Traditional Chinese medicine composition for treating AD by improving sleep disorder of AD patient and application of traditional Chinese medicine composition

Abstract

The invention relates to a traditional Chinese medicine composition for treating AD (Alzheimer's disease) by improving sleep disorder of AD patients and application thereof. The traditional Chinese medicine composition is prepared from the following components: 5-20g of pinellia ternate and 5-20g of selfheal. Pharmacological experiments prove that the traditional Chinese medicine composition has the effects of improving rhythm disorder of a rhythm disorder model mouse and further inhibiting transcription of inflammatory factors to relieve neuroinflammation by inhibiting a microglial cell REV-ERB alpha / BMAL1 pathway so as to improve memory ability and effectively control disease progression. The traditional Chinese medicine composition is applied to clinical practice of preventing and treating sleep disorders of patients with the Alzheimer's disease, has a good application basis and prospect, and is beneficial to providing a new research direction for traditional Chinese medicine prevention and treatment of the Alzheimer's disease.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to the application of a traditional Chinese medicine composition in the preparation of a medicine for improving sleep disorders in patients with Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD), also known as senile dementia, is a neurodegenerative disease with irreversible cognitive impairment and decline in daily mobility. Epidemiological studies show that the latest global survey reports that with the aging of the population, the number of AD patients is expected to increase from 55 million in 2019 to 139 million in 2050, which seriously endangers human health. The Lancet reported that sleep disorders are a key risk factor for the development of AD, and the risk of AD in people with sleep disorders is 1.55 times higher than that of people with healthy sleep.

[0003] The typical manifestations of sleep disorders in AD patients are daytime sleepiness and difficulty falling asleep at night, and the main pathological mechanism is circadian rhythm disorder. Biological rhythm is the body's internal biological clock mechanism to maintain synchronization with the external environment. Its center is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which is responsible for receiving light signals transmitted by retinal ganglion cells and coordinating the body's peripheral organ biological clocks through the rhythmic release of neurotransmitters. Studies have found that the loss of SCN neurons in AD patients causes circadian rhythm disorder, which aggravates Aβ deposition. Therefore, one of the important causes of early AD is sleep disorders mediated by rhythm disorders. Adjusting biological rhythms and improving sleep disorders through drug intervention are the key to early treatment of AD.

[0004] The following describes the pathogenesis of circadian rhythm in the early stages of AD:

[0005] The early pathological mechanism of AD is manifested in neuroinflammation mediated by the secretion of proinflammatory factors by M1 microglia. Microglia are important myeloid cells in the brain. They are highly sensitive to the environment and can rapidly transform their morphology into unique phenotypes, including quiescent state (M0), classical activation (M1), and alternative activation (M2). Among them, M1 microglia are highly enriched in the plaque area of ​​the brain of AD patients. Activated M1 microglia promote the production of proinflammatory factors, aggravate neuroinflammatory response, and thus lead to AD cognitive dysfunction. As the disease progresses, inflammation accumulation aggravates Aβ deposition and neuronal damage, further aggravating the pathological state of AD. This proves that neuroinflammation mediated by M1 microglia activation appears earlier than Aβ deposition and is a key pathological factor in the early onset of AD. The activation of M1 microglia is regulated by biological rhythms and plays a role in the occurrence and development of AD by affecting its immune, phagocytic and metabolic functions. In model mice, circadian rhythm disturbance promotes neuroinflammation mediated by microglial M1 polarization, ultimately leading to AD; while microglial clearance can alleviate AD cognitive impairment caused by circadian rhythm disturbance. Manipulating rhythmic genes can regulate microglial function, specifically manifested in the circadian oscillatory expression pattern of the main proinflammatory cytokines (TNFα, IL-6, and IL-1β) secreted by them. Therefore, it is concluded that the excessive secretion of proinflammatory factors by M1 microglia caused by early circadian rhythm disturbance in AD is the intermediate link between AD and rhythm regulation.

[0006] Biological rhythms regulate the human circadian rhythm through transcription-translation feedback loops, mainly including 10 genes such as positive feedback regulatory genes BMAL1, CLOCK and negative feedback regulatory genes PER1-3, CRY1-2, REV-ERBα. The key links are two negative feedback loops: First, during the day, the main rhythm negative feedback loop BMAL1 / CLOCK promotes clock gene transcription. At night, clock proteins translocate to the nucleus and bind to BMAL1 / CLOCK to inhibit its transcription. Second, in the REV-ERBs / BMAL1 negative feedback loop, REV-ERBs inhibit the BMAL1 / CLOCK pathway and negatively feedback regulate biological rhythms. In AD mice, the oscillations of key biological clock genes BMAL1, CLOCK, REV-ERBα, etc. in the early stage disappeared. Among them, BMAL1 knockout mice showed significant neurological disease phenotypes related to the occurrence of AD, including severe spontaneous astrogliosis, oxidative damage, and impaired learning ability and memory, which shows the importance of rhythmic gene regulation in the pathogenesis of AD. REV-ERBα and BMAL1 can regulate the function of M1 microglia proinflammatory factors and show a circadian oscillatory expression pattern. In AD mice, the microglial BMAL1 / CLOCK pathway is impaired, activating the NF-κB pathway and leading to abnormal expression of proinflammatory genes. The REV-ERBα / BMAL1 negative feedback regulatory loop may regulate neuroinflammation by affecting the phenotypic transformation of microglia. Inhibition of REV-ERBα can enhance the M2-like transformation and phagocytosis of microglia and reduce the deposition of Aβ and amyloid plaques. The core pathological mechanism of circadian rhythm disorder in the early stage of AD is that the REV-ERBα / BMAL1 negative feedback pathway regulates biological rhythms, which is specifically manifested in the activation of REV-ERBα, the promotion of microglial activation, the increase of proinflammatory gene expression, and the decline of cognitive ability; while the pharmacological effect of inhibiting REV-ERBα upregulates the transcription of BMAL1, accelerates the uptake of fAβ1-42 by microglia, and reduces the number and size of amyloid plaques. Therefore, it is concluded that the early cognitive impairment of AD is caused by targeting REV-ERBα, promoting microglial activation and increasing pro-inflammatory genes; and early effective treatment is to target antagonism of REV-ERBs to promote BMAL1 transcription and reshape the biological rhythm master transcription-translation circuit.

[0007] Research on early intervention treatment of AD with traditional Chinese medicine:

[0008] At present, there is still a lack of therapeutic drugs with satisfactory efficacy for the early treatment of AD. At present, modern medical treatment methods mainly act on different neurotransmitter systems through drugs, enhance the higher activities of the central nervous system, alleviate various symptoms that occur during the disease process, and delay the progression of dementia. Clinically, cholinesterase inhibitors and glutamate receptor agonists are mainly used to improve symptoms and some psychotropic drugs are used for short-term auxiliary treatment. However, the therapeutic effects of these drugs are very limited, and basically there are adverse reactions of varying degrees such as gastrointestinal reactions (nausea, vomiting, diarrhea), arrhythmias (conduction blocks, arrhythmias), drowsiness, blurred vision, etc. Therefore, it is urgent to find safe, effective and suitable for long-term use drugs that can improve memory and improve sleep disorders in Alzheimer's disease.

[0009] Chinese herbal medicine resources are abundant in my country and have a long history of treatment. Studies have shown that some Chinese herbal ingredients can improve memory and cognitive abilities in animal models, but the specific mechanism of action is still unclear. Traditional Chinese medicine has a long history of research on AD with sleep disorders and has a deep understanding of the pathogenesis, treatment principles and prescriptions of the disease.

[0010] Shuangxia Decoction is an important prescription. It was first seen in the prescription of Linglan Yaolan, which is composed of 15g of Pinellia ternata and 15g of Prunella vulgaris. Modern studies have found that Shuangxia Decoction treats sleep disorders through multiple pathways, but its role in the field of AD treatment needs to be further studied. It has now been found that Shuangxia Decoction can effectively treat rhythm disorders and AD in the early stage, which is manifested in that Shuangxia Decoction can quickly restore the activity-sleep cycle of model animal mice, effectively improve their learning and memory abilities, and significantly inhibit Aβ deposition in the brain. In addition, the study reported that Shuangxia Decoction was used to intervene in early AD mice for 3 months, and the results showed that: 1) Shuangxia Decoction inhibited the neuroinflammatory pathway and reduced the release of proinflammatory factors such as TNFα, IL-6, and IL-1β (confirmed by transcriptomics analysis and qPCR); 2) Shuangxia Decoction inhibited microglial M1 polarization, which was manifested by a decrease in the number of CD68+Iba1+ cells and an increase in the number of Arg1+Iba1+ cells. At the same time, the morphology of microglia changed, which was manifested by a decrease in cell size and fewer branches (cytoskeleton analysis). The current study also found that Shuangxia Decoction downregulated REV-ERBα and upregulated the expression of BMAL1 in microglia after intervention. In addition, mass spectrometry analysis results screened out that the top three active ingredients in Shuangxia Decoction were 3-(3,4-dihydroxyphenyl) lactic acid, Hainan holly glycoside D and isorosmarinic acid glycoside. Molecular docking results showed that these three active ingredients were tightly bound to REV-ERBα.

[0011] In summary, through various methods including transcriptomics, pharmacology and biological experiments, it was verified that Shuangxia Decoction, a traditional Chinese medicine composition, may inhibit the expression of microglial rhythmic gene REV-ERBα, activate the BMAL1 / CLOCK pathway, improve neuroinflammation, and reshape the circadian rhythm of AD, thereby improving sleep disorders in patients with Alzheimer's disease and treating AD at an early stage. Summary of the invention

[0012] The purpose of the present invention is to overcome the problems existing in the prior art and provide a Chinese medicine composition and its application for improving memory and improving sleep disorders in AD patients, and prove that the effective ingredients of Shuangxia Decoction, a Chinese medicine composition, improve neuroinflammation and treat AD by inhibiting the microglial REV-ERBα / BMAL1 negative feedback pathway.

[0013] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0014] A traditional Chinese medicine composition for treating AD by improving sleep disorders in AD patients, characterized by comprising the following raw materials by weight: 5-20g of Pinellia ternata and 5-20g of Prunella vulgaris.

[0015] Preferably, the composition comprises, by weight: 15 g of Pinellia ternata and 15 g of Prunella vulgaris.

[0016] The invention discloses an application of the Chinese medicine composition in preparing a pharmaceutical preparation and food for improving memory and improving sleep disorders caused by senile dementia.

[0017] Preferably, the pharmaceutical preparation is prepared by mixing the components in the composition, soaking the drug in ethanol and refluxing for extraction; filtering out the residue, concentrating under reduced pressure to dryness and weighing; evaporating the ethanol solvent, then refluxing and extracting with purified water for several times, combining the extracts, concentrating under reduced pressure to a certain density, and then pouring the fluid extract to room temperature; vacuum compressing into freeze-dried powder, weighing an appropriate amount, fully dissolving it in phosphate buffered saline solution by vortex ultrasound, filtering with a filter and preparing a liquid preparation with a concentration of 5g / kg.

[0018] Preferably, when ethanol reflux extraction is used, the extraction is performed by soaking in 10 times the amount of 95% ethanol for 1 hour; when pure water reflux extraction is used, the extraction is performed by reflux extraction in 10 times the amount of pure water for 1-3 times, each time for 1-3 hours.

[0019] Preferably, the reduced pressure concentration density is 0.76 g / mL.

[0020] The present invention provides a Chinese medicine composition product for improving memory and improving sleep disorders in AD patients. The product is prepared from the Chinese medicine composition for improving memory and improving sleep disorders in AD patients.

[0021] The beneficial effects of the present invention are:

[0022] Through transcriptomics, pharmacology and biological experiments, the present invention verifies that Shuangxia Decoction, a traditional Chinese medicine composition, inhibits the expression of microglial rhythmic gene REV-ERBα, activates the BMAL1 / CLOCK pathway, improves neuroinflammation, reshapes the AD circadian rhythm, thereby improving memory, improving sleep disorders in patients with Alzheimer's disease, and effectively preventing and treating AD in the early stages. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It shows that Shuangxia Decoction can quickly restore the circadian rhythm amplitude of mice with chronic jetlag (CJL): Figure A. Flowchart; Figure B. Real-time activity graph of mice. The activity pattern of the LD group was disrupted in the CJL group. The activity curve returned to normal on the 4th day after exposure to normal light; the activity curve of the CJL+SXD group was consistent with that of the LD group after exposure to normal light for 3 days; Figure C. Activity cycle of mice: the cycle of the LD group was about 24h, the CJL group had two cycles (20h, 25h), and the CJL+SXD group quickly recovered to 24h after exposure to normal light for 1 day; Figure D. Comparison of activity amplitude of mice in each group: the amplitude of the LD group was 12000, the amplitude of the CJL group was 6000, and the amplitude of the CJL+SXD group was 10000;

[0024] Figure 2 It shows that Shuangxia Decoction significantly improves the learning and memory ability of early AD mice: Figure A. Behavioral experiment after SXD intervention; Figure B. In the new object recognition experiment, Shuangxia Decoction significantly improved the 1h cognitive index of AD mice; Figure C. In the water maze experiment, Shuangxia Decoction significantly increased the number of times mice crossed the platform; Figure D. In the water maze experiment, Shuangxia Decoction significantly reduced the latency of AD mice; Figure E. In the water maze experiment, Shuangxia Decoction significantly increased the statistics of AD mice's platform residence time; all of the above were dose-dependent;

[0025] Figure 3 It shows that Shuangxia decoction significantly inhibits Aβ deposition in the brain: Figure A. Thioflavin S dye labeled Aβ deposition in the cerebral cortex, CA1 and DG regions of each group of mice, and the results showed that Shuangxia decoction could significantly inhibit Aβ deposition; Figures B, C, D: Bar graphs of the quantitative results of Aβ in the cerebral cortex, CA1 and DG regions of each group of mice (WT, 5×FAD, 5×FAD-SXDL / M / H mice (n=5)); the bar graphs represent mean ± SEM, *p<0.05, ***p<0.001;

[0026] Figure 4Shuangxia decoction regulates neuroinflammatory pathways and rhythmic gene expression in AD mice: Figure A. Venn diagram of differentially expressed genes in three groups (WT, 5×FAD, 5×FAD-SXD) of mice; Figure B. PCA diagram of RNAseq of three groups of mice; Figure C. DGE heat map analysis of three groups; Figure D. KEGG analysis of DGE; Figure E. Heat map analysis of inflammation-related factors in three groups; Figure F. Heat map analysis of three groups of rhythmic genes (n=4);

[0027] Figure 5 It shows that Shuangxia decoction inhibits microglial M1 polarization and increases M2 conversion: Figure A. The changes in microglial morphology were evaluated by immunofluorescence in the cortex, CA1 and DG regions of AD mice using Iba-1 antibody; Figure B. The microglial skeleton image was analyzed using the AnalyzeSkeleton plug-in in ImageJ software, which highlights the skeleton process in orange, the endpoints in blue, and the connections in purple; Figures C, D, and E. Quantitative analysis of microglial length, cell endpoints, and integrated density; Shuangxia decoction inhibits microglial activation in AD mice and promotes the conversion of microglial M2 phagocytic morphology;

[0028] Figure 6 It shows that Shuangxia decoction significantly inhibits the activation of astrocytes; Figure A. GFAP staining of AD mice, immunohistochemical analysis was used to evaluate the activation of astrocytes in the cortex, CA1 and DG regions of AD mice; Figures B, C, D. Quantitative analysis of the integrated density of astrocytes in each region; Shuangxia decoction significantly inhibits the activation of astrocytes in AD mice;

[0029] Figure 7 It was shown that Shuangxia Decoction inhibited the expression of REV-ERBα, and its effective ingredients tightly bound to REV-ERBα: Figure A. Shuangxia Decoction inhibited REV-ERBα+ and promoted the expression of BMAL1+ microglia; Figure B. Mass spectrometry analysis screened the effective monomer components of Shuangxia Decoction, and the top three were: (3-(3,4-dihydroxyphenyl)lactic acid, hainan holly glycoside D, and isorosmarinic acid glycoside); Figure C. Molecular docking found that the effective ingredients were all tightly bound to REV-ERBα; Figure D. Salviaflaside, one of the effective ingredients in Prunella vulgaris, and the target protein REV-ERBα; CETSA analysis results showed the interaction between Salviaflaside and the receptor protein REV-ERBα; as the temperature increased, the activity of REV-ERBα binding to Salviaflaside was significantly stronger than that of the DMSO group. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0031] The reagents and antibodies used in the present invention are as follows:

[0032]

[0033] The main reagents of the present invention are as follows:

[0034] 1) 1L PBS buffer: NaCl 8.5g; Na2HPO4·12H2O 2.9g; Na2HPO4 0.3g; ddH2O to 1000ml;

[0035] 2) 1L 4% paraformaldehyde: 40g paraformaldehyde; 800ml PB; 65°C, 1000 rpm, until clear, filter; dilute to 1000ml with ddH2O;

[0036] 3) 12% chloral hydrate: add 12g chloral hydrate to every 100ml normal saline;

[0037] 4) 1LPB: Na2HPO4·12H2O 4.93g; Na2HPO4 24.5g; ddH2O to 1000ml;

[0038] 5) DMSO: Mix DMSO and water in a volume ratio of 1:4 and shake well to obtain a 20% DMSO solution.

[0039] Example 1

[0040] 1.1 Alzheimer's disease mouse model:

[0041] The AD mice used in the present invention are 5×FAD heterozygotes. 5×FAD is mated with wild-type C57 / BL6J mice, and mice with PS1 gene mutations are selected through gene identification. The mice are provided by the Model Animal Research Institute of Nanjing University and are raised in the SPF-level laboratory of the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. The model mice usually begin to have amyloid plaque deposition and glial cell proliferation in the brain at 2 months of age. Amyloid plaque deposition increases rapidly at 4-6 months of age, and plaques are deposited throughout the hippocampus and cortex after 6 months. And learning working memory and spatial memory are impaired. At 9 months of age, the learning and memory disorders of mice are very obvious, with pathological manifestations similar to Alzheimer's disease. In addition, the animals are raised in SPF-level mouse houses, the animals move freely in the cages, and are given sufficient water and feed. The ambient temperature is maintained at about 22 degrees Celsius, the humidity is maintained at about 55%, 12 hours of light, 12 hours of dark, and alternating irradiation. The negative control is a male C57 / BL6J mouse of the same age. The mice with early AD were 6 months old, and the intervention time started from 3 months old. All animal experiments in the experiment complied with the regulations on experimental animal husbandry and animal use.

[0042] 1.2 CJL mouse model of Alzheimer's disease

[0043] The present invention established a chronic jet lag (CJL) model lasting for 2 weeks. The activity-sleep cycle of mice was recorded in real time using the infrared photosensor of the Clocklab system, and the activity cycle and amplitude differences of each group were compared. After the experiment, samples were collected at four time points: ZT0, ZT6, ZT12, and ZT18. The fresh brain was placed in liquid nitrogen and moved to a -80°C refrigerator for subsequent experiments. ZT0 represents light on, and ZT12 represents dark light. qPCR was used to detect the expression of rhythmic genes Bmal1, Clock, and Rev-erbα, and oscillation curves were drawn to observe the differences.

[0044] Example 2

[0045] 2.1 Extraction and preparation of a Chinese medicine composition

[0046] Shuangxia decoction is composed of 15g of Pinellia ternata and 15g of Prunella vulgaris. The drug is added to 10 times the amount of 95% ethanol and soaked for 1 hour for reflux extraction. The residue is filtered out and concentrated to dryness and weighed; the ethanol solvent is evaporated, and then refluxed and extracted twice with 10 times the amount of pure water, each time for 1.5 hours, the extract is combined, and the concentration is reduced to a density of 0.76g / mL. The fluid extract is poured to room temperature; vacuum compressed into freeze-dried powder, weighed an appropriate amount, fully dissolved in PBS by vortex ultrasound and other methods, filtered and prepared at the required concentration at 4°C for use. According to the body surface area conversion and previous experiments, the dosage of the mice in this subject is 5g / kg.

[0047] 2.2 Mouse grouping and Chinese medicine intervention

[0048] Male 5×FAD mice aged 3 months were randomly divided into a model group (model), a low-dose group of the present invention (5×FAD-SXD-L), a medium-dose group of the present invention (5×FAD-SXD-M), and a high-dose group of the present invention (5×FAD-SXD-H), and wild-type C57BL / 6 male mice of the same litter were used as a control group, with 8 mice in each group. The mice intervened by the Chinese medicine of the present invention were fed with a low-dose Chinese medicine of the present invention (2.5g / kg), a medium-dose Chinese medicine of the present invention (5g / kg), and a high-dose Chinese medicine of the present invention (7.5g / kg). The model group and the normal control group were gavaged with an equal amount of sterile water for 3 consecutive months. That is, after the mice were 6 months old, they were subjected to behavioral experiments-new object recognition experiments and Morris water maze experiments, and samples (brain tissue) were taken after the behavioral experiment detection.

[0049] 2.3 Behavioral testing (water maze / mine / new object recognition experiment / )

[0050] 2.3.1 Morris water maze

[0051] The Morris water maze experiment was designed by Morris in the UK in the early 1980s and used as an experimental method to test the spatial learning and memory ability of animals. This behavioral experiment involves a complex memory process, including the collection of information and visual information related to spatial positioning, as well as the process of information processing, memory and organization. The specific operation of the experiment is as follows: a barrel with a diameter of 100 cm and a depth of 50 cm is filled with water about 30 cm deep, and food-grade titanium dioxide is added for dyeing to enhance the black and white contrast, so that the software can track mice. The platform diameter is 5 cm and the water temperature is maintained at 18-23 ° C. The barrel is surrounded by a black curtain, and three geometric cardboards of different colors and shapes are evenly attached to the curtain as references. The indoor light is adjusted and quiet is maintained during the experiment. The barrel is divided into four quadrants, the midpoint of each quadrant is the entry point, and the platform is located at the midpoint of the second quadrant. The movement trajectory of the mouse in the pool is monitored by the EthovisionXT monitoring system. The first day was set as the visual platform experiment stage. The platform was 0.8 cm above the horizontal plane, which was convenient for mice to see the location of the platform. Starting from one of the quadrants, the mouse was placed in the water gently with the mouse facing the pool wall. It was asked to find the platform within 90 seconds, and the time when the platform was found was recorded. After staying on the platform for 30 seconds, it was taken out and placed in a separate cage. It rested for about 60 minutes before starting the next quadrant. If the mouse did not find the platform within the specified time, the experimental operator guided the mouse to the platform and let it stay on the platform for 30 seconds. After that, the platform was submerged 0.8 cm below the platform. The mice were trained to find the platform for 7-8 days, and the automatic camera synchronously recorded the movement trajectory of the mice. After the training, the platform was removed, and the number of times the mouse crossed the platform area within 90 seconds and its trajectory map were observed.

[0052] 2.3.1.1 The present invention can improve the spatial learning and memory ability of 5xFAD mice

[0053] Patients with Alzheimer's disease usually show cognitive decline and loss of memory function, and similar spatial learning and ability decline also exist in 5xFAD transgenic mice. This behavioral abnormality can be verified by Morris water maze experiment. After 3 months of intervention with the feed of the present invention on 3-month-old mice, we conducted a water maze experiment on each group of mice. The statistical results showed that in the water maze experiment, Shuangxia Decoction significantly increased the number of times mice crossed the platform ( Figure 2 C), significantly reduced the latency of AD mice ( Figure 2 D), and significantly increased the platform residence time statistics of AD mice ( Figure 2 E), all of which were dose-dependent. The above results show that the present invention can improve the spatial learning and memory ability of 5xFAD mice.

[0054] 2.3.2 Object recognition test

[0055] During the training phase of the novel object recognition experiment, two plastic toys (bottom area 5cm2, height 4cm) of exactly the same color and shape were placed in the open field. The toys were placed at a diagonal distance of 12cm from the wall of the open field, and the objects were firmly glued to prevent them from being moved at will. Mice were placed in the open field and allowed to adapt to the two identical toys for 5 minutes. After 1 hour, one of the toys was replaced with a new toy of different shape to test the learning and recognition memory ability of the mice, and the recognition behavior of the mice within 5 minutes was recorded. After 24 hours, the time and frequency of the mice recognizing the new toys were tested again. Spontaneous recognition behavior is considered to be the behavior of the experimental mice sniffing or touching the object with their front paws. Other behaviors such as mice sitting on objects or circling around objects are not considered recognition behaviors. When the mouse's nose is within 2cm of the toy, it is considered to have recognized the object. After each mouse experiment is completed, the object and the inside of the open field need to be wiped with 75% alcohol to prevent the smell from interfering with the recognition behavior of the next mouse. The discrimination index measured in the experiment refers to the ratio of the time it takes for mice to recognize new objects to the total time of recognition. The higher the discrimination index, the better the recognition memory ability of the experimental mice for objects.

[0056] 2.3.2.1 Effects of the present invention on novel object recognition in 5xFAD mice.

[0057] The novel object recognition test is a highly validated method for recognition memory. During the experiment, if the mouse has normal memory, it will take longer to explore the new object compared to the familiar object. If the time spent exploring the two objects is the same, it can be regarded as memory loss. In the novel object recognition experiment of mice, Shuangxia Decoction significantly improved the 1h cognitive index of AD mice, and the discrimination index of 6-month-old 5xFAD mice was significantly lower than that of wild-type mice (*P<0.05)( Figure 2 B).

[0058] Example 3: Immunohistochemical staining and quantification

[0059] 3.1 Fixation and sectioning of mouse brain tissue

[0060] After 5xFAD mice were fed with feed for 3 months, 12% chloral hydrate (400mg / kg) was intraperitoneally injected to quickly anesthetize the mice, and then the chest cavity was quickly opened to expose the heart, a needle was inserted into the left ventricle, the right auricle was cut open, and about 80ml of 0.9% sodium chloride solution was perfused through the heart, and then 200ml of 4% paraformaldehyde was continuously perfused, and the pump speed was first fast and then slow. The mouse skull was opened, the brain tissue was carefully peeled off, and placed in a 4% paraformaldehyde fixative overnight, and carefully transferred to a 30% sucrose solution the next day. After the brain tissue sank to the bottom, frozen sections were made, with a thickness of 30um, and gently taken with a brush and placed in a 24-well plate containing 1ml PB solution, for immunohistochemical staining of the brain tissue, and the slices were stained immediately. It can also be temporarily stored in a freezing solution.

[0061] 3.2 Thioflavine S staining (Thio-S staining)

[0062] The deposition of β-amyloid protein in the brain is an important pathological feature in AD patients, so Thio-S staining was used to detect the deposition of β-amyloid protein in the brains of each group of mice. Specific experimental steps: stick the brain slices on the imported anti-slip slides and place them in a ventilated place to dry naturally. First, dissolve 0.05g Thio-S powder in 1ml of first-class water, then take 0.5ml of the solution and mix it with 49.5ml 50% alcohol to make 0.05% Thio-S staining solution. Put the dried brain slices into the staining solution and shake them on a shaker for 8min under light-proof conditions; wash them twice with 80% alcohol, each time for 10s; then wash the brain slices with first-class water for 4 to 5 times, and then put them in high PB and incubate them on a shaker at 4℃ for 30min; wash the brain slices with first-class water for 4 to 5 times; seal the slices with fluorescent sealing agents.

[0063] 3.2.1 The present invention can reduce the deposition of β-amyloid protein in the brain of 5xFAD mice

[0064] The results of Thio-S staining were quantified using Image-J software. The results showed that no β-amyloid protein deposition was found in the brains of wild mice in the normal control group, while a large amount of β-amyloid protein deposition was found in the cortex of the brains of the 6-month-old model group mice. However, the protein plaques in the mice in the high-dose and low-dose intervention groups of the present invention were relatively reduced ( Figure 3 A), the statistical results showed that the plaque deposition in the brain of mice in the high-dose and low-dose intervention groups of the present invention was significantly less than that in the model group, and the difference was statistically significant (*P<0.05) ( Figure 3 B, C, D). The above results show that the present invention can significantly reduce the deposition of β-amyloid protein in the brain of 5xFAD mice.

[0065] 3.3 Immunohistochemical staining

[0066] If it is taken out from the freezing solution, wash it three times in PBS buffer, each time for 10 minutes.

[0067] (1) Place the brain slices in 3% H2O2 solution (6-well plate, 5 ml per well) and shake on a shaker for 30 min;

[0068] (2) Dilute Triton-X 100 to 0.1% with PB, remove the brain slices, and place them on a shaker for 5 min at room temperature;

[0069] (3) Wash with PBS three times, 10 min each time;

[0070] (4) The brain slices were placed in 1% BSA (24-well plate, 1 ml per well) and blocked for 2 h at room temperature;

[0071] (5) Transfer the brain slices into the primary antibody diluted at a certain ratio and incubate at 4°C overnight;

[0072] (6) Wash with PBS three times, 10 min each time;

[0073] (7) Transfer the brain slices into the secondary antibody and incubate on a shaker at room temperature for 2 h;

[0074] (8) Wash with PBS three times, 10 min each time;

[0075] (9) Transfer the brain slices into the tertiary antibody (HRP:BSA = 1:1000) and incubate at room temperature for 2 h;

[0076] (10) Wash with PBS three times, 10 min each time;

[0077] (11) DAB color development (DAB 100ul+PBS 4900ul+H2O2 10ul), color development for 1.5min;

[0078] (12) Wash with PBS three times, 10 min each time;

[0079] (13) Pour PB into a large glass dish, attach the patch, and let it dry;

[0080] (14) Dehydration and defatting: xylene twice, 5 min each time, 100% alcohol twice, 5 min each time, then 95%, 80%, 70%, 80%, 95% alcohol for 5 min each; 100% alcohol twice, 10 min each time, xylene twice, 10 min each time. Seal the slides with neutral gum, observe under a microscope, and take pictures. The pictures obtained were edited with Photoshop software to obtain the experimental results.

[0081] 3.3.1 Quantitative immunohistochemical analysis

[0082] ImageJ software was used for quantitative analysis. First, the image was converted into 8-bit byte format, the area for quantification was marked, the threshold was adjusted so that the immunohistochemical positive signal was selected, the pixels of the positive signal in the selected area and the area of ​​the selected area were measured, and the pixel value of the positive signal per unit area was obtained for statistical analysis.

[0083] 3.3.2 The present invention can improve the activation of glial cells in the brain of 5xFAD mice

[0084] In the brain tissues of AD patients and AD mice, the activation of glial cells in the brain is mainly based on the massive activation of microglia and astrocytes. Not only do glial cells proliferate in large numbers, but the activated glial cells show enlarged cell bodies and increased and thickened protrusions, and they mainly gather around β-amyloid plaques. In addition, overactivated glial cells secrete various proinflammatory factors, aggravating neuroinflammation, thereby further aggravating the condition of AD. Therefore, the activation of microglia and astrocytes in the brains of mice in each group after the intervention of the medicated feed of the present invention was observed.

[0085] First, the microglia in the brain were labeled with Iba-1 antibody, and the polarization of Iba-1 positive microglia M1 in the cortical region of each group of mice after the intervention of the feed of the present invention was observed. The number of polarized microglia M1 in wild-type mice was small, while the polarization of microglia M1 in mice in the model group was significantly activated and aggregated (*P<0.05). In comparison, the polarization of microglia M1 in mice in the high-dose feed intervention group of the present invention was significantly reduced (*P<0.05) ( Figure 5 A and B). The above results show that the present invention can significantly inhibit the polarization of microglial cells M1 in the brain of AD model mice.

[0086] In addition, the astrocytes in the mouse brain were labeled with GFAP antibody. The same picture showed that the number of activated astrocytes in the brain of wild-type mice was small, while the astrocytes in the brain of the mice in the model group were significantly activated (*P<0.05). In comparison, the number of astrocytes in the hippocampus of the mice in the low-dose and high-dose feed intervention groups of the present invention was significantly reduced (*P<0.05) ( Figure 6 A and B). The above results show that the present invention can significantly inhibit the activation of astrocytes in the brain of AD model mice.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Chinese medicine composition for treating AD by improving sleep disorders in AD patients, characterized in that: The raw materials include the following components by weight: 5-20g of pinellia tuber and 5-20g of selfheal.

2. The Chinese medicine composition according to claim 1, characterized in that: The composition comprises 15g of pinellia tuber and 15g of selfheal by weight.

3. Use of the Chinese medicine composition according to claim 1 or 2 in the preparation of pharmaceutical preparations and foods for improving memory and improving sleep disorders in senile dementia.

4. The use according to claim 3, characterized in that: The drug preparation is prepared by mixing the components in the composition, soaking the drug in ethanol and refluxing for extraction; filtering out the residue, concentrating under reduced pressure to dryness and weighing; evaporating the ethanol solvent, then refluxing and extracting with purified water for several times, combining the extracts, concentrating under reduced pressure to a certain density, and then pouring out the fluid extract to room temperature; vacuum compressing into freeze-dried powder, weighing an appropriate amount, fully dissolving it in a phosphate buffered saline solution through vortex ultrasound, filtering with a filter and preparing a liquid preparation with a concentration of 5g / kg.

5. The use according to claim 4, characterized in that: When ethanol reflux extraction is used, the steps are: soak in 10 times the amount of 95% ethanol for 1 hour and reflux extraction; when pure water reflux extraction is used, the steps are: reflux extraction in 10 times the amount of pure water for 1-3 times, each time for 1-3 hours.

6. The use according to claim 4, characterized in that: The density after reduced pressure concentration is 0.76 g / mL.

7. A Chinese medicine composition product for improving memory and improving sleep disorders in AD patients, characterized in that: The product is prepared from the traditional Chinese medicine composition according to claim 1 or 2.

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