CN103992985A - Application of D-erythro-Sphingosine in central nervous system - Google Patents

Abstract

The invention relates to the application of a compound sphingosine (D-erythro-Sphingosine, DES) in the central nervous system. During the pathogenesis of multiple sclerosis, many inhibitory factors are produced in the disintegrated axon myelin sheath. These inhibitors inhibit maturation and remyelination of nascent oligodendrocytes. The results of our screening of compounds prove that sphingosine can relieve the inhibition of myelin fragments on the differentiation and maturation of new oligodendrocytes, and promote the recovery of motor ability in EAE demyelinated rats, thus suggesting that the compound can be used for central nervous system demyelination. Treatment of myelin disorders.

Description

The role of sphingosine in the central nervous system

technical field

The invention relates to the application of a compound sphingosine (D-erythro-Sphingosine, DES) in the central nervous system.

Background technique

In the pathogenesis of central nervous system demyelinating diseases such as multiple sclerosis, it is accompanied by the differentiation, maturation and remyelination of OPC cells. At present, the research on the molecular mechanism of how to promote the process of remyelination in patients with multiple sclerosis is a hot spot both at home and abroad. During the pathogenesis of multiple sclerosis, many inhibitory factors are produced in the disintegrated axon myelin sheath. These inhibitors inhibit maturation and remyelination of nascent oligodendrocytes. The use of antibodies antagonizing these inhibitors was found to induce partial recovery of function in experimentally demyelinated rats. But its downstream signal transduction pathway is unknown.

Contents of the invention

One of the objectives of the present invention is to propose a use of sphingosine in the preparation of inhibiting the response of oligodendrocyte precursor cells to myelin sheath disintegration fragments.

The second object of the present invention is to propose a use of sphingosine in the preparation of a drug for promoting the maturation and remyelination of oligodendrocyte precursor cells.

D-erythro-Sphingosine has a molecular weight of 299.5, a molecular formula of C18H37NO2, and a structural formula of:

Experiments have proved that sphingosine can relieve the inhibition of myelin fragments on the differentiation and maturation of newborn oligodendrocytes, thus suggesting that the compound can be used to prepare drugs for treating demyelinating diseases of the central nervous system.

Description of drawings

Fig. 1 Immunohistochemical diagrams showing that the addition of sphingosine reversed the effect of myelin fragments on OPC cell differentiation and maturation.

Fig. 2 Statistical graphs showing that the addition of sphingosine reversed the effect of myelin fragments on OPC cell differentiation and maturation.

Detailed ways

The following examples can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.

Example 1: Culture of SD rat oligodendrocytes

The brains of SD neonatal rats were taken, and the bilateral cerebral cortex was separated, D. Wash with Hanks solution, pipette to make cell suspension, filter with 200-mesh cell mesh, centrifuge at 180×g for 5 min, discard the supernatant, add high-glucose DMEM culture medium containing 10% fetal bovine serum to resuspend the cells, and dilute to (1.0 -2.0) *106 density planted in 0.025% poly-lysine pretreated 75c㎡ culture flask and cultured in 5% CO2 for 8-11 days, after the cells were confluent and formed obvious stratification, OPCs shaking separation and purification began (the culture flask Place on a constant temperature air bath vibrating shaker, 37 degrees, 150r/min, shake for 2h to remove microglial cells, etc. D. Wash with Hanks solution, change the liquid; 5% CO2 incubator balance for 2h; 230r/min, 16-18h , 280r/min, 1-2h; collect the cell suspension, centrifuge at 400×g for 5 min, resuspend the cells in high-glucose DMEM medium containing 10% fetal bovine serum, and plant them in polylysine at a density of 1*104/cm2 on acid-pretreated coverslips). After 4-8 hours, when the cells adhere to the wall, replace with serum-free medium, the composition is DMEM/F12, containing 5mg/L insulin, 100mg/L transferrin, 6ug/L progesterone, 16mg/L putrescine, and sodium nitrite 5ug/L, PDGF 20ng/ml, bFGF 20ng/ml. Thereafter, the medium was changed every 2 days in half. OPC cell differentiation medium is Neurobasal plus B27 (1:50) plus T3 (30nM).

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Example 2: See Figures 1 and 2. We found that using myelin sheath fragments as a matrix to culture primary cultured rat OPC cells can inhibit the differentiation and maturation of OPC cells in its differentiation medium, and adding sphingosine reverses the myelin sheath Debris inhibits the differentiation and maturation of OPC cells. Figure 1 shows that the primary cultured rat OPC cells were cultured with myelin sheath fragments as a substrate and stained with MBP after 5 days of differentiation in the differentiation medium. reduce. After adding sphingosine, the OPC cells differentiated well, and the total area of their branches increased significantly. Figure 2: Statistical analysis of the total area of OPC cells grown on myelin debris in the control group (DMSO) and sphingosine treatment (DES 100 nM concentration) for 5 days, the area of the control group is 1, and the area of the treatment group is About 4 times that of the control group. The data are from three independent experiments and expressed as mean ± standard error. Comparison by Student's-t-test, p<0.001.

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Example 3: Preparation of EAE rat model and evaluation of exercise ability after treatment with sphingosine.

Strain: 20 Wistar rats, 6-8 weeks old, weighing (200 ± 20) g, female; (Shanghai Experimental Animal Center, Chinese Academy of Sciences).

Modeling method: According to the literature and previous work, MOG and Freund's adjuvant (the ratio is 1:1 by volume, each rat was dissolved with 50ul of complete Freund's adjuvant plus 50ul of 50ug of normal saline) Rat MOG (35-55 small peptide) was injected subcutaneously at multiple points to establish the model. After 3 days of immunization, 20 female Wistar rats were randomly divided into 2 groups: EAE + DMSO group (control group), EAE + intraperitoneal injection of sphingosine group [ 0.5 mg/ (kg d) ].Observe the scores of rats' exercise ability at different time points after treatment, the results are shown in Table 1:

EAE rat scoring test. EAE rat scores: 0.5 distal tail paralysis, 1 complete tail paralysis, 1.5 complete tail paralysis plus hindlimb weakness, 2.0 unilateral hindlimb paresis, 2.5 bilateral hindlimb paresis, 3.0 complete bilateral hindlimb paralysis, 3.5 complete bilateral hindlimb paralysis Paralysis plus unilateral forelimb paresis, 4.0 complete bilateral hindlimb paralysis plus bilateral forelimb paralysis, 5.0 moribund or dead.

Table 1: Comparison of neurological function scores in each experimental group at each stage (Mean±sem)

group Number of cases day 12 day 22 day 32 Day 42 EAE group 10 0.83±0.37 3.12±0.76 2.78±0.67 2.28±0.49 EAE+DES group 10 0.86±0.43 2.36±0.58* 1.67±0.52** 0.56±0.17***

*Compared with the EAE group in the same period, P < 0. 05; **Compared with the EAE group in the same period, P <0. 01; ***Compared with the EAE group in the same period, P < 0. 001

The results showed that after treatment with sphingosine, the motor ability of EAE rats was greatly enhanced at the peak of the onset and recovery period, suggesting that sphingosine can be used for the treatment of demyelinating patients in the recovery period. the

Claims (3)

1. ceramide suppresses oligodendroglia precursor cell to the application in the reaction of myelin disintegration fragment in preparation.

2. ceramide promotes oligodendroglia precursor cell maturation the application in pulpefaction sheath medicine again in preparation.

3. application according to claim 1, is characterized in that described ceramide has to be used for the treatment of clinically because of various inflammation, and damage and autoimmune disorder cause demyelinating disease of central nervous system to become.