CN116235953A - Application of low molecular weight sea cucumber glycosaminoglycan in medicine for preventing and treating senile dementia and health care and functional food - Google Patents
Application of low molecular weight sea cucumber glycosaminoglycan in medicine for preventing and treating senile dementia and health care and functional food Download PDFInfo
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- CN116235953A CN116235953A CN202310317027.2A CN202310317027A CN116235953A CN 116235953 A CN116235953 A CN 116235953A CN 202310317027 A CN202310317027 A CN 202310317027A CN 116235953 A CN116235953 A CN 116235953A
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Abstract
The invention discloses application of low molecular weight sea cucumber glycosaminoglycan in medicaments for preventing and treating senile dementia and health care and functional foods. The invention has the advantages that: in an animal in-vivo experiment, the oral administration of the low-molecular sea cucumber glycosaminoglycan significantly improves the cognitive dysfunction of a model mouse with senile dementia, and reduces Abeta accumulation, nerve synapse loss and nerve inflammation in the brain of the model mouse; in an in vitro cell experiment, the low-molecular sea cucumber glycosaminoglycan remarkably inhibits the concentration of beta amyloid 38 and beta amyloid 40 of H4 human glioma cells, has a certain inhibition effect on Tau protein phosphorylation in SH-SY5Y human neuroblastoma cells, remarkably inhibits the production of microglial cell BV-2 inflammatory factors TNF-alpha, KC/GRO and IL-6, and promotes the concentration of anti-inflammatory factors IL-2, IL-5 and IL-4; the effects show that the low molecular weight sea cucumber glycosaminoglycan is expected to be applied to medicaments for preventing and treating senile dementia and health care and functional foods.
Description
Technical Field
The invention belongs to the technical field of biological medicines and health-care foods, and in particular relates to application of low-molecular sea cucumber glycosaminoglycan in medicines for preventing and treating senile dementia and health-care and functional foods.
Background
Senile dementia (Alzheimer Disease, AD), also known as Alzheimer's disease, is a neurodegenerative disease of the nervous system, and clinical symptoms are manifested by memory loss, cognitive dysfunction, etc. In our country, senile dementia is one of the first five causes of death leading to death of residents, and in 2019, the number of senile dementia and related dementia patients in our country is over 1300 ten thousand, and the senile dementia is a serious burden for individuals, families and society, and at least 5000 ten thousand senile dementia patients are worldwide at present.
The etiology of senile dementia is heretofore unknown, and there are a variety of theories that the influence is widely exerted on the abnormal phosphorylation hypothesis of Amyloid beta (aβ) and Tau proteins. The amyloid cascade hypothesis suggests that soluble aβ oligomers and insoluble amyloid deposits in the brain form amyloid plaques due to overproduction or untimely clearance of aβ42 or other aβ polypeptide fragments. Their interaction with microglia, astrocytes, blood vessels and neurons triggers a variety of deleterious cellular responses, ultimately leading to neuronal dysfunction and death. Abnormal phosphorylation of Tau protein the hypothesis is that in the brain of Alzheimer's disease patients, tau protein is phosphorylated, whereas phosphorylated Tau protein will be converted from soluble to insoluble, while highly aggregated. This can lead to the loss of synaptic proteins function and neurodegenerative disorders, causing neurofibrillary tangles. In addition, the causes of senile dementia include microglial activation, cytokine release, astrocyte release neurotoxin, neuroinflammation, and blood microcirculation disturbance.
In terms of medicine, senile dementia lacks effective clinical medicine. In 2023, 1 month and 8 days, according to the accelerated evaluation channel, the United states Food and Drug Administration (FDA) has approved 100mg/mL of injection of Lecanemab for treating Alzheimer's disease. The medicine is a new medicine for senile dementia which is marketed in batch in the third year in 2003, and is also a second new therapy for senile dementia targeting beta amyloid. The other two therapeutic drugs are respectively: the national medicine mannite sodium capsule (GV-971) which is approved by the condition for treating mild to moderate senile dementia and improving the cognitive function of patients is just obtained in China in 2021, but the international phase III clinical study is announced to be stopped in 2022 in the last half year; the first approved drug for treating senile dementia by the FDA, a drug for monoclonal antibodies developed based on the beta-amyloid hypothesis, was a Du Nashan anti (aducanaumab), but the suspension of european market applications was declared in 2022. To sum up, the cause of senile dementia is unknown so far, and few therapeutic drugs are available. Therefore, it is of great importance to find more strategies for treating Alzheimer's disease.
Disclosure of Invention
The invention aims to provide an application of low-molecular sea cucumber glycosaminoglycan in medicines for preventing and treating senile dementia and health-care and functional foods.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the application of low molecular weight sea cucumber glycosaminoglycan in medicine for preventing and treating senile dementia and health care and functional food.
Further, the application of the low molecular weight sea cucumber glycosaminoglycan in medicines for preventing and treating senile dementia and health care and functional foods, wherein: the low molecular weight sea cucumber glycosaminoglycan is used as an inhibitor of beta-amyloid (Abeta).
Furthermore, the application of the low molecular weight sea cucumber glycosaminoglycan in medicines for preventing and treating senile dementia and health care and functional foods, wherein: beta-amyloid (aβ) includes: one or more of aβ38, aβ40 and aβ -42.
Further, the application of the low molecular weight sea cucumber glycosaminoglycan in medicines for preventing and treating senile dementia and health care and functional foods, wherein: the low molecular weight sea cucumber glycosaminoglycan is used as an inhibitor of inflammatory factors.
Furthermore, the application of the low molecular weight sea cucumber glycosaminoglycan in medicines for preventing and treating senile dementia and health care and functional foods, wherein: inflammatory factors include: one or more of tumor necrosis factor-alpha (TNF-alpha), keratinocyte chemokine/growth regulator gene protein (KC/GRO), and interleukin-6 (IL-6).
Further, the application of the low molecular weight sea cucumber glycosaminoglycan in medicines for preventing and treating senile dementia and health care and functional foods, wherein: the low molecular weight sea cucumber glycosaminoglycan is used as an activator of anti-inflammatory factors.
Furthermore, the application of the low molecular weight sea cucumber glycosaminoglycan in medicines for preventing and treating senile dementia and health care and functional foods, wherein: anti-inflammatory factors include: one or more of interleukin-2 (IL-2), interleukin-5 (IL-5), and interleukin-4 (IL-4).
Further, the application of the low molecular weight sea cucumber glycosaminoglycan in medicines for preventing and treating senile dementia and health care and functional foods, wherein: the low molecular weight sea cucumber glycosaminoglycan is used as Tau protein phosphorylation inhibitor.
Further, the application of the low molecular weight sea cucumber glycosaminoglycan in medicines for preventing and treating senile dementia and health care and functional foods, wherein: the low molecular weight sea cucumber glycosaminoglycan is used as a novel object recognition capability promoter.
Further, the application of the low molecular weight sea cucumber glycosaminoglycan in medicines for preventing and treating senile dementia and health care and functional foods, wherein: the low molecular weight sea cucumber glycosaminoglycan is used as a cognitive disorder improving agent.
Further, the application of the low molecular weight sea cucumber glycosaminoglycan in medicines for preventing and treating senile dementia and health care and functional foods, wherein: the low molecular weight sea cucumber glycosaminoglycan is used as a neurite loss inhibitor.
Further, the application of the low molecular weight sea cucumber glycosaminoglycan in medicines for preventing and treating senile dementia and health care and functional foods, wherein: the low molecular weight sea cucumber glycosaminoglycan is used as a neuroinflammation inhibitor.
A low molecular weight sea cucumber glycosaminoglycan pharmaceutical composition, wherein: the low molecular weight sea cucumber glycosaminoglycan is used as an active pharmaceutical ingredient, and pharmaceutically acceptable auxiliary materials.
A low molecular weight sea cucumber glycosaminoglycan health food composition, wherein: the low molecular weight sea cucumber glycosaminoglycan is taken as a component and auxiliary materials acceptable in food.
A low molecular weight sea cucumber glycosaminoglycan functional food composition, which: the low molecular weight sea cucumber glycosaminoglycan is taken as a component and auxiliary materials acceptable in food.
In one set of examples (examples 1-5) but not limited to this set of examples, an experiment of the effect of oral administration of low molecular weight sea cucumber glycosaminoglycans on the improvement of symptoms in Alzheimer's disease model mice (APP/PS 1 mice) was described. Three low molecular weight sea cucumber glycosaminoglycans with different molecular weights are taken as research medicines, the modeled senile dementia model mice are orally administered for 4 months continuously, the administration dosage is 500-1000 mg/Kg/d/mouse, then a new object recognition experiment and a water maze experiment are examined, and synaptophysin and various inflammatory factors are checked by immunofluorescence staining and enzyme-linked immunosorbent assay. The results show that: the recognition index of APP/PS1 mice orally administered with 4 low-molecular sea cucumber glycosaminoglycan feeds in a new object recognition test is obviously higher than that of mice in a model control group, and even reaches the same level as that of normal mice; the low molecular weight sea cucumber glycosaminoglycan can improve cognitive disorder of APP/PS1 mice in a water maze test; immunofluorescence experimental results of accumulation of Abeta show that oral administration of low-molecular-weight sea cucumber glycosaminoglycan can reduce accumulation of Abeta plaque in brain of APP/PS1 mice, and the results show that oral administration of low-molecular-weight sea cucumber glycosaminoglycan can reduce the content of soluble Abeta 42 in cortex area of APP/PS1 mice, but does not affect Abeta level of hippocampal parts; in the aspect of synaptic loss, the oral administration of the low-molecular sea cucumber glycosaminoglycan can improve the synaptic loss condition of an APP/PS1 mouse; in the aspect of neuroinflammation, oral administration of low molecular weight sea cucumber glycosaminoglycans improves astrocytes and microglial-mediated neuroinflammation in the brain of APP/PS1 mice.
In another set of examples (examples 6-8), but not limited to this set of examples, the beneficial pharmacodynamic effects of low molecular weight sea cucumber glycosaminoglycans on Alzheimer's disease-associated Abeta secretion, tau protein phosphorylation, inflammatory factors, and anti-inflammatory factors in vitro cell experiments are demonstrated. Three groups of high, medium and low concentration low molecular sea cucumber glycosaminoglycan tested drugs, a negative blank control group and a positive drug control group are respectively arranged in the experiment, and the influence on the Abeta secretion of H4 human glioma cells, the influence on Tau protein phosphorylation in SH-SY5Y human neuroblastoma cells and the influence on the LPS-induced neuroinflammation of BV-2 cells are respectively treated and examined. The results show that: the low-molecular sea cucumber glycosaminoglycan can obviously inhibit the concentration of beta amyloid 38 and 40 of H4 human glioma cells, has a certain inhibition effect on Tau protein phosphorylation in SH-SY5Y human neuroblastoma cells, obviously inhibits the production of microglial cell BV-2 inflammatory factor tumor necrosis factor-alpha (TNF-alpha), keratinocyte chemotactic factor/growth regulating gene protein (KC/GRO) and interleukin-6 (IL-6), and improves the concentration of anti-inflammatory factors interleukin-2 (IL-2), interleukin-5 (IL-5) and interleukin-4 (IL-4). Through implementation of the technical scheme, the invention has the beneficial effects that: provides the application of the low molecular weight sea cucumber glycosaminoglycan in medicaments for preventing and treating senile dementia and health care and functional foods: in an animal in-vivo experiment, the oral administration of the low-molecular sea cucumber glycosaminoglycan significantly improves the cognitive disorder of an Alzheimer's disease model mouse (APP/PS 1 mouse) and reduces Abeta accumulation, nerve synapse loss and nerve inflammation in the brain of the model mouse; in an in vitro cell experiment, the low-molecular sea cucumber glycosaminoglycan remarkably inhibits the concentration of beta amyloid 38 and beta amyloid 40 of H4 human glioma cells, has a certain inhibition effect on Tau protein phosphorylation in SH-SY5Y human neuroblastoma cells, remarkably inhibits the production of microglial cell BV-2 inflammatory factors TNF-alpha, KC/GRO and IL-6, and promotes the concentration of anti-inflammatory factors IL-2, IL-5 and IL-4; the effects show that the low molecular weight sea cucumber glycosaminoglycan is expected to be applied to medicaments for preventing and treating senile dementia and health care and functional foods.
Drawings
FIG. 1 is a graph comparing experimental results of the movement path of WT mice and APP/PS1 mice in the mine, the movement speed in the mine, the number of times of crossing the central region of the mine, the stay time in the central region, and the movement path in the central region of the mine in example 2 of the present invention.
FIG. 2 is a graph showing comparison of the results of experiments for identifying new objects of WT mice and APP/PS1 mice in example 2 of the present invention.
FIG. 3 is a graph showing comparison of the results of the water maze test between WT and APP/PS1 mice in example 3 of the present invention.
FIG. 4 is a graph showing comparison of Abeta plaque density at the cortex and hippocampal sites of WT mice and APP/PS1 mice in example 4 of the present invention.
FIG. 5 shows the levels of soluble and insoluble Aβ42 and Aβ40 in the cortex and hippocampal regions of WT mice and APP/PS1 mice in example 4 of the present invention.
FIG. 6 is a graph comparing SYN fluorescence intensity results of WT mice and APP/PS1 mice in example 4 of the present invention.
FIG. 7 is a graph showing comparison of astrocyte density measurements of WT mice and APP/PS1 mice in example 5 of the present invention.
FIG. 8 is a graph showing comparison of microglial density measurements of WT mice and APP/PS1 mice in example 5 of the present invention.
FIG. 9 is a graph showing comparison of the results of detection of the levels of inflammatory factors IL-1. Beta. And IL-6 in WT mice and APP/PS1 in example 5 of the present invention.
FIG. 10 shows the levels of Abeta 1-38, abeta 1-40 and Abeta 1-42 in the culture supernatants of H4 human glioma cells 24 hours after treatment with low-molecular sea cucumber glycosaminoglycans of the present invention at different concentrations in example 6.
FIG. 11 shows the levels of total Tau protein and pTau231 after 24h treatment of SH-SY5Y human neuroblastoma cells with low molecular weight sea cucumber glycosaminoglycans of example 7 of the present invention.
FIG. 12 shows TNF- α, KC/GRO, IL-6 and IFN- γ levels in the supernatant of BV-2 cells stimulated with LPS for 24 hours after treatment with low molecular weight sea cucumber glycosaminoglycan according to example 8 of the present invention.
FIG. 13 shows the levels of IL-5, IL-2, IL-1. Beta. And IL-12p70 in the supernatant of BV-2 cells stimulated with LPS for 24 hours after treatment with low molecular weight sea cucumber glycosaminoglycan according to example 8 of the present invention.
FIG. 14 shows the levels of IL-4 and IL-10 in the supernatant of BV-2 cells stimulated with LPS for 24 hours after treatment with low molecular weight sea cucumber glycosaminoglycan according to example 8 of the present invention.
Detailed Description
The following describes specific embodiments of the present invention with reference to the drawings and examples. It should be understood that the following examples are only for illustrating the technical scheme of the present invention, but are not intended to limit the scope of the present invention.
In one set of examples (examples 1-5) but not limited to this set of examples, an experiment of the effect of oral administration of low molecular weight sea cucumber glycosaminoglycans on the improvement of symptoms in Alzheimer's disease model mice (APP/PS 1 mice) was described. Three low molecular weight sea cucumber glycosaminoglycans with different molecular weights are taken as research medicines, the modeled senile dementia model mice are orally administered for 4 months continuously, the administration dosage is 500-1000 mg/Kg/d/mouse, then a new object recognition experiment and a water maze experiment are examined, and synaptophysin and various inflammatory factors are checked by immunofluorescence staining and enzyme-linked immunosorbent assay. The results show that: the recognition index of APP/PS1 mice orally administered with 4 low-molecular sea cucumber glycosaminoglycan feeds in a new object recognition test is obviously higher than that of mice in a model control group, and even reaches the same level as that of normal mice; the low molecular weight sea cucumber glycosaminoglycan can improve cognitive disorder of APP/PS1 mice in a water maze test; immunofluorescence experimental results of accumulation of Abeta show that oral administration of low-molecular-weight sea cucumber glycosaminoglycan can reduce accumulation of Abeta plaque in brain of APP/PS1 mice, and the results show that oral administration of low-molecular-weight sea cucumber glycosaminoglycan can reduce the content of soluble Abeta 42 in cortex area of APP/PS1 mice, but does not affect Abeta level of hippocampal parts; in the aspect of synaptic loss, the oral administration of the low-molecular sea cucumber glycosaminoglycan can improve the synaptic loss condition of an APP/PS1 mouse; in the aspect of neuroinflammation, oral administration of low molecular weight sea cucumber glycosaminoglycans improves astrocytes and microglial-mediated neuroinflammation in the brain of APP/PS1 mice.
Example 1: experimental design of effect of low molecular weight sea cucumber glycosaminoglycan on improving symptoms of Alzheimer's disease model mice (APP/PS 1 mice)
The embodiment is to examine the effect of low molecular weight sea cucumber glycosaminoglycan on improving APP/PS1 mouse symptoms, and relates to experimental design, a dosing scheme and the like, and the method comprises the following steps:
1. information of experimental animal
APP/PS1 transgenic mice can express human mutant amyloid precursor protein (APPswe) and human presenilin 1 (PS 1-dE 9), and are commonly used senile dementia model mice at present. Mice used in the experiments of this example were 3.5 months old and had a weight of 25g-35g, all male.
2. Grouping and dosing regimen for animals
Wild-type control mice and male APP/PS1 model mice were divided into the following 6 groups (table 1). APP/PS1 mice of groups 3-6 were orally administered feeds containing low molecular weight sea cucumber glycosaminoglycans at 3.5 months of age. Group 2 age-matched sex APP/PS1 mice given normal diet are model control mice. In addition, the same-age and same-sex wild-type mice (WT) were selected and given normal diet as wild-type control mice (group 1).
Table 1 dosing experimental animals group
3. Administration mode and time
The low molecular weight sea cucumber glycosaminoglycan is added into feed by oral administration, and the quantitative feed is put into a mouse feed box. Oral administration was started 3.5 months after birth of the transgenic APP/PS1 mice. Once daily for 4 months. Thus, the administration time is 3.5 months to 7.5 months after birth. The WT control and model control were orally given normal feed.
4. Data arrangement and statistical analysis
The improvement effect of low molecular sea cucumber glycosaminoglycan on APP/PS1 mouse symptoms is examined by a new object recognition experiment, a water maze experiment, immunofluorescence staining, an enzyme-linked immunosorbent assay and the like.
All data are presented as mean±sem and were statistically analyzed by SPSS software. Comparisons between the Two groups were performed using t-test, multiple groups were analyzed using One wayova, and water maze was counted using Two wayova. * Represents p <0.05; * Represents p <0.01; * Represents p <0.001.
Example 2: new object recognition experiment, an experiment of improving symptoms of low molecular weight sea cucumber glycosaminoglycan on senile dementia model mice (APP/PS 1 mice)
The exploring condition of the APP/PS1 mouse on the new and old objects is reflected through the identification index; first, to exclude the influence of the placement position of the object on the mouse, we should detect the preference index of the mouse on the object position, that is, the sum of the time the mouse explores for the object placed on one side/the time explored for the object on both sides when the same object is placed at the bottom of the experiment box in a prescribed time period of 10 min.
Formal experiments: mice were first acclimatized in boxes for three consecutive days, 10 minutes a day. Note that the sides and bottom of the box were sprayed with 75% alcohol each time the mice were changed and rubbed dry with paper. On the fourth test day, 2 different sets of objects were prepared, set A of objects were placed diagonally in box one, note that the objects were both 5cm away from both walls, and set B of objects were placed in box two. The mice were then placed in a box for testing and recorded with software for 10 minutes. After 90 minutes, the objects were exchanged in two adaptation boxes, and the same test was performed for 10 minutes, and the time and distance the mice explored for the old and new objects were recorded. Note that each time the mice were changed for testing, the boxes were sprayed with 75% alcohol and wiped dry to avoid odors interfering with the behavior of the mice. Differences in the identification index of each group of mice to new objects were recorded and analyzed, excluding the mice' position preference.
Detection time point: and (3) performing new object recognition experimental detection after the mice are 7.5 months old, namely orally administering the low-molecular sea cucumber glycosaminoglycans for 4 months.
Detecting the index:
(1) Preference index: the total of the time the mice explored for objects placed on one side/the time explored for objects on both sides, when the same objects were placed at the bottom of the laboratory box, within a prescribed 10 minute period.
(2) Identification index: the sum of the exploration time of the mice for the new object/the exploration time for the new object and the exploration time for the old object within a prescribed 10 minute time period.
Experimental results:
the low molecular weight sea cucumber glycosaminoglycan does not affect the autonomous locomotor ability and anxiety behavior of senile dementia model mice (as shown in figure 1). In fig. 1, a represents the movement path of the mice in the mine; b represents the speed of movement of the mice in the mine; c represents the number of times the mouse crosses the central area of the mine; d represents the time the mice stay in the central region; e represents the movement path of the mice in the central area of the mine; n=11-16 mice; differences in data between groups were examined with One way Anova, data representing mean±sem;
the number of times the mice cross the central zone, the time spent in the central zone and the route were used to evaluate the anxiety behaviour of the mice. As shown in figures 1C-E, compared with a WT mouse, the number of times the APP/PS1 mouse passes through the central area, the stay time and the movement distance in the central area are not changed, the number of times the APP/PS1 mouse passes through the central area, the stay time and the movement distance in the central area are not influenced by the administration of the four low-molecular sea cucumber glycosaminoglycans, the APP/PS1 mouse is not provided with anxiety behaviors, and the four low-molecular sea cucumber glycosaminoglycans are not provided with anxiety behaviors.
The experiment of the embodiment researches the improvement effect on the short-term learning and memory capacity of mice after oral administration of the low-molecular sea cucumber glycosaminoglycan through a new object recognition experiment. FIG. 2 is a graph showing comparison of a new object recognition experiment of WT mice and APP/PS1 mice, wherein in FIG. 2, A represents the position preference index of the mice to the objects; b represents the identification index of the mouse to the new object; n=11-16 mice; differences in data between groups were examined with One way Anova, data representing mean±sem; * P <0.01, p <0.001. In the first experiment, i.e., when two identical objects were placed, the experimental results showed that there was no difference in the position preference index of each group of mice, i.e., there was no preference of the mice for the placement of the objects (as shown in fig. 2A). In the second experiment, namely when one object is replaced by another new object, the recognition index of the APP/PS1 mouse is obviously smaller than that of the WT mouse, and the APP/PS1 mouse is not capable of distinguishing the new object from the old object, so that the short-term learning and memory capacity is damaged. The recognition index of new objects of the oral administration of 4 low molecular weight sea cucumber glycosaminoglycans is obviously higher than that of APP/PS1 mice, and even reaches the same level as that of WT mice (as shown in figure 2B);
the results show that oral administration of the low-molecular sea cucumber glycosaminoglycan can improve short-term memory dysfunction of senile dementia model mice.
Example 3: water maze test, an experiment of improving symptoms of low molecular weight sea cucumber glycosaminoglycan on senile dementia model mice (APP/PS 1 mice)
Experimental principle: the mouse naturally swims well but extremely dislike to stay in water, when the mouse is placed in a circular water pool, the mouse can choose to escape from the water environment, at the moment, a hidden platform with the diameter of 10cm is arranged at the position of 1cm under water, and the hidden platform is placed in one quadrant of a water bucket, so that the mouse is guided to the platform. The diameter of the water maze drum is 1.6m, the periphery of the water maze drum is covered by a blue curtain, and the periphery of the water maze drum is marked by hanging objects with different shapes and colors. Mice were randomly grouped and placed in a behavioural test room, one hour prior to the experiment, to suit the surrounding environment. At the beginning of the experiment, training experiments are firstly carried out, a mouse is trained to find an underwater platform, and the time, distance and swimming speed of the mouse found the platform are recorded by an instrument. Mice were trained 4 times a day for 5 consecutive days. The pool was divided into four halves, named first, second, third, and fourth quadrants, and in each session, mice were placed in the water half-randomly from the middle of each quadrant. Each training was continued for 60s or until the mouse found the platform, i.e. the hidden target platform was found or the time exceeded 60s, if the mouse did not find the platform beyond 60s, the mouse was gently picked up to the platform for 10s and then returned. On day 6, the platform was removed and the number of times the mice traversed the original platform location was analyzed. After the end of the experiment, each group of mice was counted and analyzed for latency (time from entry into pool to platform finding) and path (swimming distance from starting point to platform) for 5 consecutive days. Data were collected using the Anymaze software and the number of passes across the platform and swim speed were analyzed for differences between groups using One wayANOVA. Latency and path differences between groups were analyzed using the Two way anova method. After the water maze experiment is finished, the mice are placed for about 2 weeks, a water maze reverse experiment is carried out, the platform is moved to the opposite side quadrant, the mice are trained for 3 days by the method, and the time, the distance and the swimming speed of the platform are recorded by the instrument. The learning and memory ability of the mice was evaluated by the time they found the platform.
Detection time point: and (3) when the mice are 7.5 months old, namely after oral administration of the low-molecular sea cucumber glycosaminoglycan for 4 months, carrying out water maze experiment detection after the new object identification experiment is finished.
Detecting the index:
(1) Escape Latency (Escape Latency): the time from entry into the pool to the arrival of the mouse swimming to the platform;
(2) Swimming Path (Path Length): the mice swim the distance from the starting point of swimming into the pool to the point where the platform was found.
(3) Swimming Speed (Speed): mice were trained on day 6 from the average swimming speed into the pool of water 60 s.
Experimental results:
the water maze test of this example was used to examine the spatial learning memory ability of mice.
As shown in FIG. 3, FIG. 3 is a graph showing the comparison of the results of the water maze test between WT mice and APP/PS1 mice. Wherein A represents the time taken by the mice to find the platform after continuous training for 5 days; b represents the path length of the mouse finding the platform after continuous training for 5 days; c represents the average swimming speed of the mice; d represents the time taken by the mouse to find the platform in the reverse experiment; n=11-16 mice; checking the difference (C) in speed between groups with One way Anova, and detecting the difference between the time (A and D) used for finding the platform and the swimming path length (B) data with Two way Anova; data represent mean±sem; * p <0.05, < p <0.01, < p <0.001. Compared with the WT mice, the time taken for the APP/PS1 mice to find the platform (FIG. 3A) and the total distance to swim for the platform found (FIG. 3B) were significantly higher than the WT mice, indicating that there was a learning memory impairment in the APP/PS1 mice. Oral administration of 4 low molecular weight sea cucumber glycosaminoglycans did not improve the time and swimming distance taken to find the platform during the spatial learning phase compared to APP/PS1 mice. However, oral administration of GAG-2 significantly reduced latency to find the platform in APP/PS1 mice during the reverse learning phase (fig. 3D). To exclude the effect of swimming speed on the results, we analyzed the swimming speeds of the groups of mice, and the results showed that the swimming speeds of the groups of mice were not different (fig. 3C), indicating that the difference between the groups of mice in the above results was not due to the difference in swimming speeds.
These results demonstrate that oral administration of low molecular weight sea cucumber glycosaminoglycans can improve spatial learning and memory in APP/PS1 transgenic mice.
Example 4: immunofluorescence staining of low molecular sea cucumber glycosaminoglycan for improving senile dementia model mice (APP/PS 1 mice) symptom
Each mouse is injected with 500 mu L of 3.6% chloral hydrate for anesthesia, sterilized by 75% alcohol, wiped off the alcohol with paper, then the scissors are broken, the brain is taken out, put into PBS which is precooled in advance for cleaning, put into 4% PFA for fixing for 4-6 hours, dehydrated by 15% sucrose, dehydrated and precipitated, changed into 30% sucrose for continuous dehydration, and after the mouse is submerged, the mouse is embedded by OCT embedding agent and frozen for slicing. Coronal slices were 20 μm thick and serial sagittal slices were 30 μm thick. Placed on a gelatin-coated glass slide, air-dried overnight and stored at-20 ℃. Taking a corresponding slide glass during immunofluorescence staining, airing at room temperature, washing with 1 XPBS three times for 5 minutes each time, punching holes for 20 minutes by penetrating a film with 0.3% -0.5% Triton-100, and sealing for 1 hour by 10% FBS at room temperature; incubating the primary antibody at 4 ℃ overnight; every other day, 1×pbs was washed three times for 5 minutes each. Incubating with corresponding fluorescent secondary antibodies at room temperature for 1 hour; washing with 1×pbs three times for 5 minutes each; seal with DAPI-containing seal tablet and examine with a microscope.
The technique was used to analyze the fluorescence intensity of synaptorin protein in the CA3 region of the hippocampus of mice, the density of astrocytes and microglial cells in the brain of mice, the area and number of aβ plaques in the brain of mice.
Detection time point: after the end of the behaviours, the mice were about 8.5 months old.
Experimental results:
this example experiment uses immunofluorescent staining to detect aβ plaque density in the cortex and hippocampus of each group of mice. As shown in fig. 4, fig. 4 is a graph comparing aβ plaque density at the cortex and hippocampal sites of WT mice and APP/PS1 mice. Wherein A represents the area and the number representative graph of the Abeta plaque at the cortex part; b represents an area and number representative map of aβ plaques at the hippocampal site; c represents a total area statistical graph D of the Abeta plaque at the cortical site; d represents a statistical graph of the number of Abeta plaques at cortical sites; e represents a total area statistical graph D of Abeta plaque at the hippocampal site; f represents a statistical plot of the number of aβ plaques at the hippocampal site; n=15 sections (from 3 mice); testing the difference of the data between groups by using One way Anova; data represent mean±sem; * p <0.05, < p <0.01, < p <0.001; NS is not statistically significant.
The results show that the area, number (FIG. 4 ACD) and number (FIG. 4 BF) of Aβ plaques in the cortex region and the Aβ plaques in the hippocampal region of APP/PS1 mice administered orally with low molecular weight sea cucumber glycosaminoglycans (GAG-2, GAG-3 and GAG-4) are significantly reduced compared to WT mice, and that the area of Aβ plaques in the hippocampal region of APP/PS1 mice administered orally with 4 low molecular weight sea cucumber glycosaminoglycans is significantly reduced (FIG. 4 BE), indicating that oral administration of low molecular weight sea cucumber glycosaminoglycans can reduce the accumulation of Aβ plaques in the brain of APP/PS1 mice (as shown in FIG. 5), FIG. 5 is the level of soluble and insoluble Aβ 42 and Aβ 40 in the cortex and hippocampal regions of WT mice and APP/PS1 mice in example 4 of the present invention; wherein a-D represents the level of cortex and hippocampal soluble and insoluble aβ40; E-H represents the level of cortex and hippocampal soluble and insoluble Aβ42; I-L represents the cortex and hippocampal soluble and insoluble Abeta 40/Abeta 42 ratio; n=3 mice; testing the difference of the data between groups by using One way Anova; data represent mean±sem; * p <0.05, < p <0.01, < p <0.001, ns is not statistically significant.
Synaptopsin (SYN) is a vesicle-adsorbing protein closely related to synaptic structure and function, which is widely present at all nerve terminals of the body and is specifically distributed on presynaptic vesicle membranes, and is involved in the release of calcium-dependent neurotransmitters and circulation of synaptic vesicles, and is a recognized important marker of synaptic occurrence and synaptic remodeling. The APP/PS1 model mice used in this study showed significant synaptic loss at 6-7 months. Since the CA3 region of the hippocampal region of mice is rich in synapses, synaptic loss can be reflected by detecting the expression of synaptocins at this region. In the experiment of the embodiment, the brain slices of six groups of mice are subjected to immunofluorescence staining by using a synaptorin antibody, and then, whether the synaptorin loss condition in the brain of the APP/PS1 mice can be improved by orally taking the low-molecular sea cucumber glycosaminoglycan through a fluorescence microscope to shoot and count CA3 synaptorin fluorescence density of the hippocampal parts of the mice of each group. The results show a significant decrease in synaptic fluorescence density in the CA3 region of the APP/PS1 mice compared to the WT mice, indicating synaptic loss in the CA3 region of the APP/PS1 mice. Compared with the model control group APP/PS1 mice, the synaptic fluorescence density of CA3 region of APP/PS1 mice orally administered with low molecular weight sea cucumber glycosaminoglycans (GAG-2, GAG-3, GAG-4) is significantly increased; whereas the synaptic fluorescence density of the CA3 region of APP/PS1 mice orally administered with low molecular weight sea cucumber glycosaminoglycan GAG-1 was unchanged (as shown in FIG. 6); FIG. 6 is a graph comparing SYN fluorescence intensity results of WT mice and APP/PS1 mice, wherein A represents a representative graph of SYN fluorescence intensity at CA3 site of hippocampus; b represents the statistical result of SYN fluorescence intensity detection; n=15 sections (from 3 mice); testing the difference of the data between groups by using One way Anova; data represent mean±sem, p <0.05, p <0.01, p <0.001, ns is not statistically significant.
These results suggest that oral administration of low molecular weight sea cucumber glycosaminoglycans can improve synaptic loss in APP/PS1 mice.
Example 5: enzyme-linked immunosorbent assay (ELISA) of low molecular weight sea cucumber glycosaminoglycan for improving symptoms of Alzheimer's disease model mice (APP/PS 1 mice)
The method comprises the steps of (1) pouring a heart into a heart by using physiological saline, flushing clean blood, cutting off the head by using scissors, taking the brain, putting the brain into precooled PBS, then dividing the brain into left and right half brains along a middle sagittal line by using a blade, taking the left half brain, separating the whole sea horse (about 30 mg) and cortex tissue (200-250 mg) by using forceps and scissors, weighing and recording the weight, adding TBS into 400 mu L/100mg tissue, homogenizing for 10 minutes on ice, centrifuging at 13200g at the rotating speed of 4 ℃ for 15 minutes, collecting supernatant to obtain a soluble component, adding 5M guanidine hydrochloride with the same volume as TBS into a precipitate, performing room temperature pyrolysis for 3 hours, centrifuging at the rotating speed of 13200g at the rotating speed of 4 ℃ for 15 minutes, and collecting the supernatant to obtain a non-soluble component. The amounts of aβ40 and 42 in the soluble and insoluble fractions of cortical and hippocampal tissues were then detected by ELISA kit (KHB 3441 and KHB3481, invitrogen) for aβ40 and 42, respectively, according to the ELISA instructions. The method comprises the following steps: diluting the sample with a standard diluent buffer; diluting standard products, and preparing 1 Xrabbit anti-IgG HRP and 1 Xcleaning buffer; adding 50 mu L of standard substance/control/sample into a proper hole, adding 50 mu L of HUA beta detection antibody, and incubating for 3 hours at room temperature; thoroughly aspirate the solution and wash with 1 Xwash buffer 5min X4 times; adding 100 mu L of rabbit anti-IgG HRP, and incubating for 30 minutes at room temperature; thoroughly aspirate the solution and wash with 1 Xwash buffer 5min X4 times; adding 100 mu L of stable chromogen, starting to turn blue by a substrate solution, and incubating for 30 minutes at room temperature in a dark place; 100. Mu.L of stop solution was added and the solution changed from blue to yellow; and detecting the absorbance at 450nm to generate a standard curve. The data were analyzed for differences between groups using One way ANOVA statistical methods.
Using the right half brain of the mice perfused with physiological saline hearts in the above-mentioned portions, the whole hippocampus and cortical tissue were isolated, weighed and recorded, then lysates were added in 400. Mu.L/100 mg of tissue, homogenized on ice for 10 minutes, centrifuged at 13200g at 4℃for 15 minutes, and supernatants were collected, and IL-1 beta and IL-6 levels in the cortex and hippocampus tissues were detected using ELISA kits (BMS 6002 and BMS603-2, ebioscience) for IL-1 beta and IL-6, respectively, according to the ELISA specification guidelines. The data were analyzed for differences between groups using One waydanova statistical methods.
Experimental results:
neuroinflammation plays an important role in the pathological process of senile dementia. Neuroinflammation is mainly involved by astrocytes and microglia. The density of both in the brain may reflect the extent of neuroinflammation within the brain. GFAP is glial fibrillary acidic protein, a marker of astrocyte activation. Iba1 is a 17kDa chiral protein that is expressed in microglia and increases in its expression during activation. Therefore, the GFAP antibody and the Iba-1 antibody are respectively adopted to carry out immunofluorescence staining on the brain slices of the mice, and the proportion of two glial cells in the brain slices of the mice in each group is counted, so that the change of neuroinflammation is analyzed.
Immunofluorescent staining of GFAP to detect astrocyte density of each group as shown in fig. 7, fig. 7 is a graph comparing astrocyte density detection results of WT mice and APP/PS1 mice, wherein a and B represent representative graphs of astrocyte density in each group of mouse cortex (a) and hippocampus (B), respectively; c and D represent statistical plots of astrocyte densities in the cortex (C) and hippocampus (D) of each group of mice; n=15 sections (from 3 mice); testing the difference of the data between groups by using One way Anova; data represent mean±sem, p <0.05, p <0.01, p <0.001, ns is not statistically significant.
The results show that the density of both APP/PS1 mouse cortex and hippocampal GFAP+ cells was significantly increased compared to WT mice, oral administration of low molecular weight sea cucumber glycosaminoglycans (GAG-3 and GAG-4 may) significantly reduced the density of astrocytes at the cortex site of APP/PS1 mice (FIGS. 7A and C), and oral administration of 4 LHGs significantly reduced the density of astrocytes at the hippocampal site of APP/PS1 mice (FIGS. 7B and D). It is suggested that oral administration of low molecular weight sea cucumber glycosaminoglycan can significantly improve astrocytes increased in brain of mice model for senile dementia. Immunofluorescent staining of Iba1 to examine the density of each group of microglial cells showed that the density of APP/PS1 mouse cortex and hippocampal iba1+ cells were significantly increased compared to WT mice, and oral administration of low molecular weight sea cucumber glycosaminoglycans (GAG-2, GAG-3 and GAG-4) significantly reduced the density of APP/PS1 mouse cortex and hippocampal microglial cells (as shown in fig. 8), fig. 8 is a graph comparing the results of microglial cell density examination of WT mice and APP/PS1 mice, wherein a and B represent representative graphs of the density of microglial cells in each group of mouse cortex (a) and hippocampus (B), respectively; c and D represent statistical plots of microglial cell density in the cortex (C) and hippocampus (D) of each group of mice; n=15 sections (from 3 mice); testing the difference of the data between groups by using One way Anova; data represent mean±sem; * P <0.01, p <0.001, ns has no statistical significance.
It is suggested that oral administration of low molecular weight sea cucumber glycosaminoglycan can significantly improve microglial cells increased in brain of mice with senile dementia model. These results indicate that oral administration of low molecular weight sea cucumber glycosaminoglycans ameliorates astrocyte-and microglial-mediated neuroinflammation in the brain of APP/PS1 mice.
The Elisa method detects the levels of IL-1 beta and IL-6, which are related factors of the inflammation in the brains of each group of mice. As shown in FIG. 9, FIG. 9 is a graph comparing the results of detection of the levels of inflammatory factors IL-1β and IL-6 in WT mice and APP/PS1, wherein A and B represent statistical graphs of IL-1β levels in the cortex (A) and hippocampus (B) of each group of mice, respectively; c and D represent statistical graphs of IL-6 levels in the cortex (A) and hippocampus (B) of each group of mice; n=3 mice; differences in data between groups were checked with One way Anova. Data represent mean±sem, p <0.05.
The results showed that APP/PS1 mice had an elevated cortical IL-1β level and a tendency to elevate IL-1β at the hippocampal site compared to WT mice, whereas oral administration of low molecular weight sea cucumber glycosaminoglycans had a tendency to lower cortical and hippocampal IL-1β (fig. 9A and B). There was no difference in IL-6 levels in each group (FIGS. 9C and D).
These results suggest that oral administration of low molecular weight sea cucumber glycosaminoglycans ameliorates astrocyte-and microglial-mediated neuroinflammation in the brain of APP/PS1 mice in terms of neuroinflammation.
In another set of examples (examples 6-8), but not limited to this set of examples, the beneficial pharmacodynamic effects of low molecular weight sea cucumber glycosaminoglycans on Alzheimer's disease-associated Abeta secretion, tau protein phosphorylation, inflammatory factors, and anti-inflammatory factors in vitro cell experiments are demonstrated. Three groups of high, medium and low concentration low molecular sea cucumber glycosaminoglycan tested drugs, a negative blank control group and a positive drug control group are respectively arranged in the experiment, and the influence on the Abeta secretion of H4 human glioma cells, the influence on Tau protein phosphorylation in SH-SY5Y human neuroblastoma cells and the influence on the LPS-induced neuroinflammation of BV-2 cells are respectively treated and examined. The results show that: the low-molecular sea cucumber glycosaminoglycan can obviously inhibit the concentration of beta amyloid 38 and 40 of H4 human glioma cells, has a certain inhibition effect on Tau protein phosphorylation in SH-SY5Y human neuroblastoma cells, obviously inhibits the production of microglial cell BV-2 inflammatory factor tumor necrosis factor-alpha (TNF-alpha), keratinocyte chemotactic factor/growth regulating gene protein (KC/GRO) and interleukin-6 (IL-6), and improves the concentration of anti-inflammatory factors interleukin-2 (IL-2), interleukin-5 (IL-5) and interleukin-4 (IL-4).
Example 6: influence of low molecular weight sea cucumber glycosaminoglycan on secretion of Abeta by H4 human glioma cells
H4 human glioma cells were thawed in Opti-MEM medium containing 10% FCS, 1% penicillin/streptomycin, 200. Mu.g/mL hygromycin B and 2.5. Mu.g/mL blasticidin S, counted, cells seeded in 96-well plates, number 2X 10 4 Individual cells/wells.
The following day, cells in 96-well plates were treated with different concentrations of low molecular weight sea cucumber glycosaminoglycans (500. Mu.M, 25. Mu.M, and 5. Mu.M), positive controls (DAPT 400 nM) and saline blank, all groups were repeated 6 times, and incubation was continued for 24 hours. Collecting cell culture supernatant, diluting 10 times, and using
96 well->
6E10 plates were read on a Sector Imager 2400 and assayed for the content (secretion level) of Abeta 1-38, abeta 1-40 and Abeta 1-42 in pg/mL. />
Experimental results:
aβ1-40 is the most important Aβ, and FIG. 10 shows the levels of Aβ1-38, Aβ1-40 and Aβ1-42 in H4 human glioma cell culture supernatants 24 hours after treatment with low molecular weight sea cucumber glycosaminoglycans at different concentrations, as compared to normal saline control group, wherein (A) is Aβ1-38 evaluated in terms of MSD V-plex given in pg/mL; (B) Aβ1-40 and (C) Aβ1-4; VC: solvent control; t.i.: low molecular weight sea cucumber glycosaminoglycan; testing the difference of the data between groups by using One way Anova; * p <0.05, < p <0.01, < p <0.001. While the levels of various Abeta in DAPT (gamma secretase inhibitor) were significantly reduced in the positive control group (FIG. 10A-C), the levels of Abeta 1-38 were significantly reduced in the 500. Mu.M group and 1. Mu.M group (FIG. 10A), the effects of Abeta 1-40 and Abeta 1-38 in the 25. Mu.M group were not significant (FIG. 10B), and furthermore, no difference in the expression levels of Abeta 1-42 was observed in the test group (FIG. 10C).
Example 7: effect of low molecular sea cucumber glycosaminoglycans on phosphorylation of Tau protein in SH-SY5Y human neuroblastoma cells
The mutant SH-SY5Y-hTau441 (V337M/R406W) is an important research human brain Tau protein transport cell model and is used for investigating the influence of low-molecular sea cucumber glycosaminoglycan on Tau protein phosphorylation.
The experiment of this example is as follows: the above human neuroblastoma cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% NEAA, 1% L-glutamine, 100. Mu.g/mL gentamicin, and 300. Mu.g/mL Geneticin g-418 for about 2 days to 80% -90% cell fusion. Differentiation culture with medium containing 10. Mu.M Retinoic Acid (RA) was continued for 5 days, with medium replacement every 2-3 days. Counting, inoculating cells onto 48-well plate, number 1×10 5 Individual cells/wells.
The test sets up different concentrations of low molecular weight sea cucumber glycosaminoglycans (500. Mu.M, 25. Mu.M and 5. Mu.M), positive control (LiCl 20 mM) and saline control blank treatments, all groups were repeated 6 times and incubation was continued for 24 hours. Cells on 48 well plates were collected, washed with pre-chilled PBS, and harvested with 60. Mu.L of IPA buffer (50 mM Tris, 150mM NaCl, 1% Nonidet P40, 0.25% sodium deoxycholate, 1. Mu.M NaF, 0.2mM sodium orthovanadate, 80. Mu.M glycerophosphate and protease (Calbiochem) and phosphatase (Sigma) inhibitors, pH 7.4). Protein concentration was measured using the BCA protein assay kit of Thermo Scientific, adjusted to the same concentration, and the levels of total Tau protein and phosphorylated Tau protein (pTau 231) in human neuroblastoma cell RIPA extract were determined by immunoadsorption using the ELISA kit instructions, total Tau protein expressed in pg/mL, and pTau231 in arbitrary units.
Experimental results:
overall, the total Tau protein and phosphorylated Tau protein pTau231 are at normal levels. As shown in FIG. 11, FIG. 11 shows the levels of total Tau protein and pTau231 after 24h treatment of SH-SY5Y human neuroblastoma cells with medium-low molecular weight sea cucumber glycosaminoglycans, wherein (A, B) total Tau protein was evaluated with MSD; (C, D) pTau231; the total Tau protein unit is pg/μg; pTau231 is an Arbitrary Unit (AU); (E, F) ratio of pTau231 to total Tau protein; VC: saline control; t.i.: low molecular weight sea cucumber glycosaminoglycan; statistical analysis: (a, C, E) unpaired t-test (B, D, F); data represent mean±sem (n=6 per group) and differences in data between groups were examined with One way Anova, <0.05. Positive control group human neuroblastoma cells treated with LiCl showed no effect on total Tau protein level (fig. 11A), and pTau231 (phosphorylation of Tau protein at residue Thr 231) showed a reduced trend (p=0.08) compared to normal saline control group (fig. 11C), corrected to total Tau protein level, reaching significant level (fig. 11D). There were no significant differences in total Tau protein (fig. 12B) and pTau231 (fig. 11D) levels in the three low molecular weight sea cucumber glycosaminoglycan drug groups, no significant increases in pTau231 for the 1 μm group, and no differences in concentrations for the other two groups, compared to the saline placebo group (fig. 11F).
Example 8: influence of low molecular weight sea cucumber glycosaminoglycan on BV-2 cells induced by LPS
In this example, experiments were performed using LPS to stimulate microglial cells BV-2 to induce neuroinflammation, and the experiments were performed with different concentrations of low molecular weight sea cucumber glycosaminoglycans (500. Mu.M, 25. Mu.M, and 5. Mu.M), positive controls (dexamethasone), and saline blank, all groups were repeated 6 times, and cell culture supernatants were collected and evaluated for cytokine levels using electrochemiluminescence (MSD) assays.
The experiment is as follows:
mouse microglial cells BV-2 were cultured in Opti-MEM medium containing 10% FCS, 1% penicillin/streptomycin and 2mM L-glutamine, counted, and cells were seeded into 96-well plates in 5000 cells/well. After 48 hours, the medium was changed to a treatment medium (DMEM medium containing 5% FCS, 2mM L-glutamine) and the culture was continued.
Different concentrations of low molecular weight sea cucumber glycosaminoglycans (500. Mu.M, 25. Mu.M and 5. Mu.M), positive controls (LPS+positive drug (10. Mu.M of dexamethasone)) and saline placebo and control groups treated with LPS alone were set up for 1 hour prior to LPS stimulation and treated in the same manner, respectively. BV-2 cell supernatants were collected 24 hours after LPS induction, diluted 1-fold, and the levels of 10 cytokines (IFN-. Gamma., IL-1β, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, and TNF-. Alpha.) were assayed by ELISA kit protocol, and the results were expressed in pg/mL.
Experimental results:
as shown in FIG. 12, FIG. 12 shows the levels of TNF- α, KC/GRO, IL-6 and IFN- γ in the supernatant of BV-2 cells stimulated by LPS for 24 hours after low molecular weight holothurian glycosaminoglycan treatment; wherein BV-2 cells were treated with 3 concentrations of low molecular weight sea cucumber glycosaminoglycans for 24 hours and the level of cytokine release in the supernatant was assessed by MSD; data units are pg/mL and are shown as bar graphs, mean±sem (n=6 per group); VC: saline control, t.i.: low molecular weight sea cucumber glycosaminoglycans, r.i.: a positive control; one-way analysis of variance was performed with the LPS stimulated control group, with One way Anova to examine the differences in data between groups, p <0.01, p <0.001.
FIG. 13 shows the levels of IL-5, IL-2, IL-1β and IL-12p70 in BV-2 cell supernatants stimulated by LPS for 24 hours after low molecular weight sea cucumber glycosaminoglycan treatment; wherein BV-2 cells were treated with 3 concentrations of low molecular weight sea cucumber glycosaminoglycans for 24 hours and the level of cytokine release in the supernatant was assessed by MSD; data units are pg/mL, shown as bar graphs, mean±sem (n=6 per group); differences in data between groups were examined with One way Anova, p <0.001.
FIG. 14 shows the levels of IL-4 and IL-10 in BV-2 cell supernatants stimulated by LPS for 24 hours after low molecular weight sea cucumber glycosaminoglycan treatment; wherein BV-2 cells were treated with 3 concentrations of low molecular weight sea cucumber glycosaminoglycans for 24 hours and the level of cytokine release in the supernatant was assessed by MSD; data units are pg/mL, shown as bar graphs, mean±sem (n=6 per group); differences in data between groups were examined with One way Anova p <0.001.
A number of cytokine (TNF- α, KC/GRO, IL-6, IL-4 and IL-10) levels are within the detection response range, while IFN- γ, IL-5, IL-2, IL-1β and IL-12p70 values are near or below the limit of quantitation (LOQ).
Upon 24-hour stimulation induction of LPS, the levels of cytokines TNF- α, KC/GRO and IL-6 were significantly increased (FIG. 12), and conversely, the levels of IL-4 were significantly decreased (FIG. 14A). No significant effect was observed with LPS treatment on other cytokines as the detection values were near or below the limit of quantification (LOQ). In the positive control group, the levels of TNF- α, KC/GO and IL-6 were reduced due to the use of the positive drug dexamethasone (FIG. 12). Whereas the three groups of low molecular weight sea cucumber glycosaminoglycans at different concentrations had significantly reduced levels of TNF- α factor and were dose dependent (fig. 12A) compared to the LPS-treated control group alone, whereas the levels of KC/GRO factor (fig. 12B) and IL-6 (fig. 14C) were significantly reduced in the 500 μm and 25 μm groups, while there was no effect in the low concentration 1 μm group. Furthermore, the detection value of cytokine IFN-. Gamma.was below the limit of quantification (LOQ), and thus, no difference was observed (FIG. 12D).
The detection values of the IL-5 levels of the most groups were close to or below the limit of quantification (LOQ), and the low molecular weight sea cucumber glycosaminoglycans of the high concentration group (500. Mu.M) showed a significant increase in IL-5 levels compared to the LPS control, but no difference was observed in the medium and low concentration groups (FIG. 13A). The levels of detection of IL-2 (FIG. 13B), IL-1β (FIG. 13C) and IL-12p70 (FIG. 13D) were also near or below the limit of quantitation (LOQ), and no significant difference in these factors was observed for the three concentration groups of low molecular weight sea cucumber glycosaminoglycan treatments compared to the LPS control. In addition, both anti-inflammatory cytokines IL-4 and IL-10 were within the detection range, and high and medium concentration groups (500. Mu.M and 25. Mu.M) of low molecular weight sea cucumber glycosaminoglycan-treated BV-2 cells showed a significant increase in IL-4 levels (FIG. 14A), but no significant increase in IL-10 levels was observed (FIG. 14B) compared to the LPS control.
In the comprehensive examples 1-5, in vivo experiments, oral administration of low molecular weight sea cucumber glycosaminoglycan significantly improves cognitive dysfunction of senile dementia model mice (APP/PS 1 mice), and reduces Abeta accumulation, nerve synapse loss and nerve inflammation in the brains of the model mice. In addition, in the in vitro cell culture of the comprehensive examples 6-8, the low-molecular sea cucumber glycosaminoglycan remarkably inhibits the concentration of beta amyloid 38 and beta amyloid 40 of H4 human glioma cells, has a certain inhibition effect on Tau protein phosphorylation in SH-SY5Y human neuroblastoma cells, remarkably inhibits the production of microglial cell BV-2 inflammatory factors TNF-alpha, KC/GRO and IL-6, and promotes the concentration of anti-inflammatory factors IL-2, IL-5 and IL-4. Therefore, the low molecular weight sea cucumber glycosaminoglycan is expected to be used as a medicine or health food for preventing and treating the related senile dementia diseases.
The invention has the advantages that: provides the application of the low molecular weight sea cucumber glycosaminoglycan in medicaments for preventing and treating senile dementia and health care and functional foods: in an animal in-vivo experiment, the oral administration of the low-molecular sea cucumber glycosaminoglycan significantly improves the cognitive disorder of an Alzheimer's disease model mouse (APP/PS 1 mouse) and reduces Abeta accumulation, nerve synapse loss and nerve inflammation in the brain of the model mouse; in an in vitro cell experiment, the low-molecular sea cucumber glycosaminoglycan remarkably inhibits the concentration of beta amyloid 38 and beta amyloid 40 of H4 human glioma cells, has a certain inhibition effect on Tau protein phosphorylation in SH-SY5Y human neuroblastoma cells, remarkably inhibits the production of microglial cell BV-2 inflammatory factors TNF-alpha, KC/GRO and IL-6, and promotes the concentration of anti-inflammatory factors IL-2, IL-5 and IL-4; the effects show that the low molecular weight sea cucumber glycosaminoglycan is expected to be applied to medicaments for preventing and treating senile dementia and health care and functional foods.
Finally, it should be noted that the above description is only a preferred embodiment of the present invention, and it should be noted that the scope of the present invention is not limited to the description of the above embodiments, and that it should be apparent to those skilled in the art that several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the scope of the invention.
Claims (15)
1. The application of low molecular weight sea cucumber glycosaminoglycan in medicine for preventing and treating senile dementia and health care and functional food.
2. The use of the low molecular weight sea cucumber glycosaminoglycan according to claim 1 in medicines for preventing and treating senile dementia and health and functional foods, characterized in that: the low molecular weight sea cucumber glycosaminoglycan is used as an inhibitor of beta-amyloid (Abeta).
3. The use of the low molecular weight sea cucumber glycosaminoglycan according to claim 2 in medicaments for preventing and treating senile dementia and health care and functional foods, characterized in that: beta-amyloid (aβ) includes: one or more of aβ38, aβ40 and aβ -42.
4. The use of the low molecular weight sea cucumber glycosaminoglycan according to claim 1 in medicines for preventing and treating senile dementia and health and functional foods, characterized in that: the low molecular weight sea cucumber glycosaminoglycan is used as an inhibitor of inflammatory factors.
5. The use of the low molecular weight sea cucumber glycosaminoglycan according to claim 4 in medicines for preventing and treating senile dementia and health and functional foods, characterized in that: inflammatory factors include: one or more of tumor necrosis factor-alpha (TNF-alpha), keratinocyte chemokine/growth regulator gene protein (KC/GRO), and interleukin-6 (IL-6).
6. The use of the low molecular weight sea cucumber glycosaminoglycan according to claim 1 in medicines for preventing and treating senile dementia and health and functional foods, characterized in that: the low molecular weight sea cucumber glycosaminoglycan is used as an activator of anti-inflammatory factors.
7. The use of the low molecular weight sea cucumber glycosaminoglycan according to claim 6 in medicines for preventing and treating senile dementia and health and functional foods, characterized in that: anti-inflammatory factors include: one or more of interleukin-2 (IL-2), interleukin-5 (IL-5), and interleukin-4 (IL-4).
8. The use of the low molecular weight sea cucumber glycosaminoglycan according to claim 1 in medicines for preventing and treating senile dementia and health and functional foods, characterized in that: the low molecular weight sea cucumber glycosaminoglycan is used as Tau protein phosphorylation inhibitor.
9. The use of the low molecular weight sea cucumber glycosaminoglycan according to claim 1 in medicines for preventing and treating senile dementia and health and functional foods, characterized in that: the low molecular weight sea cucumber glycosaminoglycan is used as a novel object recognition capability promoter.
10. The use of the low molecular weight sea cucumber glycosaminoglycan according to claim 1 in medicines for preventing and treating senile dementia and health and functional foods, characterized in that: the low molecular weight sea cucumber glycosaminoglycan is used as a cognitive disorder improving agent.
11. The use of the low molecular weight sea cucumber glycosaminoglycan according to claim 1 in medicines for preventing and treating senile dementia and health and functional foods, characterized in that: the low molecular weight sea cucumber glycosaminoglycan is used as a neurite loss inhibitor.
12. The use of the low molecular weight sea cucumber glycosaminoglycan according to claim 1 in medicines for preventing and treating senile dementia and health and functional foods, characterized in that: the low molecular weight sea cucumber glycosaminoglycan is used as a neuroinflammation inhibitor.
13. The low molecular weight sea cucumber glycosaminoglycan pharmaceutical composition is characterized in that: the low molecular weight sea cucumber glycosaminoglycan is used as an active pharmaceutical ingredient, and pharmaceutically acceptable auxiliary materials.
14. The low molecular weight sea cucumber glycosaminoglycan health food composition is characterized in that: the low molecular weight sea cucumber glycosaminoglycan is taken as a component and auxiliary materials acceptable in food.
15. The low molecular weight sea cucumber glycosaminoglycan functional food composition is characterized in that: the low molecular weight sea cucumber glycosaminoglycan is taken as a component and auxiliary materials acceptable in food.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102218076A (en) * | 2011-04-14 | 2011-10-19 | 易杨华 | Apostichopus japonicus polysaccharides, and application thereof in preparation of medicament or food for preventing and treating senile dementia |
| CN110776578A (en) * | 2019-11-12 | 2020-02-11 | 苏州颐华生物医药技术股份有限公司 | Low-molecular sea cucumber glycosaminoglycan and application thereof |
| CN115813946A (en) * | 2021-09-16 | 2023-03-21 | 苏州颐华生物医药技术股份有限公司 | Application of low-molecular sea cucumber glycosaminoglycan in preparation of drugs for preventing and treating coronavirus related diseases |
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|---|---|---|---|---|
| CN102218076A (en) * | 2011-04-14 | 2011-10-19 | 易杨华 | Apostichopus japonicus polysaccharides, and application thereof in preparation of medicament or food for preventing and treating senile dementia |
| CN110776578A (en) * | 2019-11-12 | 2020-02-11 | 苏州颐华生物医药技术股份有限公司 | Low-molecular sea cucumber glycosaminoglycan and application thereof |
| CN115813946A (en) * | 2021-09-16 | 2023-03-21 | 苏州颐华生物医药技术股份有限公司 | Application of low-molecular sea cucumber glycosaminoglycan in preparation of drugs for preventing and treating coronavirus related diseases |
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