阅读说明：本技术 硫酸乙酰肝素在防治阿尔兹海默症中的应用 (Application of heparan sulfate in prevention and treatment of Alzheimer's disease ) 是由 王凤山 吴丽丹 刘冬科 赵娜 于 2020-11-04 设计创作，主要内容包括：本发明提供硫酸乙酰肝素在防治阿尔兹海默症中的应用,属于医药研发技术领域。本发明通过体外和体内试验均证明HS具有预防和治疗AD活性,具体的,HS在体外AD模型中能够增强神经细胞的活力,抑制Aβ_(1–42)对神经细胞带来的损伤,如降低神经炎症因子水平,减少细胞凋亡。同时还发现HS与Aβ_(1–42)共同孵育能够改变减少寡聚体的状态,将寡聚体转变为毒性较弱的纤维体；体内试验亦证实HS在小鼠体内能够发挥改善小鼠认知损伤的作用,从而为进一步药物研发奠定基础,因此具有良好的实际应用之价值。(The invention provides application of heparan sulfate in prevention and treatment of Alzheimer's disease, and belongs to the technical field of medicine research and development. In vitro and in vivo experiments prove that HS has the activity of preventing and treating AD, and specifically, HS can enhance the activity of nerve cells and inhibit Abeta in an in vitro AD model 1–42 The damage to nerve cells, such as reducing the level of nerve inflammatory factors and reducing apoptosis. HS and Abeta were also found 1–42 Co-incubation can alter the state of oligomer reduction, converting oligomers to less toxic fibers; in vivo experiments also prove that HS can play a role in improving cognitive impairment of mice in vivo, thereby laying a foundation for further drug research and development, and having good practical application value.)

1. Application of heparan sulfate in preparing products for preventing and/or treating Alzheimer's disease.

2. A product characterized in that the active ingredient of the product comprises heparan sulfate.

3. The product according to claim 2, which has the effect of preventing and/or treating alzheimer's disease, in particular, it has any one or more of the following uses:

(a) improving the activity of nerve cells;

(b) prevention of A beta_1-42Damage to the cell morphology of nerve cells;

(c) inhibition of A beta_1-42Apoptosis of nerve cells;

(d) reducing the level of a neuro-inflammatory factor;

(e) mixing A beta_1-42The A beta is reduced by converting oligomer with stronger toxicity into fibrous body with less toxicity_1-42Toxicity in vivo;

(f) improvement of A beta_1-42Resulting in learning cognitive impairment in the subject;

(g) decrease of factor Abeta_1-42Resulting in elevated levels of inflammatory factors in the hippocampus of the subject;

(h) reduction of peripheral Abeta_1-42Horizontal;

(i) increasing A beta in peripheral neutrophils to peripheral circulation_1-42Phagocytosis of (a);

(j) the brain of the subject is protected.

4. A product according to claim 3, wherein in said use (d), said inflammatory factors include, but are not limited to, TNF- α, IL-6;

in the use (g), the inflammatory factor includes, but is not limited to TNF- α, IL-6.

5. The product of claim 3, wherein the subject is a human or a non-human mammal.

6. The product of claim 5, wherein the non-human mammal comprises a mouse, rat, guinea pig, rabbit, dog, monkey, and chimpanzee.

7. The product of claim 3, wherein the product is a medicament.

8. The product of claim 7, wherein the active ingredient of the medicament comprises heparan sulfate; or comprises at least one other substance having an effect of preventing and/or treating Alzheimer's disease.

9. The product of claim 7, wherein said medicament further comprises pharmaceutically acceptable carriers, excipients and diluents.

10. Use of heparan sulphate in the preparation of any one or more of the following:

(1) an MMP2 inhibitor;

(2) an MMP9 inhibitor;

(3) a TLR4 inhibitor;

(4) inhibitors of pNF- κ B;

(5) inhibitors of PJUN;

(6) a TNF-alpha inhibitor;

(7) an IL-6 inhibitor;

(8) caspase 3 inhibitors;

(9) caspase8 inhibitors;

(10) a Bax inhibitor;

(11) inhibitors of GSK3 α/β protein;

(12) tau protein inhibitors.

Technical Field

The invention belongs to the technical field of medicine research and development, and particularly relates to application of heparan sulfate in prevention and treatment of Alzheimer's disease.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Glycosaminoglycans (GAGs) are linear polysaccharides present on the cell surface, in the extracellular matrix (ECMs) and in the basement membrane. GAGs bind to many core proteins to form Proteoglycans (PGs), which mediate a variety of physiological functions, such as cell adhesion, cell proliferation, anticoagulation, and the like.

Heparan Sulfate (HS) is a linear polysaccharide formed from repeating units of uronic acid and D-glucosamine disaccharide linked by 1-4 glycosidic linkages. Various variable substitutions of the disaccharide unit such as N-sulfate, O-sulfate, N-acetyl groups and the like form complex sequences. HS, due to its highly sulfated regions, often binds to some proteins in vivo to form proteoglycans. Heparan Sulfate Proteoglycans (HSPGs) formed by HS-protein binding have a variety of biological functions, including regulation of embryonic development, inflammatory responses, coagulation, viral/bacterial infections, tumor metastasis, and the like.

However, the inventors have found that the physiological functions of HS are not completely understood at present due to the complexity and diversity of HS structure.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of heparan sulfate in preventing and treating Alzheimer's Disease (AD). In vitro and in vivo tests prove that HS has the activity of preventing and treating AD, so that a foundation is laid for further drug research and development, and the method has good practical application value. Based on the above results, the present invention has been completed.

Specifically, the invention relates to the following technical scheme:

in a first aspect of the invention, the application of heparan sulfate in preparing a product for preventing and/or treating Alzheimer's disease is provided.

The composition ratio of the disaccharide of the heparan sulfate is D2S6, D0S6/D2S0, D2A6, D0A6/D2A0, D0S0, D0A0, 5:2:85:1:6: 9.

According to the present invention, the concept of "prevention and/or treatment" means any measure suitable for the treatment of diseases associated with alzheimer's disease, or the prophylactic treatment of such manifested diseases or manifested symptoms, or the avoidance of further development of such diseases, such as the further development of the disease after the end of a treatment period or the treatment of symptoms of an already established disease, or the prevention or inhibition or reduction of the occurrence of such diseases or symptoms with prior intervention.

In a second aspect of the invention, a product is provided, the active ingredient of the product comprises heparan sulfate, and the product has the effect of preventing and/or treating alzheimer disease, and specifically, the product has any one or more of the following purposes:

(a) improving the activity of nerve cells;

(b) prevention of A beta_1-42Damage to the cell morphology of nerve cells;

(c) inhibition of A beta_1-42Apoptosis of nerve cells;

(d) reducing the level of a neuro-inflammatory factor;

(e) mixing A beta_1-42The A beta is reduced by converting oligomer with stronger toxicity into fibrous body with less toxicity_1-42Toxicity in vivo;

(f) improvement of A beta_1-42Resulting in learning cognitive impairment in the subject;

(g) decrease of factor Abeta_1-42Resulting in elevated levels of inflammatory factors in the hippocampus of the subject;

(h) reduction of peripheral Abeta_1-42Horizontal;

(i) increasing A beta in peripheral neutrophils to peripheral circulation_1-42Phagocytosis of (a);

(j) the brain of the subject is protected.

Wherein, in said use (d), said inflammatory factor includes, but is not limited to TNF- α, IL-6;

in said use (g), said inflammatory factor includes, but is not limited to TNF- α, IL-6;

the subject of administration can be human and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, and the like.

In yet another embodiment of the invention, the product may be a medicament. The active ingredient of the medicine comprises heparan sulfate; or comprises other at least one substance for preventing and/or treating Alzheimer's disease;

the composition ratio of the disaccharide of the heparan sulfate is D2S6, D0S6/D2S0, D2A6, D0A6/D2A0, D0S0, D0A0, 5:2:85:1:6: 9.

The medicine further comprises a pharmaceutically acceptable carrier, an excipient, a diluent and the like. Further, the composition can be prepared into oral preparations such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and spray, external preparations, suppositories, and sterile injectable solutions according to a conventional method.

Such pharmaceutically inactive ingredients, which may include carriers, excipients and diluents, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.

In still another embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In yet another embodiment of the present invention, the medicament of the present invention may be administered into the body by known means. For example, by intravenous systemic delivery, local injection delivery, or oral delivery into the body. Optionally via intravenous, transdermal, intranasal, mucosal, oral, or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

In a third aspect of the present invention, there is provided the use of heparan sulphate as described above in the preparation of any one or more of the following:

(1) an MMP2 inhibitor;

(2) an MMP9 inhibitor;

(3) a TLR4 inhibitor;

(4) inhibitors of pNF- κ B;

(5) inhibitors of PJUN;

(6) a TNF-alpha inhibitor;

(7) an IL-6 inhibitor;

(8) caspase 3 inhibitors;

(9) caspase8 inhibitors;

(10) a Bax inhibitor;

(11) inhibitors of GSK3 α/β protein;

(12) tau protein inhibitors.

The beneficial technical effects of one or more technical schemes are as follows:

the technical scheme reports the application of HS in preventing and treating AD for the first time. Both in vitro and in vivo experiments prove that HS has the activity of preventing and treating AD, and specifically, HS can enhance the activity of nerve cells and inhibit Abeta in an in vitro AD model_1–42Damage to nerve cells, such as reduction of nerve inflammatory factor levels, reduction of apoptosis. HS and Abeta were also found_1–42Co-incubation can alter the state of oligomer reduction, converting oligomers to less toxic fibers; in vivo experiments also prove that HS can play a role in improving cognitive impairment of mice in vivo, thereby laying a foundation for further drug research and development, and having good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 shows the effect of HS on SH-SY5Y cytotoxicity and cellular activity in example 1 of the present invention.

FIG. 2 shows the effect of HS on SH-SY5Y cell morphology in example 1 of the present invention; wherein A is a control group, B is a model group, and C is HS-25 mug/mL; d is 50 mu g/mL; e is 100 mu g/mL; magnification 40 ×;

FIG. 3 is a graph showing the effect of HS on SH-SY5Y apoptosis in example 1 of the present invention; wherein A is a control group, B is a model group, and C is HS-25 mug/mL; d is 50 mu g/mL; e is 100 mu g/mL; f is a histogram;^**p<0.001,^#p<0.05；

FIG. 4 is a graph showing the effect of HS on SH-SY5Y cellular inflammatory factor levels in example 1 of the present invention.^*p<0.05,^**p<0.001,^#p<0.05,^##p<0.001,^###p<0.0001；

FIG. 5 is a graph showing the effect of HS on MMP2, MMP9, TLR4, and phosphorylated JNK levels in example 1 of the present invention.^*p<0.05,^**p<0.001,^#p<0.05,^##p<0.001；

FIG. 6 is a graph showing the effect of HS on the level of apoptotic factors in example 1 of the present invention; wherein, A is the relative expression level of Caspase8, B is the relative expression level of Caspase 9, C is the relative expression level of Bax, and D is the relative expression level of Bcl-2;

FIG. 7 shows the ratio of HS to A.beta.in example 1 of the present invention_1-42Influence of oligomer morphology; wherein, A is A beta after 1 day of incubation_1-42Oligomeric morphology; b is Abeta after 1 day incubation with 25. mu.g/mL HS_1-42Oligomeric morphology; c is Abeta after 3 days incubation_1-42Oligomeric morphology; d is Abeta after 3 days incubation with 25. mu.g/mL HS_1-42Oligomeric morphology; e is Abeta after 5 days incubation_1-42Oligomeric morphology; f is Abeta after 5 days incubation with 25. mu.g/mL HS_1-42Oligomeric morphology;

FIG. 8 is a time chart of the administration and experimental procedures in example 2 of the present invention;

fig. 9 is a time chart of dosing and experimental procedures in example 2 of the present invention, 1, 4, 7, 10, 13, 16, 20, 22, 24, 26, 28, 30, showing decapitation on days 1, 4, 7, 10, 13, 16, 20, 22, 24, 26, 28, 30 of dosing and 16# of decapitation on day 16 of the experiment;

FIG. 10 shows the ratio of HS to A β in example 2 of the present invention_1–42Influence of model mouse water maze experiment; wherein A is a statistical graph of escape latencies of each group; b isTime statistical graphs of each group in the target quadrant; c is a swimming track diagram of each group of mice in the directional navigation test and the space exploration test; wherein, in the C, a is a control group, b is a model group, C is a pseudo-operation group, d is HS-25mg/kg, and e is HS-100 mg/kg; f is HS-400 mg/kg;^*p<0.05,^**p<0.001；

FIG. 11 is a graph showing the effect of HS on the autonomic activity of Abeta model mice in the open field experiment in example 2 of the present invention;^#p<0.05,^*p<0.05；

FIG. 12 shows the morphological changes of HS to hippocampal CA1 region of cerebral cortex of mice model A beta in example 2 of the present invention; wherein, a is a cortical area, and b is a hippocampal CA1 area; in a and B, A is a control group, B is a model group, C is a pseudo-operation group, D is HS-25mg/kg, E is HS-100 mg/kg; f is HS-400 mg/kg; the scale bar is 100 μm;

FIG. 13 is a graph showing the effect of HS on the cell structure of mouse hippocampus in example 2 of the present invention; wherein A is a control group, B is a model group, C is a pseudo-operation group, D is HS-25mg/kg, E is HS-100 mg/kg; f is HS-400 mg/kg; the scale bar is 1.2 mu m;

FIG. 14 is a graph showing the effect of HS on IL-6 and TNF- α in mouse hippocampus in example 2 of the present invention;

FIG. 15 is a graph showing the effect of HS on mouse hippocampal inflammatory factors in example 2 of the present invention; wherein, A is TLR 4; b is PNF-KB; c is PJNK;

FIG. 16 is a graph showing the effect of HS on apoptosis factor in mouse hippocampus in example 2 of the present invention; wherein A is Caspase 3, B is Caspase8, C is Bax;

FIG. 17 shows the changes in levels of HS on PGSK and Ptau in mouse hippocampus in example 2 of the present invention;

FIG. 18 is a tissue distribution of HS in mice in example 2 of the present invention;

FIG. 19 is a graph of A β in plasma at the week of month one or more in example 2 of the present invention_1-42Changes in levels and Abeta in various groups of brains_1-42A change in level;

FIG. 20 is a graph of the effect of HS on the ratio of monocyte to neutrophil subpopulations in the peripheral circulation of mice in example 2 of the present invention;

FIG. 21 shows HS in example 2 of the present inventionFor A beta in peripheral circulation_1-42The effect of the clean-up.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. Unless otherwise indicated, the experimental procedures of the specific conditions in the following detailed description are generally in accordance with conventional methods and conditions of biology within the skill of the art, which are fully explained in the literature.

The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1

1. Experimental methods

1.1 disaccharide composition identification and analysis of HS

1.1.1 preparation of recombinant heparinases I, II, III

Activation of recombinant heparinase I, II and III bacterial solutions: 30. mu.L of each of the three types of bacterial suspension stored at-80 ℃ was added to 30mL of LB liquid medium (50. mu.g/mL of ampicillin, 15. mu.g/mL of kanamycin, 12.5. mu.g/mL of tetracycline, 20. mu.g/mL of chloramphenicol to the heparinase I medium, and 50. mu.g/mL of ampicillin to the heparinases II and III), and cultured overnight at 37 ℃ and 225 r/min.

And (3) amplification culture of recombinant heparinase I, II and III bacterial solutions: transferring 5mL of activated bacteria liquid to 500mL of LB liquid culture medium for amplification culture the next day, adding antibiotics with corresponding concentration into the culture medium, culturing at 37 deg.C and 225r/min to OD₆₀₀0.6-0.8, and cooling to room temperature for induction, wherein the induction method comprises the steps of adding L-arabinose with the final concentration of 1mg/mL into heparinase I, and adding IPTG with the final concentration of 0.2mM after 15 min. And adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.2mM into the heparinases II and III for induction, adjusting the culture temperature to 22 ℃, continuing culture, and culturing for 18-24 h. Taking out the bacterial liquid, centrifuging at 8000r/min for 25min, and pouring off the supernatant.

Extracting recombinant heparinase I, II and III bacterial liquids: dissolving the bacterial liquid in buffer solution A (25mM Tris, 0.5M sodium chloride, 30mmol/L imidazole, pH 7.5), crushing with an ultrasonic crusher at 4 deg.C until the bacterial liquid becomes clear, centrifuging at 12000r/min at 4 deg.C for 35min, collecting supernatant, and filtering with 0.22 μ M filter head.

Purifying the recombinant heparanases I, II and III: purifying with Ni Sepharose 6B affinity column, balancing with buffer solution A, dripping sample, and washing with buffer solution A to OD of effluent₂₈₀<0.1, eluting with buffer B (25mmol/L Tris, 0.5mol/L sodium chloride, 250mmol/L imidazole, pH 7.5), collecting the eluate with an EP tube, and combining OD₂₈₀>0.6 of eluent is added with 15 to 20 percent of glycerin and then is subpackaged for standby at minus 80 ℃.

1.1.2 HS polysaccharide degradation

1.1.3 analysis of disaccharides

1.2 cytotoxicity assays

The frozen tube of SH-SY5Y cells is quickly thawed in water bath at 37 ℃, centrifuged at 1000r/min for 5min, and the supernatant is discarded. Resuspending the precipitated cells in DMEM medium containing 10% FBS, gently pumping to mix well to form a cell suspension, and passing through a cell counting plateCounting cells, inoculating the cell suspension into a culture flask, and placing in CO₂Constant temperature incubator (5% CO)₂Culturing for 24 hours at 37 ℃, changing the culture solution every 2-3 days, taking out the cells when the adherent growth of the cells accounts for about 80% of the cell culture bottle, pouring out the original cell culture solution, washing the cells once with PBS, pouring out the PBS, adding 1mL of 0.25% pancreatin solution for digestion, observing the state of the cells by an inverted microscope, adding 4mL of DMEM culture medium containing 10% FBS to stop digestion when the cell gaps become larger and the cells become round and bright, and repeatedly blowing and beating the cells on the wall of the culture bottle. Cells were formed into a cell suspension and counted on an inverted microscope at 10⁵cells were seeded at cell/mL density in new sterile 96-well plates, CO₂Culturing in a constant temperature incubator. After 24h, discarding the supernatant from the administration well, adding HS solution (12.5,25,50,100,200, 400. mu.g/mL, diluting with DMEM medium, setting 3 parallel wells for each concentration), changing the solution in the blank group of wells, culturing for 24h, carefully sucking the supernatant, centrifuging, discarding the supernatant, adding fresh DMEM medium 200. mu.L, adding MTT solution 5. mu.L into each well, continuing to culture for 4h at 37 ℃, terminating the culture, adding DMSO 150. mu.L, shaking and mixing uniformly, measuring the absorbance value of each well (the measurement wavelength is 570nm, the reference wavelength is 630nm) by using a microplate reader, and measuring the survival rate of the cells.

1.3 cell viability assay

Cells without any treatment served as blank, A.beta._1-42The treated wells were model groups and the MTT method was used to investigate the inhibition of HS by Abeta_1-42Inducing cell damage generated by SH-SY5Y cells, and the specific experimental steps are as follows;

after 24h, the drug treatment is given, and A beta is added in the administration group_1-42Mixing with HS solution (12.5,25,50,100,200, 400. mu.g/mL, diluting with DMEM medium, setting 3 parallel wells for each concentration), culturing for 24h, carefully sucking supernatant, centrifuging, discarding supernatant, adding fresh DMEM medium 200. mu.L, adding MTT solution 5. mu.L for each well, culturing for 4h at 37 ℃, terminating culturing, adding DMSO 150. mu.L, shaking and mixing, measuring absorbance value (measuring wavelength is 570nm, reference wavelength is 630nm) for each well with microplate reader, and measuring cell survival rate.

1.4 changes in cell morphology

Cells with 10 percent⁵cell/mL density cells were plated on 96-well plates, and after cells were attached to the wall, the cells were treated by dosing, HS (25,50, 100. mu.g/mL, diluted with DMEM medium, 3 parallel wells per concentration) was added to the dosing group for 12 hours, and the wells of the blank group and the model group were changed, and after 12 hours, the model group was added to give 30. mu. mol/L A. beta. in the case of the model group_1-42The culture was continued for 24 hours, and the morphology of the cells was observed under an inverted microscope and photographed.

1.5 apoptosis assay

Cells with 10 percent⁵cells are inoculated in a 6-well plate at the cell/mL density, 25,50 and 100 mu g/mL HS is added for 12 hours after the cells are attached to the wall, and the cells are added into a model group for 30 mu mol/L A beta after 12 hours_1-42Culturing for 24h, digesting with pancreatin without EDTA, centrifuging at 1000r/min for 5min after digestion, discarding supernatant, rinsing cells with cold PBS for 2 times, detecting according to annexin V-FITC apoptosis kit, adding appropriate amount of 1 xnexin V-FITC conjugate, suspending and counting, and adjusting cell concentration to 10⁶cell/mL, adding 5 μ L of 20 μ g/mL annexin V-FITC staining solution into the cell suspension, mixing gently, incubating at 4 deg.C in the dark for 15min, adding 10 μ L of 5 μ g/mL Propidium Iodide (PI) staining solution, mixing gently, incubating at 4 deg.C in the dark for 5min, measuring with BD flow cytometer, measuring wavelength at 488nm, and calculating 10 μ L of each sample⁴cells. The proportion of normal cells, apoptotic cells and late apoptotic and necrotic cells in the total number of all cells was determined by flow cytometry.

1.6 extraction and quantification of Total protein

1.7 Western-blot analysis

(1) SDS-PAGE electrophoresis

(2) Rotary film

(3) Sealing of

(4) Anti-bonding

(5) Binding of secondary antibody

(6) Film washing development

(7) Data analysis

1.8 enzyme-linked immunosorbent assay kit for detecting tumor necrosis factor alpha (TNF-alpha) and interleukin 6(IL-6)

1.9 HS vs. Abeta_1-42Effect of oligomer formation in vitro

Accurately weighing Abeta_1-425mg, dissolved in sterile PBS, mixed well and filtered through sterile 0.22 μm filter. Filtering the A beta_1-42The solution is divided into 6 aseptic centrifuge tubes, three of the 6 aseptic centrifuge tubes are added with the aseptic HS solution for incubation at 37 ℃, and two centrifuge tubes are taken out for photographing and observation under a transmission electron microscope on the 1 st day, the 3 rd day and the 5 th day of incubation respectively.

Negative dyeing sample preparation: adsorbing the sample on the front surface of the copper net for 1min, adsorbing excessive sample on the carrier net, attaching the front surface of the carrier net to 2% phosphotungstic acid (pH 6.8) for 2min, adsorbing excessive dye on the carrier net, and drying at room temperature. And observing the photographing record under a transmission electron microscope.

1.10 statistical analysis

One-way anova Statistics with SPSS Statistics software were performed and p <0.05 was considered a significant difference.

2. Results of the experiment

2.1 HS disaccharide composition and proportion

TABLE 1 HS structural formulas and disaccharide compositions thereof

From the results in the table, the ratio of disaccharides is:

D2S6, D0S6/D2S0, D2A6, D0A6/D2A0, D0S0, D0A0 are 5:2:85:1:6: 9.

2.2 determination of cytotoxicity

The MTT result in figure 1 shows that the apoptosis rate of the HS administration group of 12.5,25,50,100,200,400 mu g/mL has no significant difference with the blank group, which indicates that HS has no toxic effect on the nerve cell SH-SY 5Y.

2.3 cell viability assay

As shown in FIG. 1, A.beta.was administered alone to SH-SY5Y cells, which are nerve cells_1-42The survival rate of the cells is obviously reduced by stimulation, the survival rate of the model group is only 72 percent, and the survival rates of the HS administration group are 83 percent +/-0.06, 88 percent +/-0.02, 91 percent +/-0.03, 85 percent +/-0.03, 91 percent +/-0.03 and 72 percent +/-0.06 respectively from the high concentration of 400 mu g/mL to the low concentration of 12.5 mu g/mL. Of these, 200, 100, 25. mu.g/mL are significant (P)<0.05) improving the activity of the cells.

2.4 changes in cell morphology

As can be seen from FIG. 2, SH-SY5Y cells in the blank group are fusiform, and the cells have filaments; the cell morphology of the model group is mostly circular and has no filaments. There was no significant change in cell morphology after early administration of HS. This indicates that HS is able to prevent A.beta._1-42Damage to the cell morphology of the nerve cells SH-SY 5Y.

2.5 apoptosis assay

Apoptosis is one of the major ways a β induces neuronal cell damage. In order to investigate whether HS can inhibit apoptosis induced by Abeta on nerve cells, the present example uses the annexin V-FITC apoptosis kit for detection. As can be seen from FIG. 3, 30. mu. mol/LA. beta. is_1-42Can induce apoptosis of cells, and the apoptosis rate is 12.24% +/-0.62. The occurrence of the apoptosis phenomenon can be remarkably inhibited by the early HS treatment, and the apoptosis rate of HS of 50,100 mu g/mL is reduced to 4.52 +/-1.28 percent and 5.77 +/-0.95 percent (P)<0.05). From the above results, it is presumed that HS significantly suppresses Abeta_1-42Apoptosis of SH-SY5Y nerve cells.

2.6 HS vs. Abeta_1-42Effect of inducing changes in the levels of factors associated with inflammatory pathways in SH-SY5Y neurons

Neuroinflammation plays a crucial role in the development of AD. Over-expression of inflammatory factors can lead to neurodegeneration, and up-regulation of inflammatory factor expression can also promote the progression of AD.

The significant difference between the TNF-alpha and IL-6 contents in the model group and the blank group is increased ((19.6 +/-0.91 vs 3.2 +/-0.49 and 7.98 +/-0.01 vs 2.78 +/-0.008, p)<0.0001), which indicates that a β is administered_1-42Treatment can cause damage to SH-SY5Y cells. The administration of HS can obviously reduce the inflammatory factor TNF-Levels of alpha, IL-6 (6.7 + -0.18 and 2.96 + -0.019, 1.73 + -0.024, p<0.0001)。

From grey value analysis, it can be seen that the levels of MMP2, MMP9, TLR4 and PJUN were all significantly elevated for the model group (1.43 ± 0.120vs 0.56 ± 0.109, 0.47 ± 0.036vs 0.31 ± 0.032, 1.12 ± 0.121vs 0.63 ± 0.059 and 1.26 ± 0.116vs 0.80 ± 0.042), HS treatment was able to reduce the levels of MMP2, MMP9, TLR4 and PJUN.

2.7 HS vs. Abeta_1-42Influence of the change of the expression level of the SH-SY5Y nerve cell apoptosis related protein.

Aβ_1-42Can induce the increase of Caspase8, 9 and Bax levels (0.45 +/-0.041 vs 0.28 +/-0.012 and 1.18 +/-0.220 vs 0.39 +/-0.049) of SH-SY5Y nerve cells and reduce the level of Bcl-2(0.69 +/-0.047 vs 1.03 +/-0.088). This result is consistent with the flow cytometry detection of apoptosis. Shows A.beta._1-42Can induce apoptosis of cells. The administration of SH-SY5Y nerve cell HS treatment can improve the abnormality of apoptosis related factor level, thereby inhibiting the apoptosis of the cell.

2.8 HS vs. Abeta_1-42Effect of oligomer formation in vitro.

As can be seen from the transmission electron microscope results, Abeta_1-42The forms of the cells do not greatly differ within 5 days of single incubation, and the cells are all filamentous oligomer structures with loose structures, however, A beta_1-42Incubation with HS (25. mu.g/mL) showed that on the third day the structure had transformed into a more structurally dense aggregate precipitate, at which point the state was no longer oligomeric but rather into fibrous bodies. The oligomer has strong neurotoxicity in brain, can affect the conduction of nerve synapses, prevent the formation of ion channels, destroy the membrane structure of cells, promote the apoptosis of nerve cells and the like. Reacting HS with Abeta_1-42Co-incubation enables the formation of A beta_1-42The A beta is reduced by converting oligomer with stronger toxicity into fibrous body with less toxicity_1-42Toxicity in vivo.

Aβ_1-42Can induce SH-SY5Y cells to have certain pathological changes in vitro, including affecting the elongation of nerve cell axons, reducing cell viability, and increasing cell soma to have inflammatory response, thereby inducing nerve cellsAnd (4) apoptosis. From the above results, HS was able to inhibit a β in an in vitro AD model_1-42Damage to nerve cell morphology, increase cell activity, and reduce cell apoptosis. In order to research how HS plays a role, research on the level of cytokines shows that HS can reduce the levels of inflammatory factors TNF-alpha and IL-6 of cells, MMP2, MMP9, TLR4 and PJUK for regulating inflammation-related pathways. And simultaneously regulates the level of apoptosis factors Caspase 9, Bax and Bcl-2. To investigate whether HS could affect A.beta._1-42The formation of oligomers, both dissolved in PBS, incubated at 37 deg.C, found that heparan sulfate can promote A beta_1-42The low-toxicity oligomer is converted into the low-toxicity fiber. Therefore, it is presumed that HS can convert Abeta_1-42Conversion of oligomers to fibrillar bodies by alteration of A.beta._1-42To reduce A beta_1-42Has reduced Abeta_1-42Inducing the generated inflammatory reaction and inhibiting the apoptosis of nerve cells.

This example demonstrates that HS is capable of inhibiting A β in an in vitro model_1-42For SH-SY5Y induced neurotoxicity, HS can enhance the activity of nerve cells, reduce the inflammatory reaction of nerve cells and further reduce the apoptosis of SY5Y cells.

Example 2

1. Experimental methods

1.1 animal grouping and administration methods

After the experimental mice are adapted for five days after being purchased, the experimental mice are grouped according to the experimental results of the water maze. The platform is not removed from the water maze, and the mice are grouped according to the time for finding the platform, so that the mean value of the time for finding the platform by the mice in each experimental group is consistent. Eight patients were divided into a blank group, a sham group, a model group, a low dose group (25m/kg), a medium dose group (100m/kg) and a high dose group (400 m/kg). The administration groups are respectively irrigated with stomach at the ratio of 0.1mL/10g body weight 1 time per day, and the experimental flow is shown in figure 8.

1.2 modeling

Two weeks after gavage, the mice were injected into the lateral ventricle of the brain for molding. Administration group and model group mice were injected with Abeta in lateral ventricle_1-42Go on to makeAnd (5) molding. Anaesthetizing the mouse by using a Ravord anaesthesia machine, fixing the mouse on a ventricular locator, shaving the mouse, cutting the top skin of the mouse head, exposing the front halogen after disinfection, locating according to the ventricular locator, and finding an injection site: 0.5mm on the back side of the front bittern, 1.0mm on the left and right sides, and depth of 2.5mm (AP-0.5 mm; ML +/-1.0 mm; DV-2.5mm) below the surface of the skull, injecting 5 μ L of the injection into each mouse, injecting for 3min at constant speed, leaving the needle for 5min, slowly pulling out the injector, and suturing the skin after disinfection. The sham-operated experimental group was operated in accordance with the model group, and only the same volume of sterile saline was injected. After the mice had a rest for three days after the operation, the administration was continued for one week, and the water maze experiment and the open field experiment were performed. After the test experiment, the mouse is cut off the head and the brain is taken out, the cortex and the hippocampus are separated, and the sample is frozen and stored at minus 80 ℃.

1.3 behavioral testing: morris Water maze experiment (MWM) and open field experiment (OFT)

1.4 Aβ_1-42TNF-alpha and IL-6 enzyme linked immunosorbent assay kit detection

1.5 immunohistochemistry

1.6 alteration of mouse Hippocampus ultrastructure

1.7 Total protein extraction quantification

1.8 Western blot analysis

1.9 in vivo tissue distribution in HS mice

The heart, liver, spleen, lung, kidney, stomach, intestine and brain tissues of the blank mouse are respectively weighed.

By intragastric administration of HS conjugated with FITC fluorescent probe to mice, mice were dislocated and sacrificed at 0, 0.5, 1, 2, 4, 6, 8, 12h after administration, and heart, liver, spleen, lung, kidney, gastrointestinal and brain tissues and blood were collected in a dark place. After homogenization, fluorescence absorbance was measured. The fluorescence absorption values of the tissues were calculated from the standard curve.

1.10 peripheral Abeta of mice_1-42Change of level

To study peripheral A.beta.during dosing in mice_1-42The level of change, experimental design as shown in FIG. 9, and experimental groups were divided into a blank group, a sham-operated group, a model group and a drug-administered group (HS-25 mg/kg). Each group had 8. The administration group was administered for two weeks (0 days to 14 days),mice were bled by tail-off on days 1, 4, 7, 10, and 13 of the administration, respectively. The molding operation was performed on day 16. Mice were rested for 3 days post-surgery, continued gavage of HS on days 20 to 27, and tail-biting ischemia was performed on days 20, 22, 24, 26, 28, 30. On day 30, blood was collected and then sacrificed by dislocation, and whole brain was collected. The operation method of tail-breaking blood sampling specifically comprises the following steps: mouse method in mouse fixer, the mouse tail was heated by hand, cut off by surgical scissors for 10mm, and blood was collected by EDTA-2Na sealed centrifuge tube.

The blood thus obtained was centrifuged at 12000r/min at 4 ℃ for 15 min. Taking supernatant plasma, and detecting Abeta in peripheral plasma of mice by using kit_1-42The content of (a).

1.11 statistical treatment

The experimental result adopts SPSS19.0 software to carry out statistical analysis on the data, the comparison between groups adopts LSD to carry out pairwise comparison in the groups, the data is expressed by mean +/-S.D, and the difference is expressed when p is less than 0.05 or p is less than 0.01, so that the statistical significance is realized.

2. Results of the experiment

2.1 behavioural test results

2.1.1 Water maze experiment

The experimental results are shown in fig. 10: in a directional navigation experiment, compared with a blank group, the escape latency of the model group is obviously prolonged from the next day; compared with the model group, the HS medium and high dose can be obviously shortened to the escape latency, and the escape latency is obviously different in the low dose group on the fifth day of the directional navigation test compared with the model group. Compared with the blank group and the sham operation group, the time of the model group in the target quadrant is obviously reduced, and the HS-100mg/kg is obviously different from the model group. The swimming tracks of the mice in each group of the directional navigation test and the space exploration test can be seen, in the space exploration test, the proportion of the mice in the model group in the target quadrant is obviously reduced compared with that in the blank group, and the administration group has no significant difference compared with the blank group.

2.1.2 open field experiments

As can be seen from the experimental results of the open field experiment, the total walking route of the mice between the groups has no significant difference, which indicates that the lateral ventricle injection of the mice does not influence the motor function of the mice.

2.2 cortical and hippocampal HE staining test results

FIG. 12 shows that the cortical region of the mice in the blank group and the sham-operated group has intact nerve cells and uniform cell staining. The cells in the cerebral cortex area of the model group mice have the phenomena of a large amount of cell nucleus fixation and uneven staining, and the cell morphology of the low-dose group and the high-dose group is normal. The cells of the blank group and the false operation group in the hippocampal CA1 area are arranged neatly and tightly, the cells of the model group are arranged loosely and disorderly, and a large number of cells have the phenomenon of cell nucleus shrinkage and have dark brown pigment deposits. The cells in the CA1 area of the hippocampus were relatively normal and no morphological abnormality was observed in the mice given HS.

2.3 alteration of mouse Hippocampus ultrastructure

From FIG. 13, it can be seen that the blank and sham operated groups had intact nuclear membranes, dense cytoplasm, normal endoplasmic reticulum, and normal mitochondrial energy organelle structure. The cells of the model group begin to have nuclear changes, nuclear membrane invagination, cytoplasm sparseness and cytoplasm vacuolation. No abnormal phenomenon was observed in HS-100mg/kg and HS-400mg/kg cells in the administered group.

2.4 Effect of inflammatory factor levels

Through ELISA analysis, it can be seen that mice injected with Abeta in lateral ventricle_1–42TNF-alpha and IL-6 in the hippocampal region of the posterior mice were significantly increased compared to the blank and sham operated groups. The levels of TNF-alpha and IL-6 in the hippocampus of mice were reduced following administration of HS.

Western-blot analysis shows that mouse lateral ventricle is injected with Abeta_1–42TLR4 (p) of the hippocampal region of the posterior mouse<0.01), and NF-kB (p)<0.05) and JNK (p)<0.001) was significantly different from those of the blank group and the sham group. The administration of HS can reduce the level of TLR4, pNF-kB and pJNK.

2.5 changes in apoptosis factor levels

Western-blot analysis shows that mouse lateral ventricle is injected with Abeta_1–42Caspase 3, 8 and Bax in the hippocampal region of the posterior mice were all significantly increased compared to the blank and sham groups. Levels of Caspase 3, 8 and Bax can be reduced following HS administration.

2.6 changes in PGSK and Ptau protein levels

Mouse lateral ventricle injection of Abeta_1-42Can induce the phosphorylation of GSK3 alpha/beta (216, 279) in the hippocampal region of the mouse to increase, activate the phosphorylation of tau protein, further induce the apoptosis of nerve cells in the brain of the mouse and cause nervous disorder. The administration of HS to mice can inhibit the activity of GSK3 alpha/beta and reduce A beta_1-42Damage to the brain nerves of mice.

2.7 in vivo distribution of HS

According to the ethological discovery, HS can improve A beta_1–42The composition can be used for treating mouse induced brain injury, and can inhibit the change of mouse hippocampal tissue apoptosis related factor and inflammatory factor level. Therefore, in order to further investigate whether HS can cross a blood brain barrier and enter the brain of a mouse to play a role, the HS and FITC are connected by a chemical method and are perfused into the stomach, and fluorescence value analysis of various organs of the mouse at different time points shows that the HS cannot penetrate the blood brain barrier, but passes through the stomach, is partially excreted through the intestine, and is partially excreted through the kidney. After 12h in vivo, the blood-activating agent is mainly distributed in spleen, kidney and blood. There are reports in the literature of highly toxic soluble Abeta for the brain_1–42Can reach the periphery through the blood brain barrier, and the result of the experiment suggests that the hypothesis that HS can not penetrate the blood brain barrier is that the HS can play the drug effect in the peripheral circulation. Example 1 transmission electron microscopy results also demonstrate that HS is capable of converting the more toxic A beta_1–42The oligomers convert to less toxic fibers. Therefore, it is assumed that the site of action of HS in mice with drugs is the periphery.

2.8 HS vs peripheral Abeta of mice_1–42Influence of level

During dosing, to study multiple mouse peripheral a β during HS dosing_1–42The influence of the level, the mode of tail breaking and blood drawing is adopted for the mice, and the real-time monitoring of Abeta is realized by adopting ELISA_1–42And (4) content. As can be seen from the line graph in FIG. 19, A.beta.in the periphery of the mice in the HS-administered group on the first day of administration_1–42Levels were lower than those of the blank, sham and model groups. One week after molding, model group Abeta_1–42The level is obviously higher than that of the other three groups, namely the HS group and the peripheral Abeta_1–42The level is still at the lowest level. This indicates HS to peripheral A β_1–42Has a certain cleaning function. Three weeks after HS administration, mice were dislocated at the end of the month to harvest brains. For mouse brain Abeta_1–42The detection is carried out, and the model group mouse brain Abeta is found to be compared with the sham operation group_1–42Has obvious increase, and can be used for intragastric administration of HS to mice and A beta in peripheral blood plasma_1–42The level decreases. It is presumed that although HS is unable to permeate the blood-brain barrier, it is able to reduce peripheral A.beta.by peripheral decrease in the peripheral_1–42Levels, and thus, A.beta.of mouse brain_1–42And (3) reducing brain damage and improving cognitive damage of mice.

2.9 Effect of HS on the ratio of the respective subsets of monocytes and neutrophils in the peripheral circulation of mice.

Aβ_1–42Can activate monocytes and increase F4/80⁺/CD11b⁺The proportion of subtypes, causing an inflammatory response. But has less effect on neutrophils. After administration of HS, A beta can be reduced_1–42Activation of monocytes. And it can be seen that when no HS treatment was given, Abeta_1–42Is mainly phagocytically cleared by monocytes, and after administration of HS, Abeta_1–42Is mainly cleared by neutrophils. Abeta (beta)_1–42Can reduce the level of NO in leucocytes and increase the level of TNF-alpha.

2.10 HS on A β in peripheral circulation_1-42Influence of cleaning

Flow results show that administration of HS treatment can reduce monocyte F4/80⁺/CD11b⁺Subtype and neutrophil CD11b⁺/LY6G⁺The proportion of subtypes. This indicates that HS is able to reduce inflammatory responses in the peripheral circulation. Simultaneous administration of HS groups to Abeta_1-42The phagocytic clearance of the compound is more than that of the model group.

The experimental results of example 1 demonstrate that HS can enhance the activity of nerve cells and inhibit A beta in an in vitro AD model_1–42The damage to nerve cells, such as reducing the level of nerve inflammatory factors and reducing apoptosis. HS and Abeta were also found_1–42Co-incubation can alter oligomer reduction status and convert oligomers to less toxic fibersAnd (3) a body.

To further study the activity of HS in mice, this example employed lateral intracerebroventricular injection of Abeta in mice_1–42And (5) molding. The cognitive function of the mice is evaluated through a water maze experiment and an open field experiment, and the mice are injected with Abeta in the lateral ventricle_1–42The method can enable the mice to generate cognitive disorder, and shows that the method can simulate the pathological process of the Alzheimer disease. After the mice are perfused with HS, the escape latency of the mice is obviously shortened in a directional navigation test, and the mice in the administration group are found to have obviously increased time in a target quadrant compared with a model group in a space exploration test in the last day. HS is proved to play a role in improving cognitive impairment of mice in vivo.

In the embodiment, HS is connected with fluorescein FITC, and the distribution of HS in the body of the mouse is judged according to the fluorescence value of each tissue of the mouse, so that the HS can not directly reach the brain to play a role by penetrating the blood brain barrier and can play an anti-AD activity in the peripheral circulation. A beta in brain is reported in literature_1–42Can penetrate blood brain barrier to reach periphery, and realize brain and periphery circulation. Finding that most of HS is located in the intestine kidney and peripheral blood through fluorescence distribution, and detecting the peripheral Abeta of the mouse during the HS perfusion period by an ELISA (enzyme-linked immunosorbent assay) detection method_1–42The level is detected in real time, and HS is found to reduce peripheral Abeta_1–42In the presence of a reducing amount of peripheral Abeta_1–42Content of the A beta in the brain of the mouse_1–42The content of (A) is also reduced.

The conclusion is as follows:

(1) the behavioral test result of the mouse shows that lateral ventricle is injected with Abeta_1–42Can induce the cognitive disorder of the mice, and after the administration of HS, the low, medium and high doses all improve the learning cognitive disorder of the mice to a certain extent.

(2) Lateral ventricle injection of Abeta_1–42Can cause the level of mouse brain inflammatory factors TNF-alpha and IL-6 to be increased, and cause the level of apoptosis factors Caspase 3 and Bax to be increased. After intervention of HS, the level of mouse brain inflammatory factors TNF-alpha and IL-6 can be reduced, and the level of apoptosis factors Caspase 3 and Bax can be reduced. Thereby exerting an aim atThe mouse brain has a certain protection effect.

(3) Although HS does not function directly into the brain, it can act peripherally by reducing peripheral a β_1–42Horizontal indirect reduction of brain Abeta_1–42And further, the effect of improving cognitive impairment of mice is achieved.

(4) HS can increase A beta in peripheral circulation by peripheral neutrophils_1–42Phagocytosis of (4). However, the phagocytic capacity of the E.coli by the neutrophils in the HS administration group is not obviously enhanced. It was thus concluded that HS may act as a precipitant, promoting A β, in the peripheral circulation_1–42The oligomer is converted into fiber, so as to reduce the concentration at the periphery, weaken the inflammatory reaction caused by the oligomer in the peripheral circulation and further reduce the brain A beta_1–42The concentration and inflammatory reaction indirectly protect the brain nerves.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.