阅读说明：本技术 高丽槐素在制备用于抗神经炎症药物中的应用 (Application of Korean sophoricoside in preparing medicine for treating neuritis ) 是由 李雪彤 郑希 张蓝月 李春莲 蔡秋仰 谭泽楷 张伟诚 吴显仪 赵凯 黄泽彬 于 2021-08-31 设计创作，主要内容包括：本发明属于医药技术领域,涉及高丽槐素的应用。本发明提供了高丽槐素的应用,实验结果表明,高丽槐素能够安全有效控制神经炎症反应,从而改善脑组织的认知功能、记忆障碍和运动协调能力,可用于预防和治疗阿尔茨海默症、帕金森症等。高丽槐素应用于制备预防和治疗神经炎症药物或保健品,具有很好的应用价值和开发前景。(The invention belongs to the technical field of medicines, and relates to application of Korean sophoricoside. The invention provides application of the Korean sophoricoside, and experimental results show that the Korean sophoricoside can safely and effectively control neuroinflammatory reaction, so that the cognitive function, dysmnesia and motor coordination capability of brain tissues are improved, and the Korean sophoricoside can be used for preventing and treating Alzheimer's disease, Parkinson's disease and the like. The Korean sophoricoside is applied to preparing the medicines or health care products for preventing and treating neuroinflammation, and has good application value and development prospect.)

1. Application of Korean sophoricoside in preparing medicine for inhibiting neuroinflammation is provided.

2. The use of claim 1, wherein the medicament is used for preparing medicaments and health products for preventing and treating neurodegenerative diseases.

3. Use according to claim 2, wherein the neurodegenerative disease is selected from alzheimer's disease, parkinson's disease, depression, cerebral stroke, postoperative complications of the nervous system, amyotrophic lateral sclerosis or multiple sclerosis.

4. The use according to any one of claims 1 to 3, wherein the medicament further comprises a pharmaceutically acceptable excipient.

5. The use according to any one of claims 1 to 3, wherein the medicament is in the form of an oral preparation or an injection.

6. A product for inhibiting neuroinflammation, comprising Korean sophoricine.

Technical Field

The invention belongs to the technical field of medicines, and relates to application of Korean sophoricoside.

Background

Inflammatory responses are a complex cascade of processes of defense of the body against infection and injury, the pathological changes of which are manifested by overproduction of inflammatory factors, extensive infiltration of inflammatory cells and necrotic breakdown of tissues, whereas neuroinflammation is a specific immune response occurring in the nervous system. Under normal conditions, microglia and astrocytes are inactive and have the effect of maintaining normal tissue homeostasis in the central nervous system. Upon infection or injury to the brain, these cells are activated, mount an immune response and tissue repair processes, and return to a quiescent state once the infection or injury is recovered. Generally, inflammation disappears rapidly, but when inflammation progresses chronically, neurodegenerative diseases (NDDs) such as Alzheimer's Disease (AD), Parkinson's Disease (PD), Multiple Sclerosis (MS), and Amyotrophic Lateral Sclerosis (ALS) may be caused.

At present, a plurality of chemical compounds for treating neuroinflammation exist, but the chemical compounds can only improve symptoms, can rarely effectively reverse the state of illness, and even have side effects, so that the search for new medicaments and methods for treating neuroinflammation is particularly important.

Clinical and experimental results prove that the Chinese herbal medicine has good anti-inflammatory effect, the history of treating inflammation by using the Chinese herbal medicine is long in China, and the Chinese herbal medicine resources are rich, so that the advantages of exact curative effect, safety and no toxicity in screening and developing natural products are achieved. The radix millettiae speciosae has been reported for treating rheumatic arthritis, chronic bronchitis and chronic hepatitis, so that the radix millettiae speciosae can be used for developing a medicament for preventing and treating neuroinflammation such as Alzheimer's disease by utilizing the characteristic of the anti-inflammatory effect of the radix millettiae speciosae. The main component, namely the Maackianin (MAA), separated from the beautiful millettia root belongs to the isoflavanone compounds, and researches show that the Maackianin has various pharmacological effects of oxidation resistance, allergy resistance, inflammation resistance, bacteria resistance, tumor resistance and the like. The invention mainly researches the effect and the action mechanism of the Korean sophoricoside on resisting neuritis.

Disclosure of Invention

The invention aims to provide application of Korean sophoricoside in inhibiting neuroinflammation. After the mice are subjected to modeling and drug administration treatment, a water maze experiment is carried out, and the memory and the movement coordination ability of the mice are detected. Then, the brain tissues of the mice of each group are subjected to transcriptome sequencing, and the way of the function of the Korean sophoricine in the aspect of resisting neuritis is researched. The result shows that the Korean sophoricoside can control neuroinflammation, improve dysmnesia and motor coordination ability, and does not produce obvious side effect.

The specific technical scheme of the invention is as follows:

application of Korean sophoricoside in preparing medicine for inhibiting neuroinflammation is provided.

The invention carries out water maze experiments on mice of all groups after the experiments, records the action tracks of the mice in the water maze positioning navigation and space exploration experiments, and aims to detect the change of the memory of the mice before and after the treatment by the Korean sophoricine. The invention randomly divides experimental animals into 5 groups, which are respectively as follows: normal Control group (Control), LPS group (LPS, 250. mu.g/kg/d), positive drug group (LPS + positive drug, TTP488, 5mg/kg/day), low dose kukosenicin group (LPS + low dose kukoicin, MAA-L, 25mg/kg/day) and high dose kukoicin group (LPS + high dose kukoicin, MAA-H, 50mg/kg/day), 7 per group. Each experimental group was administered first by concentration and then LPS injection in week 1. On week 2, each experimental group was dosed daily by concentration. After 2 weeks, the memory function of the mice was examined using the water maze test, the experimental data are expressed as Mean ± SD, all the experimental data were statistically analyzed using GraphPad Prism 8 software, the inter-group parameters of each group of mice were examined using ANOVA, and P < 0.05 was considered statistically significant.

Extracting total RNA before and after the neuroinflammation of mice induced by LPS (lipopolysaccharide) treated by the Korean sophoricide according to a method of an instruction, and sending a total RNA sample (containing biological repetition) qualified by quality inspection to Huada gene for transcriptome sequencing (RNA-seq) analysis. The sequencing original data are preprocessed to obtain effective transcriptome data of each sample before and after the neuroinflammation of the mice induced by the Korean sophoricone treatment LPS, a Q value is adopted in data analysis, namely a corrected P value, and the Q value is less than 0.05, so that the obvious difference is considered.

The invention also provides the application of the Korean sophoricoside in preparing medicines and health-care products for preventing and treating degenerative nerve diseases.

In the present invention, the neurodegenerative disease is a disease associated with neuroinflammation, and when the Korean sophoricoside is used for preventing and treating the neurodegenerative disease, the administration dose is preferably 25mg/kg/d to 50 mg/kg/d.

Preferably, the neurodegenerative disease is selected from alzheimer's disease, parkinson's disease, depression, cerebral stroke, postoperative complications of the nervous system, amyotrophic lateral sclerosis or multiple sclerosis.

Preferably, the medicament also comprises pharmaceutically acceptable auxiliary materials.

Preferably, the dosage form of the medicament is oral preparation or injection.

In the invention, the oral preparation is selected from capsules, microcapsules, pills, tablets, decoction, granules, paste, dispersion powder, distillate oral preparation, dripping pills or liposome;

the injection is powder injection or injection.

The invention also provides a product for inhibiting neuroinflammation, which comprises the Korean sophoricoside which is an effective component of the product for inhibiting neuroinflammation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a diagram showing the results of the positioning navigation experiment in the water maze of the mouse in example 1;

FIG. 2 is a graph of the experimental results of the water maze space exploration of the mouse in example 1;

FIG. 3 is a graph showing the expression of COX-2 protein in the brain tissue of a mouse in example 2;

FIG. 4 is a graph showing the sequencing results of mouse brain tissue transcriptome in example 3.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the examples, the starting materials and reagents used are all commercially available. The experimental animal adopts SPF-grade 8-week-old male C57 mice with the weight of 20-22 g, which are purchased from the center of the experimental animal of Zhongshan university and have the license number of SCXK (Guangdong) 2016-. Mouse feed was purchased from the laboratory animal center of traditional Chinese medicine university, Guangzhou. The experimental animals are raised in a clean-grade laminar flow frame, the temperature and air condition of the raising environment are controlled to be 23 +/-2 ℃, the relative humidity is controlled to be 75 +/-10%, and the illumination time is 12h/d (7: 00-19: 00).

Example 1 Water maze test to test the ameliorating effect of Korean Sophora extract on LPS-induced neuroinflammatory memory disorders in mice

The experimental animals were randomly divided into 5 groups in this example, which were: normal Control group (Control), LPS group (LPS, 250. mu.g/kg/d), positive drug group (LPS + positive drug, TTP488, 5mg/kg/day), low dose kukosenicin group (LPS + low dose kukoicin, MAA-L, 25mg/kg/day) and high dose kukoicin group (LPS + high dose kukoicin, MAA-H, 50mg/kg/day), 7 per group. Each experimental group was administered first by concentration and then LPS injection in week 1. On week 2, each experimental group was dosed daily by concentration. After 2 weeks, the memory function of the mice was examined using the water maze test, the experimental data are expressed as Mean ± SD, all the experimental data were statistically analyzed using GraphPad Prism 8 software, the inter-group parameters of each group of mice were examined using ANOVA, and P < 0.05 was considered statistically significant. Referring to fig. 1, the results of the water maze positioning navigation experiment are shown, and referring to fig. 2, the results of the water maze space exploration experiment are shown.

Long-term intraperitoneal injection of LPS can cause inflammation of brain tissues and damage to brain nerves, thereby reducing learning and memory abilities. Therefore, this example examines whether the effect of the Korean sophoricide on the memory and cognitive impairment induced by LPS in mice was improved by the water maze experiment. In the water maze positioning navigation experiment, each mouse must search for a hidden platform from one, two, three and four quadrants. FIG. 1 is a water maze positioning navigation experiment result diagram of a mouse of example 1, wherein (A) a movement track diagram (hidden platform) of a positioning navigation experiment mouse, and (B) the escape latency (hidden platform) of the positioning navigation experiment mouse in each quadrant. Fig. 2 is a graph of the results of the water maze spatial exploration experiment performed by the mouse of example 1, (a) a graph of the movement trajectory of the spatial exploration experimental mouse (platform removal), (B) the number of times the spatial exploration experimental mouse spans the original platform region (platform removal), and (C) the percentage of time the spatial exploration experimental mouse spends in the target quadrant of the original platform (platform removal). In the example, the latency time is recorded, and it can be seen that in 4 quadrants of the localization navigation experiment, compared with the Control group, after LPS is injected to the abdominal cavity of the mouse in the LPS group, the latency period of the mouse in the water maze localization navigation experiment is obviously prolonged, and the result of the motion trajectory of the mouse is consistent with the result of the latency period (fig. 1A and 1B). Meanwhile, the frequency and time proportion of entering the target quadrant in the space exploration experiment of the mouse are reduced (fig. 2B and 2C), which indicates that the learning and memory functions of the mouse are damaged due to continuous intraperitoneal injection of LPS. While the memory function of the mice in the experimental group given low and high doses of the high dose of the low dose of the high dose of the mice was observed. In the space exploration experiment, the frequency and time ratio of the mice in the low-dose and high-dose kubinin groups entering the target platform are increased (fig. 2B and 2C), and the result of the movement track of the mice is shown in fig. 2A, which indicates that the kubinin can relieve the influence of the neuroinflammation induced by LPS on the learning and memory functions of the mice.

Example 2 Effect of Korean Sophora extract on LPS-induced inflammatory response in brain tissue of neuroinflammatory mice

In this example, experimental grouping and administration protocol were the same as in example 1, mice were sacrificed after administration, intact brain tissue was taken out and paraffin sections were prepared, immunohistochemistry was performed on the sections, see fig. 3 for results, and then immunohistochemical staining was performed to determine the expression level of inflammatory factor COX-2 in the cortical region, the experimental data were expressed as Mean ± SD, all experimental data were statistically analyzed using GraphPad Prism 8 software, the inter-group parameters of each group of mice were examined by ANOVA, and P < 0.05 was considered to be statistically different.

The main steps for making paraffin sections include washing tissue and fixing in 4% paraformaldehyde for 1 week → washing away excess fixative with tap water → gradient dehydration with different concentration gradients (70%, 80%, 90%, 95% and 100%) of alcohol → soaking in gradient xylene (50% and 100%) for transparency after dehydration → soaking the transparent sample in paraffin in molten state for 4h → embedding (pouring embedded paraffin to solidification) → section (section in 5 μm thickness) → spread sheet → roast sheet → dewaxing and rehydration.

Immunohistochemical staining was performed on the tissue sections to detect the expression level of the inflammatory factor COX-2. Baking the cut paraffin sections (baking the cut paraffin sections in an oven at the temperature of 62-65 ℃ for 1h, dewaxing and hydrating in a xylene ethanol solution) → antigen retrieval (placing the tissue sections in a retrieval box containing EDTA antigen retrieval buffer solution with pH8.0, performing antigen retrieval in a microwave oven) → blocking endogenous peroxidase (using a 3% hydrogen peroxide solution, incubating away from light at room temperature for 25min, and then washing with PBS) → serum blocking (3% BSA incubating for 30min) → primary antibody incubation for overnight (dropwise adding a primary anti-TNF-alpha, 1: 100, 1 XPBS dilution) → primary anti-washing → secondary antibody incubation for 50min at room temperature (dropwise adding a secondary antibody corresponding to the primary antibody, 1: 100, 1 XPBS dilution) → secondary antibody washing → DAB color (controlling the color development time under a microscope, the positive is brownish yellow, washing the color development of tap water stops), air-dried, neutral gum block) → xylene dehydrated transparent → microscopic photography (the section was observed under a microscope and the image was collected), and as a result, see fig. 3.

Under normal conditions, COX-2 has very low activity in normal tissue cells, and when cells are stimulated by inflammation and the like, the expression level of COX-2 in inflammatory cells can be increased to 10-80 times of the normal level, resulting in inflammatory response and tissue damage. FIG. 3 is a graph showing the expression of COX-2 in mouse brain tissue in example 2, wherein (A) is an immunohistochemistry graph, the first line is a mouse brain tissue cross-section graph, the second line is a brain cortical region, and (B) is a tissue COX-2 expression quantitative analysis of a brain cortical region Image using Image-Pro plus 8 software, FIG. 3 shows that characterization of the expression level of COX-2 in the cortex (FIG. 3A) and Image analysis quantification of the expression level of COX-2 in the cortex (FIG. 3B) in combination with immunohistochemical sections, and that after LPS injection, overexpression of the inflammatory factor COX-2 is caused, and after administration of Korean, the concentration of the inflammatory factor COX-2 is significantly reduced.

The results show that the Korean sophoricoside can effectively inhibit the secretion of inflammatory factor COX-2 and control neuroinflammation reaction, thereby relieving the subsequent neuron damage and improving the dysmnesia and the motor coordination ability.

Example 3 transcriptome sequencing (RNA-seq) analysis of the differences in Gene expression before and after treatment of Korean Sophora elements with LPS-induced neuroinflammation in mice

Extracting total RNA before and after the neuroinflammation of mice induced by LPS (lipopolysaccharide) treated by the Korean sophoricide according to a method of an instruction, and sending a total RNA sample (containing biological repetition) qualified by quality inspection to Huada gene for transcriptome sequencing (RNA-seq) analysis. And preprocessing the sequencing original data to obtain effective transcriptome data of each sample before and after the Korean sophoricoside treats the mouse neuroinflammation induced by the LPS.

FIG. 4 is a graph showing the sequencing results of mouse brain tissue transcriptome in example 3, wherein (A) is a graph showing the gene expression levels Venn among Control group, LPS group and MAA group, and RNA-seq data analysis showed that the number of genes co-expressed in Control group, LPS group and MAA group was 21365, 23698 genes were detected in Control group, 24056 genes were detected in LPS group and 22933 genes were detected in MAA group. In order to further research which genes are influenced by MAA intervention, the differentially expressed genes between the LPS group and the Control group and between the LPS group and the MAA group are intersected and analyzed, and 161 differentially expressed genes which are commonly expressed by the LPS group and the Control group and between the LPS group and the MAA group are found from (B) Venn diagrams of the differentially expressed genes of different comparison groups of the LPS group and the Control group and between the LPS group and the MAA group, so that the MAA mainly influences the expression of the genes, and further the neuroinflammation is improved. (C) The different gene expression levels of the LPS group and the Control group and the LPS group and the MAA group in different comparison groups can be seen from the quantity of the different genes between the LPS group and the Control group and between the LPS group and the MAA group, and the MAA group has 723 gene expression up-regulation and 468 gene expression down-regulation compared with the LPS group. The KEGG pathway enrichment analysis can provide further information on gene function and interaction thereof, and thus KEGG enrichment analysis is performed on up-and down-regulated genes of the LPS and Control groups, LPS and MAA groups. From (D) KEGG enriched bubble map of LPS vs Control upregulated genes, it can be seen that the upregulated genes are mainly enriched in endoplasmic reticulum Protein processing (Protein processing in endoplasmic reticulum), peroxisomes (peroxisomes), Pyrimidine metabolism (pyrimida metabolism), Endocytosis (Endocytosis), Drug metabolism-other enzymes (Drug metabolism-other enzymes) in the Control group samples compared to the LPS group samples. From (E) the KEGG enriched bubble map of LPS vs Control downregulated genes, it can be seen that the downregulated genes in the Control group samples were mainly enriched in Neuroactive ligand-receptor interaction (Neuroactive ligand-receptor interaction), Adipocytokine signaling pathway (adipocyte signaling pathway), and adult diabetes of young people (diabetes on set diabetes of the young) compared to the samples of the LPS group. From (F) the KEGG enriched bubble map of LPS vs MAA upregulation genes, it can be seen that the upregulation genes were mainly enriched in spliceosomes (helicosomes), Lysine degradation (Lysine degradation), Herpes simplex infection (Herpes simplex infection), mRNA monitoring pathways (mRNA monitoring pathway), Cushing syndrome (Cushing syndrome), Glycosylphosphatidylinositol (GPI) -anchored biosynthesis (glycosylphosphatidylinositol) (GPI) -and polymerases (RNA polymerase) in the samples of the MAA group compared to the samples of the LPS group. From the KEGG enrichment bubble map of (G) LPS vs MAA downregulated genes, it can be seen that the downregulated genes in the MAA group samples were mainly enriched in Arginine biosynthesis (Arginine biosynthesis), Alanine, aspartate, glutamate metabolism (Alanine and glutamate metabolism), Tyrosine metabolism (Tyrosine metabolism) compared to the LPS group samples. It can be seen that the sophoricoside controls neuroinflammation mainly by regulating amino acids.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.