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Application of holomycin in inhibition of activation of NLRP3 inflammasome

A technology of inflammasomes and panmycin, applied in anti-inflammatory agents, digestive system, organic active ingredients, etc., can solve the undiscovered problems of inhibiting inflammasome activation and IL-1β secretion

Active Publication Date: 2018-11-16
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Holomycin (Holomycin, referred to as HL in this article) is an antibiotic produced by Streptomyces, which has broad-spectrum antibacterial activity, but it has not been found to inhibit the activation of inflammasome and the secretion of IL-1β.

Method used

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  • Application of holomycin in inhibition of activation of NLRP3 inflammasome
  • Application of holomycin in inhibition of activation of NLRP3 inflammasome
  • Application of holomycin in inhibition of activation of NLRP3 inflammasome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] This example is used to illustrate that under the stimulation of different NLRP3-specific agonists, holomycin can significantly inhibit the processing, maturation and release of IL-1β or IL-18 after activation of NLRP3 inflammasome.

[0061] ①. Cultivation and differentiation of mouse bone marrow-derived macrophages (BMDM): take about 10 weeks old C57BL / 6 mouse bone marrow cells, and use RPMI 1640 medium + 10% inactivated serum + 100ng / ml MSCF (macrophage Cell colony-stimulating factor, Peprotech) was cultured, and the medium was changed every three days, and mature BMDM could be obtained after culturing for 7 days.

[0062] ②. Inoculate 2.5×10 5 cells / hole into a 24-well plate, RPMI 1640 medium + 10% inactivated serum without MCSF was cultured overnight, then 100ng / mL lipopolysaccharide (LPS, Invivogen Company, tlrl-pb5lps) stimulated BMDM for 6h, and then added different concentrations of Panmycin (HL, 0nM, 5nM, 10nM, 25nM, 50nM, 100nM, 250nM; TRC Company, H458490) w...

Embodiment 2

[0068] This example is used to illustrate that panmycin can inhibit the enzyme activity of Caspase-1 in the NLRP3 inflammasome and the pyroptosis caused by its activation.

[0069] ①. Acquisition of BMDM: Same as step ① in Embodiment 1.

[0070] ②. Inoculate 5×10 6 Cells were cultured overnight in RPMI 1640 medium+10% inactivated serum without MCSF in a 60mm (non-treated) culture dish, then 100ng / mL lipopolysaccharide (LPS, Invivogen, tlrl-pb5lps) stimulated BMDM for 6h, and then added whole Mycin (HL, 100nM; TRC Company, H458490) was treated for 2h, the full mycin was removed, the cells were collected into a sterile flow tube, and the FAM-YVAD-FMK substrate fluorescent probe (ImmunoChemistry Company, FAM-FLICA TM Caspase 1 analysis kit, 97), washed once with PBS, stimulated with 20 μM nizhrinidine for different times (0 min, 15 min, 30 min, 45 min), washed once with PBS, and then detected the fluorescence intensity of substrate binding by flow cytometry. The result is as ...

Embodiment 3

[0074] This example is used to illustrate that panmycin can specifically inhibit the multimerization of ASC during the activation of NLRP3 inflammasome.

[0075] ①. Acquisition of BMDM: Same as step ① in Embodiment 1.

[0076] ②. Place 75% ethanol-sterilized coverslips in a 24-well plate, and then inoculate 5×10 5 Cells / well were cultured overnight in RPMI1640 medium + 10% serum without MCSF, then 100 ng / mL lipopolysaccharide was used to stimulate BMDM for 6 h, then holomycin (HL, 100 nM; TRC company, H458490) was added for 2 h, and holomycin was withdrawn. Add 20μM nizhrinidine to stimulate for 45min, fix the cells with 4% paraformaldehyde, and detect the formation of ASC spots by immunofluorescence. The result is as Figure 7-8 shown. *** p<0.0001.

[0077] During the activation of inflammasomes, the spatial structure of ASC multimerizes and forms spots, which can be observed under confocal laser microscopy by immunofluorescence. Figure 7-8 The results showed that holo...

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Abstract

The invention relates to the field of biology and medicine and discloses an application of holomycin in inhibition of the activation of an NLRP3 inflammasome. The holomycin can inhibit NLRP3 protein self-deubiquitination under agonist stimulation, binding of NLRP3 to an ASC protein, formation of ASC spots and activation of Caspase-1, and finally inhibit IL-1 beta and IL-18 secretion. The holomycincan inhibit monosodium urate-induced peritonitis in vivo. Therefore, holomycin can be used a specific drug for treatment on NLRP3 abnormal activation-related diseases.

Description

technical field [0001] The invention relates to the fields of biology and medicine, in particular to the application of holomycin in the preparation for inhibiting the activation of NLRP3 inflammasome, the application of holomycin in inhibiting the deubiquitination of NLRP3 protein, and the application of holomycin in inhibiting NLRP3 and ASC The application of protein binding, the application of holomycin in inhibiting the activation of Caspase-1 protein, the application of holomycin in the inhibition of NLRP3 inflammasome secretion of IL-1β and / or IL-18, the application of holomycin in the use of Application of a drug for preventing and / or treating diseases related to abnormal activation of NLRP3 inflammasomes, a method for inhibiting activation of NLRP3 inflammasomes, a method for preventing and / or treating diseases related to abnormal activation of NLRP3 inflammasomes, and overall Mycin. Background technique [0002] Innate immunity is the body's first line of defense t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/407A61P29/00A61P31/04A61P19/06A61P19/02A61P9/10A61P11/00A61P1/16
CPCA61K31/407A61P1/16A61P9/10A61P11/00A61P19/02A61P19/06A61P29/00A61P31/04
Inventor 尹荣华任广明杨晓明汪煜张洁张文马文兵李长燕于淼葛常辉詹轶群陈慧
Owner ACADEMY OF MILITARY MEDICAL SCI
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