CN118078826A - Application of clioquinol in preparation of NLRP3 inflammation small inhibitor and medicine for treating or preventing related diseases - Google Patents
Application of clioquinol in preparation of NLRP3 inflammation small inhibitor and medicine for treating or preventing related diseases Download PDFInfo
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- CN118078826A CN118078826A CN202410289278.9A CN202410289278A CN118078826A CN 118078826 A CN118078826 A CN 118078826A CN 202410289278 A CN202410289278 A CN 202410289278A CN 118078826 A CN118078826 A CN 118078826A
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Abstract
The invention discloses application of clioquinol in preparing an NLRP3 inflammation small body inhibitor and a medicine for treating or preventing related diseases. According to the research, chloroiodoxyquine can be used as a specific targeting NLRP3 inhibitor, can inhibit activation of classical inflammation bodies induced by bacterial lipopolysaccharide combined with ATP, nigericin and microcrystalline sodium urate, and has remarkable inhibition effects in iBMDM, BMDM and THP-1 cells. In addition, chloroiodoxyquine can prevent peritonitis and gouty arthritis induced by microcrystalline sodium urate and has a good prevention effect on bacterial lipopolysaccharide-induced infectious shock. The clioquinol has good prevention and treatment effects on NLRP3 related inflammatory diseases, and shows that the clioquinol can be used as a therapeutic drug for NLRP3 related diseases, thereby providing a new choice for screening and recycling clinical drugs.
Description
Technical Field
The invention relates to the field of medicines, in particular to application of clioquinol in preparing an NLRP3 inflammation small body inhibitor and a medicine for treating or preventing related diseases.
Background
NLRP3 inflammatory corpuscles are complexes consisting of NLRP3 (NOD-like receptor family, pyrin domain containing) protein, ASC (apoptosis-associated speck-like protein containing a CARD) protein and caspase-1 protein. NLRP3 protein is a receptor capable of sensing a series of stimulus signals within cells, such as changes in intracellular potassium ion concentration, increases in intracellular calcium ion concentration, mitochondrial damage, oxidative stress, etc. When these stimulus signals are perceived by NLRP3 proteins, they bind to ASC proteins to form NLRP3 inflammatory corpuscles. Subsequently, NLRP3 inflammatory small molecules activate caspase-1 proteins, which in turn trigger inflammatory responses.
Cell death is a form of cell death caused by activation of inflammatory bodies, during which caspase-1 is activated to cleave and activate a range of pro-inflammatory cytokines, such as IL-1 beta and IL-18. The release of these pro-inflammatory cytokines initiates an inflammatory response, which further activates immune cells to enhance the inflammatory response. In addition, caspase-1 is also capable of cleaving a protein called stomatin (GASDERMIN D) to form the N-terminal domain of stomatin, which in turn leads to cell membrane disruption and cell scorch.
NLRP3 inflammatory corpuscles and cell coke apoptosis play an important role in the occurrence and progression of various diseases, and inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease and the like are all associated with activation and cell coke apoptosis of NLRP3 inflammatory corpuscles. In addition, NLRP3 inflammatory bodies and cell death are involved in the development and progression of a variety of infectious diseases, such as infectious pneumonia, tuberculosis, and the like. Studies have shown that some microbial infections activate NLRP3 inflammatory bodies, triggering cell death, and thus enhancing host clearance of microorganisms.
Chloroiodoquinoline (Clioquinol), hereinafter referred to as CQ, is a broad-spectrum antimicrobial drug commonly used to treat skin and eye infections. The clioquinol is an antibacterial drug which is rapidly absorbed by oral administration and has lasting effect, and is suitable for treating infectious diseases caused by various sensitive bacteria. It has antibacterial and antifungal effects, and can kill or inhibit the growth of various bacteria and fungi. CQ is also used in the form of topical preparations, and can be used as a component of ointments, eye drops or creams. It can be used for treating various skin infections such as eczema, furuncle, acne, tinea pedis, etc. In addition, it can be used to treat ocular infections such as conjunctivitis and keratitis. In recent years, researchers have also found that CQ plays an important role in the occurrence and development of neurodegenerative diseases and cancers. Several studies have shown that CQ can reverse the progression of alzheimer's disease, parkinson's disease and huntington's disease. However, whether CQ can affect NLRP3 inflammatory bodies and cell apoptosis is currently unclear and requires further investigation.
Summarizing the above, NLRP3 inflammatory bodies and cell death are important mediators in cellular immune and inflammatory responses. They play an important role in the development and progression of a variety of diseases, including inflammatory diseases, infectious diseases, and tumors. Deep research on NLRP3 inflammatory corpuscles and cell apoptosis is helpful for revealing the role of the NLRP3 inflammatory corpuscles and cell apoptosis in the occurrence mechanism of diseases, and provides a new thought and target point for the treatment of related diseases. The development and re-application of the old medicine have the significance of saving cost, improving curative effect, increasing medicine selection and the like, and have important roles in promoting the development of the medicine field and improving the treatment effect of patients.
Disclosure of Invention
The invention aims to provide a novel application of chloroiodoquinoline in preparing an inhibitor specifically targeting NLRP3 inflammatory corpuscles.
The invention also aims to provide a novel application of the chloroiodoquinoline in preparing medicaments for preventing and treating diseases related to NLRP3 inflammatory body activation, in particular to a novel application of the chloroiodoquinoline in inhibiting inflammatory diseases related to cell apoptosis.
The chloroiodoxyquine has a molecular formula of C 9H5C11 NO, and a chemical formula structure shown as follows:
The technical scheme provided by the invention is as follows:
use of clioquinol in the preparation of an inhibitor specifically targeting NLRP3 inflammatory bodies.
Meanwhile, the invention provides application of chloroiodoxyquine in preparing a medicament for treating or preventing NLRP3 inflammatory small-scale related diseases.
The invention also provides application of the clioquinol in preparing an NLRP3 inflammatory pathway inhibitor and application of the clioquinol in preparing an NLRP3 inflammatory corpuscle induced cell apoptosis inhibitor.
Further, the NLRP3 inflammatory small body related disease refers to peritonitis induced by microcrystalline sodium urate.
The NLRP3 inflammatory small-scale related disease refers to gouty arthritis induced by microcrystalline sodium urate.
The NLRP3 inflammatory small body related disease refers to lipopolysaccharide induced sepsis/septic shock.
In particular, the clioquinol is used for inhibiting activation of NLRP3 inflammatory corpuscles induced by bacterial lipopolysaccharide and ATP in combination.
The clioquinol is used for inhibiting activation of NLRP3 inflammatory corpuscles induced by combination of bacterial lipopolysaccharide and nigericin.
The chloroiodoxyquine is used for inhibiting activation of NLRP3 inflammatory corpuscles induced by combination of bacterial lipopolysaccharide and microcrystalline sodium urate.
Compared with the prior art, the invention has the beneficial effects that:
The invention provides application of chloroiodoquinoline as an NLRP3 inflammation corpuscle inhibitor, and researches show that the chloroiodoquinoline can inhibit bacterial lipopolysaccharide combined with ATP, nigericin and microcrystalline sodium urate induced classical inflammation corpuscle activation, has obvious inhibition effects in iBMDM, BMDM and THP-1 cells, and can reduce Caspase-1p20 and IL-1 beta p17 production caused by NLRP3 inflammation pathway activation. And experiments and researches show that the chloroiodoquinoline does not inhibit AIM2 inflammatory body passage stimulated by bacterial lipopolysaccharide and Poly (dA: dT), and NLRC4 inflammatory body passage stimulated by bacterial lipopolysaccharide and flagellin, and proves the specific inhibition effect on NLRP3 inflammatory body.
The invention provides application of chloroiodoquinoline in preventing and treating NLRP3 inflammatory small body Guan Yanzheng diseases, in particular: the chloroiodoquinoline can prevent and treat peritonitis induced by microcrystalline sodium urate, and reduce the level of inflammatory cells and inflammatory factors; the chloroiodoquinoline can prevent and treat gouty arthritis induced by microcrystalline sodium urate, reduce joint swelling degree and reduce inflammatory factor release; the chloroiodoquinoline can reduce the lethality of bacterial lipopolysaccharide-induced infective shock and reduce the level of inflammatory factors in serum.
Therefore, the invention proves that chloroiodoxyquine can be used as a therapeutic drug for NLRP3 related diseases for the first time, and provides a new choice for screening and recycling clinical drugs.
Drawings
FIG. 1 shows that CQ can inhibit NLRP3 inflammatory corpuscle activation, demonstrating the effect of CQ on NLRP3 inflammatory corpuscle activation in iBMDM and THP-1 cells: FIGS. 1A and B are the results of a bacterial lipopolysaccharide and ATP combined stimulation of cell lactate dehydrogenase and the inflammatory factor interleukin-1 beta in iBMDM cells, respectively; FIGS. 1C and D are results of a combination of bacterial lipopolysaccharide and Nigericin stimulation of cell lactate dehydrogenase and interleukin 1 beta in iBMDM cells, respectively; in FIG. 1, E and F are the results of the measurement of inflammatory factor interleukin-1 beta in THP-1 cells stimulated by bacterial lipopolysaccharide and nigericin/microcrystalline sodium urate combination, respectively; CQ is administered in uM;
Fig. 2 is a complement to the result of fig. 1: immunoblotting determination results of relevant proteins NLRP3, GSDMD-NT, caspase-1, IL-1β, ASC, caspase-1p20 and IL-1βp17 after activation of NLRP3 inflammatory corpuscles by CQ; wherein the administration concentration unit of CQ is uM, LYS represents intracellular protein of cell lysate, SN represents cell supernatant protein, NIGERICIN represents Nigericin, and MSU represents microcrystalline sodium urate; FIG. 2A shows the immunoblotting results of related proteins NLRP3, GSDMD-NT, caspase-1, IL-1β, ASC, caspase-1p20, IL-1βp17 after the combined stimulation of bacterial lipopolysaccharide and ATP to human NLRP3 inflammatory bodies of mice iBMDM; FIG. 2B shows the immunoblotting measurement results of related proteins NLRP3, GSDMD-NT, caspase-1, IL-1β, ASC, caspase-1p20, IL-1βp17 after the combined stimulation of bacterial lipopolysaccharide and nigericin on human cells NLRP3 inflammatory bodies of mice iBMDM; FIG. 2C shows the results of immunoblotting assays of related proteins NLRP3, GSDMD-NT, caspase-1, IL-1β, ASC, caspase-1p20, IL-1βp17 after combined stimulation of human THP-1 cell NLRP3 inflammatory body activation by CQ with bacterial lipopolysaccharide and Nigericin; FIG. 2D shows the immunoblotting results of related proteins NLRP3, GSDMD-NT, caspase-1, IL-1β, ASC, caspase-1p20, IL-1βp17 after the combined stimulation of bacterial lipopolysaccharide and microcrystalline sodium urate on human THP-1 cell NLRP3 inflammatory corpuscle activation.
Fig. 3 shows that CQ can act as an inhibitor of NLRP3 inflammatory bodies, demonstrating the effect of CQ on NLRP3 inflammatory body activation in BMDM cells: FIG. 3A is a graph showing the results of a bacterial lipopolysaccharide and ATP/nigericin combination stimulation of cellular lactate dehydrogenase in BMDM cells; FIG. 3B is a graph showing the results of a bacterial lipopolysaccharide and ATP/nigericin combination stimulation of the inflammatory factor interleukin IL-1 beta in BMDM cells; FIG. 3C shows the results of immunoblotting assays of CQ on NLRP3 inflammatory body activated related proteins NLRP3, GSDMD-NT, caspase-1, IL-1β, ASC, caspase-1p20, IL-1βp17; wherein CQ is administered in uM, LYS represents intracellular protein in cell lysate, SN represents cell supernatant protein, NIGERICIN represents Nigericin.
FIG. 4 shows that CQ acts as a specific inhibitor of NLRP3 inflammatory corpuscles, demonstrating the effect of CQ on AIM2 and NLRC4 inflammatory corpuscle activation, and FIG. 4A is the measurement result of cell lactate dehydrogenase after stimulation by five stimulators ATP/NIGERICIN/MSU/poly (dA: dT)/FLAGELLIN in BMDM cells; FIG. 4B is a graph showing the results of an inflammatory factor interleukin-1 beta assay after stimulation of five stimulators ATP/NIGERICIN/MSU/poly (dA: dT)/FLAGELLIN in BMDM cells; FIG. 4C shows the immunoblotting results of the related proteins NLRP3, GSDMD-NT, caspase-1, IL-1β, ASC, caspase-1p20, IL-1βp17 after the activation of the NLRP3 inflammatory corpuscle by the three stimulators ATP/NIGERICIN/MSU in BMDM cells; FIG. 4D shows the results of immunoblotting assays of CQ on two stimulators poly (dA: dT)/FLAGELLIN on NLRP3 inflammatory bodies after activation of the relevant proteins NLRP3, GSDMD-NT, caspase-1, IL-1β, ASC, caspase-1p20, IL-1βp17 in BMDM cells; wherein CQ is administered in uM, LYS represents intracellular protein in the cell lysate, SN represents cell supernatant, NIGERICIN represents Nigericin, FLAGELLIN represents flagellin.
FIG. 5 is the effect of CQ on NLRP3 inflammatory small domain related proteins ASC and NEK 7: FIG. 5A is the effect of CQ on ASC self-assembly in iBMDM cells, FIG. 5B is the effect of CQ on NLRP3 oligomers in iBMDM cells, and FIG. 5C is the effect of CQ on NLRP3 and NEK7 interactions in iBMDM cells.
FIG. 6 shows the prophylactic and therapeutic effects of CQ on sodium microcrystalline urate-induced peritonitis model mice, wherein A in FIG. 6 represents the relative proportion of leukocytes in total cells collected by flow cytometry, B in FIG. 6 represents the absolute number of leukocytes in total cells collected by flow cytometry, C in FIG. 6 represents the relative proportion of neutrophils in leukocytes collected by flow cytometry, D in FIG. 6 represents the absolute number of neutrophils in leukocytes collected by flow cytometry, E in FIG. 6 represents the relative proportion of monocytes in leukocytes collected by flow cytometry, F in FIG. 6 represents the absolute number of neutrophils in leukocytes collected by flow cytometry; wherein UN represents untreated mice, MSU represents mice injected with microcrystalline sodium urate alone, CQ 5mg represents mice injected with CQ at a dose of 5mg/kg, CQ 10mg represents mice injected with CQ at a dose of 10mg/kg, MCC950 10mg represents mice injected with MCC950 at a dose of 10 mg/kg.
FIG. 7 shows the prophylactic and therapeutic effects of CQ on model mice with microcrystalline sodium urate-induced gouty arthritis and bacterial lipopolysaccharide-induced sepsis, wherein A in FIG. 7 represents the effect of CQ on mouse foot pad thickness in gouty arthritis and B in FIG. 7 represents the effect of CQ on mouse survival in sepsis; wherein UN represents untreated mice group, MSU represents mice group injected with microcrystalline sodium urate only, LPS represents mice injected with bacterial lipopolysaccharide only, CQ 5mg represents mice group injected with CQ at 5mg/kg dose, CQ 10mg represents mice group injected with rocq at 10mg/kg dose, MCC950 mg represents mice group injected with MCC950 at 10mg/kg dose.
Detailed Description
The above-described matters of the present invention will be further described in detail by way of examples, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples, and all techniques realized based on the above-described matters of the present invention are within the scope of the present invention.
The experimental methods used in the examples below are conventional methods, and the reagents, methods and apparatus used are conventional in the art, unless otherwise indicated.
Inflammatory diseases in the present invention include, but are not limited to, gout, neurodegenerative diseases, sepsis, septic shock, ulcerative colitis, diseases such as multiple sclerosis, alzheimer's disease, parkinson's disease, atherosclerosis and the like.
The material and the method in the invention are as follows:
1.1 sources and dosages of the drugs
The clioquinol and MCC950 used in the present invention are available from Selleck, inc., CQ (S4601) and MCC950 (S8930).
In vitro cell experiments, the following experimental groups were shared: (1) cells which have not been subjected to any treatment. (2) Only LPS (lipopolysaccharide) -stimulated cells (which may not be set depending on the experimental situation). (3) Cells stimulated by LPS and other stimulating agents (ATP/Nigeriin/MSU/poly (dA: dT)/flagellin) in combination. (4) Cells treated with LPS and other stimulating agents in combination with CQ.
In an in vivo animal experiment, 50mg/ml CQ stock solution was diluted with PEG300, tween80 and ddH 2 O to a concentration of 1mg/ml (0.5 mg/ml) and 200uL CQ (0.1 or 0.2 mg) was administered to each mouse by intraperitoneal injection. The following experimental groups were shared: (1) Mice (UN) that were not treated or injected with PBS alone. (2) non-CQ injected model mice (LPS/MSU/DSS). (3) model mice (cQ 5 mg) given CQ at 5 mg/kg. (4) The model mice (cQ 10 mg) given CQ at 10 mg/kg. (5) Molded mice (MCC 95010 mg) were given MCC950 at a dose of 10 mg/kg.
1.2 Cell Source and culture
IBMDMs (mouse macrophage cell line, RAW 264.7), THP-1 (human monocytic leukemia cell, THP-1)TIB-202 TM) and HEK293T (human embryonic kidney 293 cells, ATCC, CRL-11268) were obtained from ATCC.
1.3 Laboratory animals
C57BL/6 mice were obtained from university of south kyo model animal research centers and housed in a sterile environment certified by the international laboratory animal care and assessment association, all animal experiments followed the laboratory animal guidelines and were approved by the animal care committee of the south kyo drummer hospital.
Reagents and materials
DMEM medium (Gibco, 31985-070); RPMI 1640 medium (Gibco, 11875119); fetal bovine serum (Gibco, 10091141); bacterial lipopolysaccharide LPS (Sigma-Aldrich, L2630); ATP (Sigma-Aldrich, A6419); nigericin NIGERICIN (Sigma-Aldrich, 481990);
Microcrystalline sodium urate MSU(Invitrogen,Tlrl-MSU);Poly(dA:dT)(CA,6249-42-01);LipofectamineTM3000(Invitrogen,L3000075); flagellin FLAGELLIN (Beyotime, P7388); cell lactate dehydrogenase LDH assay kit (Beyotime, C10007); CCK8 cell activity assay kit (Beyotime, C0039); NP-40 lysate (Beyotime, P0013F); PMSF (Beyotime, ST 506); protein quantification kit (Bioworld, BD 0028); denatured protein loading buffer (5 x) (Beyotime, P0015L); non-denatured protein loading buffer (5 x) (Beyotime, P0016); a PVDF membrane (Amersham Bioscience); ECL hypersensitive glow solution (Tanon, 180-5001);
one-step gel rapid preparation kit (Nanjinopran); 180kDa pre-dye protein marker (Vazyme, MP 102-01/02); ASC antibodies (CELL SIGNALING Technology, CST-67824); caspase-1 antibody (Adipogen, AG-20B-0042); GAPDH antibody (CELL SIGNALING Technology, 5174S); IL-1 beta antibody (R & D, AB-401-NA); NLRP3 antibody (CELL SIGNALING Technology, CST-15101); GSDMD (GSDMD-NT) antibodies (abcam, ab219800, ab 209845);
Goat anti-rabbit IgG (h+l) -HRP (Bioworld, BS 13278); goat anti-mouse IgG (h+l) -HRP (Bioworld, BS 12478); rabbit anti-sheep IgG (h+l) -HRP (Bioworld, BS 30503); DSS crosslinking agents (selleck, S0657); all ELISA kits were purchased from Baao.
Example 1
2.1 Cell culture
IBMDM in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin; THP-1 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin; BMDM was grown in DMEM medium supplemented with 10% L929 first day culture supernatant, 10% L929 second day culture supernatant, 10% L929 third day culture supernatant, 10% fetal bovine serum, and 1% penicillin/streptomycin; cells were grown in culture in a constant temperature cell incubator at 37℃and 5% CO 2.
2.2 Extraction of bone marrow derived macrophage BMDM
C57BL/6 cervical dislocation of 8-10 weeks old is killed and then soaked in 75% ethanol for 5 minutes, and the operation is required to be carried out in a cell super clean bench; taking femur and tibia marrow cells of a mouse to culture in the special induction DMEM culture medium prepared by the method, and changing half of old culture medium and adding half of new culture medium on the third day; observing the condition of cells on the seventh day, scraping the cells with a cell scraper and centrifuging, and then spreading the cells on a corresponding cell culture plate for culturing overnight, wherein the cells can be used after adhering to the wall on the next day.
2.3 Classical inflammation small body pathway stimulation
Inoculating the cells on a culture plate overnight in advance, and using the cells after the cells cling to the wall the next day; adding 100ng/ml of DMEM medium containing LPS into the cell plate, culturing for 4 hours, and adding CQ according to the required concentration for further culturing for one hour; the whole culture medium is replaced, ATP is added according to the concentration of 4mM/ml for continuous culture for 20 minutes, or nigericin is added according to the concentration of 10mM/ml for continuous culture for 90 minutes, or microcrystalline sodium urate is added according to the concentration of 200mg/ml for 6-8 hours, so that NLRP3 inflammatory corpuscles can be stimulated to induce cell apoptosis.
2.4 Non-classical inflammatory pathway stimulation
2.4.1AIM2 stimulation of inflammatory corpuscles
Inoculating the cells on a culture plate overnight in advance, and using the cells after the cells cling to the wall the next day; adding 100ng/ml of DMEM medium containing LPS into the cell plate, culturing for 4 hours, and adding CQ according to the required concentration for further culturing for one hour; the whole culture medium is replaced, and poly (dA: dT) is added according to the concentration of 0.1ug/ml with the aid of Lipo3000 transfection reagent, and the culture is continued for 6 hours, so that AIM2 inflammatory corpuscle induced cell apoptosis can be stimulated.
2.4.2NLRC4 stimulation of inflammatory corpuscles
Inoculating the cells on a culture plate overnight in advance, and using the cells after the cells cling to the wall the next day; adding 100ng/ml of DMEM medium containing LPS into the cell plate, culturing for 4 hours, and adding CQ according to the required concentration for further culturing for one hour; the whole culture medium is replaced, FLAGELLIN is added according to the concentration of 0.1ug/ml with the aid of Lipo3000 transfection reagent, and the culture is continued for 2.5 hours, so that NLRC4 inflammatory bodies can be stimulated to induce cell apoptosis.
2.5 Measurement of Lactate Dehydrogenase (LDH) content in cells
To assess the extent of cell death, the measurement of cellular lactate dehydrogenase content was used to assess: after stimulating cells to activate inflammatory bodies according to the above-mentioned method, collecting cell supernatant, adding 60uL of cell supernatant to each well of a 96-well plate, adding 30uL of assay mixture to each well according to the instructions given in practice, incubating for 10-30 minutes at 37℃in the absence of light, and measuring absorbance at 490nm with CytoTox 96Non-radio, each set being set with 4 replicates; cellular lactate dehydrogenase was purchased from Biyundian corporation, and the cellular lactate dehydrogenase was assayed according to the method described in the instructions of the reagent manufacturer, and the test data was statistically analyzed using software GRAPHPAD PRISM 7.0.0.
The following measurement steps were performed:
(1) Cell supernatants were collected, appropriate volumes of supernatant were added to 96-well plates, and 4 replicates were set for each group;
(2) Adding the reaction solution in the Biyun Tian cell lactic dehydrogenase kit into a test tube, and uniformly mixing;
(3) The reaction is carried out at a suitable temperature, typically 37 degrees
(4) After a certain time, the absorbance in the test tube is measured by using a spectrophotometer, and is generally 450nm;
(5) The lactate dehydrogenase activity in the sample is calculated according to a standard curve provided by the kit.
2.6 Cell Activity CCK8 assay
To assess whether different drug concentrations produce toxic effects on cells, the CCK8 cell activity assay kit was used to evaluate: cells were inoculated in advance in 96 well plates for incubation overnight, after medium exchange, drugs of different concentrations were added for 4 hours, and then CCK8 reaction solution was added for incubation at 37 ℃ in the dark for 1-4 hours, absorbance was measured at 450nm using CytoTox 96 Non-radio, and at least 4 replicates were set per group.
2.7 Interleukin-1 beta inflammatory factor assay
(1) Preparing a sample: collecting a sample to be assayed, such as serum, cell supernatant, etc.; centrifuging the sample and collecting the supernatant;
(2) Sample dilution: according to the experimental requirements, properly diluting the sample to ensure that the measurement result is within the standard curve range;
(3) Preparing a reagent: diluting a dilution buffer solution in the Baao ELISA assay kit according to the requirements of the specification;
(4) Pretreatment: adding the diluted sample and the standard substance into a pretreatment plate hole, adding a pretreatment liquid, uniformly mixing, and incubating for a period of time;
(5) Washing: pouring out the liquid in the hole, washing the bottom of the hole with a washing buffer solution, and repeating the step for 3 times;
(6) Adding a detection antibody: adding diluted detection antibody into the hole, and incubating for a period of time;
(7) Washing: repeating the step 5;
(8) Adding a substrate: adding a substrate into the well, and incubating for a period of time;
(9) The reaction was stopped: adding stopping solution to stop the reaction;
(10) And (3) measuring: the absorbance was measured using a microplate reader and the concentration of IL-1β in the samples was calculated from the standard curve.
2.8 Protein gel electrophoresis
Cells or tissues were lysed with NP-40 lysate mixed with 2% PMSF on ice with shaking for 30 min; protein quantification can be performed using BCA protein quantification kit, if desired, with specific quantification methods referred to manufacturer's instructions; afterwards, adding a 5X denatured protein sample loading buffer or a non-denatured PAGE protein loading buffer (5X) in a corresponding proportion to the lysed species; placing the mixed solution on a metal bath with the temperature of 100 ℃ and boiling for 5-10 minutes; according to experimental requirements, 6%, 7.5%, 10%, 12.5% or 15% of protein separation gel is prepared, gel electrophoresis separation is carried out by using a Tanon EPS-300 digital display type voltage and current stabilizing electrophoresis apparatus, 120V is carried out for 60-100 minutes, and 250-350mA is transferred to a PVDF membrane for 50-120 minutes; blocking with 5% skimmed goat milk for 1.5-2 hours, and incubating the primary antibody on a four degree shaker overnight; the next day, the secondary antibody was incubated on a shaker at room temperature for 2 hours after washing off the primary antibody, and then imaged on Tanon TM ECL chemiluminescent substrate in a Tanon full-automatic chemiluminescent image analysis system.
2.9 Co-immunoprecipitation reactions
Cells or tissues were lysed with NP-40 lysate mixed with 2% PMSF on ice with shaking for 30 min, after which protein quantification was performed using the BCA protein quantification kit; subsequently, nonspecific protein binding was first removed with 50% protein A/G agarose bead solution; after that, specific antibodies (2. Mu.g) were added to the lysate and the shaker was inverted overnight at 4 ℃; the next day, 100% protein A/G agarose column solution is added, after the shaking table is turned over for 1-2 hours at room temperature, 12,000g is centrifuged for 10 minutes, and the supernatant is removed; washing with pre-cooled 1 XPBS for one time, and centrifuging again; adding NP-40 lysate and protein loading buffer solution (5 x) in a certain proportion after centrifugation, and carrying out metal bath at 100 ℃ for 5-10 minutes; after that, the protein samples were immunoblotted.
Example 2
3.1 Model of sodium microcrystal urate-induced gouty arthritis
C57BL/6 male mice with the age of 8-10 weeks are used, CQ is injected once in advance by intraperitoneal injection in the evening, cQ is injected for the second time after 8-12 hours from the 1 st injection, 0.6mg MSU is injected at the joint part of the foot after one hour of drug injection, MSU is injected only at the left foot of the mice, and no solution is injected at the right foot of the mice as a negative control; the thickness of each mouse foot pad is measured by a vernier caliper every hour, and the difference value between the thickness of the left foot and the thickness of the right foot is used for evaluating the onset degree of arthritis and is continuously monitored for 6 hours; subsequently, mice were euthanized and perfused with PBS and 4% pfa, and the joints of the mice were used for caspase-1 activity assessment by caspase-1 activity assay kit, IL-1 beta ELISA assay and H & E staining; CQ concentration and experimental grouping have been mentioned above.
3.2 Microcrystalline sodium urate-induced peritonitis model
C57BL/6 male mice with the age of 8-10 weeks are used, CQ is injected intraperitoneally once a night in advance, cQ is injected intraperitoneally for the second time after 8-12 hours from the 1 st injection, and 0.8mg of MSU solution is injected intraperitoneally to each mouse after one hour of drug injection; six hours later, lavage the mouse abdominal cavity with pre-chilled 1 XPBS solution, collect and centrifuge at 4deg.C 1,500 g; cells were used for flow cytometry and supernatants were used for ELISA assays for IL-1 beta and IL-6.
3.3 Bacterial lipopolysaccharide-induced sepsis model
C57BL/6 male mice with the age of 8-10 weeks are used, CQ is injected intraperitoneally once a night in advance, cQ is injected intraperitoneally for the second time after 8-12 hours from the 1 st injection, and 0.3mg of LPS solution is injected intraperitoneally to each mouse after one hour of drug injection; after 12 hours, reaching the peak inflammatory phase, the orbit was first collected, peripheral blood was used for flow cytometry, and serum was used for ELISA assays for IL-1 beta, IL-6 and TNF-alpha; mice from which blood was collected were continuously perfused with 1x PBS and 4% pfa, and lungs and livers were collected for H & E staining.
3.4 Flow cytometry
The cell suspension collected by different methods of animal model is flushed at least twice with precooled PBS (1X), if the cell suspension containing red blood cells also needs to be lysed with red blood cell lysate, 1-3 times until the sediment contains no red blood cells; adding the antibody into PBS solution according to the proportion, adding 100-200uL of the mixed solution into an EP tube containing cell sediment, uniformly mixing, dyeing at 4 ℃ for 30 minutes in a dark place, centrifuging at 1, 500rpm for 5 minutes, washing with PBS for 1-3 times, centrifuging to collect sediment, finally re-suspending cells with PBS according to the proper proportion, detecting with a flow cytometer (BD Fortessa), and analyzing the obtained data with FlowJo_v10.6.2 software.
4. Experimental results
4.1 Explore whether CQ can inhibit activation of NLRP3 inflammatory corpuscles
First, for the results of FIG. 1, it was shown that CQ can significantly reduce the release of cellular lactate dehydrogenase, and the amount of inflammatory factor IL-1. Beta. In iBMDM cells stimulated by the combination of bacterial lipopolysaccharide and ATP/nigericin; at the same time, this inhibition was also effective in THP-1 cells, indicating that CQ is a potent NLRP3 inflammatory corpuscle inhibitor.
4.2 Western Blotting immunoblotting detection
FIG. 2 is a complement to the results of FIG. 1, and for the results of FIG. 2, the levels of related NLRP3 inflammatory small proteins in iBMDM and THP-1 cells, including NLRP3, GSDMD-NT, caspase-1, IL-1 beta, ASC, in cell lysates were examined using Western Blotting immunoblotting techniques with GAPDH as a reference protein; caspase-1p20 and IL-1 beta p17 content in cell supernatant; the results show that CQ reduces Caspase-1p20 and IL-1 beta content in cell supernatants in a dose-dependent manner, indicating that CQ as an inhibitor of NLRP3 can reduce inflammatory factor cleavage.
4.3 Exploration of inhibitory Effect of CQ on NLRP3 on Primary BMDM cells
FIG. 3 is a further extension of the results of FIG. 1 and FIG. 2, with the results of FIG. 3, bone marrow-derived macrophages in the mouse tibia were extracted to investigate the inhibitory effect of CQ on NLRP3 on primary BMDM cells, as consistent with the results in iBMDM and THP-1 cells: CQ can inhibit bacterial lipopolysaccharide and NLRP3 inflammatory body activation induced by ATP/nigericin stimulator, and has reduced lactate dehydrogenase, inflammatory factor IL-1β and cleavage of Caspase-1p20 and IL-1βp17 in supernatant protein.
4.4 Explore whether CQ has specificity for inhibition of NLRP3 inflammatory corpuscles
For the results of FIG. 4, the same cells were co-stimulated with five stimulators ATP/NIGERICIN/MSU/poly (dA: dT)/FLAGELLIN, where ATP/NIGERICIN/MSU stimulated the classical inflammatory small body pathway, poly (dA: dT) stimulated the non-classical AIM2 inflammatory small body pathway, and FLAGELLIN stimulated the non-classical NLRC4 inflammatory small body pathway; the results show that CQ specifically reduced the content of lactate dehydrogenase and IL-1β in the cells of the classical inflammatory pathway group, while the poly (dA: dT) and FLAGELLIN-stimulated cell groups were not significantly reduced; the results of protein immunoblotting are consistent with the above, further demonstrating that CQ is specific for inhibition of NLRP3 inflammatory bodies.
4.5 Exploration of the mechanism of CQ to inhibit NLRP3 inflammatory corpuscles
For the results of fig. 5, we explored whether CQ could inhibit the autopolymerization of NLRP3 domain protein ASC, showing that CQ could significantly reduce the autopolymerization of ASC adapter protein; meanwhile, the influence of CQ on NLRP3 oligomerization is detected by using a non-denaturing gel electrophoresis technology, which shows that the CQ can also reduce the degree of NLRP3 oligomerization; the immunoprecipitation results further demonstrate that CQ can affect the interaction between NLRP3 and NEK7 proteins.
4.6 In vivo experiments to verify the effect of CQ
For the results of fig. 6, we collected cells from peritoneal lavage of peritonitis mice for flow cytometry using the NLRP3 classical inhibitor MCC950 as a control drug; the results showed that the percentage and absolute numbers of leukocytes, neutrophils, monocytes, and monocytes in the peritoneal lavage fluid of mice were significantly reduced when CQ was administered at 10mg/kg in vivo compared to mice without CQ drug, and were not statistically significantly different from MCC 950.
4.7 Verification of efficacy in gouty arthritis and sepsis
For the results of fig. 7, the footpad of mice given 10mg/kg CQ had significantly lower footpad swelling levels within 6 hours after injection of microcrystalline sodium uric acid than the non-administered group, and had no statistically significant difference from MCC 950; in addition, sepsis mice given CQ 10mg/kg had significantly greater survival than control mice given no CQ, showing longer survival times; the prophylactic effect of CQ 5mg/kg in sepsis was not significant with MCC950, and the control group without dosing was not statistically significant different.
The present invention is not limited to the preferred embodiments, and any simple modification, equivalent replacement, and improvement made to the above embodiments by those skilled in the art without departing from the technical scope of the present invention, will fall within the scope of the present invention.
Claims (10)
1. Use of clioquinol in the preparation of an NLRP3 inflammasome inhibitor.
2. The use according to claim 1, characterized in that: the chloroiodoxyquine is used as an NLRP3 inflammatory pathway inhibitor.
3. The use according to claim 1, characterized in that: the chloroiodoxyquine is used as an inhibitor of cell apoptosis induced by NLRP3 inflammatory corpuscles.
4. The use according to claim 1, characterized in that: the chloroiodoxyquine is used for inhibiting activation of NLRP3 inflammatory corpuscles induced by bacterial lipopolysaccharide and ATP.
5. The use according to claim 1, characterized in that: the clioquinol is used for inhibiting activation of NLRP3 inflammatory corpuscles induced by combination of bacterial lipopolysaccharide and nigericin.
6. The use according to claim 1, characterized in that: the chloroiodoxyquine is used for inhibiting activation of NLRP3 inflammatory corpuscles induced by combination of bacterial lipopolysaccharide and microcrystalline sodium urate.
7. Use of clioquinol in the manufacture of a medicament for treating or preventing a disease associated with NLRP3 inflammatory minibody.
8. The use according to claim 7, characterized in that: the NLRP3 inflammatory small-scale related disease refers to peritonitis induced by microcrystalline sodium urate.
9. The use according to claim 7, characterized in that: the NLRP3 inflammatory small-scale related disease refers to gouty arthritis induced by microcrystalline sodium urate.
10. The use according to claim 7, characterized in that: the NLRP3 inflammatory small body related disease refers to lipopolysaccharide induced sepsis/septic shock.
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| WO2001082911A2 (en) * | 2000-04-28 | 2001-11-08 | Gerolymatos P N Sa | TREATMENT OF PATHOLOGICAL CONDITIONS INFLUENCED BY THE ACTION OF MATRIX METALLOPROTEINASES (MMPs) USING CLIOQUINOL |
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