CN108498523B - Preparation method and application of ganglioside derivative containing unsaturated fatty acid chain - Google Patents
Preparation method and application of ganglioside derivative containing unsaturated fatty acid chain Download PDFInfo
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- CN108498523B CN108498523B CN201710102185.0A CN201710102185A CN108498523B CN 108498523 B CN108498523 B CN 108498523B CN 201710102185 A CN201710102185 A CN 201710102185A CN 108498523 B CN108498523 B CN 108498523B
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- fatty acid
- unsaturated fatty
- ganglioside
- chain
- derivative
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Abstract
The invention relates to the use of a group of ganglioside derivatives containing unsaturated fatty acid chains for promoting neurite outgrowth. The ganglioside derivative library with unsaturated fatty acid chain is synthesized by a sphingolipid ceramide N-deacylase enzyme method, and is combined with a classical cell model for promoting neurite growth, the ganglioside derivative with unsaturated fatty acid chain has obviously improved biological activity for promoting neurite growth, and the difference of the carbon chain number of ganglioside sphingosine base has obvious influence on the biological activity for promoting neurite growth.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a high-efficiency preparation method of a group of ganglioside derivatives containing unsaturated fatty acid and application of the derivatives in promoting neurite growth.
Background
Gangliosides are a class of glycosphingolipids containing sialic acid residues, are typical components of the plasma membrane of nerve cells, and have important biological functions. Ganglioside GM1 is one of the representative important gangliosides, and has multiple neurotrophic effects, and can promote cell differentiation, induce neurite generation, protect neuron damage, and regulate nerve plasticity. GM1 has been widely used clinically for the treatment of vascular or traumatic central nervous system injury, parkinson's disease, alzheimer's disease, and the like.
Gangliosides are present in large amounts in the mammalian brain, with GM1, GD1a, GD1b, GT1b, etc. being the major components, and about 97% of the total gangliosides in the human brain. The compound has complex structure, and the main method for obtaining the nervone is to extract the nervone from natural organisms, and gangliosides are widely distributed in other vertebrates, plants, eukaryotes and procaryotes besides mammal tissues, and have various types and structures. It has been reported that in marine echinoderms, gangliosides with pharmaceutical activities are isolated and extracted, for example, gangliosides with neurite growth promoting activity are isolated from sea cucumbers and starfish, wherein in vitro experiments of gangliosides such as SJG-2, LLG-5, LLG-3, GAA-7 and the like show that the gangliosides have higher neurite growth promoting activity than GM1. However, many gangliosides are very low in biological content and have limited sources, and the naturally extracted gangliosides may have microscopic heterogeneity and be contaminated by pathogens. Therefore, it is difficult to extract and screen substances having specific pharmaceutical activities from various kinds of natural gangliosides by a natural extraction method.
The chemical total synthesis of the neuro-glycoside with complex structure is particularly difficult, and involves many steps such as protecting, deprotecting and stereoselectivity of genes, for example, the total chemical synthesis of LLG-3 requires 30 steps of reaction, byproducts are easy to generate in the process of modifying the chemical method, the yield is low, and a large amount of strong base and organic solvent polluting the environment are needed, so that the ganglioside with medicinal activity is difficult to obtain and screen by the chemical total synthesis method.
There is growing evidence that differences in fatty acid chains in ganglioside ceramide groups have a significant impact on their biological activity, particularly the inclusion of unsaturated fatty acid chains, can significantly alter ganglioside structure, and may also bring about substances of different medicinal value. The fatty acid chain of gangliosides can be replaced by using the catalytic property of sphingolipid ceramide N deacylase (Sphingolipid ceramide N-deacylase, SCDase) to build a ganglioside derivative library containing unsaturated fatty acid chains. However, the SCDase autocatalytically reversible reaction often results in a low conversion rate of the synthesis reaction.
Neurite outgrowth is a critical process for neural differentiation and renewal, which has a repair effect on impaired nerve function caused by injury or neurodegenerative diseases. The ganglioside GM1 has been shown to have neurite-growth activity and to promote neurite-growth in cells of neuronal origin. The fatty acid chains of gangliosides greatly affect the biological activity of gangliosides by interacting with lipid rafts and membrane-associated proteins, and no systematic study of ganglioside derivatives containing unsaturated fatty acid chains has been performed. Whether unsaturated fatty acid chains affect neurite outgrowth activity of gangliosides has not been reported in the related studies.
Therefore, there is a need to obtain a library of ganglioside derivatives containing unsaturated fatty acid chains by a simple and efficient method, and further study the role of the related derivatives in neurite outgrowth on the basis of the library.
Disclosure of Invention
The invention utilizes an SCDase enzyme method synthesis system to obtain a series of ganglioside derivatives with unsaturated fatty acid chains, and applies the ganglioside derivatives to biological function screening for promoting neurite growth, and researches show that the ganglioside derivatives with unsaturated fatty acid chains are beneficial to the growth and development of nerve cells.
In a first aspect of the invention, there is provided the use of a ganglioside derivative in the promotion of neurite outgrowth, said ganglioside comprising a glycosyl group, a sphingosine base and a fatty acid chain, wherein the sphingosine base is linked to the fatty acid chain by an amide bond to form a ceramide, the glycosyl group is linked to the ceramide by a glycosidic bond, said fatty acid being an unsaturated fatty acid;
in a preferred embodiment, the fatty acid is a monounsaturated fatty acid;
in another preferred embodiment, the fatty acid is a polyunsaturated fatty acid;
the carbon chain of the unsaturated fatty acid contains 12-24 carbon atoms, and the unsaturated bond is a double bond or a triple bond;
preferably, the fatty acid is a linear olefine acid selected from the group consisting of a fatty acid having fourteen carbon atoms, a fatty acid having sixty-six carbon atoms, a fatty acid having eighteen carbon atoms, and a fatty acid having twenty-two carbon atoms;
preferably, the olefinic acid has 1 to 6 unsaturated double bonds, comprising at least one or more double bonds selected from:
(1) The double bond is located between the 3 rd and 4 th carbon atoms from the methyl end of the fatty acid chain; or (b)
(2) The double bond is located between the 5 th and 6 th carbon atoms from the methyl end of the fatty acid chain; or (b)
(3) The double bond is located between the 6 th and 7 th carbon atoms from the methyl end of the fatty acid chain; or (b)
(4) The double bond is located between the 7 th and 8 th carbon atoms from the methyl end of the fatty acid chain; or (b)
(5) The double bond is located between the 9 th and 10 th carbon atoms from the methyl end of the fatty acid chain; or (b)
(6) The double bond is located between the 12 th and 13 th carbon atoms from the methyl end of the fatty acid chain; or (b)
(7) The double bond is located between the 15 th and 16 th carbon atoms from the methyl end of the fatty acid chain; or (b)
(8) The double bond is located between the 18 th and 19 th carbon atoms from the methyl end of the fatty acid chain.
Preferably, the alkenoic acid is selected from omega-3, omega-6, omega-7, omega-9 unsaturated fatty acids; the omega carbon atom refers to one carbon atom which is farthest from the carboxyl in the linear olefine acid; the omega-3, omega-6, omega-7, omega-9 unsaturated fatty acids refer to the first double bond present on the third, sixth, seventh and ninth carbon atoms from the omega carbon atom.
Preferably, the monounsaturated fatty acid is selected from cis-9-tetradecenoic acid (C14:1, n-9), cis-9-hexadecenoic acid (C16:1, n-9), cis-9-octadecenoic acid (C18:1, n-9); the polyunsaturated fatty acid is selected from cis-4, 7,10,13,16, 19-docosahexaenoic acid (DHA);
the glycosyl is an oligosaccharyl group, comprises 2-10 glycosyl residues and has at least one sialic acid residue;
preferably the oligosaccharide is selected from the group consisting of monosialyllactose, monosialotrihose and monosialotetrahexose;
more preferably, the glycosyl is monosialotetrahexose;
the carbon chain of the sphingosine base comprises 12-24 carbon atoms and is selected from a sphingosine long-chain base, a dihydrosphingosine long-chain base or a phytosphingosine long-chain base;
the sphingosine type long-chain base is long-chain sphingosine containing 1, 3-dihydroxyl-2-amino-4-olefin;
the dihydrosphingosine long-chain base is long-chain sphingosine containing 1, 3-dihydroxy-2-amino alkane;
the phytosphingosine long-chain base is long-chain sphingosine containing 1,3, 4-trihydroxy-2-amino alkane;
preferably, the sphingosine base is a sphingosine long-chain base, the number of carbon atoms is 18 or 20, and the sphingosine base comprises 1, 3-dihydroxyl-2-amino-4-octadecene or 1, 3-dihydroxyl-2-amino-4-eicosene.
More preferably, the ganglioside derivative with an unsaturated fatty acid chain is selected from cis-9-tetradecenoic acid-GM 1, cis-9-hexadecenoic acid-GM 1, cis-9-octadecenoic acid-GM 1 or cis-4, 7,10,13,16, 19-docosahexaenoic acid-GM 1.
The application of the ganglioside derivative in promoting neurite growth is the application of the ganglioside derivative in preparing a pharmaceutical composition or preparation for treating diseases such as neurodegenerative diseases, nerve injury, nerve repair or nerve regeneration and the like;
the pharmaceutical composition or preparation is meant to include a ganglioside derivative with an unsaturated fatty acid and a pharmaceutically acceptable carrier.
In a second aspect of the present invention, there is provided a process for producing ganglioside derivatives containing unsaturated fatty acid chains, using a synthesis reaction system of sphingolipid ceramide N-deacylase, the reaction system comprising (1) a buffer system for controlling pH in the range of 6.0 to 8.0; (2) a lysoganglioside; (3) unsaturated fatty acids; (4) an unsaturated fatty acid co-solvent; and (5) a sphingolipid ceramide N-deacylase;
wherein the concentration ratio of the hemolytic ganglioside to the unsaturated fatty acid is 1:1-4; the temperature of the system reaction is controlled between 25 and 38 ℃ and the reaction time is 12 to 24 hours; the reaction system does not contain metal ions and surfactant.
Preferably, the buffer system can be PBS buffer solution, HEPES buffer solution, tris buffer solution and other buffer systems which can maintain the pH stability of the solution; the cosolvent of the unsaturated fatty acid is preferably 10% of ethylene glycol dimethyl ether; the reaction temperature is preferably 37 ℃;
preferably, the reaction system is built in a buffer system with pH of 7.2-7.8;
more preferably, the reaction system is built in a buffer system at pH 7.5.
Compared with the prior art, the synthesis reaction system provided by the invention is optimized in reaction conditions, so that the reaction steps are simpler and more convenient, particularly when the pH value is maintained at 7.5, the sphingolipid ceramide N-deacylase has relatively high synthesis activity and relatively low hydrolysis activity, and in the optimized reaction system, the addition of metal ions and surfactants for inhibiting the synthesis reaction is limited, and the synthesis direction of the sphingolipid ceramide N-deacylase is effectively controlled; meanwhile, a cosolvent for promoting the dissolution of unsaturated fatty acid is added into the system, and is very easy to remove, so that the separation and purification of subsequent compounds are not affected. The step of separation and purification is simpler because no surfactant is used, the step of cleaning the surfactant is avoided, and the synthesis reaction liquid can be directly purified on a solid-phase extraction column (such as Sep Pak tC18 of Waters company in the United states), so that the purification step and the purification time are greatly shortened. Therefore, the synthesis reaction system provided by the invention is more efficient and simpler than the prior art.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
FIG. 1 shows a schematic structure of ganglioside GM1.
FIG. 2A scheme for enzymatic synthesis of GM1 derivatives
FIG. 3 shows the enzymatic synthesis of 10 GM1 derivatives containing unsaturated fatty acid chains
FIG. 4 comparison of neurite outgrowth promoting activity of GM1 and its derivatives. After stimulating Neuro2a cells with 25. Mu.M (A) or 50. Mu.M (B) GM1 or its derivative for 24 hours, the proportion of cells containing the projections was counted.
FIG. 5 phase contrast photograph of GM1 derivative with unsaturated fatty acid chain inducing differentiation of Neuro2a cells on a scale of 100. Mu.m
FIG. 6 effects of GM1 derivatives containing different unsaturated fatty acid chains and sphingosine bases on the growth of cellular neurites. (A-B) proportion of cells containing the projections. (C-D) longest protrusion length. (E-F) number of protrusions.
FIG. 7 cytotoxicity test of GM1 derivatives with unsaturated fatty acid chain and different sphingosine bases
Detailed Description
The inventor utilizes the bidirectional catalytic function of SCDase, further optimizes the synthesis reaction condition on the basis of fully hydrolyzing ganglioside to obtain hemolytic ganglioside, synthesizes a series of ganglioside derivatives with unsaturated fatty acid chains, combines a cell model for neurite growth on the basis, and screens out a batch of ganglioside derivatives with obviously improved activity for promoting neurite growth.
Gangliosides of the present invention, which are exemplified in fig. 1, are composed of sugar chains, sphingosine bases and fatty acid chains; the sugar chain is an oligosaccharide containing at least one sialic acid, and as shown in the typical representation of ganglioside of GM1 in FIG. 1, the sugar chain part is monosialotetrahexose, the sphingosine base part is 1, 3-dihydroxy-2-amino-4-octadecene, and the fatty acid chain part is octadecanoic acid.
The ganglioside derivative with unsaturated fatty acid chain can be the product synthesized by SCDase, or can be the product obtained from lipid extract obtained from animals or plants or directly from chemical synthesis. Ganglioside derivatives as described above may be modified. By modifying the ganglioside derivative of the invention, the biological activity can be improved, and the improvement of the activity is applicable to the application of the ganglioside derivative in promoting the growth of neurites.
The ganglioside derivative of the invention can be used as a single substance or a mixture or can be used together with other lipids under the condition that the biological activity of the ganglioside derivative is not affected by the other lipids.
The present invention also provides a pharmaceutical composition or preparation capable of promoting neurite outgrowth, comprising a ganglioside derivative having an unsaturated fatty acid chain as an active ingredient. The pharmaceutical composition comprises: the ganglioside derivative with unsaturated fatty acid chain and pharmaceutically acceptable carrier.
The ganglioside derivative with unsaturated fatty acid chain provided by the invention can promote neurite growth and accelerate synapse formation in the processes of nerve repair and regeneration due to peripheral or central nervous system disorder, injury and neurodegenerative diseases.
A "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity), i.e., with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such agent carriers: they are not per se essential active ingredients and are not overly toxic after administration.
Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. A sufficient description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub.Co., N.J.1991). The pharmaceutically acceptable carrier in the composition may contain a liquid such as water, phosphate buffer, ringer's solution, physiological saline, balanced salt solution, glycerol or sorbitol, etc. In addition, auxiliary substances such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and stabilizers, such as albumin, may also be present in these carriers.
In use, a safe and effective amount of the ganglioside derivative having an unsaturated fatty acid chain of the present invention is administered to a mammal (e.g., a human) in consideration of the route of administration, the health of the patient, and the like, which are within the skill of a skilled practitioner.
The invention also provides an injection drug delivery device (such as an injection needle), a drug box or a kit, which comprises: said ganglioside derivative having an unsaturated fatty acid chain or said pharmaceutical composition.
For convenience of clinical application, the pharmaceutical composition of the present invention may be contained in an injection applicator (e.g., an injection needle) in which a single dose of the pharmaceutical composition may be contained. The injection applicator may be contained in a kit for convenient storage and use.
Instructions for use may also be included in the kits or kits of the invention to facilitate proper use by those skilled in the art.
The cis-unsaturated fatty acid disclosed by the invention is a fatty acid with an unsaturated bond, and hydrogen atoms are distributed on the same side of the unsaturated bond. Taking cis-9-tetradecenoic acid as an example, it means that the fatty acid is composed of 14 carbon atoms to form a straight-chain cis-olefine acid, the unsaturated bond of which is located between the carbon atom at the 9 th position and the carbon atom at the 10 th position from the carboxyl terminal, abbreviated as (C14:1, n-9), other unsaturated fatty acids and the like.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
General description of the sources of the biochemical materials described in the present invention:
1. unsaturated fatty acids were purchased from carbofuran technology (Shanghai) and alaa Ding Shiji (Shanghai), respectively; sep-Pak tC18 cartridge was purchased from Waters, USA, and all reagents used were analytically pure.
2. Neuro2a cells were taken from the university of Shanghai transportation institute of biomedical engineering Jin Weilin real teacher laboratory; DMEM medium was purchased from Corning corporation; fetal Bovine Serum (FBS) and pancreatin were purchased from Gibco; non-essential amino acids and green streptomycin (diabody) were purchased from Invitrogen company; MTT was purchased from Sigma-Aldrich (St. Louis, USA).
Example 1 enzymatic Synthesis of ganglioside derivative library with unsaturated fatty acid chain
Since the neurite outgrowth promoting effect of ganglioside GM1 is most clear at present, we first establish a derivative library of GM1 with unsaturated fatty acid chain, and specific embodiments are as follows:
the route for enzymatic synthesis of GM1 derivatives containing unsaturated fatty acid chains is shown in figure 2.
First, the preparation of the sphingolipid ceramide N-deacylase required for the catalytic synthesis reaction and the substrate lysoganglioside GM1 (Lyso-GM 1) required for the synthesis of the GM1 derivative were carried out according to the proprietary of our previous application (application number: CN201410508221. X) and published articles (J Lipid Res,2015, 56:1836-1842).
The synthesis reaction system: the reaction system was 20ml of 50mM Tris-HCl (pH 7.5) containing about 10% ethylene glycol dimethyl ether (DME), about 20mg of lyso-GM1,1.5mM unsaturated fatty acid. 1ml of 1mg/ml SCDase was added to the reaction system every 8-12 hours and reacted for 24 hours, and the completion of the reaction was detected by TLC. Developing agent of TLC is chloroform, methanol, 0.02% CaCl 2 (5:4:1), the chromogenic reagent is CAM reagent (10 g/L cerium sulfate, 50g/L ammonium molybdate tetrahydrate, 10% sulfuric acid), and the conversion rate of the synthesis reaction is 60-90%.
The 10 GM1 derivatives containing unsaturated fatty acid chains obtained in the above synthesis system, the corresponding unsaturated fatty acid chains are respectively: cis-5-dodecenoic acid (C12: 1, n-5), cis-9-tetradecenoic acid (C14: 1, n-9), cis-11-eicosenoic acid (C20: 1, n-11) and cis-4, 7,10,13,16, 19-docosahexaenoic acid (DHA). Cis-9-hexadecenoic acid (C16:1, n-9), trans-2-hexadecenoic acid (2 HA), and cis-5, 8, 11, 14, 17-eicosapentaenoic acid (EPA), cis-9-octadecenoic acid (C18:1, n-9), cis-13-docosenoic acid (C22:1, n-13), cis-15-tetracosenoic acid (C24:1, n-15), the structures of which are shown in FIG. 3.
Other ganglioside derivatives containing unsaturated fatty acid chains are also enzymatically synthesized according to the method shown in figure 2. First, the different gangliosides were hydrolyzed with SCDase to prepare lysogangliosides. 100ml of the reaction system (pH 5.8) containing 0.4-0.8% (w/v) of surfactant TDC), 10. Mu.l of enzyme solution, and different gangliosides (GM 1, GM2, GM3, GD1, GD2, GD3, GT1, GT2, GT3, etc.) with final concentrations of 0.2-10 mM, 100mM of metal ions Ca 2+ The reaction mixture was incubated at 37℃for 12 hours to fully hydrolyze gangliosides, and the degree of ganglioside hydrolysis was detected by TLC. Developing agent of TLC is chloroform, methanol, 0.02% CaCl 2 (5:4:1), the chromogenic reagent is a CAM reagent (10 g/L cerium sulfate, 50g/L ammonium molybdate tetrahydrate, 10% sulfuric acid).
Then the prepared lysoganglioside reacts with unsaturated fatty acid to synthesize ganglioside derivative with unsaturated fatty acid chain under the catalysis of SCDase. The reaction system was about 10% DME, about 40mg of lysoganglioside, and 1 to 4mM of unsaturated fatty acid in 40ml of 50mM PBS (pH 6.0 to 8.0). 1ml of 1mg/ml SCDase was added to the reaction system every 8-12 hours and reacted for 12-24 hours, and the completion of the reaction was detected by TLC. Developing agent of TLC is chloroform, methanol, 0.02% CaCl 2 (5:4:1), the chromogenic reagent is a CAM reagent (10 g/L cerium sulfate, 50g/L ammonium molybdate tetrahydrate, 10% sulfuric acid)
The unsaturated fatty acids used may be selected from the group consisting of alkenoic or alkynoic acids, including omega-3, omega-6, omega-7, omega-9 unsaturated fatty acids.
Example 2: isolation and purification of ganglioside derivative library with unsaturated fatty acid chain
After the catalytic synthesis reaction is completed, the derivative with unsaturated fatty acid chain ganglioside needs to be separated and purified from the reaction mixture. Here, the separation and purification were carried out using a Sep-Pak tC18 solid-phase extraction column (1 column of 500mg, 1 column of 5 g) from Waters. Because the catalyst does not contain a surfactant, the synthesis reaction liquid can be directly purified on a solid phase extraction column: the reaction mixture was passed through a small Sep-Pak tC18 solid phase extraction column (500 mg), then the flow through was passed through a large Sep-Pak tC18 solid phase extraction column (5 g), washed with water 50ml, then 50ml with 50% methanol, finally the derivative was washed with 100% methanol, concentrated by rotary evaporation and lyophilized. Alternatively, the reaction mixture may be first passed through a large Sep-Pak tC18 solid phase extraction column (5 g), washed with water, 50% methanol, then 90-100% methanol to wash down the mixture, concentrated by rotary evaporation and lyophilized, then dissolved in water, passed through a small Sep-Pak tC18 solid phase extraction column (500 mg), and washed with water to obtain the purified GM1 derivative. It was found experimentally that if an organic solvent such as methanol is present in the solution, the loading of the GM1 derivative on the column is increased, so that the latter method, after lyophilization, re-dissolution in water, allows the GM1 derivative to be less loaded to the column to the maximum extent, thereby increasing its yield. By the above-described method of purifying GM1 derivatives, the synthesized 10 GM1 derivatives were all easily purified in yields ranging from 16-67% (fig. 3).
Each isolated and purified ganglioside derivative was confirmed by HPLC-MS and High Resolution Mass Spectrometry (HRMS). Since gangliosides contain sphingosine bases differing by two carbon atoms, C18-sphingosine base (d 18) and C20-sphingosine base (d 20), respectively. Thus, each ganglioside derivative thus synthesized also contains both sphingosine bases.
Example 3: determination of neurite growth promoting Activity of ganglioside derivatives with unsaturated fatty acid chains
Neuro2a cells were seeded in 24-well plates at 10% confluency, after 24h of incubation, GM1 or its derivatives were added at the indicated concentrations and incubation was continued for 24h, and after photographing under a phase contrast microscope, the proportion of cells containing projections was counted using Image Pro-Plus software for at least 300 cells/well. In this experiment, cells with a protrusion length greater than 1 time the cell length are defined as cells containing a protrusion. In addition, the longest protrusion length of the cells is at least 30 protruding cells counted as the number of protrusions. To compare differences between experimental groups, independent sample t-tests were performed on each experimental group using SPSS software (IBM, chicago, IL, USA) with P <0.05, P <0.01, P <0.001.
As shown in FIGS. 4 and 5, compared with GM1, the Neuro2a cells treated with the derivatives C14:1-GM1, C16:1-GM1, C18:1-GM1 and DHA-GM1 had significantly more cells containing the protrusions, and in particular, the treated cells contained the protrusions at the following ratios: 23.6% and 23.2%, whereas the corresponding GM1 treated cells contained 9.2% of the protruding cells. Furthermore, DHA-GM1 was also active, and the treated cells contained 13.9% of protruding cells. The results also show that GM1 containing different unsaturated fatty acid chains has different neurite-promoting activity (FIGS. 4, 5) with C16:1-GM1 and C18:1-GM1 having higher neurite-promoting activity than GM1.
Other gangliosides (e.g., GD1a, GD1b, GT1b, etc.) may also exhibit relatively enhanced neurite outgrowth promoting activity when modified with omega-3, omega-6, omega-7, omega-9 unsaturated fatty acid chains, such as cis-11-tetradecenoic acid (C16:1, n-11), cis-7-hexadecenoic acid (C16:1, n-7), and 13,16,19-docosatrienoic acid.
Example 4 Effect of ganglioside derivatives containing different sphingosine bases on neurite outgrowth Activity
According to the results of example 2, GM1 gangliosides contained two sphingosine bases, so that we further determined the neurite outgrowth promoting activity of each of the components at 25. Mu.M and 50. Mu.M after separation of each of GM1, C16:1-GM1, and C18:1-GM 1. As shown in the results of FIG. 6, each component exhibited different neurite outgrowth-promoting activities, even though the fatty acid chains were identical, if the sphingosine bases were different, the neurite outgrowth-promoting activities were different, and the two components GM1 (d 18) and GM1 (d 20) comprising GM1, GM1 (d 18) were higher in activity than GM1 (d 20) at 25. Mu.M or 50. Mu.M. Consistent with the previous results, the two components C16:1-GM1 and C18:1-GM1, C16:1-GM1 (d 18), C16:1-GM1 (d 20), C18:1-GM1 (d 18) and C18:1-GM1 (d 20), were all more active than the two components of GM1 (FIG. 6), especially C16:1-GM1 (d 18), and the treated Neuro2a cells contained the highest proportion of protruding cells. And simultaneously counting the longest protrusion length and the number of protrusions of the Neuro2a cells containing the protrusions after the treatment of each component. As shown in FIG. 6, the C16:1-GM1 (d 18) -treated cells also had significantly more longest protrusion length and number of protrusions than the GM1 (d 18) and GM1 (d 20) -treated cells, as compared to the two components of GM1.
EXAMPLE 5 cytotoxicity assay with unsaturated fatty acid ganglioside derivative
Toxicity of GM1 (d 18), GM1 (d 20), C16:1-GM1 (d 18), C16:1-GM1 (d 20), C18:1-GM1 (d 18), and C18:1-GM1 (d 20) to the Neuro2a cells was examined by the MTT method. As shown in FIG. 7, the median cytotoxicity concentrations (CC 50) of C16:1-GM1 (d 18), C16:1-GM1 (d 20), C18:1-GM1 (d 18), C18:1-GM1 (d 20), GM1 (d 18) and GM1 (d 20) on Neuro2a cells were 61.56, 349.8, 285.8, 2420, 2447 and 3645. Mu.M, respectively.
EXAMPLE 6 ganglioside derivatives with unsaturated fatty acids promote the expression of proteins that play an important regulatory role in nerve growth and development
For comparative analysis of the reason for the enhanced activity of ganglioside derivatives with unsaturated fatty acids, we performed transcriptomic sequencing analysis. First, the Neuro2a cells treated with final concentrations of 25. Mu.M of C16:1-GM1 (d 18) and control Neuro2a cells, have a down-regulated expression of negative regulators, such as DNA binding inhibitors 2and 3 (Inhibitor of DNA binding protein 2and 3,Id2and Id3), during transcription, which have a down-regulated effect on neural differentiation, compared to the GM1 treated group and control group. Meanwhile, the expression of transcription factor mitosis-related enhancer 5 (Hairy and enhancer of split, hes 5) which plays a negative regulation role in neural differentiation is down-regulated, and research shows that the expression of the protein is inhibited to promote neural differentiation. While the expression of transcription factors early growth reaction protein 1 (Early growth response protein, egr 1) and Kruppel-like factor 7 (klf 7) with important regulation and control functions in nerve growth are up-regulated. Among them, egr1 is a downstream gene of extracellular signal-regulated kinase (Etracellular regulated kinase, erk), which has been shown to promote neurite outgrowth, while Klf7 has an important regulatory role in neural differentiation and expression of TrkA gene. C16:1-GM1 (d18:1) also promotes the expression of proteins that play an important regulatory role in nerve growth development, such as the membrane protein Ephrin type A receptor 7 (Ephrin type-A receptor 7, epha 7), and cytoplasmic cracking pay protein 10 (Dedicator of cytokinesis, dock 10). Wherein Epha7 mediates Erk activation; dock10 activates the cell division controlling protein 42 (Cell division control protein, cdc 42) protein, which promotes the growth of nerve cell processes.
Similar regulation and control rules are also found for other protein expression of ganglioside derivatives with unsaturated fatty acid chains, which are observed to have improved neurite outgrowth activity, and the role of the ganglioside derivatives with unsaturated fatty acid chains in promoting the development of the nervous system is further verified.
All documents mentioned in this application are incorporated by reference as if each was individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (2)
1. Use of a ganglioside derivative in the preparation of a medicament or formulation for promoting neurite outgrowth, said ganglioside derivative comprising a glycosyl group, a sphingosine base and a fatty acid chain, wherein the sphingosine base is linked to the fatty acid chain by an amide bond to form a ceramide, and the glycosyl group is linked to the ceramide by a glycosidic bond, wherein said fatty acid is an unsaturated fatty acid selected from the group consisting of a fatty acid having sixteen carbon atoms and a fatty acid having eighteen carbon atoms;
and the ganglioside derivative has the structure shown below:
wherein the method comprises the steps of
2. The use according to claim 1, wherein the medicament or formulation is for neurodegenerative diseases, nerve damage, nerve repair or nerve regeneration diseases.
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