CN114807032A

CN114807032A - Application of dexamethasone in preparation of anti-inflammatory drugs for inducing extracellular vesicles produced by neutrophils

Info

Publication number: CN114807032A
Application number: CN202210548483.3A
Authority: CN
Inventors: 黄振; 肖�琳; 崔芷莹; 陈江宁; 张峻峰
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2022-05-20
Filing date: 2022-05-20
Publication date: 2022-07-29
Anticipated expiration: 2042-05-20
Also published as: CN114807032B

Abstract

The invention discloses application of dexamethasone in inducing neutrophil to generate extracellular vesicles. Also discloses application of the dexamethasone to preparation of anti-inflammatory drugs for inducing the extracellular vesicles produced by the neutrophils. Dexamethasone stimulation can lead the expression level of anti-inflammatory factors IL-10 and TGF-beta in the neutrophils to be obviously up-regulated; and after the neutrophil granulocytes are stimulated by dexamethasone, the vesicle preparation yield is at least 10 times higher than that of an untreated neutrophil group. Therefore, dexamethasone has the effect of promoting the secretion of vesicles from the neutrophil, and the vesicles generated by the neutrophil are stimulated by the dexamethasone, so that the content of IL-10 and TGF-beta is remarkably improved, the dexamethasone has better vascular endothelial adhesion capability and anti-inflammatory activity, is expected to be developed into a novel biological treatment technology, and can be used for treating clinically refractory inflammatory related diseases.

Description

Application of dexamethasone in preparation of anti-inflammatory drugs for inducing extracellular vesicles produced by neutrophils

Technical Field

The invention relates to application of dexamethasone to preparation of a medicine for treating inflammation, wherein the dexamethasone induces extracellular vesicles produced by neutrophils.

Background

Extracellular Vesicles (EVs) are nanoscale Vesicles secreted by cells and have membrane structures, can carry various biomolecules such as proteins, lipids, miRNA, lncRNA and DNA, and can effectively protect the activity of corresponding biomolecules due to the closed membrane structure. Meanwhile, EVs can inherit the receptors and ligands of parent cells, and due to the nanometer-scale size of the EVs, the EVs are beneficial to crossing various physiological barriers in vivo and can be enriched in specific organ and tissue cells. Therefore, the extracellular vesicles can regulate the functions of target cells through the encapsulated biomolecules and are an important intercellular communication mode. In addition, as a natural biological vesicle, EVs also has the advantages of low toxicity, low immunogenicity, good biocompatibility and the like, so that the biological therapy based on the extracellular vesicle has good clinical application prospect.

Neutrophils, which are an important immune cell population in the body, play an important role in inflammation-related diseases and defense against pathogen infection because of their strong chemotaxis and ability to reach the focal site across vascular endothelial cells in early onset. The migration of neutrophils across the blood vessel to the lesion site requires first the vascular endothelial cells that adhere to the lesion site, followed by cell deformation out of the vessel to the site of the inflamed tissue. The neutrophil-derived extracellular vesicle can inherit the property of a neutrophil membrane, for example, the surface of the neutrophil-derived extracellular vesicle is also rich in Integrin beta 2, which suggests that the neutrophil-derived extracellular vesicle also has better targeting performance for inflammatory tissues. However, the preparation efficiency of extracellular vesicles is always a key factor limiting the large-scale application of the extracellular vesicles. Therefore, it is very important to find a new stimulant which can effectively improve the production efficiency of the vesicle while ensuring the vesicle to inherit the characteristics of the parent cell.

Disclosure of Invention

The invention aims to provide application of dexamethasone in inducing neutrophil to generate extracellular vesicles.

Another objective of the invention is to provide the application of dexamethasone to the preparation of the medicine for treating inflammation diminishing, wherein the dexamethasone induces the extracellular vesicles produced by the neutrophils.

Has the advantages that: dexamethasone stimulation can lead the expression level of anti-inflammatory factors IL-10 and TGF-beta in the neutrophils to be obviously up-regulated; and after the neutrophil granulocytes are stimulated by dexamethasone, the vesicle preparation yield is at least 10 times higher than that of an untreated neutrophil group. Therefore, dexamethasone has the effect of promoting the secretion of vesicles from the neutrophil, and the vesicles generated by the neutrophil are stimulated by the dexamethasone, so that the content of IL-10 and TGF-beta is remarkably improved, the dexamethasone has better vascular endothelial adhesion capability and anti-inflammatory activity, is expected to be developed into a novel biological treatment technology, and can be used for treating clinically refractory inflammatory related diseases.

Drawings

FIG. 1 is a transmission electron microscope analysis view of neutrophil-derived extracellular vesicles.

FIG. 2 is a diagram of Nanoparticle Tracking Analysis (NTA) analysis of neutrophil-derived extracellular vesicles.

FIG. 3, the vascular endothelial adhesion ability of the vesicles was examined in vitro.

FIG. 4, in vitro evaluation of the anti-inflammatory effect of the vesicles on vascular endothelial cells.

Figure 5, in vitro evaluation of the anti-inflammatory activity of the vesicles on macrophages (qRT-PCR).

Figure 6, in vitro evaluation of the anti-inflammatory activity (ELISA) of the vesicles on macrophages.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

A method of isolating extracellular vesicles from dexamethasone-induced neutrophils, comprising the steps of:

1) dexamethasone stimulated neutrophils for two days;

2) centrifuging the cells at room temperature of 300g for 5min, and removing the cells;

3) taking the supernatant, centrifuging at room temperature of 2000g for 10min, and removing cell debris;

4) taking the supernatant, centrifuging at 160000g for 1h at 4 ℃, and obtaining the extracellular vesicles from white micro flocculent precipitates at the bottom of a centrifuge tube.

S1, characterizing morphological features of the above-obtained neutrophil-derived extracellular vesicles, and analyzing morphology and particle size of the vesicles by Transmission Electron Microscopy (TEM) (see fig. 1) and Nanoparticle Tracking Analysis (NTA) (see fig. 2).

S2, detecting the vascular endothelial adhesion capability of the vesicle. Firstly, culturing a monolayer of human umbilical vein endothelial cells HUVEC in a pore plate, activating the cells by using LPS, and then fixing the cell morphology; the adhesion of the DiI dye-labeled vesicles to the immobilized HUVEC cells was observed by immunofluorescence after incubation for 6h or 12 h. The experimental results demonstrate that the significant increase of ICAM-1 expression on the surface of HUVEC cells after activation by LPS stimulation will help the neutrophil-derived vesicles to adhere to vascular endothelial cells, so that more vesicles can be observed to adhere to LPS-activated cells compared to normal umbilical vein endothelial cells, and the amount of vesicles adhering to the surface of activated HUVEC cells gradually accumulates as the incubation time is prolonged (see fig. 3). Relevant results show that the separated vesicle from the neutrophil has stronger vascular adhesion capability and can be more effectively adhered to vascular endothelial cells in an inflammatory state, which is particularly important for the subsequent targeting and enrichment of EVs at inflammatory sites.

S3, the inflammation inhibition effect of EVs on HUVEC cells activated by LPS is also determined while the vesicle vascular adhesion capability is detected. And (3) incubating NC-EVs and Dex-EVs with the activated cells for 24h, and collecting cells to detect the expression condition of related genes. The experimental result shows that the expression of inflammatory factors is up-regulated after LPS activation, such as IL-1 beta, IL-6, TNF-alpha, monocyte chemotactic protein 1 (MCP-1) and the like, wherein the MCP-1 gene is obviously increased by about 12 times. Meanwhile, compared with NC-EVs, Dex-EVs have obvious inflammation inhibition effect on activated HUVEC cells and obviously reduce the level of relevant proinflammatory factors (figure 4).

S4, the anti-inflammatory effect of EVs on LPS-activated Raw264.7 cells was evaluated by qRT-PCR and ELISA assays. The inflammation inhibition effect of Dex-EVs on activated macrophages is evaluated from the mRNA level of the gene, and the result shows that the mRNA level of IL-10 is up-regulated to about 20 times of that of a Control group after the Dex-EVs are absorbed by macrophages activated by LPS, and meanwhile, the expression levels of proinflammatory factors such as IL-1 beta, IL-6, TNF-alpha and the like are obviously down-regulated (figure 5); the secretion of inflammatory cytokines in the culture supernatant of Raw264.7 cells is also detected, and the result is shown in the figure, the content of the proinflammatory mediators in the cell supernatant taking Dex-EVs is obviously reduced, and the IL-10 secretion is greatly improved to about 4000pg/ml, which is critical for the subsequent Raw264.7 cells to exert anti-inflammatory activity and even eliminate inflammatory response (figure 6). In contrast, the levels of inflammatory factors were not significantly different after Raw264.7 cells phagocytosed NC-EVs compared to LPS alone treated groups.

Claims

1. Use of dexamethasone for inducing neutrophil production of extracellular vesicles.

2. Application of dexamethasone in preparing medicine for treating inflammation and inducing extracellular vesicle generated by neutrophil is provided.