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Application of estrogen up-regulation endothelial system protection molecule sphingosine-1-phosphate

A molecular sphingosine and endothelial system technology, which is applied in the field of application where estrogen upregulates the level of endothelial system protective molecule sphingosine 1-phosphate, and can solve the problems of poorly understood associations

Inactive Publication Date: 2013-12-04
TAISHAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although both estrogen and S1P have been shown to function as important protective molecules in the vascular endothelial system, little is known about their association

Method used

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  • Application of estrogen up-regulation endothelial system protection molecule sphingosine-1-phosphate
  • Application of estrogen up-regulation endothelial system protection molecule sphingosine-1-phosphate
  • Application of estrogen up-regulation endothelial system protection molecule sphingosine-1-phosphate

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Ten 22-week-old C57BL / 6 mice, half male and half male. After fasting for 4 hours, blood was collected from the orbital venous plexus and centrifuged at 3000 rpm for 10 minutes to obtain plasma.

[0028]Plasma S1P is prepared by alcohol precipitation method, that is, add 4 times the amount of plasma methanol or ethanol for ultrasonic extraction for 20 minutes, centrifuge at 12,000×g for 10 minutes to remove insoluble particles, and perform liquid phase tandem mass spectrometry analysis; the lipid extraction steps in the medium are as follows: Add chloroform, methanol, and 3 mol / L sodium hydroxide to the culture medium at a volume ratio of 1:1:1:0.1 (pH=12), mix well, and centrifuge at 3000×g for 10 minutes. Transfer the upper aqueous phase to a new tube, add chloroform and 6mol / L hydrochloric acid (pH=3), centrifuge at 3000×g for 10 minutes, dry the lower organic phase with nitrogen, redissolve in methanol, and centrifuge at 12,000×g for 10 minutes The insoluble particl...

Embodiment 2

[0030] 108 participants, aged 3-90 years (47 females, 61 males). Divided into three groups according to age, 3-15 years old, 18-55 years old and 56-90 years old. Plasma samples were collected the next day after a 12-hour fast, and were centrifuged at 3000 rpm for 10 minutes to obtain plasma. Sphingosine 1-phosphate (S1P) extract was prepared from plasma by methanol precipitation method, and the extract was centrifuged at 12000×g for 10 minutes and analyzed by liquid phase tandem mass spectrometry. Experimental results such as figure 2 and image 3 Shown: S1P levels decline with age. In the 3-15-year-old group, the mean ages of men and women were 8.2±3.4 and 8.2±3.6 years, respectively, and the plasma S1P levels of men and women were: 146.5±28.3 and 150.4±35.6ng / mL; The mean ages were 37.5±13.2 and 37.8±11.1 years old, respectively, and the plasma S1P levels of men and women were 110.9±20.4 and 130.1±30.8ng / mL. Women were significantly higher than men; in the 19-55 age gro...

Embodiment 3

[0032] After recovery, human umbilical vein endothelial cells (EA.hy926) were seeded into 6-well plates and cultured in DMEM medium. The temperature of the incubator is 37°C, and the concentration of carbon dioxide is 5%. After the cells adhered to the wall, they were treated with PBS, sphingosine or sphingosine combined with estradiol (both 1uMol / L), and continued to incubate in a 37°C incubator, and the 0 minutes, 2 minutes, 5 minutes, 10 Cells were harvested at 1 min, 20 min, 30 min, 60 min, 120 min and 180 min for mRNA expression analysis; cells and media were collected at 0 hr, 0.5 hr, 1 hr, 2 hr and 3 hr for S1P levels Determination and protein level analysis. The extraction steps of sphingosine 1-phosphate in the culture medium are as follows: add sodium hydroxide of chloroform, methanol, 3mol / L to the culture medium, the volume ratio is 1:1:1:0.1 (pH=12), mix, Centrifuge at 3000 x g for 10 minutes. Transfer the upper aqueous phase to a new tube, add chloroform and 6...

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Abstract

The invention discloses application of an estrogen up-regulation endothelial system protection molecule, sphingosine-1-phosphate and discloses novel applications of estrogen, namely promoting synthesis and excretion of endothelial system sphingosine-1-phosphate (S1P). The endogenous form, estradiol stimulated endothelial cell (EA. Hy926), of the estrogen is used for a certain period of time; then, the level of S1P in the endothelial cell and culture medium is increased significantly. The specific activator to the endothelial cell sphingosine kinase 2 is found. The novel technical means of up-regulating the level of S1P in the cell or plasma is provided. The novel molecular mechanism for cardiovascular protection of the estrogen is provided. The fact that the estrogen can activate a signal channel of a S1P / S1P receptor (S1P / S1PR) is found, and the signal channel is proved to participate in various cardiovascular protections.

Description

technical field [0001] The invention relates to the new endothelial protective function of estrogen, in particular to the application of estrogen to up-regulate the level of endothelial system protective molecule sphingosine 1-phosphate. Background technique [0002] Atherosclerotic cardiovascular disease is a chronic inflammatory disease with the highest morbidity and mortality worldwide, and protecting the endothelial system is one of the important strategies to prevent and treat atherosclerosis. In the 1990s, people tried to use estrogen replacement therapy to curb cardiovascular disease in women, but after more than ten years, it was found that the expected effect did not appear. The reason may be related to the important defects in the molecular mechanism of estrogen anti-atherosclerosis discovered in the experiment. Therefore, re-examining the vascular protection mechanism and molecular targets of estrogen may provide new ideas for estrogen replacement therapy. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K31/566A61K31/565A61P9/14
Inventor 秦树存郭守东于杨
Owner TAISHAN MEDICAL UNIV
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