CN111265662A

CN111265662A - Use of intervention 14-3-3 in the treatment of sepsis

Info

Publication number: CN111265662A
Application number: CN202010086925.8A
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: 2020-02-11
Filing date: 2020-02-11
Publication date: 2020-06-12
Anticipated expiration: 2040-02-11
Also published as: CN111265662B

Abstract

The invention belongs to the technical field of immunotherapy, and discloses a medicament for treating sepsis, which is characterized in that the medicament can inhibit 14-3-3 protein. The invention uses 14-3-3 molecule as target point to treat pyemia for the first time. The targeted 14-3-3 method can realize the immunotherapy of the sepsis, has the advantages of improving the survival rate of the sepsis, relieving inflammation and the like, is suitable for the comprehensive therapy of the sepsis, and is suitable for clinical popularization and application.

Description

Use of intervention 14-3-3 in the treatment of sepsis

Technical Field

The invention relates to the technical field of immunotherapy, in particular to application of intervention 14-3-3 in treatment of sepsis.

Background

Sepsis refers to Systemic Inflammatory Response Syndrome (SIRS) caused by infection, and clinically confirmed presence of bacteria or highly suspicious foci of infection is a major cause of death in critically ill patients. They can be classified according to their severity into sepsis, severe sepsis and septic shock. The pathogenesis of the disease is mainly pathological damage of the organism caused by 'inflammatory storm' generated by immune cells.

Although a great deal of manpower, material resources and financial resources are invested in various countries to treat the sepsis, the death rate is still high, and one of the main reasons is lack of early effective drug treatment.

Disclosure of Invention

Aiming at the technical defect that the prior art is lack of a drug treatment scheme for developing early sepsis, the invention firstly provides a drug for treating sepsis.

A second object of the invention is to provide a target for immunotherapy of sepsis.

The third purpose of the invention is to provide the application of the BV02 inhibitor in preparing the medicine for treating sepsis.

The technical scheme adopted by the invention is as follows:

a medicament for the treatment of sepsis, which medicament is capable of inhibiting the binding of 14-3-3 protein to its target protein.

The invention discloses a research concept that a 14-3-3 protein family is a regulatory protein family with complex functions and composed of a plurality of highly conserved but specific members, which mainly play a role by combining with a phosphorylated serine motif, firstly researches the role played by the 14-3-3 protein in sepsis, and as a result, discovers that 14-3-3 can interact with NLRP3 inflammasome, the interaction is increased along with the development of sepsis, the interaction between 14-3-3 and NLRP3 inflammasome is positive, NLRP3 inflammasome promotes the maturation and secretion of IL-1 β so as to initiate a strong inflammatory reaction in vivo, namely aggravated sepsis, and then discovers that the CLP sepsis model of a 14-3-3 myeloid cell-deficient mouse is built through experiments, the survival rate of the 14-3-3 gene-deficient mouse is increased, the inflammatory reaction is slowed down, and the pathological lung injury is relieved, thereby determining that the 14-3-3 protein is a target point for treating sepsis.

Therefore, the combination of the 14-3-3 protein and the NLRP3 protein is inhibited, so that the sepsis is relieved, and the aim of treating the sepsis is fulfilled.

There are several substances that inhibit 14-3-3 proteins, such as 2-5 protein, BV02 inhibitor, FOBISIN, etc., which bind to specific sites of 14-3-3 protein and thereby prevent interaction of 14-3-3 protein with the target protein (NLRP 3 protein), and in some embodiments, the agents of the present invention include BV02 inhibitors.

In some specific embodiments, the sepsis comprises early sepsis, severe sepsis, and septic shock.

Therefore, the invention also provides the application of the BV02 inhibitor in preparing a medicament for treating sepsis.

In some embodiments, the pharmaceutical dosage form is an intraperitoneal dosage form; by intraperitoneally infusing BV02 inhibitor, the interaction of 14-3-3 and target protein is blocked, and the activation of NLRP3 inflammasome is weakened.

The invention has the beneficial effects that:

the invention takes 14-3-3 as the target point of immunotherapy sepsis for the first time. The 14-3-3 inhibitor, namely the BV02 inhibitor and the immunotherapy method thereof can realize the immunotherapy of the sepsis, have the advantages of improving the survival rate, slowing down the inflammatory factor storm, the pathological damage of the lung and the like, and are suitable for the comprehensive therapy of the sepsis.

Drawings

FIG. 1A survival rate change of caecum ligation sepsis model in 14-3-3 gene deficient mice; FIG. 1B is a microscope picture of 14-3-3 gene defective mouse caecum ligation sepsis model lung tissue section; FIG. 1C shows CD11b in 14-3-3 gene defect mouse caecum ligation sepsis model⁺And F4/80⁺Expression of IL-1 β in cells.

FIG. 2A is the effect of BV02 inhibitor on the survival rate of sepsis model mice, FIG. 2B is the microscope image of BV02 inhibitor treated cecal ligation sepsis model lung tissue section, FIG. 2C is the effect of BV02 inhibitor on the expression of L-1 β in sepsis model mice, and FIG. 2D is the effect of BV02 inhibitor on CD11B in sepsis model mice⁺Ly6G⁺The number of cells in (c).

Detailed Description

For a more clear understanding of the technical features, objects and effects of the present invention, embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

SPF-grade female C57BL/6 mice of the invention, 4-6 weeks old, were purchased from the center of Guangdong province animals; the methods used in the present invention are conventional in the art unless otherwise specified.

Example 114-3-3 study of mice deficient in myeloid lineage cells

A model of caecal ligation sepsis (CLP) was constructed in a WT mouse and a 14-3-3 gene deficient mouse using a 4-6 week SPF-grade female C57BL/6 mouse. Mouse survival was observed and recorded and the results are shown in figure 1A.

A mouse caecal ligation sepsis model (caecal ligation sepsis model mouse is constructed according to a conventional method in the field) is constructed by using SPF grade female C57BL/6 mice for 4-6 weeks. Lavage liquid of mouse lung and abdominal cavity is taken. The lung leaflets were fixed in 4% paraformaldehyde, and the lung tissue sections were stained with H & E and observed under a microscope, and the results are shown in fig. 1B.

The remaining lung tissue was milled, filtered, and subjected to specific flow antibody staining with peritoneal lavage fluid to detect antibodies CD11b +, F4/80+, and IL-1 β, respectively, as shown in fig. 1C.

The results in FIG. 1A show that the survival rate of the caecum ligation sepsis model of the 14-3-3 gene deficient mouse is increased compared with that of the wild mouse, which indicates that the 14-3-3 gene deficiency effectively improves the survival rate of the mouse.

FIG. 1B it can be seen that septal wall thickening and pulmonary alveolar hyperemia and edema were evident in septic mice. And the symptoms of septum wall thickening and pulmonary alveolus hyperemia and edema of the pulmonary alveolus of the 14-3-3 gene defect mouse sepsis model are obviously relieved.

The results in FIG. 1C show that the expression of macrophage IL-1 β in lung and abdominal cavity lavage fluid of a mouse in a sepsis model of a 14-3-3 gene defective mouse is obviously reduced.

In summary, it can be concluded that: 14-3-3 gene defect effectively improves survival rate of mice and slows down inflammatory factor storm and lung pathological injury, namely, reduces the content of 14-3-3 in vivo, and is beneficial to improving survival rate and slowing down inflammatory factor storm and lung pathological injury.

Example 214-3-3 Effect of inhibitor BV02 on sepsis model mice

After 12 hours, a mouse caecum ligation sepsis model is constructed by respectively injecting BV02 and DMSO (both 10mg/kg) into a tail vein of a 4-6 week SPF-grade female C57BL/6 mouse, and the survival rate of the mouse is observed and recorded, and the structure is shown in FIG. 2A.

A4-6-week SPF-grade female C57BL/6 mouse is injected with BV02 and DMSO (both 10mg/kg) in equal amounts in tail vein, 12 hours later, a mouse caecum ligation sepsis model is constructed, and mouse lung and peritoneal lavage fluid is taken. Grinding lung tissue, filtering, performing specific flow antibody staining together with peritoneal lavage solution, and detecting antibody CD11b +, F4/80+, ly6G⁺And IL-1 β, the results are shown in FIG. 2B, FIG. 2C and FIG. 2D.

The results in fig. 2A show that the survival rate of mice in the caecal ligation sepsis model injected with BV02 inhibitor was greatly improved compared to the survival rate of mice in the DMSO injected group. FIG. 2B it can be seen that septal wall thickening and pulmonary alveolar hyperemia and edema were evident in septic mice. And the symptoms of septum wall thickening in alveoli and alveolar congestion edema of a mouse sepsis model injected with the BV02 inhibitor are obviously relieved.

FIG. 2C results show that IL-1 β expression was significantly reduced in mice from the caecal ligation sepsis model injected with a BV02 inhibitor FIG. 2D results show CD11b in mice from the sepsis model injected with a BV02 inhibitor⁺Ly6G⁺The number of the cells is obviously reduced, namely the infiltration ratio of the neutrophils in the lung lavage fluid and the peritoneal lavage fluid is obviously reduced.

In summary, it can be concluded that: the 14-3-3 inhibitor BV02 is effective in improving mouse survival rate, and slowing down inflammatory factor storm and lung neutrophil infiltration.

While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A medicament for the treatment of sepsis, wherein said medicament is capable of inhibiting 14-3-3 protein.

2. A medicament as claimed in claim 1 for the treatment of sepsis, which comprises a BV02 inhibitor.

3. A medicament for the diagnosis of sepsis according to claim 2, characterized in that the sepsis comprises early sepsis, severe sepsis and septic shock.

Use of a BV02 inhibitor in the manufacture of a medicament for the treatment of sepsis.

5. The use of claim 4, wherein the medicament is in the form of an intraperitoneal administration.