阅读说明：本技术 一类组合物在神经损伤保护领域的应用 (Application of composition in nerve injury protection field ) 是由 朱明� 马毅涛 其他发明人请求不公开姓名 于 2020-05-15 设计创作，主要内容包括：本发明涉及一类对神经损伤有保护作用的组合物,这类组合物是一类神经节苷脂组合物,涉及神经节苷脂的抗神经细胞调亡作用及对缺氧/复氧损伤的保护作用。神经节苷脂有多种类型,在神经细胞的生理活动中协同产生作用,协调神经细胞的分化、发育、神经元的可塑性、突触的传递和对增加创伤后的神经细胞的树突生成,因而单一的使用某一种神经节苷脂来治疗神经细胞损伤有所不足。本发明涉及神经节苷脂组合物在预防和治疗神经细胞损伤药物和食品领域的应用,发明所涉及的神经节苷脂组合物成分中包含但不限于GM1、GD1a、GD1b、GT1b。本项发明属于神经细胞损伤的保护和修复领域和生物化学及生物制药领域。(The present invention relates to a kind of composition with the action of protecting nerve injury, and the composition is a kind of ganglioside composition, and relates to the action of ganglioside in resisting nerve cell apoptosis and protecting against anoxia/reoxygenation injury. Gangliosides are of various types and act synergistically in the physiological activities of nerve cells to coordinate the differentiation and development of nerve cells, neuronal plasticity, synaptic transmission and dendritic formation to increase post-traumatic nerve cells, and thus a single use of a certain ganglioside for the treatment of nerve cell injury is insufficient. The invention relates to application of ganglioside composition in the fields of medicine and food for preventing and treating nerve cell injury, and the ganglioside composition comprises but is not limited to GM1, GD1a, GD1b and GT1 b. The invention belongs to the field of protection and repair of nerve cell injury and the fields of biochemistry and biopharmaceuticals.)

1. The invention relates to application of a composition in the field of nerve injury protection.

2. A class of compositions as claimed in claim 1 is a class of ganglioside compositions comprising GM1, GD1a, GD1b, GT1b, and also any other ganglioside known to the person skilled in the art.

3. The ganglioside composition of claim 2 wherein the ratio of GM1, GD1a, GD1b, GT1b is GM 1: 20% -23%; GD1 a: 40% -43%; GD1 b: 15% -17%; GT1 b: 17% -21% and can be in any proportion.

4. The ganglioside composition of claim 2 can be prepared into pharmaceutically acceptable solid preparation, liquid preparation, biological product, slow release particle, target preparation and medical device according to known pharmaceutical technology.

5. The ganglioside composition of claim 2 is used as medicine or effective component of medicine composition for protecting and repairing nerve cell damage of human body, and also can be used as nutrient substance of nerve cell of human body.

6. The ganglioside composition of claim 2 for use in the prevention and treatment of central nerve injury, acute spinal cord injury, stroke, parkinson's disease, diabetic nerve injury, alzheimer's disease, neurogenic pain and peripheral nerve injury.

7. The ganglioside composition of claim 2 is used in the medicine and food for preventing and treating nerve cell damage.

8. The ganglioside composition of claim 2, wherein pharmaceutically acceptable components and carriers can be added, and any pharmaceutically acceptable components known to be effective in preventing and treating nerve cell damage can be added.

9. An effective amount of the ganglioside composition of claim 2 as an effective ingredient of a medicament or pharmaceutical composition for preventing and treating nerve cell damage.

1. Field of the invention

The present invention relates to a kind of composition with the action of protecting nerve injury, and the composition is a kind of ganglioside composition, and relates to the action of ganglioside in resisting nerve cell apoptosis and protecting against anoxia/reoxygenation injury. The invention relates to application of ganglioside composition in the fields of medicine and food for preventing and treating nerve cell injury, and the ganglioside composition comprises but is not limited to GM1, GD1a, GD1b and GT1 b. The invention belongs to the field of protection and repair of nerve cell injury and the fields of biochemistry and biopharmaceuticals.

2. Background of the invention

Gangliosides are a class of membrane glycolipids of complex structure whose molecule consists of a lipophilic ceramide moiety, which is formed by the acidification of the amino group of sphingosine with fatty acids, and a hydrophilic sialyloligosaccharide group. Currently, the method of Sevennerholm naming is universally adopted internationally for gangliosides, and gangliosides are classified into different classes according to the position of sialic acid residues and the length of the oligosaccharide core region.

Gangliosides are present in the cell membranes of animals, with the most abundant gangliosides in the central nerve cells of mammals, with GM1, GD1a, GD1b (disialogangliosides a and b), GT1b (trisialoganglioside b), GQ1b (tetrasialoganglioside b), followed by GM2 and GD3 in the grey brain matter; the white matter is mainly GM1 and GM 4. Gangliosides are present in the neuronal cell bodies at slightly below average levels and in the synaptic bodies at higher levels, GT1, GD1b and GM1 are concentrated in the postsynaptic membrane. Ganglioside is located at the outer layer of double-layer structure of neuron cell membrane, one end of ceramide is embedded into cell membrane, and one end of oligosaccharide chain is extended out of cell membrane and projected into external environment. This asymmetric distribution of gangliosides and their differences in chemical properties make them particularly susceptible to interaction with various extracellular messages, thereby playing an important role in cell membrane activity.

Experiments prove that the exogenous ganglioside can be embedded into a nerve cell membrane, imitate certain functions of endogenous ganglioside, regulate membrane-mediated cell functions, stimulate a potential replacement mechanism after central nervous system injury to prevent damage development, protect undamaged nerve tissues, influence the growth and activity of neurons cultured in vitro, and promote survival and sprouting of the neurons. The main physiological functions of gangliosides in the nervous system are summarized as promoting the growth of nerve cells, differentiation and nerve regeneration, participating in synaptic transmission, maintaining the normal function of the brain, participating in various learning and memory activities, mediating in the interaction process between cells, cells and microorganisms, and cells and substrates, and regulating various protein functions in cell membranes, such as ion channels, epidermal growth factor receptors, etc.

The localization of gangliosides in the outer layers of the cell membrane suggests that they play an important role in cell recognition, growth and differentiation. Gangliosides and other bound glycolipid components on the cell surface are known to undergo changes during differentiation and during cell maturation.

Gangliosides play a role in causing plastic changes in neurons, which promote axonal growth in cell culture and in the central and peripheral nervous systems. Studies have shown that not only the development of the nervous system, but also the repair of the nervous system is controlled by extracellular signals of molecules acting on the cell surface. Gangliosides appear to play a prominent role in regulating neuronal development and repair. During neuronal development, the properties of the brain gangliosides undergo significant quantitative and qualitative changes. The exogenous ganglioside can enhance the action of trophic factors such as NGF, promote nerve regeneration, and reduce cell death around focus. The exogenous ganglioside can promote nerve growth, increase the number of neurons, the length of synapse, the number of branches and neurotrophic effect, and has various physiological functions such as neurotrophic effect and central nervous system repairing effect.

Various gangliosides are currently extracted from animal brain tissue (bovine brain and porcine brain). The ganglioside GM1 has been shown in preclinical and clinical studies at home and abroad that clinical indications include central nerve injuries such as acute spinal cord injury and cerebral apoplexy and Parkinson's disease, and potential clinical indications include Alzheimer's disease, neurogenic pain and peripheral nerve injury. GM1 injection is available in many products and is used clinically in many countries throughout the world.

The invention discloses an anti-nerve cell apoptosis effect of ganglioside GD1b and an application thereof in the institute of biophysics of Chinese academy of sciences (Chinese patent invention, publication number: CN1634090A), relates to an anti-nerve cell apoptosis effect of ganglioside GD1b for regulating potassium ion channel activity, and also relates to an application of ganglioside GD1b in preparing an anti-nerve cell apoptosis medicine for preventing and treating neurodegenerative diseases and an application in preparing a medicine for preventing and treating potassium ion channel diseases.

The application of ganglioside GM3 in preparing medicine for treating atherosclerosis (Chinese patent publication No. CN110507663A) at Nanchang university provides an application of ganglioside GM3 in preparing medicine for treating cardiovascular and cerebrovascular diseases, and an effective amount of ganglioside GM3 is used as an effective component in preparing medicine or pharmaceutical composition for treating cardiovascular and cerebrovascular diseases, and experiments in vitro and animals prove that ganglioside GM3 has an effect on resisting atherosclerosis.

The present invention discloses the use of natural gangliosides or their derivatives to stabilize and maintain the biological activity of nerve growth factor (Chinese patent publication No. CN1052868A), relates to the use of protein and ganglioside to form a new stable and biologically active complex, i.e., a stable and biologically active complex formed from β subunit of nerve growth factor (β NGF) and natural ganglioside or its semisynthetic analogs such as carboxylic esters and amides, and pharmaceutical compositions containing such a complex, wherein the ganglioside described in this invention is GM 1.

Researchers have used sialidase (VCS) as a probe to study the permeability of ganglioside on cell membranes, VCS is sensitive to substrates of ganglioside, the more GD1a component, the lower the reactivity to VCS, Protein Kinase C (PKC) is not activated, the activity is not expressed, the permeability of ganglioside on cell surfaces is not influenced, the content of GD1a and GD1b on cell membranes is changed, the permeability of ganglioside on cell membranes is influenced, and the fact that the PKC activity and the content of GD1a and GD1b are changed is the key of ganglioside permeability change.

The effects of yangyi and liu rui zhen on primary culture neuron ischemia reperfusion injury, the study on the injury and apoptosis conditions of neuron simple ischemia reperfusion groups is performed in the 10 th volume, 2009 in the journal of cardiovascular and cerebrovascular diseases by combining traditional Chinese medicine and western medicine, and the effects have obvious neuron protection effects after the addition of GM1, and the effects have statistical significance (P is less than 0.01) at any time point. This neuronal protection effect of GM1 is largely achieved by an anti-apoptotic effect.

Liu Yan and Zhang Yingdong research on action of ganglioside induced bone marrow mesenchymal stem cells to differentiate into nerve cells, clinical neurology journal 2009, Vol.22, No. 2, ganglioside GM1 induced bone marrow mesenchymal stem cells (BMSCs) to differentiate into nerve cells, GM1 induced group BMSC s rounded cell bodies, increased protrusions, and part of protrusions connected to form a net, NSE positive expression cells were (29.47 + -3.26)%, GFAP positive expression cells were (2.32 + -0.18)%, FBS group and blank control group were (6.97 + -0.56)% and (10.6 + -0.75)% of cells NSE expression positive, (1.41 + -0.35)% and (1.21 + -0.35)% of cells GFAP expression positive, respectively. Levels of NGFmRNA and BDNFmRNA in the induction group are obviously higher than those of the FBS control group (mean P is less than 0.01) and the blank control group (mean P is less than 0.05). GM1 was shown to induce differentiation of BMSCs into neuron-like cells in vitro.

Ningna and Chennahong biological Activity of ganglioside in physiological science progress 2009, volume 40, No. 1, shows that ganglioside GM1 not only can promote normal expression of brain higher functions, but also can improve and protect impaired brain higher functions. Glycoconjugates such as ganglioside GT1b can be transported to the tail end of a neuron through axial plasma transport, after the tail end of the neuron and target cell binding protein, namely a possibly existing specific carbohydrate receptor, are received, intracellular protein kinase such as CaMK II is activated through the modes of mobilizing intracellular storage calcium and the like, a series of signal transduction such as activation of cdc42 is initiated, the quantity of synaptophyte formed by polymerized filamentous pseudopodia actin in cytoskeleton is increased, the contact area of front and rear membranes is increased, and the condition of GT1b and the like with a certain concentration can influence long-term memory to promote the development of the neuron generating dendrites and maintain the normal shape and function of a nervous system.

Gangliosides have been shown in a number of studies to play an important role in the differentiation, development of nerve cells, neuronal plasticity, synaptic transmission and in increasing dendritic production of post-traumatic nerve cells. Neurons of the central nervous system first synthesize the lipophilic portion of gangliosides at the endoplasmic reticulum, and then link glucose at the endoplasmic reticulum and golgi apparatus to form neutral carbohydrate backbones of gangliosides. Then under the action of glycosyl and sialyl convertase, neutral sugar and sialic acid are added to synthesize the series ganglioside.

The exogenous ganglioside can be embedded into nerve cell membrane, and can reduce toxicity of Excitatory Amino Acid (EAAS) on isolated brain granule nerve cell under normal, anoxic or sugarless condition, and GM1 is found in cortex and retina neuron cultured in vitro to relieve cytotoxicity caused by EAAS. Studies of the molecular mechanisms indicate that GM1 does not affect the functional properties of EAAS receptors to gate the passage of calcium ions while fighting the cytotoxic processes caused by overstimulation of EAAS receptors. GM1 can limit the neurotoxic effects of EAAS in early cerebral ischemia to slow or prevent the progression of acute neuronal injury without affecting the normal physiological processes mediated by EAAS, allowing the normal function of its regulatory cells to be maintained and restored. The exogenous ganglioside can prevent the toxic action of some compounds, not only can raise the tolerance of dorsal root ganglion cell to adverse environmental condition, but also can raise the function of taking exogenous dopamine by in vitro juvenile mouse brain cell, and can raise the activity of mesencephalon and striatum tyrosine hydroxylase of aged rat and the content of dopamine and 3, 4-dihydroxy phenylacetic acid. When the exogenous ganglioside is incorporated into the nerve cell membrane of the rat prepared separately, Na + -K + ATPase can be activated, and the activity of the nerve cell membrane can be influenced by inducing the change of the microenvironment of the cell membrane or directly acting on a special site on the membrane to cause the change of the conformation of the sodium pump.

Because the acyl sphingosine protein is positioned in the double lipid layer of the cell membrane, and the polarity of the carbohydrate is towards the outside of the cell, the physical asymmetry and the difference of the chemical structure thereof enable the ganglioside substances to be different from various extracellular signalsThe interactions are close. The concentration of cell membrane gangliosides depends on the dynamic action of the polar groups of gangliosides, Ca²⁺Concentration and cell surface glycoprotein content, which can lead to local cell membrane structure changes. Cell membrane gangliosides can also affect glycoproteins and cell intercalating proteins on the surface of cell membranes. Therefore, the change of ganglioside substances of the nerve cell membrane has important significance for regulating neuron to transmit information inside and outside cells.

Gangliosides are of various types, have different molecular compositions and similar molecular structures, and all structures contain a certain number of sugar rings, fat chains and sialic acid groups, which have a synergistic effect in the physiological activities of nerve cells, and coordinate the differentiation and development of nerve cells, the plasticity of neurons, the synaptic transmission and the dendritic generation of nerve cells after trauma, so that the single use of a certain ganglioside for treating nerve cell injury is insufficient. Scientific experiments prove that the four main types of natural ganglioside in human brain are GM1, GD1a, GD1b and GT1b, and the proportion of the above components in bovine brain and pig brain is very close to the data of human brain. The functions of GM1, GD1a, GD1b and GT1b in gangliosides in neuronal protection and injury repair are of great significance. The ganglioside GM1, GD1a, GD1b and GT1b composition is significant in preparing medicine and food for preventing and treating nerve cell injury.

3. Summary of the invention

Defining:

as used herein, the following terms have the following meanings:

"ganglioside (G L S)" refers to any ganglioside known to those skilled in the art.

"GM 1, GD1a, GD1b, GT1 b" refers to four single gangliosides known to those skilled in the art, each having a specific molecular composition and molecular structure, named according to the severnerholm nomenclature, all containing a sugar ring, an aliphatic chain and a sialic acid group.

"other gangliosides" refers to any ganglioside known to those skilled in the art to remove GM1, GD1a, GD1b, GT1b, as named by the severnerholm nomenclature.

Modes for carrying out the invention

Through a large number of research experiments, researchers adopt various methods to extract and prepare exogenous ganglioside, and research the application of the ganglioside in treating and repairing cerebral apoplexy nerve cell injury, central nerve injury, peripheral nerve injury, senile dementia and Parkinson's disease of people. The physicochemical properties of GM1 in gangliosides and its mechanism of action on models of neuronal cell injury have been studied extensively since the seventies of the last century internationally, with late nineties being approved as a drug for clinical use. Researchers in China systematically research the properties and action mechanisms of GM1 from the nineties, and enter clinical research at the end of the nineties, and in 2005, the researchers in China are approved as drugs to enter clinical application, and many pharmaceutical enterprises are preparing and producing GM1 drugs.

In vitro culture of neuron cell strain Neuro-2A, establishment of hypoxia/reoxygenation injury model, and investigation of the protective effect of ganglioside composition on neurons. The results show that the ganglioside composition has a certain effect on protecting Neuro-2a cells damaged by hypoxia/reoxygenation.

The ganglioside composition comprises 20-23% of GM1, 40-43% of GD1a, 15-17% of GD1b and 17-21% of GT1b, the ganglioside composition is prepared by self-made method and has the same quality standard as GM1 injection, and GM1, GD1a, GD1b and GT1b contrast products are purchased from alexis (bovine source), the purity is more than 98% (T L C), and the composition is prepared into 5mg/ml, 10mg/ml and 15mg/ml aqueous solution for test.

Example 1

Mouse brain neuron cell line Neuro-2a cell (purchased from Beijing collaborating and medical university cell bank) culture and experimental grouping, and the mouse brain neuron cell line Neuro-2a cell is cultured in complete culture medium (MEM, FBS, 100U/ml)^-1Penicillin, 0.1g/l^-1Streptomycin, 2mmol/l^-1L-Glutamine). Neuro-2a cells in logarithmic growth phase were digested and blown into single cell suspension, and cell density was adjusted to 4 × 10⁵One/ml was inoculated in 96-well plates overnight.

Cell grouping and processing were as follows:

① control group, replacement of complete culture broth with MEM, CO₂Normally culturing in an incubator for 4h, and then replacing with complete culture solution to culture for 24 h.

② Damage model group complete culture solution was replaced with balanced salt solution D-Hank's filled with 95% N₂-5％CO₂The anaerobic chamber of the mixed gas is incubated for 4h, and then replaced by the whole culture solution for 24 h.

③ ganglioside composition group, the complete culture solution was replaced with D-Hank's solution containing ganglioside compositions of different concentrations (5mg/ml, 10mg/ml, 15mg/ml) filled with 95% N₂-5％CO₂The anaerobic chamber with mixed gas was incubated for 4h and then replaced with complete medium containing different concentrations of ganglioside composition (5mg/ml, 10mg/ml, 15mg/ml) for 24 h.