CN114807032B

CN114807032B - Application of dexamethasone-induced neutrophil-produced extracellular vesicles in preparation of anti-inflammatory drugs

Info

Publication number: CN114807032B
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: 2023-09-12
Anticipated expiration: 2042-05-20
Also published as: CN114807032A

Abstract

The invention discloses an application of dexamethasone in inducing neutrophils to generate extracellular vesicles. Also discloses the application of dexamethasone in inducing the extracellular vesicles produced by neutrophils in preparing anti-inflammatory drugs. Dexamethasone stimulation significantly up-regulates the expression levels of the anti-inflammatory factors IL-10 and TGF-beta in neutrophils; and the vesicle preparation yield is at least 10-fold higher than that of the untreated neutrophil group after the neutrophil group is stimulated by dexamethasone. Therefore, the dexamethasone has the effect of promoting the secretion of vesicles by the neutrophils, and the dexamethasone stimulates the vesicles produced by the neutrophils, so that not only the IL-10 and TGF-beta content is obviously improved, but also the vascular endothelial adhesion capability and anti-inflammatory activity are better, and the dexamethasone is expected to develop into a novel biological treatment technology and can be used for treating clinically refractory inflammatory related diseases.

Description

Application of dexamethasone-induced neutrophil-produced extracellular vesicles in preparation of anti-inflammatory drugs

Technical Field

The invention relates to an application of dexamethasone to the preparation of anti-inflammatory drugs for inducing extracellular vesicles produced by neutrophils.

Background

Extracellular vesicles (Extracellular Vesicles, EVs) are nanoscale vesicles secreted by cells and having membrane structures capable of carrying various biomolecules such as proteins, lipids, mirnas, lncRNA, DNA and the like, and the closed membrane structures of the extracellular vesicles can effectively protect the activity of the corresponding biomolecules. While EVs are capable of inheriting the receptors and ligands of the parent cell and, due to their nanoscale dimensions, facilitate their crossing of various physiological barriers in vivo and their enrichment in specific organ and tissue cells. So extracellular vesicles can regulate the function of target cells through the encapsulated biomolecules, and are an important intercellular communication mode. In addition, as a natural biological vesicle, the 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 are an important immune cell population of the organism, and can reach focus positions through vascular endothelial cells in early stage of onset due to strong chemotaxis, so that the neutrophils play an important role in inflammation-related diseases and resisting pathogen infection. Migration of neutrophils across the blood vessel to the lesion first requires vascular endothelial cells to adhere to the lesion, and then the cells deform out of the blood vessel to the site of inflamed tissue. Neutrophil-derived extracellular vesicles can inherit the properties of neutrophil membranes, for example, the surface of neutrophil-derived extracellular vesicles is also rich in Intergrin beta 2, suggesting that the neutrophil-derived extracellular vesicles also have better inflammatory tissue targeting performance. However, the efficiency of extracellular vesicle production has been a key factor limiting its large-scale application. Therefore, it is important to find new stimulators to ensure that vesicles inherit the characteristics of parent cells and to effectively improve their production efficiency.

Disclosure of Invention

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

Another object of the invention is to provide the use of dexamethasone for inducing the production of extracellular vesicles by neutrophils in the preparation of a medicament for the treatment of inflammation.

The beneficial effects are that: dexamethasone stimulation significantly up-regulates the expression levels of the anti-inflammatory factors IL-10 and TGF-beta in neutrophils; and the vesicle preparation yield is at least 10-fold higher than that of the untreated neutrophil group after the neutrophil group is stimulated by dexamethasone. Therefore, the dexamethasone has the effect of promoting the secretion of vesicles by the neutrophils, and the dexamethasone stimulates the vesicles produced by the neutrophils, so that not only the IL-10 and TGF-beta content is obviously improved, but also the vascular endothelial adhesion capability and anti-inflammatory activity are better, and the dexamethasone is expected to develop into a novel biological treatment technology and can be used for treating clinically refractory inflammatory related diseases.

Drawings

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

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

FIG. 3 in vitro test of vascular endothelial adhesion capacity of the vesicles.

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

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

FIG. 6 in vitro evaluation of anti-inflammatory Activity (ELISA) of the vesicles on macrophages.

Detailed Description

The invention will be further illustrated with reference to specific examples. These examples are only for illustrating the present invention and are not intended to limit the scope of the present 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) Centrifuging the supernatant at 2000g for 10min at room temperature to remove cell debris;

4) Taking the supernatant, centrifuging at 4 ℃ for 1h with 160000g, and obtaining white micro flocculent precipitate at the bottom of the centrifuge tube, namely the extracellular vesicles.

S1, characterizing morphological characteristics of the obtained neutrophil-derived extracellular vesicles, and analyzing morphology and particle size of the vesicles by a Transmission Electron Microscope (TEM) (shown in figure 1) and a nanoparticle tracking analysis technology (NTA) (shown in figure 2).

S2, detecting vascular endothelial adhesion capacity of the vesicle. First, culturing a monolayer of human umbilical vein endothelial cells HUVEC in an orifice plate, activating the cells with LPS, followed by fixing the cell morphology; diI dye-labeled vesicles were incubated with immobilized HUVEC cells for 6h or 12h and their adhesion was observed by immunofluorescence. Experimental results demonstrate that significant increases in ICAM-1 expression on the surface of HUVEC cells following LPS-stimulated activation will facilitate neutrophil-derived vesicle adhesion to vascular endothelial cells, thus allowing the observation that LPS-activated cells adhere more vesicles than normal umbilical vein endothelial cells, and that the amount of vesicles adhered to the surface of activated HUVEC cells gradually accumulates over time (FIG. 3). The related results indicate that isolated neutrophil-derived vesicles have a strong vascular adhesion capacity and can adhere more effectively to vascular endothelial cells in inflammatory states, which is particularly important for subsequent targeting and enrichment of EVs at the site of inflammation.

S3, detecting the vascular adhesion capability of vesicles and simultaneously determining the inflammatory inhibition effect of EVs on HUVEC cells activated by LPS. After incubating NC-EVs and Dex-EVs with activated cells for 24 hours, the cells were collected to detect the related gene expression. Experimental results show that inflammatory factor expression is up-regulated after LPS activation, such as IL-1 beta, IL-6, TNF-alpha, monocyte chemotactic protein 1 (monocyte chemotactic protein 1, MCP-1) and the like, wherein the MCP-1 gene is remarkably increased by about 12 times. Simultaneously, dex-EVs had a significant anti-inflammatory effect on activated HUVEC cells compared to NC-EVs, significantly down-regulating proinflammatory factor levels (FIG. 4).

S4, evaluating the anti-inflammatory effect of EVs on LPS activated Raw264.7 cells through qRT-PCR and ELISA detection means. The inflammatory inhibition effect of Dex-EVs on activated macrophages is evaluated from the mRNA level of the genes, and the result shows that after the LPS activated macrophages take the Dex-EVs, the mRNA level of IL-10 is up-regulated by about 20 times of that of a Control group, and the expression level of pro-inflammatory factors such as IL-1 beta, IL-6, TNF-alpha and the like is significantly down-regulated (figure 5); secretion of inflammatory cytokines in raw264.7 cell culture supernatants was also examined, and as shown, the content of the pro-inflammatory mediators in cell supernatants taken up by Dex-EVs was significantly reduced, while the secretion of IL-10 was greatly increased, about 4000pg/ml, which was critical for the subsequent anti-inflammatory activity of raw264.7 cells and even elimination of inflammatory responses (fig. 6). In contrast, there was no significant difference in inflammatory factor levels 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.