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Application of thiolutin in inhibiting nlrp3 inflammasome activation

A technology of thiolutin and inflammasome, applied in anti-inflammatory agents, organic active ingredients, non-central analgesics, etc., can solve problems that have not been reported in research

Active Publication Date: 2019-03-19
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Thiolutin (also referred to as THL in this paper) is a sulfur-based microbial antibiotic produced by Streptomyces, which has broad-spectrum antibacterial activity, and its research on inflammation has not been reported yet.

Method used

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  • Application of thiolutin in inhibiting nlrp3 inflammasome activation
  • Application of thiolutin in inhibiting nlrp3 inflammasome activation
  • Application of thiolutin in inhibiting nlrp3 inflammasome activation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] This example is used to illustrate the inhibitory effect of thiolutin on the processing, maturation and release of IL-1β downstream of NLRP3 inflammasome activation.

[0060] ①. Acquisition of mouse bone marrow-derived macrophages (BMDM): take C57B6 / L mouse bone marrow cells, and press 1×10 6 Inoculate at the density of cells / ml, culture with RPMI 1640 medium (adding 10% serum and 20% L929 cell supernatant) for 9 days, and change fresh medium every 3 days.

[0061] ②. In a 24-well plate, press 2.5×10 5 Inoculate BMDM at a density of 1 cell / well, pretreat with 500ng / mL lipopolysaccharide (LPS, Invivogen, tlrl-pb5lps) for 6h, add different concentrations of thiolutin (THL, 0nM, 50nM, 100nM, 250nM, 500nM, 1000nM Cayman Company, 11350) was treated for 1 h, the supernatant was removed, and 2 mM ATP (Invivogen Company, tlrl-atp) was added to stimulate for 1 h, the supernatant was collected, and the IL-1β level was detected using CBA microspheres (BD Company, 560232). The re...

Embodiment 2

[0067] This example is used to illustrate the inhibitory effect of thiolutin on the activation of Caspase1, a downstream event of NLRP3 inflammasome activation.

[0068] ①. Acquisition of BMDM: Same as step ① in Embodiment 1.

[0069] ②. In a 12-well plate, press 1×10 6 Inoculate BMDM (serum-free RPMI1640 medium) at a density of 1 cell / well, pretreat with 500ng / mL LPS for 6h, add 100nM thiolutin for 1h, remove the supernatant, and load the FAM-YVAD-FMK substrate fluorescent probe ( ImmunoChemistry, FAM-FLICA TM Caspase 1 analysis kit, 97), washed once with PBS, added 2mM ATP to stimulate for different times (0min, 30min, 60min, 90min), collected cells, and detected the fluorescence intensity of substrate binding by flow cytometry. The result is as Figure 5 shown.

[0070] ③. In a 96-well plate, press 1×10 5 Inoculate BMDM (serum-free RPMI 1640 medium) at a density of 1 cell / well, pretreat with 500ng / mL LPS for 6h, add different concentrations of thiolutin (0nM, 25nM, 50n...

Embodiment 3

[0073] This example is used to illustrate the inhibitory effect of thiolutin on ASC multimerization, a downstream event of NLRP3 inflammasome activation.

[0074] ①. Acquisition of BMDM: Same as step ① in Embodiment 1.

[0075] ②. In a 24-well plate, press 2.5×10 5 BMDM was inoculated at a density of 1 cell / well, BMDM was pretreated with 500ng / mL LPS for 6h, 100nM Thioletin was added for 1h, the supernatant was removed, 20μM Nigericin was added for 1h, and the formation of ASC spots was detected by immunofluorescence. The result is as Figure 7-8 shown.

[0076] Depend on Figure 7-8 It can be seen from the results that thiolutin can significantly reduce the formation of ASC spots in BMDM treated with the NLRP3 inflammasome agonist Nigericin, without affecting the ASC speckle formation induced by the AIM2 inflammasome agonist Poly(dA:dT) stimulation. The formation of spots indicated that thiolutin could specifically inhibit the multimerization of ASC downstream of NLPR3 infl...

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Abstract

The invention relates to the field of medicine and discloses application of thiolutin in inhibition of NLRP3 inflammasome activation. It is discovered by the inventor that thiolutin can inhibit the NLRP3 inflammasome activation by agonists of different attributes, such as ATP (adenosine triphosphate), Nigericin and MSU (monosodium urate) and cannot affect the activation of inflammasomes such as NLRC4 and AIM2 (absent in melanoma 2); thiolutin can inhibit the assembly of NLRP3 inflammasome, including NLRP3a and ASC (apoptosis-associated speck like protein containing a CARD) combination and ASCspot formation, can inhibit deubiquitinating of NLRP3 protein, and can also inhibit MSU-induced peritonitis and LPS-induced (lipopolysaccharide-induced) sepsis. Therefore, thiolutin can act as a potential drug to prevent and / or treat diseases related to abnormal activation of NLRP3 inflammasomes.

Description

technical field [0001] The present invention relates to the fields of biology and medicine, in particular to the application of thiolutin in the preparation for inhibiting the activation of NLRP3 inflammasome, the application of thiolutin in inhibiting the deubiquitination of NLRP3 protein, and the use of thiolutin Application of thiolutin in inhibiting the binding of NLRP3 to ASC protein, application of thiolutin in a drug for preventing and / or treating diseases related to abnormal activation of NLRP3 inflammasome, a method for inhibiting activation of NLRP3 inflammasome, A method for preventing and / or treating diseases related to abnormal activation of NLRP3 inflammasome, and thiolutin. Background technique [0002] The inflammasome is a multiprotein complex assembled from receptor protein, adapter protein ASC (apoptosis-associated speck like protein containing a CARD, apoptosis-associated speck like protein) and Caspase1 as key proteins, and accepts immunogen stimulation ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/407A61P7/00A61P29/00A61P31/00
CPCA61K9/0019A61K31/407A61P7/00A61P29/00A61P31/00
Inventor 杨晓明尹荣华任广明肖阳张煊宜张文汪煜李长燕于淼葛常辉詹轶群陈慧
Owner ACADEMY OF MILITARY MEDICAL SCI
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