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Application of NALP3-ASC inflammation complex and activator inhibitor thereof in terms of preparation of medicines for treating prion diseases

A technology of NALP3-ASC and prions, which is applied in the direction of antiviral agents, drug combinations, and microbial measurement/testing, and can solve problems such as not clarifying the activated state of neuroglial cells

Inactive Publication Date: 2013-10-02
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although a large number of literatures have reported that Prion induces neuroglial cells to release IL-1β, the mechanism by which prions activate microglial cells and release IL-1β has not been elucidated.

Method used

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  • Application of NALP3-ASC inflammation complex and activator inhibitor thereof in terms of preparation of medicines for treating prion diseases
  • Application of NALP3-ASC inflammation complex and activator inhibitor thereof in terms of preparation of medicines for treating prion diseases
  • Application of NALP3-ASC inflammation complex and activator inhibitor thereof in terms of preparation of medicines for treating prion diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 The role of NALP3-ASC inflammatory complex in prion-induced cell release of IL-1β

[0051] 1. Materials and methods

[0052] 1.1 Reagents

[0053] Rabbit anti-mouse caspase1, NALP3 and ASC antibodies were purchased from BioVision, Abcam and Santa Cruz, respectively; rabbit anti-mouse NF-kappaB p65 antibody, βactin and Max antibodies were purchased from Beyontian; LPS and N-acetylcysteine ​​(NAC ) was purchased from Sigma, IL-1β detection kit and protein rapid precipitation and concentration kit were purchased from Wuhan Boster Company; goat anti-rabbit secondary antibody, Bay 11-7082 were purchased from Beyond Biotechnology Company.

[0054] 1.2 Isolation and culture of microglia

[0055] The isolation of primary cells was carried out according to the existing literature (Kingham PJ, Cuzner ML, Pocock JM. 1999. Apoptotic pathways mobi-lized in microglia and neurones as a consequence of chromogranin A-induced microglial activation. J Neurochem73: 538-547) op...

Embodiment 2

[0091] Example 2K + Solution and reactive oxygen species (ROS) inhibitor NAC reduces IL-1β secretion by inhibiting the expression of NALP3 and ASC

[0092] Using high concentration (130mM) of KCL and ROS inhibitor NAC at a concentration of 10μM to act on the cells respectively, and detecting the effect of BV2 cells on the NALP3 inflammatory complex after being challenged by PrP106-126 under the action of these two reagents influences.

[0093] 100 μM PrP106-126 co-stimulated the cells with 130 mM KCL and 10 μM NAC, respectively. After 6 hours, the supernatant of the cells was taken to detect IL-1β. ELISA results showed that both KCL and NAC could significantly inhibit the production of IL-1β ( Figure 8 ), in addition, the total RNA of cells after treatment was taken, and the changes of NALP3 and ASC were detected by qPCR. The primers used were:

[0094] NALP3, upstream: 5'-ATTACCCGCCCGAGAAAGG-3',

[0095] Downstream: 5'-TCGCAGCAAAGATCCACACAG-3';

[0096] ASC, upstream: 5...

Embodiment 3

[0100] Example 3 Screening of NALP3-ASC Inflammatory Complex Activation Inhibitors

[0101] 1. Processing of samples to be tested

[0102] Take KCL and NAC chemical analysis pure grade powder, dilute with PBS to the working concentration, respectively 130mM and 10μM.

[0103] 2. Screening method establishment

[0104] Stimulate the BV2 cells with the sample to be tested and the prion neurotoxic fragment PrP106-126, extract the total RNA of the treated cells, use qPCR to detect the NALP3-ASC inflammatory complex, and judge the mRNA expression of NALP3 and ASC according to the PCR amplification results Inhibition situation, screening the test samples that can inhibit the expression of NALP3 and ASC is the anti-Prion disease drug.

[0105] The primers used to detect NALP3 and ASC by qPCR method are:

[0106] NALP3, upstream: 5'-ATTACCCGCCCGAGAAAGG-3',

[0107] Downstream: 5'-TCGCAGCAAAGATCCACACAG-3';

[0108] ASC, upstream: 5'-GCAACTGCGAGAAGGCTAT-3',

[0109] Downstream: 5'...

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Abstract

The invention discloses application of an NALP3-ASC inflammation complex and an activator inhibitor thereof in terms of preparation of medicines for treating prion diseases. Experiments show that prion infects microgliacyte, NALP3-ASC inflammation complex enhanced caspase-1 and IL-1beta activation are formed. The inflammation complex NALP3 inhibitor can suppress NALP3 and ASC expression, and then suppresses activation and release of the caspase-1 and IL-1beta. The invention provides the application of the NALP3-ASC inflammation complex inhibitor for screening medicines for treating the prion diseases, by means of cell co-stimulation for a sample to be detected and the prion, total RNA (ribonucleic acid) of treated cells is extracted, NALP3 and ASC are detected by a qPCR (quantitative polymerase chain reaction) method, expression suppression conditions of the sample to mRNA (messenger ribonucleic acid) of the NALP3 and the ASC are judged according to PCR (polymerase chain reaction) amplification results, accordingly medicines for resisting Prion diseases are screened out, and effects of preventing, treating or controlling the prion diseases are realized.

Description

technical field [0001] The present invention relates to the pharmaceutical use of the NALP3-ASC inflammatory complex and its activation inhibitor, in particular, the use of the NALP3-ASC inflammatory complex and its activation inhibitor in the preparation of medicines for prion-related diseases. Background technique [0002] Prion disease, also known as transmissible spongiform encephalopathies (TSEs), is a lethal neurodegenerative disease characterized by brain vacuolation, neuron death, and neuroglial proliferation. The disease is caused by cellular prion protein (PrP C ) conformational changes, into the pathological type (PrP Sc ) caused by. PrP Sc It is protease resistant and contains more beta sheets instead of the normal alpha helix. Abnormally morphological prion proteins (PrP Sc ) accumulation is the main cause of the disease. [0003] In recent years, scholars at home and abroad have done a lot of research on the screening of targeted drugs for Prion disease. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K45/00A61P31/12A61P25/00C12Q1/68
Inventor 杨利峰赵德明师福山王继红姚皓周向梅尹晓敏赵化粉
Owner CHINA AGRI UNIV
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