRP3 oligomerization. The results are shown in FIG. 9.

After the NLRP3 protein is oligomerized to form a complex, the complex can be assembled with ASC and pro-Caspase 1. Under the condition that 293T cells over-express NLRP3 protein, as can be seen from FIG. 9, capsaicin can inhibit self-oligomerization of NLRP3 protein, and the inhibition effect of capsaicin on the assembly process of NLRP3-ASC protein complex is illustrated.

Example 6

This example illustrates the effect of capsaicin on K content in BMDM cells.

(1) Acquisition of BMDM cells: same as in step (1) of example 1.

(2) In 24-well plates, according to 5X 10⁵BMDM was inoculated at a density of individual cells/well overnight, treated the next day with 100ng/ml LPS for 3h, then with capsaicin (0, 10, 20, 40. mu.M) for 1h, treated with 5. mu.M nigericin for 30min, and the supernatant removed. PBS buffer (containing NaH) without K ions was used₂PO₄、NaCl、Na₂HPO₄.2H₂O and dd H₂O, KH in the formula₂PO₄Replacement by NaH₂PO₄KCl is replaced by NaCl) for 3 times, washing liquid is washed out, 1ml of concentrated nitric acid with the mass concentration of 70% is added into each hole, and blowing and sucking are carried out repeatedly for 5-10 times.

(3) And (3) transferring the lysate obtained in the step (2) into a beaker, adding 2 ml/hole of concentrated nitric acid to wash a 6-hole plate, uniformly putting the lysate into the beaker, heating to 200 ℃, boiling to dryness, adding concentrated nitric acid into the beaker, boiling to dryness, repeating for a plurality of times until the powder is light yellow or white after boiling to dryness (if the powder is brown or tan, adding the nitric acid to boil continuously), and dripping 100 mu l of concentrated nitric acid to dissolve the powder (the concentrated nitric acid used in the step is 70% by mass of the concentrated nitric acid).

(4) The beaker was rinsed with 3ml of double distilled water, pipetted into a 5ml volumetric flask to constant volume, and 2ml was taken into an EP tube and sent for measurement of K ion content (LPS alone, and LPS + Nigericin as controls). The results are shown in FIG. 10.

K ion outflow is an upstream event in the activation process of NLRP3 inflammasome, and it can be seen from fig. 10 that capsaicin can inhibit K ion outflow, thereby achieving the effect of inhibiting NLRP3 inflammatory pathway.

Example 7

This example is presented to illustrate the effect of capsaicin on the Cl content of BMDM cells.

(1) Acquisition of BMDM cells: same as in step (1) of example 1.

(2) In 24-well plates, according to 5X 10⁵BMDM was inoculated at a density of individual cells/well overnight, treated the next day with 100ng/ml LPS for 3h, then with capsaicin (0, 10, 20, 40. mu.M) for 1h, treated with 5. mu.M nigericin for 30min, and the supernatant removed. Adding double distilled water for cracking, placing at 37 ℃ for 30min, repeatedly purging the gun head for 20 times, and transferring into a 1.5ml EP tube.

(3) The supernatant was placed in a refrigerator at-80 ℃ for repeated freeze-thawing 2 times, and then centrifuged at 8000prm for 5min, and the supernatant was taken and placed in a new EP tube, and 50. mu.M of Cl ion dye MQAE (Chloride ion fluorescence probe, available from Thermo Fisher, cat. No. E3101) was added to determine the fluorescence value using a multifunctional microplate reader. The results are shown in FIG. 11.

The outflow of Cl ions is one of the upstream events of NLRP3 inflammasome activation, and as can be seen from figure 11, capsaicin had no effect on Cl content in BMDM, and therefore capsaicin did not inhibit NLRP3 inflammasome activation through the Cl ion pathway.

Example 8

This example illustrates that capsaicin promotes mice against LPS-induced sepsis.

C57BL/6 mice (purchased from shanghai south model organism, SPF grade 8 week old male mice) were intraperitoneally injected with LPS for the establishment of a sepsis model:

a. intraperitoneal injection of 0.75mg/kg Capsaicin, injection of 20mg/kg LPS (LPS + Capsaicin) after 1 hour, blood sampling from eyeball after 6 hours, and detection of IL-1 beta cytokine concentration in serum are carried out, wherein C57BL/6 mice without LPS injection are used as a blank control (Vehicle), and mice with LPS injection are used as a positive control (LPS). The results are shown in FIG. 13;

b. the peritoneal cavity was injected with 0.75mg/kg capsaicin, 20mg/kg LPS was injected after 1 hour, the peritoneal cavity was cut off after 6 hours, PBS was used to wash the peritoneal cavity to obtain neutrophils in the peritoneal cavity, the IL-1. beta. cytokine concentration in the peritoneal cavity was measured, and the infiltration of neutrophils was measured by flow cytometry, and mice injected with LPS alone were used as a control (Vehicle) and mice injected with LPS were used as a positive control (LPS). The results are shown in fig. 12 and 14.

From the results in fig. 13 and 14, it can be seen that LPS treatment causes release of IL-1 β in immune cells in the abdominal cavity, systemic inflammatory factor storm in mice leads to sepsis, capsaicin can reduce the level of IL-1 β in serum and ascites of mice, and from fig. 12, capsaicin also improves neutrophil infiltration in the abdominal cavity, thereby promoting mice to resist sepsis. The results show that the capsaicin can effectively inhibit the activation of NLRP3 inflammatory bodies in vivo and promote mice to resist septicemia caused by abnormal activation of the inflammatory bodies.

As can be seen from the results of examples 1-8 above, capsaicin inhibited the activation of the Nigericin, MSU and Alum-induced NLRP3 inflammasome, but did not affect the activation of the NLRC4 inflammasome; capsaicin inhibits the generation of downstream ROS by inhibiting the outflow of K ions, and further inhibits the central event of the generation of NLRP3-ASC-Pro caspase1 complex. Meanwhile, capsaicin also inhibits LPS-induced sepsis. Therefore, the capsaicin can be used as a potential medicament for treating diseases related to NLRP3 inflammatory body abnormal activation.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. Use of capsaicin for inhibiting the activation of NLRP3 inflammasome, characterized by: the environment of the application is an in vitro environment.

2. Use according to claim 1, characterized in that: the activation of NLRP3 inflammasome is NLRP3 inflammasome activation induced by NLRP3 inflammasome activation activator or activation of NLRP3 inflammasome induced by lipopolysaccharide.

3. Use according to claim 2, characterized in that: the activation of the NLRP3 inflammasome is the activation of the NLRP3 inflammasome induced by at least one of nigericin, sodium urate, alum and lipopolysaccharide.

4. Use according to claim 3, characterized in that: the sodium urate is microcrystalline sodium urate.