CN112168966A

CN112168966A - Application of interfering NLRP3 inflammasome in preparing medicine for treating novel coronavirus pneumonia

Info

Publication number: CN112168966A
Application number: CN202010872412.XA
Authority: CN
Inventors: 黄曦; 曹灿; 吴永坚
Original assignee: Fifth Affiliated Hospital of Sun Yat Sen University
Current assignee: Fifth Affiliated Hospital of Sun Yat Sen University
Priority date: 2020-08-26
Filing date: 2020-08-26
Publication date: 2021-01-05

Abstract

The invention belongs to the technical field of immunotherapy, and particularly relates to application of an intervention NLRP3 inflammatory corpuscle in preparation of a medicine for treating novel coronavirus pneumonia. The specific inhibitor of NLRP3 inflammasome and the immunotherapy method thereof can realize the immunotherapy of the neocoronary pneumonia, have the advantage of slowing down the storm of inflammatory factors, and are suitable for the comprehensive therapy of the neocoronary pneumonia.

Description

Application of interfering NLRP3 inflammasome in preparing medicine for treating novel coronavirus pneumonia

Technical Field

The invention relates to the technical field of immunotherapy, in particular to application of intervening NLRP3 inflammatory bodies in preparing a medicine for treating novel coronavirus pneumonia.

Background

Coronavirus disease 2019(COVID-19) is a respiratory disease caused by acute respiratory syndrome coronavirus 2(SARS-CoV-2), and has become a pandemic. Most covi-19 patients exhibit mild to moderate symptoms, but about 15% progress to severe pneumonia, and about 5% eventually develop Acute Respiratory Distress Syndrome (ARDS), septic shock, and/or multiple organ failure. The main content of clinical treatment includes symptomatic management and oxygen therapy, and provides mechanical ventilation for patients with respiratory failure. Although several antiviral drugs, including the nucleotide analog remdesivir, are being actively tested for their effect against covd-19, no effective drug for treating covd-19 exists.

The pathogenesis of the new coronavirus pneumonia mainly lies in that: the 'inflammatory storm' produced by immune cells causes pathological damage to the organism. The research shows that: the NLRP3 inflammasome promotes the maturation and secretion of IL-1 beta and the apoptosis of cells, plays an important role in the process of amplifying the storm of the new coronavirus inflammatory factor, but the specific mechanism of activation is not clear. The novel coronavirus infection endangers the world at present, the cumulative confirmed cases of the new coronary pneumonia break through 766 ten thousand and the cumulative death cases exceed 42 ten thousand. One of the most significant reasons is the lack of early effective drug therapy. Therefore, the development of a drug treatment regimen for treating the novel coronavirus pneumonia is urgent.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide a target for treating the novel coronavirus pneumonia.

The second purpose of the invention is to provide a medicine for treating the novel coronavirus pneumonia.

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

a target for treating novel coronavirus pneumonia, wherein the target is NLRP3 inflammasome.

The research concept of the invention is as follows: the NLRP3 protein is an important member of NOD-like receptor family and is an important receptor for identifying pathogen-related molecular patterns and danger signal-related molecular patterns in cells. NLRP3 recruits pro-caspase-1 to self-cleave to form mature caspase-1(p20) and further cleave pro-IL-1 β to mature IL-1 β (p20) by recruiting ASCs to form active inflammasome complexes. At the same time, activation of NLRP3 inflammasome induces cellular inflammatory cell death, cell apoptosis. Plays an important role in autoimmune and inflammatory diseases. The invention firstly researches the function of the novel coronavirus M protein in the activation of NLRP3 inflammasome, and as a result, the novel coronavirus M protein is found to be capable of interacting with NLRP3, the interaction of the novel coronavirus M protein and the NLRP3 inflammasome is positive, and the NLRP3 inflammasome promotes the maturation and secretion of IL-1 beta and the scorching of cells, so that a strong inflammatory reaction in vivo is initiated, namely the condition of a patient with the novel coronavirus pneumonia is aggravated.

The inventor constructs macrophages with over-expressed new coronary M protein through cell experiments, and then finds that the secretion of cytokine IL-1 beta over-expressed new coronary M protein is increased, the cell apoptosis is increased, and therefore the NLRP3 inflammatory corpuscle is determined to be a target for treating the novel coronavirus pneumonia.

Accordingly, the present invention also provides a medicament for the treatment of novel coronavirus pneumonia, which is for inhibiting the activation of NLRP3 inflammasome.

There are several substances that inhibit the activation of NLRP3 inflammasome, such as CY-09, MCC950 and Tranilast, among others, and in some embodiments, the medicaments of the present invention include MCC950 inhibitors.

Therefore, the invention also provides application of the MCC950 inhibitor in preparing a medicament for treating novel coronavirus pneumonia.

Compared with the prior art, the invention has the beneficial effects that:

the invention takes the NLRP3 inflammasome as a target spot for immunotherapy of the neocoronary pneumonia for the first time. The specific inhibitor of NLRP3 inflammasome and the immunotherapy method thereof can realize the immunotherapy of the neocoronary pneumonia, have the advantage of slowing down the storm of inflammatory factors, and are suitable for the comprehensive therapy of the neocoronary pneumonia.

Drawings

FIG. 1A shows the interaction of 293T cell neocoronavirus structural proteins S, E, M, N with NLRP 3; figure 1B shows the interaction of a549 cells neocoronavirus structural protein M with NLRP 3; FIG. 1C shows immunofluorescence co-localization of 293T cell neocoronavirus structural protein M with NLRP 3; FIG. 1D is a graph of the effect of THP1 cell structural proteins E, M, N of the neocoronavirus on the activation of NLRP3 inflammasome; FIG. 1E shows the effect of THP1 cellular neocoronavirus structural proteins E, M, N on cellular lactate dehydrogenase release;

FIG. 2A is a graph of the effect of a MCC950 inhibitor of THP1 cells on the activation of inflammatory bodies by a novel coronavirus M protein; FIG. 2B is a graph of the effect of an MCC950 inhibitor of THP1 cells on the induction of cellular lactate dehydrogenase release by the novel coronavirus M protein; FIG. 2C is a graph of the effect of 293T cell MCC950 inhibitor on activation of inflammatory bodies by novel coronavirus M protein; figure 2D is a graph of the effect of MCC950 inhibitors in a549 cells on ASC aggregation induced by the new coronavirus M protein.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 investigation of the Effect of the structural protein of the novel coronavirus on the inflammasome

293T cells were transfected with plasmids S, E, M, N expressing different novel coronavirus proteins. Collecting cell lysate, incubating the cell lysate with Flag beads, incubating for four times overnight, eluting, detecting and analyzing WB, and obtaining a result shown in FIG. 1A, wherein the result shown in FIG. 1A shows that the M protein of the new coronavirus directly interacts with NLRP3, and other structural proteins do not directly interact with NLRP 3.

A549 cells are transfected with a plasmid for expressing the new crown protein M, Flag beads and cell lysate are used for co-incubation, four-day overnight incubation and elution are carried out, WB is detected and analyzed, the result is shown in figure 1B, and the result in figure 1B shows that the new crown protein M expressed in the A549 cells can interact with NLRP 3.

293T cells were transfected with a plasmid expressing Flag-tagged neocoronavirus protein M and a plasmid expressing NLRP3 protein with HA-tag. Different immunofluorescence secondary antibodies are used for staining, the result is observed by a confocal microscope, the result is shown in figure 1C, and the immunofluorescence result in figure 1C shows that NLRP3 and the new crown M protein exist in co-localization.

THP1 cells were transfected with plasmids E, M, N expressing different novel coronavirus proteins. Cell lysate and cell culture supernatant are collected, WB is used for detecting the expression of different molecules, and the result is shown in figure 1D, and the result in figure 1D shows that the neocoronary M protein in THP1 cells can activate NLRP3 inflammasome and promote the maturation and secretion of caspase-1 and IL-1 beta. At the same time, the increased splicing of GSDMDM-N, i.e. the neocoronal M protein, activates NLRP3 inflammatory bodies to promote apoptosis of cells.

THP1 cells were transfected with plasmids E, M, N expressing different novel coronavirus proteins. Collecting cell culture supernatant, detecting the release of lactate dehydrogenase by using the kit, wherein the result is shown in figure 1E, and the result of figure 1E shows that the new crown M protein promotes the release of the THP1 cell lactate dehydrogenase, namely the new crown M protein obviously promotes cell death.

In summary, it can be concluded that: the new crown M protein can directly interact with NLRP3, and promotes the activation of NLRP3 inflammasome, the maturation and release of IL-1 beta cytokines and the scorching of cells.

Example 2 Effect of NLRP3 inhibitor MCC950 on activation of NLRP3 inflammatory bodies by New coronavirus M protein

THP1 cells were transfected with a plasmid expressing the new coronavirus M protein, treated with MCC950 inhibitor. The cell lysate and cell culture supernatant are collected, WB detects the expression of different molecules, and the results are shown in FIG. 2A, and the results in FIG. 2A show that the MCC950 inhibitor inhibits NLRP3 inflammatory bodies activated by new coronary M protein, and inhibits the maturation and release of cytokine IL-1 beta and cell apoptosis.

THP1 cells were transfected with a plasmid expressing the new coronavirus M protein, treated with MCC950 inhibitor. The cell culture supernatant is collected, the kit detects the release of lactate dehydrogenase, and the result is shown in fig. 2B, and the result in fig. 2B shows that the MCC950 inhibitor inhibits the release of lactate dehydrogenase induced by the new coronatine M protein, i.e., the MCC950 inhibits the death of cells induced by the new coronatine M protein.

293T cells express NLRP3 inflammatory corpuscle different components NLRP3, caspase-1, IL-1 beta and ASC, and an NLRP3 inflammatory corpuscle system is reconstructed, and an MCC950 inhibitor is added. Cell lysates and cell culture supernatants were collected, WB was used to detect the expression of different molecules, and the results are shown in fig. 2C, and fig. 2C shows that MCC950 inhibitor inhibited NLRP3 inflammasome activated by neocoronin, and inhibited the maturation and release of cytokine IL-1 β and apoptosis of cells in 293T cells.

A549 cells were transfected with a plasmid expressing the novel coronavirus M protein, with the addition of an MCC950 inhibitor. Cell lysates were collected, Dextran Sodium Sulfate (DSS) was added for crosslinking at 37 ℃, WB was used to detect ASS aggregation, and the results are shown in fig. 2D, where fig. 2D shows that the new crown M protein in a549 cells promotes ASC aggregation, and MCC950 inhibitor inhibits ASC aggregation induced by M protein.

In summary, it can be concluded that: MCC950 inhibitors inhibit activation of the new crown M protein-induced NLRP3 inflammasome, maturation and release of IL-1 β, cell apoptosis, and aggregation of ASCs.

Claims

1. A target for the treatment of novel coronavirus pneumonia, wherein the target is NLRP3 inflammasome.

2. A medicament for the treatment of novel coronavirus pneumonias, wherein said medicament is for inhibiting the activation of NLRP3 inflammasome.

3. The medicament for treating novel coronavirus pneumonia according to claim 2, wherein the medicament comprises CY-09, MCC950 and Tranilast.

Use of an MCC950 inhibitor for the preparation of a medicament for the treatment of a novel coronavirus pneumonia.