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Cell glycocalyx protection effect of anisodamine

An anisodamine, cell technology, applied in the field of medicine, health food or food

Inactive Publication Date: 2020-12-25
CHENGDU FIRST PHARMACEDTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently no drugs that directly act on the glycocalyx

Method used

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  • Cell glycocalyx protection effect of anisodamine
  • Cell glycocalyx protection effect of anisodamine
  • Cell glycocalyx protection effect of anisodamine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1: the effect of anisodamine hydrobromide on LPS-induced hCMEC / D3 cell proliferation

[0062] 1.1 Cell culture and treatment

[0063] Immortalized human brain microvascular endothelial cells hCMEC / D3 were obtained from Beijing Beinan Chuanglian Institute of Biotechnology (BNBIO), China. hCMEC / D3 endothelial cells were incubated with endothelial cell medium (ECM; cat.1001, Sciencell, USA) at 37°C and 5% CO 2 cultivated under conditions. Anisodamine hydrobromide was obtained from Chengdu First Pharmaceutical Co., Ltd., Chengdu, China, dissolved in 20 mg / mL distilled water, and filtered through a 0.22 μm membrane filter. Prepare anisodamine hydrobromide at final concentrations of 10, 20, 40, and 80 μg / mL using ECM. LPS was dissolved in distilled water at 2 mg / mL, and 1, 10, 20, 50, and 100 μg / mL of LPS were prepared with ECM. hCMEC / D3 cells were treated with LPS and anisodamine hydrobromide.

[0064] 1.2 Cell proliferation detection

[0065] Cell prolifera...

Embodiment 2

[0069] Example 2: Effect of anisodamine hydrobromide on glycocalyx and adhesion of vascular endothelial cells

[0070] 2.1 Experimental grouping: normal control group, LPS treatment group (50 μg / mL), low concentration administration group (LPS 50 μg / mL + anisodamine hydrobromide 10 μg / mL), high concentration administration group (LPS 50 μg / mL + hydrogen bromide Anisodamine 20 μg / mL).

[0071] 2.2 Immunofluorescence staining and confocal microscopy

[0072] After the cells were fixed and permeabilized, they were blocked with 2% goat serum, using mouse heparan sulfate (HS) monoclonal antibody (1:100, 10E4 epitope, AMS Biotechnology, UK) and rabbit cadherin polyclonal antibody (1:100 , A0734, Abclonal, China) and Alexa Fluor 488 goat anti-mouse (1:400, A11001, Thermo Fisher), Alexa Fluor 568 donkey anti-rabbit (1:400, A10042, Thermo Fisher) secondary antibodies to label the cells. Cell nuclei were labeled with DAPI and detected under a confocal microscope on LSM710 after mounti...

Embodiment 3

[0075] Example 3: Effect of anisodamine hydrobromide on LPS disrupted endothelial permeability

[0076] 3.1 Experimental grouping: same as Example 2

[0077] 3.2 Endothelial permeability assay

[0078] cells (5×10 4 Inoculate the upper well of Transwell (0.4 μm, Corning, USA) in 200 μL ECM, add 200 μL ECM to the lower well, and inoculate at 37°C and 5% CO 2 Incubate for 3 days in an incubator until 90% confluent. The medium in the upper wells was replaced with fresh ECM at 50 μg / mL LPS with or without anisodamine hydrobromide (10 or 20 μg / mL), and the medium in the lower wells was replaced with fresh ECM. After 24 hours, 10 μL of fluorescein isothiocyanate (FITC)-dextran (40 kDa; Cat. 46944, Sigma-Aldrich, USA) was added to the upper well. After 40 min, remove 100 µL of medium from the lower chamber. Measured fluorescence values ​​at wavelengths of 490nm and 525nm. The leakage of FITC-dextran was calculated to assess the permeability of the endothelial cell layer.

[00...

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Abstract

The invention relates to application of anisodamine or a pharmaceutically acceptable salt thereof in preparation of a cell glycocalyx protective agent, in particular to application of the anisodamineor the pharmaceutically acceptable salt thereof serving as the cell glycocalyx protective agent in recovery and protection of cell glycocalyx and cell adhesion. The application of the anisodamine or the pharmaceutically acceptable salt thereof in preparation of the cell glycocalyx protective agent also has the effect of protecting endothelial cell structures and functions.

Description

technical field [0001] The present disclosure relates to the field of medicine, health food or food, and in particular, the present disclosure relates to the restoration and protection of anisodamine on glycocalyx, cadherin and permeability of endothelial cells. Background technique [0002] Vascular diseases are closely related to vascular endothelial dysfunction. Serum lipopolysaccharide (LPS) is closely related to atherosclerosis. High-density lipoproteins (HDLs) can bind LPS. The sharp decrease of HDL levels in atherosclerosis, stroke and sepsis may lead to a decrease in the body's ability to clear LPS. [0003] LPS can inhibit the formation of endothelial adherens junctions by down-regulating VE-cadherin in pulmonary endothelial cells, destroying the integrity of pulmonary vessels and aggravating inflammatory injury. LPS can damage the endothelium and promote thrombus formation. In addition, LPS inhibits the expression of endothelial nitric oxide synthase (eNOS), bu...

Claims

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Application Information

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IPC IPC(8): A61K31/46A61P9/14A61P9/00
CPCA61K31/46A61P9/14A61P9/00
Inventor 曾烨吴庆江岳永花冷为军
Owner CHENGDU FIRST PHARMACEDTICAL CO LTD
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