Use of Xinshubao tablets in preparing medicine for treating vascular dementia

Abstract

The present invention discloses the use of Xinshubao tablets in preparing a medicine for treating vascular dementia, confirms the therapeutic effect of Xinshubao tablets on typical pathological damage in vascular dementia model mice from multiple perspectives such as cerebral blood flow, cognitive function, white matter damage, glial cell activation, nerve regeneration and neuronal apoptosis, and reveals its key mechanism of action, providing a scientific basis for expanding the clinical indications of Xinshubao tablets.

Description

Technical Field

[0001] The present invention specifically relates to the use of Xinshubao tablets in preparing medicines for treating vascular dementia. Background Art

[0002] Vascular dementia (VaD) refers to clinical or subclinical cerebrovascular damage caused by ischemic stroke, hemorrhagic stroke and other cerebrovascular diseases, which leads to a clinical syndrome with impaired brain cognition and memory function as the main symptoms. Currently, cholinesterase inhibitors and non-competitive N-methyl-D-aspartate receptor antagonists are mostly used to treat VaD in clinical practice. Although long-term medication does not cause serious adverse reactions, some patients experience gastrointestinal reactions such as nausea and decreased appetite. In addition, these two types of drugs are expensive, and VaD patients need long-term medication treatment. Therefore, the development of effective and affordable drugs for the treatment of VaD has broad market prospects and huge economic value.

[0003] Xinshubao tablets are composed of salvia miltiorrhiza, white peony root, curcuma, acanthopanax senticosus and hawthorn. Among them, salvia miltiorrhiza promotes blood circulation and removes blood stasis, clears the heart and relieves restlessness, curcuma promotes blood circulation and relieves pain, promotes qi and relieves depression, white peony root replenishes blood and restrains yin, softens the liver and relieves pain, acanthopanax senticosus replenishes qi and strengthens the spleen, replenishes the kidney and calms the mind, and hawthorn promotes qi and disperses blood stasis, removes turbidity and reduces fat. The combination of all the medicines has the effects of promoting blood circulation and removing blood stasis, invigorating qi and relieving pain. At present, it is mainly used clinically to treat coronary heart disease, chest tightness caused by qi deficiency and blood stasis, angina pectoris, and hypertension, hyperlipidemia and arteriosclerosis. There is no application research on the treatment of dementia, especially vascular dementia, on Xinshubao tablets. Summary of the invention

[0004] To solve the above problems, the present invention provides a use of a composition in preparing a drug for treating vascular dementia; the composition is a preparation prepared from salvia miltiorrhiza, white peony root, curcuma, acanthopanax senticosus and hawthorn as raw materials and pharmaceutically acceptable excipients.

[0005] Furthermore, the drug is a drug for improving cerebral blood flow.

[0006] Furthermore, the drug is a drug for improving cognitive function.

[0007] Furthermore, the drug is a drug for improving pathological damage of brain tissue.

[0008] Furthermore, the drug is a drug that improves ultrastructural damage of hippocampal neurons and / or promotes hippocampal neurogenesis.

[0009] Furthermore, the drug is a drug that improves white matter damage in brain regions and / or inhibits excessive activation of astrocytes.

[0010] Furthermore, the drug is a drug that inhibits apoptosis of nerve cells in the cortex and hippocampus.

[0011] Furthermore, the drug is a drug that regulates synaptic transport, neuronal cell apoptosis, neurotransmitter secretion, neuronal cell differentiation, vascular system development and / or immune function.

[0012] Furthermore, the drug is a drug that inhibits excessive activation of the hippocampal NF-κB signaling pathway.

[0013] Furthermore, the mass ratio of salvia miltiorrhiza, white peony root, curcuma, acanthopanax senticosus and hawthorn in the composition is 150-250: 1500-2500: 200-400: 700-1300: 1500-2500.

[0014] Furthermore, the mass ratio of salvia miltiorrhiza, white peony root, curcuma, acanthopanax senticosus and hawthorn in the composition is 200:2000:300:1000:2000.

[0015] Furthermore, the preparation is preferably Xinshubao tablets.

[0016] The use of the Xinshubao tablets of the present invention in preparing a drug for treating vascular dementia has been proved through animal experiments that the Xinshubao tablets can significantly improve cerebral blood flow and cognitive function by improving brain tissue pathological damage, promoting hippocampal neurogenesis and other mechanisms, thereby improving the learning and memory functions of mice with vascular dementia. The application of Xinshubao tablets in treating vascular dementia has broad application prospects.

[0017] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0018] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Laser speckle system detected the cerebral blood flow of mice in each group (n=6; A: typical graph of cerebral blood flow of mice in each group, B: statistical graph of cerebral blood flow of mice in each group; ## compared with Sham group p<0.01; * compared with BCAS group p<0.05)

[0020] Figure 2Morris water maze test results (n=12; A: Typical swimming trajectories of mice in each group, B: Escape latency statistics of mice in each group, C: Target quadrant residence time percentage statistics of mice in each group, D: Number of platform crossings of mice in each group, E: Average swimming speed statistics of mice in each group; #, ## Compared with Sham group p<0.0, p<0.01; *, * Compared with BCAS group p<0.05, p<0.01)

[0021] Figure 3 HE staining and Nissl staining results (n=3; A: typical HE staining image, B: typical Nissl staining image, C: Nissl body statistical image; # compared with the Sham group p<0.05; * compared with the BCAS group p<0.05)

[0022] Figure 4 Ultrastructure of mouse hippocampal CA1 neurons (n=3; A: typical image of mitochondria, B: typical image of synapses, C: statistical image of mitochondrial diameters, D: statistical image of the number of damaged mitochondria, E: statistical image of the number of synapses. #, ## compared with the Sham group p<0.05, p<0.01; * compared with the BCAS group p<0.05);

[0023] Figure 5 White matter damage in mice in each group (n=3; A: LFB detection of white matter damage in five brain regions, B: Immunofluorescence assay to detect MBP protein expression in corpus callosum of mice in each group, C: MBP protein expression statistical graph, D: Transmission electron microscopy to detect typical images of corpus callosum myelin sheath in mice in each group, E: Scatter plot of G ratio and axon diameter, and linear regression to obtain the best fitting line; F: Schematic diagram of G ratio calculation method; G: Axon diameter in each group; H G ratio of myelinated axons in corpus callosum. #, ## compared with Sham group p<0.05, p<0.01; * compared with BCAS group p<0.05)

[0024] Figure 6 Results of Iba and GFAP immunofluorescence staining (A: Typical immunofluorescence image of Iba-1, B: Statistical graph of Iba-1 positive cell area, C: Typical immunofluorescence image of GFAP, D: Statistical graph of GFAP positive cell area. #, ## compared with Sham group p<0.05, p<0.01; *, * compared with BCAS group p<0.05, p<0.01)

[0025] Figure 7Sox-2 and NeuN immunofluorescence staining results (A: Sox-2 immunofluorescence typical images, B: Sox-2 positive cell area statistics, C: NeuN immunofluorescence typical images in the cortex, D: NeuN immunofluorescence typical images in the CA1 region, E: NeuN immunofluorescence typical images in the DG region, F: NeuN positive cell number statistics in the cortex, G: NeuN positive cell number statistics in the CA1 region, H: EdU / NeuN double positive cell number statistics in the CA1 region, I: EdU / NeuN double positive cell number statistics in the DG region. #, ## compared with the Sham group p<0.05, p<0.01; *, * compared with the BCAS group p<0.05, p<0.01)

[0026] Figure 8 TUNEL staining results of the cortex and hippocampus of mice in each group (A: typical results of TUNEL staining, B: statistical graph of the number of TUNEL-positive cells in the cortex, C: statistical graph of the number of TUNEL-positive cells in the CA1 region of the hippocampus, D: statistical graph of the number of TUNEL-positive cells in the CA3 region of the hippocampus, E: statistical graph of the number of TUNEL-positive cells in the DG region of the hippocampus. #, ## compared with the Sham group p<0.05, p<0.01; *, * compared with the BCAS group p<0.05, p<0.01; ns: p>0.05)

[0027] Fig. 9 RNA-seq analysis results (A: Venn diagram of each group of genes detected, B: Cluster analysis of common genes, C: Principal component analysis of common genes, D: Volcano map of gene expression of Xinshubao tablets and model group, E: Enrichment analysis of genes upregulated by Xinshubao tablets; F: Enrichment analysis of genes downregulated by Xinshubao tablets)

[0028] Fig.10 Regulatory effect of Xinshubao tablets on NF-κB signaling pathway (A: PPI protein interaction network diagram, B: PPI protein interaction diagram of the top 20 proteins, C: Q-PCR detection of key gene expression regulated by Xinshubao tablets, D: Key protein expression band diagram of NF-κB signaling pathway (in the cytoplasm), E: IKKα protein expression statistical diagram, F: p-IKKα / β protein expression statistical diagram, G: IκBα protein expression statistical diagram, H: p-IκBα protein expression statistical diagram, I: p65 protein expression statistical diagram, J: p-p65 protein expression statistical diagram, K: Key protein expression band diagram of NF-κB signaling pathway (in the nucleus), L: p65 protein expression statistical diagram in the nucleus, M: p-p65 protein expression statistical diagram in the nucleus; #, ## compared with Sham group p<0.05, p<0.01; *, * compared with BCAS group p<0.05, p<0.01) DETAILED DESCRIPTION

[0029] The raw materials, equipment and reagents used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products. Among them, Xinshubao tablets are provided by Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd., which are prepared from Salvia miltiorrhiza, White Peony Root, Curcuma Radix, Acanthopanax Senticosus and Crataegus Pinellia with a mass ratio of 200:2000:300:1000:2000 as raw materials.

[0030] Example 1 Study on the application of Xinshubao tablets in treating vascular dementia

[0031] 1. Methods

[0032] 1. Preparation of vascular dementia mouse model

[0033] The vascular dementia (VaD) mouse model was prepared by causing chronic cerebral hypoperfusion by bilateral common carotid artery stenosis. Male C57BL / 6J mice, 6-8 weeks old, were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital. A 1 cm incision was made along the midline of the neck, and the mucosa and muscle were bluntly separated to expose the right common carotid artery, which was placed in a microcoil. After 30 minutes, the left common carotid artery was placed in the microcoil by the same method and sutured. The specifications of the microcoil were: inner diameter 0.18 mm, pitch 0.5 mm, and total length 2.5 mm. Drug administration began the day after surgery.

[0034] 2 Grouping and Dosing

[0035] The experiment was divided into sham group (sham, bilateral common carotid artery dissection, but no stenosis treatment), model group (BCAS, vascular dementia mice), and three dose groups of Xinshubao tablets (XSB), with 12 mice in each group. The sham and model groups were gavaged with pure water, and the three dose groups of Xinshubao tablets were gavaged with 3.75g / kg, 7.5g / kg and 15g / kg (calculated according to the amount of raw medicine) to mice with vascular dementia. The drugs were given by gavage continuously for 7 weeks. Cerebral blood flow was detected on the 7th day of gavage, and Morris water maze test was performed on the 42nd day of gavage to detect the learning and memory function of mice in each group, and then the brain was taken out, paraffin sections were prepared for pathological staining and immunostaining experiments; the hippocampus was taken out, RNA was extracted for transcriptomic sequencing analysis and Q-PCR experiment; the brain was taken out, protein was extracted for Western blot experiment.

[0036] 3 Laser speckle detection of cerebral blood flow in mice

[0037] On the 7th day of XSB administration, laser speckle was used to detect the cerebral blood flow of mice. The mice were anesthetized with 1.5% isoflurane, and the skull was exposed through a midline incision of the scalp, which was washed with sterile saline. The cerebral blood flow was continuously recorded and captured for 10 seconds, and the cerebral blood flow of each group of mice was analyzed.

[0038] Morris water maze to detect learning and memory function in mice

[0039] On the 42nd day of XSB administration, the Morris water maze test was used to detect the learning and memory abilities of mice in each group. ① Space exploration experiment: The escape platform was 1 cm below the water surface, and the water temperature was 20-21°C. Each of the four quadrants was trained once a day. During the training, the mice were gently placed in the water from 1 / 2 arc of any quadrant, facing the pool wall. If the mice could not find the platform within 60 seconds, they were led to the platform and stayed for 20 seconds. The training was continued for 6 days. The escape latency of each group of mice before going on stage was recorded to evaluate the learning ability of the mice. ② Positioning navigation experiment: On the 7th day, the platform was removed, and the mice were placed in the water from the quadrant opposite to the original platform facing the pool wall. The number of times the mice crossed the platform within 60 seconds and the percentage of time they stayed in the quadrant where the platform was located were detected and analyzed to evaluate the spatial memory ability of the mice.

[0040] 5. Preparation of Paraffin Sections

[0041] After the water maze experiment, three mice were taken from each group and anesthetized with 50 mg / kg sodium pentobarbital. The heart was exposed and 20 mL of normal saline and 4% paraformaldehyde solution were perfused through the left ventricle in sequence. The brain was taken out and fixed with 4% paraformaldehyde for 8 h. The brain was dehydrated, immersed in wax, and embedded in paraffin to prepare paraffin tissue blocks. The brain was cut into 3 μm sections using a paraffin slicer and stored in a refrigerator at 4°C.

[0042] 6H&E staining experiment

[0043] Brain tissue paraffin sections were baked for 2 h, dewaxed conventionally, stained with hematoxylin (60°C) for 60 s, washed off with running water for 5-10 s, differentiated with 1% hydrochloric acid ethanol for 3 s, washed with water for 1-2 s, rinsed with running water for 10 min, stained with 0.5% eosin solution for 30-60 s, washed with distilled water for 1-2 s, 80% ethanol for 1-2 s, 95% ethanol for 1-2 s, anhydrous ethanol I for 1-2 s, anhydrous ethanol II for 1-2 s, xylene I for 5 min, xylene II for 5 min, sealed with neutral gum, observed under a microscope and photographed.

[0044] 7Nissl staining experiment

[0045] Brain tissue paraffin sections were baked for 2 h, routinely dewaxed, stained with Nissl staining solution for 10 min (37°C, protected from light), washed with distilled water for 5 s, 95% ethanol for 2 min, xylene I for 5 min, xylene II for 5 min, sealed with neutral gum, observed under a microscope and photographed.

[0046] 8LFB myelin staining experiment

[0047] Brain tissue paraffin sections were baked for 2 h, routinely dewaxed, washed with distilled water for 5 s, dehydrated with 95% ethanol for 5 s, stained with LFB solution at 60 °C for 2 h, washed with 95% ethanol to remove excess staining solution, washed with distilled water for 5 s, separated with fast blue differentiating solution for 15 s, separated with 70% ethanol for 30 s, washed with water, observed under a microscope until the outlines of gray and white matter were clear, counterstained with tar violet staining solution for 30 s, washed with water, dehydrated with 95% and 100% ethanol, made transparent with xylene, sealed with neutral gum, observed under a microscope and photographed.

[0048] 9. Immunofluorescence detection of MBP / GFAP / Iba-1 expression

[0049] Brain tissue paraffin sections were baked for 2 hours, dewaxed conventionally, washed 5 times with PBS, permeabilized with 0.5% Triton×100 for 30 minutes, blocked with 5% BSA at room temperature for 1 hour, washed 5 times with PBS, and added with Rabbit anti-GFAP (1:100), Rabbit anti-Iba (1:100) and Rabbit anti-MBP (1:100) antibodies respectively. The sections were incubated at 4°C overnight, washed 5 times with PBS, and Goat anti-rabbit secondary antibody labeled with Alexa Fluor488 was added. The sections were incubated at room temperature in the dark for 1.5 hours, washed 5 times with PBS, DAPI was added to counterstain the cell nuclei, anti-fluorescence quencher was added, the sections were sealed, and the sections were observed and photographed under a fluorescence microscope.

[0050] 10 Immunofluorescence detection of hippocampal neurogenesis

[0051] Brain tissue paraffin sections were baked for 2 hours, dewaxed conventionally, washed 5 times with PBS, labeled with EdU according to the instructions of the kit, and then subjected to immunofluorescence staining: 0.5% Triton×100 permeabilization for 30 minutes, 5% BSA blocking for 1 hour at room temperature, washed 5 times with PBS, Rabbit anti-NeuN (1:100) antibody was added, incubated overnight at 4°C, washed 5 times with PBS, 488-labeled Goat anti-rabbit secondary antibody was added, incubated at room temperature in the dark for 1.5 hours, washed 5 times with PBS, DAPI was added to counterstain the cell nucleus, anti-fluorescence quencher was added, the sections were sealed, and observed and photographed under a fluorescence microscope.

[0052] 11TUNEL staining to detect neuronal apoptosis in the cortex and hippocampus

[0053] According to the kit, brain tissue sections were dewaxed and hydrated, incubated in proteinase K solution at 37°C for 20 min, and then immersed in 0.1% Triton X-100 0.1% sodium citrate at room temperature for 20 min. After that, the sections were incubated in TdT incubation buffer at 37°C for 1 h, then counterstained with DAPI solution for 8 min, and photographed under a fluorescence microscope.

[0054] 12 Transmission electron microscopy to detect ultrastructural damage of hippocampal neurons

[0055] After 7 weeks of XSB treatment, mice were deeply anesthetized with sodium pentobarbital and then perfused with PBS. The hippocampus was isolated from the brain and fixed in 2% glutaraldehyde (pH = 7.2). The CA1 region of the hippocampus was selected and cut into 1.0 × 1.0 × 1.0 mm 3 The tissues were blocked and fixed with 1% osmium tetroxide for 2 h at room temperature. After dehydration, infiltration, embedding, and polymerization, the tissues were cut into 70 nm sections and stained with 2% uranyl acetate saturated alcohol solution and 2.6% lead citrate in sequence. Finally, the sections were observed and photographed under TEM (HT7700, Hitachi, Tokyo, Japan). Image J software was used to analyze mitochondrial size and synaptic number.

[0056] 13 Transcriptome sequencing analysis of key genes and protein targets regulated by Xinshubao

[0057] Total RNA was extracted from the hippocampus of each group of mice, and the purity of RNA was detected by nanophotometer (IMPLEN, CA, USA). The concentration and integrity of RNA samples were determined by Agilent 2100RNA nano 6000 detection kit (Agilent Technologies, CA, USA). RNA-seq libraries were constructed using Illumina's VAHTS Universal V6 RNA-seq Library Prep Kit (NR604-01 / 02). The library was sequenced on the Illumina platform, generating 150bp paired-end reads. Cluster generation and sequencing were performed on the NovaSeq 6000S4 platform using the Novasek 6000S4 kit. The reads were aligned with the GRCm38 mouse reference genome and mapped. Low-abundance mRNAs (FPKM<1) were removed. Differentially expressed genes (DGEs) were defined as FPKM fold changes ≥1.2 and p-values ​​<0.05. The https: / / www.bioinformatics.com.cn platform was used to draw volcano maps, heat maps, and principal component analysis diagrams. The Metascape database was used for GO annotation and KEGG pathway enrichment analysis. The PPI network was constructed using the https: / / string-db.org / platform and visualized using the Cytoscape software.

[0058] 14Q-PCR assay to detect key genes regulated by Xinshubao

[0059] Total RNA from the hippocampus of each group of mice was extracted, and cDNA was synthesized for PCR amplification. The PCR conditions were as follows: pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and a total of 40 cycles. -△Ct The relative mRNA expression was calculated by the comparative method and normalized using the internal reference gene Gapdh. Primer information is shown in Table 1

[0060]

[0061] 15 Western blot assay to detect the expression of key proteins in the NF-κB signaling pathway

[0062] Total hippocampal protein was extracted and the protein concentration was determined by BCA method. Loading buffer was added and boiled for 5 min. Protein was separated by SDS-PAGE gel electrophoresis and wet transfer was performed at 250 mA for 80 min. The membrane was blocked with 5% skim milk powder at room temperature for 1 h. The primary antibodies were incubated: IKKα, IKKβ, phospho-IKKα / β (Ser176 / 180), IκBα, phospho-IκBα (Ser32), p65, phospho-p65 (Ser536), β-tubulin and Histone H3 at 4°C overnight. Then the corresponding secondary antibodies were incubated. The gel imager was used to take pictures. The grayscale value was analyzed by Image J and normalized with the internal reference to compare the protein expression of each group.

[0063] 16 Statistical analysis

[0064] Data were expressed as mean ± standard deviation (mean ± sd), and statistical analysis was performed using GraphPad Prism 5.0 software. The escape latency data in the water maze experiment were analyzed using repeated measures two-way ANOVA combined with Bonferroni post-hoc test; other data were compared between multiple groups using one-way ANOVA combined with Tukey's post-hoc test, and two-tailed Students' test was used for comparison between two groups. P < 0.05 indicated that the difference was statistically significant. Cell counts and fluorescence intensity were counted and quantified using Image J software.

[0065] 2. Results

[0066] 1Xinshubao Tablets Improve Cerebral Blood Flow in VaD Model Mice

[0067] One week after Xinshubao tablets were administered, the cerebral blood flow of mice in each group was detected using a laser speckle system. Figure 1As shown in the figure, the cerebral blood flow of the model mice was significantly reduced, which was significantly different from the sham operation group. After the administration of Xinshubao, the cerebral blood flow of the mice was restored. The high-dose group had the best effect, which was significantly different from the model group. This shows that Xinshubao tablets can improve the cerebral blood flow of VaD mice. The results of the cerebral blood flow experiment are shown in Figure 1 .

[0068] 2Xinshubao tablets improve the learning and memory function of VaD model mice

[0069] After 6 weeks of Xinshubao tablet administration, the Morris system was used to detect the learning and memory functions of mice in each group. Figure 2 As shown, the model mice had a longer swimming trajectory, a significantly prolonged escape latency, and a significantly reduced percentage of time spent in the target quadrant and the number of times they crossed the platform, indicating that the model mice had significant damage to their learning and memory functions. After drug administration, the escape latency of the mice was shortened. On the 6th day of training, there were significant differences between the medium-dose and high-dose groups and the model group. In the spatial exploration experiment, the percentage of time spent in the target quadrant and the number of times they crossed the platform in the drug-treated group increased, and there were significant differences between the medium-dose and high-dose groups and the model group. The above results indicate that Xinshubao tablets can improve the learning and memory functions of VaD mice. High-dose Xinshubao tablets (15 g / kg) have the most obvious effect in improving cerebral blood flow and cognitive function, so this dose was selected for subsequent pathological and mechanism studies. The results of the Morris water maze experiment are shown in Figure 2 .

[0070] 3 Structural damage of mouse brain tissue

[0071] After the water maze experiment, HE staining and Nissl staining were used to detect brain tissue damage in each group of mice. Figure 3 As shown in A, the neurons in the cortex and hippocampus of the model group were arranged loosely, the nuclei were shrunken and darkly stained, and inflammatory cell infiltration was visible. After the administration of Xinshubao tablets, the neurons were arranged regularly and tightly, the nuclei were round, the staining was reduced, and the inflammatory cell infiltration was reduced. Figure 3 The cortex and hippocampus of the mice in the model group shown in B were loosely arranged and the number was reduced. After the administration of Xinshubao tablets, the Nissl bodies were compactly arranged, the number of Nissl bodies increased, and the structure of the CA1 and DG regions of the cortex and hippocampus was effectively restored. The above results show that Xinshubao tablets can effectively improve the pathological damage of brain tissue in VaD model mice. HE and Nissl staining results are shown in Figure 3 .

[0072] 4 Xinshubao tablets improve the ultrastructure of hippocampal neurons in VaD model mice

[0073] Transmission electron microscopy was used to observe the ultrastructure of hippocampal CA1 neurons in each group of mice. Figure 4As shown in the figure, the double membrane structure of the nuclear membrane of neurons in the model group was unclear, mitochondrial cristae were missing, mitochondrial vacuolization was obvious, the mitochondrial diameter was shortened, and the number of synapses was reduced, indicating that the ultrastructure of neurons in the model group was damaged. After the administration of Xinshubao, the mitochondrial morphology returned to the typical long spindle shape, the mitochondrial cristae were clearly visible, the bilateral nuclear membrane structure was intact, and the number of synapses increased. The above results show that Xinshubao tablets can effectively improve the ultrastructural damage of hippocampal neurons in VaD model mice. Transmission electron microscopy results are shown in Figure 4 .

[0074] Xinshubao tablets alleviate white matter damage in VaD model mice

[0075] Firstly, LFB staining was used to detect the white matter damage in five brain regions of mice, including Paramedian, Medial, caudate putamen, internal capsule and optic nerve bundle; then immunofluorescence experiment was used to detect the expression of myelin basic protein (MBP) in the corpus callosum; finally, transmission electron microscopy was used to observe the myelin sheath of the corpus callosum, and the white matter damage of mice in each group was comprehensively evaluated ( Figure 5 ).like Figure 5 As shown in A, the blue-stained white matter bundles in the five brain regions of the model group were sparse and had a large number of holes, while the white matter bundles in the drug-treated group were tightly arranged and had fewer holes. The results of immunofluorescence showed that the expression of MBP in the corpus callosum of the model group was significantly reduced, which was significantly different from the blank group, and the expression level of MBP increased significantly after drug administration compared with the model group. The results of transmission electron microscopy showed that the myelin sheath structure of the sham operation group was clear and complete, and the myelin sheath was thicker, while the myelin sheath of the model group became thinner and the structure was incomplete, and the myelin sheath became thicker and the structure was complete after drug administration. The above results show that Xinshubao tablets can effectively improve white matter damage in VaD model mice.

[0076] 6Xinshubao tablets inhibit the overactivation of cortical and hippocampal glial cells in VaD model mice

[0077] Iba is a marker protein of microglia, and microglia can be identified by detecting Iba. The results of Iba immunofluorescence staining showed that compared with the sham operation group, the Iba in the hippocampus of the model group mice was higher than that in the sham operation group. + / DAPI positive cells increased significantly, and the cell bodies became larger, indicating that microglia were activated; compared with the model group, Iba + The number of DAPI-positive cells decreased and the cell bodies became smaller, similar to the sham operation group, indicating that Xinshubao tablets can inhibit the excessive activation of microglia in mice with vascular dementia. GFAP is a marker protein of astrocytes, and astrocytes can be identified by detecting GFAP. The results of GFAP immunofluorescence staining showed that compared with the sham operation group, the astrocytes (GFAP + / DAPI) cell bodies became larger and their number increased, indicating that astrocytes were activated; compared with the model group, GFAP + The number of DAPI-positive cells decreased and the cell bodies became smaller, similar to the sham operation group, indicating that Xinshubao tablets can inhibit the overactivation of astrocytes in mice with vascular dementia. The results of Iba and GFAP immunofluorescence staining are shown in Figure 6 .

[0078] 7Xinshubao tablets promote neurogenesis in the hippocampus of VaD model mice

[0079] Sox-2 is a marker protein for neural stem cells in the DG region of the hippocampus. Sox-2 can be detected to identify neural stem cells in the DG region of the hippocampus. EdU is a nucleotide analog that can be incorporated into dividing cells and can identify dividing cells. NeuN is a marker protein for mature neurons. NeuN can be detected to identify mature neurons. Figure 7 As shown in AB, there is a certain amount of Sox-2 in the DG region of the sham operation group + / DAPI positive cells, while Sox-2 + / DAPI positive cells decreased significantly, and increased after drug administration, with significant difference compared with the model group. Figure 7 CI shows that NeuN in the cortex of mice in the model group + The number of mature neurons with positive DAPI / DAPI was significantly reduced, but increased after drug administration. + The number of mature neurons positive for DAPI and EdU + / NeuN + The number of mature neurons derived from the proliferation and differentiation of neural stem cells with double positive PD-1 / DAPI was significantly reduced, but increased significantly after drug administration. The above results show that Xinshubao tablets can promote the proliferation and differentiation of neural stem cells in the hippocampus of VaD model mice, that is, promote hippocampal neurogenesis. The results of Sox-2 and NeuN immunofluorescence staining are shown in Figure 7 .

[0080] 8Xinshubao tablets inhibit apoptosis of neural cells in VaD model mice

[0081] TUNEL staining was used to detect the apoptosis of cortical and hippocampal neurons in each group of mice. Figure 8As shown in the figure, there were few TUNEL-positive cells in the cortex and hippocampus of the sham-operated group, while obvious TUNEL-positive cells were observed in the cortex, hippocampal CA1, CA3 and DG of the model group, indicating that apoptosis occurred in the neurons of the cortex and hippocampus of the mice in the model group. There were a certain number of TUNEL-positive cells in the cortex, hippocampal CA1, CA3 and DG of the Xinshubao-treated group, but they were significantly reduced compared with the model group. The above results show that Xinshubao tablets can effectively inhibit the apoptosis of neurons in the cortex and hippocampus of VaD model mice. TUNEL staining results are shown in Figure 8 .

[0082] Xinshubao tablets regulate the gene expression profile of the hippocampus in VaD model mice

[0083] RNA transcriptome sequencing technology was used to analyze the effect of Xinshubao tablets on the gene expression profile of the hippocampus of VaD model mice and to explore the key genes and protein targets regulated by Xinshubao tablets. Fig. 9 As shown in Venn diagram A, 12999 genes were detected in the sham operation group, 13198 genes were detected in the model group, and 13066 genes were detected in the Xinshubao tablets group. There were 12657 common genes in the three groups. These common genes were input into the microbial information platform for cluster analysis and principal component analysis, as shown in Fig. 9 B and Fig. 9 As shown in C, the model group was far away from the sham operation group, while the Xinshubao group was closer to the sham operation group, suggesting that Xinshubao tablets had a certain correction effect on the hippocampal gene spectrum of model mice. Further analysis found that compared with the model group, 109 genes were upregulated and 77 genes were downregulated in the Xinshubao group ( Fig. 9 D). These differentially expressed genes were input into the Metascape database for enrichment analysis, and it was found that Xinshubao tablets mainly regulate synaptic transport, neuronal apoptosis, neurotransmitter secretion, neuronal differentiation, vascular system development and immune function, and play an anti-VaD role ( Fig. 9 ).

[0084] Xinshubao tablets inhibit the over-activation of NF-κB signaling pathway in the hippocampus of VaD model mice

[0085] In order to further explore the key genes and protein targets regulated by Xinshubao tablets, the differentially expressed genes were input into the String database for PPI protein network interaction analysis, and then the obtained network information was input into the Cytoscape software for analysis and visualization. Fig.10 As shown in AB, Xinshubao tablets mainly affect proteins related to the NF-κB signaling pathway, which was further verified by Q-PCR. Fig.10As shown in C, the expression of genes Tnfaip3, Nfkbia, Zbtb7b, and Mdm2 that inhibit the NF-κB signaling pathway in the hippocampus of model mice was reduced, while the expression of genes Mapk14 and Sgk1 that activate the NF-κB signaling pathway was increased, indicating that the NF-κB signaling pathway was overactivated in the hippocampus of model mice. After the administration of Xinshubao, the expression of the above genes can be significantly adjusted back, indicating that Xinshubao tablets can inhibit the overactivation of the NF-κB signaling pathway in the hippocampus of model mice. Then, we used Western blot experiments to detect the expression of key proteins in the NF-κB signaling pathway to further clarify the regulatory effect of Xinshubao tablets on the NF-κB signaling pathway. Fig.10 As shown in DJ, the expression of p-IKKα / β, which activates the NF-κB signaling pathway, in the hippocampus of the model group mice increased, while the expression of IκBα, which inhibits the NF-κB signaling pathway, decreased, and the expression of p-IκBα increased. At the same time, it can be seen that p65 in the cytoplasm decreased, while p65 in the nucleus increased. Phosphorylation of p65 is conducive to the nuclear translocation of p65, so we see an increase in p-p65 in the cytoplasm, and at the same time, p-p65 in the nucleus also increased, indicating that the NF-κB signaling pathway in the hippocampus of the model group mice was overactivated. After the administration of Xinshubao, the expression of p-IKKα / β decreased, the expression of IκBα increased, the expression of p-IκBα decreased, and the p65 in the cytoplasm increased while the p65 in the nucleus decreased. Both p-p65 in the cytoplasm and the nucleus decreased. The above results fully demonstrate that Xinshubao tablets can inhibit the overactivation of the NF-κB signaling pathway in the hippocampus of model mice. The results of the regulatory effect of Xinshubao tablets on the NF-κB signaling pathway are shown in Fig.10 .

[0086] In summary, the present invention proves through animal experiments that Xinshubao tablets can significantly improve cerebral blood flow and cognitive function by improving brain tissue pathological damage, promoting hippocampal neurogenesis and other mechanisms, thereby improving the learning and memory functions of vascular dementia rats. Xinshubao tablets have broad application prospects in the treatment of vascular dementia.

Claims

1. Use of a composition in the preparation of a medicament for treating vascular dementia, characterized in that: The composition is a preparation prepared by using salvia miltiorrhiza, white peony root, curcuma, acanthopanax senticosus and hawthorn as raw materials and adding pharmaceutically acceptable excipients; The mass ratio of salvia miltiorrhiza, white peony root, curcuma, acanthopanax senticosus and hawthorn in the composition is 200:2000:300:1000:2000.

2. The use according to claim 1, characterized in that: The drug is a drug that improves cerebral blood flow.

3. The use according to claim 1, characterized in that: The drug is a drug for improving cognitive function.

4. The use according to claim 1, characterized in that: The medicine is a medicine for improving pathological damage of brain tissue.

5. The use according to claim 4, characterized in that: The drug is a drug for improving ultrastructural damage of hippocampal neurons and / or promoting hippocampal neurogenesis.

6. The use according to claim 4, characterized in that: The drug is a drug that improves white matter damage in brain regions and / or inhibits excessive activation of astrocytes.

7. The use according to claim 4, characterized in that: The drug is a drug that inhibits apoptosis of nerve cells in the cortex and hippocampus.

8. The use according to claim 4, characterized in that: The drug is a drug that regulates synaptic transport, neuronal cell apoptosis, neurotransmitter secretion, neuronal cell differentiation, vascular system development and / or immune function.

9. The use according to claim 4, characterized in that: The drug is a drug that inhibits excessive activation of the hippocampal NF-κB signaling pathway.

10. The use according to claim 1, characterized in that: The preparation is Xinshubao tablets.

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