WO2020253179A1 - Application of small molecule compound in preparation of drug for inhibiting tau protein expression - Google Patents

Abstract

An application of a small molecule compound in preparation of a drug for inhibiting Tau protein expression. An application of 2',3'-adenosine dialdehyde (ADDA) or an in vivo metabolite, a tautomer, a stereoisomer, a pharmaceutically acceptable salt, a hydrate and/or a solvate thereof in preparation of an Tau protein inhibitor. The invention relates to a drug for treatment of a central nervous system disease. The invention clarifies for the first time the function of the compound ADDA in inhibiting the expression of human Tau protein, and provides a candidate drug for treatment of Alzheimer disease (AD) and Parkinson's disease.

Description

Application of a small molecule compound in the preparation of drugs for inhibiting Tau protein expression Technical field

The present invention relates to the field of medicine. Specifically, the present invention relates to the application of a small molecule compound in the preparation of a medicine that inhibits the expression of Tau protein.

Background technique

Alzheimer's disease (Alzheimer disease, AD), also called senile dementia, is a degenerative disease of the central nervous system with an insidious onset and a chronic progressive course. It is the most common type of senile dementia. [Burns A et al, BMJ., 338:467-471, 2009; WHO, "Dementia Fact sheet N°362", 2015] Mainly manifested as progressive memory impairment, cognitive impairment, personality change and language impairment Mental symptoms seriously affect social, professional and life functions [National Institute on Aging, "About Alzheimer's Disease: Symptoms", 2012]. With the aging of the population, the incidence of AD is increasing year by year, seriously endangering the physical and mental health and quality of life of the elderly, causing serious suffering to patients, and bringing a heavy burden to the family and society. It has become a serious social problem. The general concern of governments and medical circles of various countries.

The etiology and pathogenesis of AD are complex and are currently not very clear. It is generally considered to be related to gene mutations, Aβ deposition, cholinergic defects, Tau protein hyperphosphorylation, mitochondrial defects, neuronal apoptosis, oxidative stress, free radical damage and infections, poisoning, brain trauma, and hypoglycemia. Risk factors for AD include age, gender (females are higher than males), education level, brain trauma, AD is also related to genetics, hypothyroidism, exposure to heavy metals, toxic chemicals and organic solvents, and others such as cerebrovascular disease, diabetes And the first depression in old age is also a risk factor for AD [Querfurth HW, LaFerla FM, NEJM, 362(4):329–44, 2010)].

In 1993, the FDA approved the marketing of tacrine, which inhibits acetylcholinesterase, making it the first drug to treat Alzheimer's disease. However, due to the excessive side effects of the drug, it was withdrawn from the US market in May 2012 [tacrine (Discon-tinued)-Cognex". Medscape Reference. WebMD, Retrieved 8 October 2013]. The disease progression of Alzheimer's disease is very slow. So far, there are four main hypotheses for the pathogenesis of diseases: the amyloid cascade hypothesis, APOE4, Tau protein and ASC (PYCARD) protein hypothesis. In 1992, John Hardy of University College London, UK, proposed a bold approach to the origin of the disease. Hypothesis: Amyloid cascade hypoth-esis. Hardy believes that the disease begins with the formation of amyloid beta in the brain, and tangles, neuronal cell death, memory decline, and dementia are all amyloid effects on the brain. A secondary event caused by internal destruction. The continuous accumulation of amyloid fragments produced by neurons outside the cell can lead to the formation of plaques. The volume of plaques will gradually increase over time and gradually begin to affect normal neuronal cells. The communication, which affects the normal function of neurons, triggers the formation of intraneuronal tangles, and ultimately leads to the death of neurons. [Hardy JA, Higgins GA, Science, 256(5054): 184-5, 1992]. The theory There is a lot of evidence to support. For example, in 1995, scientists confirmed that amyloid plaques are produced in the brains of mice carrying human APP mutant genes and cognitive function will also decline. The amyloid cascade hypothesis is not only relatively reasonable for the course of disease More importantly, it provides a feasible target for the development of new drugs. [Octave JN, Rev Neurosci., 6(4): 287-316, 1995]

But in fact, not everyone agrees with the amyloid hypothesis. Allen Roses and colleagues found that APOE4 carriers have a significantly higher risk of early-onset and late-onset Alzheimer's disease. Carrying one copy of the gene will increase the risk by 4 times, while carrying two gene copies will increase the risk by 12 times. APOE4 will affect the normal intake of blood sugar in the brain, leaving the brain in a state of energy deficiency. Compared with APOE2 and APOE3 carriers, APOE4 carriers have a lower blood glucose metabolism rate. Due to the long-term energy shortage of the brain, the function of neurons will be damaged, which will lead to the formation of plaques and tangles, and finally lead to neuronal apoptosis. Since neurons are difficult to regenerate, the process is not reversible, and neurons can only die one by one. APOE4 theory has a clearer explanation of the origin of diseases than amyloid theory. Unfortunately, compared with the amyloid hypothesis, it is difficult to develop new drugs based on the APOE4 hypothesis. 【Roses A,et al.,Alzheimer’s&Dementia,12(6):687-694,2016】

There are many defects in the above two theories, which provides the possibility for the proposal of the Tau protein theory. One of the main functions of Tau protein (Tubulin associated unit) is to maintain the stability of axon microtubules. The hyperphosphorylation of Tau protein will lead to the formation of tangles in neurons, causing microtubules to fall off and affect the transport of neurotransmitters and other substances in neurons, and gradually cause synapses to degenerate, axons disappear, and only left The remnant cell body of a neuron. The uniqueness of the Tau protein theory is that it does not describe the cause of the disease. Whether it is amyloid or APOE4 or other factors that cause the disease, they will make neurons move toward a common fate: abnormal phosphorylation of Tau protein, synaptic degeneration and neuronal death. Although the Tau protein hypothesis can also provide a target for the development of new drugs, a major flaw of the Tau protein hypothesis is that there is currently little genetic evidence supporting the hypothesis. 【Mudher M, Lovestone S, Trends in Neurosciences, 25:22-6, 2002.】

However, due to the high edge of the amyloid hypothesis, the Tau protein hypothesis has not received the same attention as the amyloid hypothesis for a long time. But when the amyloid hypothesis encountered numerous obstacles, some proponents of the amyloid hypothesis began to turn to the Tau hypothesis. Therapies supported by the Tau protein hypothesis so far mainly have the following directions: targeting tau protein-related kinase/phosphatase, or active and passive immunity, and reducing Tau protein phosphorylation through strategies such as anti-Tau protein aggregation inhibitors. Or inhibit Tau protein aggregation.

In 2002, TauRx Therapeutics, a company focused on the study of Tau protein aggregation inhibition, was established, and in 2004, the first drug, methylene blue, was advanced to clinical trials. In 2008, TauRx announced the results of phase II clinical trials at a conference, but the results caused a lot of controversy: the data showed that the two low-dose regimens of the drug are effective for moderate patients (non-early patients), and the clinical design The plan was also seriously questioned, and the company did not publish complete data on clinical trials after the meeting. In 2016, the company announced that two of its clinical trials had failed. Tau protein aggregation inhibition has not made good progress.

The abnormal phosphorylation state caused by the imbalance between the phosphorylation and dephosphorylation of Tau protein may play a very important role in the formation of neurofibrillary tangles, which can cause the Tau protein to detach from the microtubules and then aggregate with each other. The abnormal phosphorylation state may be caused by increased kinase activity or decreased phosphatase activity. GSK-3 plays a key role in the phosphorylation of Tau protein under physiological and pathological conditions. However, the development of GSK-3 inhibitors is extremely difficult, and it is difficult to obtain compounds with high selectivity. Due to too many GSK-3 substrates, it is difficult to control toxic and side effects.

Nevertheless, some inhibitors have entered clinical research. Noscira's Tideglusib clinical phase IIa trial showed good safety, but the subsequent phase IIb trial did not reach the clinical trial endpoint. Although the development of phosphatase (such as PP2A) agonists may theoretically make up for the failure of the development of kinase inhibitors, no drugs have entered clinical trials.

Since the detachment of microtubules and Tau protein will cause abnormal function of microtubules, the material transport process between cell bodies and axons will be blocked and the synaptic function will eventually be damaged. Therefore, the development of microtubule stabilizers has become a potential treatment for the disease. Drug. Microtubule stabilizers are commonly used drugs in tumor chemotherapy. Similar to GSK-3 inhibitors, the side effects of these drugs are also difficult to control. In 2012, Bristol-Myers Squibb (BMS) started a phase I clinical trial to evaluate the safety of the microtubule stabilizer epothilone D, but after the clinical trial, BMS terminated the development of this indication.

Another theory is to use the immune system to eliminate pathogenic Tau protein. Tau protein immunotherapy is also divided into active immunity and passive immunity. Active immunization uses antigens to activate human immune cells to produce antibodies against a certain type of pathogenic Tau protein, while passive immunization uses monoclonal antibodies directly. So far, active immunotherapy has entered the clinic with two vaccines: Axon Neuroscience SE's AADvac-1, and Janssen's ACI-35. ACI-35 is undergoing a phase Ib clinical trial, and AADvac-1 will complete the clinical trial for effectiveness verification in 2019. AbbVie's monoclonal antibody ABBV-8E12 in passive immunotherapy is undergoing phase II clinical trials. After the failure of Verubecestat, Merck purchased the rights to a Tau monoclonal antibody from Teijin. Biogen purchased BMS-986168 from BMS, which is also in phase II clinical trials.

Parkinson's disease (PD) is another chronic central nervous system degenerative disease that mainly affects the motor nervous system. The disease is named after the British doctor James Parkinson, who published the book "An Essay on the Shaking Palsy" in 1817, which first detailed the symptoms of Parkinson's disease. . The symptoms of Parkinson’s disease usually appear slowly over time. The most obvious early symptoms are tremor, limb stiffness, hypokinesia, and abnormal gait. There may also be cognitive and behavioral problems [Parkinson's Disease Information Page.NINDS.2016-06 -30]. Dementia is quite common in severely ill patients, and major depressive disorder and anxiety disorders also occur in more than one-third of cases. Other possible symptoms include perception, sleep, and emotional problems [Shulman JM et al, Annual Review of Pathology, 6:193–222, 2011].

In 2015, about 6.2 million people worldwide suffered from Parkinson's disease and 117,000 people died [GBD, Lancet, 388(10053):1459-1544, 2016]. Parkinson's disease usually occurs in people over 60 years old, and about 1% of the elderly suffer from the disease; men are more likely to get Parkinson's disease than women. If the patient is younger than 50 years of age, it is called early-onset Parkinson's disease. The expected life expectancy after Parkinson’s disease is confirmed is about 7-14 years [Sveinbjornsdottir S, Journal of Neurochemistry, 139:318-324, 2016].

The cause of Parkinson's disease is still unclear, but it is generally believed to be related to genetic and environmental factors. People in the family with Parkinson's disease are more likely to get this disease, and those who have been exposed to certain pesticides and have had head trauma are also at higher risk. The main motor symptoms caused by Parkinson's disease are collectively called Parkinson's syndrome. The main motor symptom of Parkinson's disease is caused by the death of substantia nigra cells of the midbrain, resulting in insufficient dopamine in the relevant brain areas of the patient. The cause of cell death is currently poorly understood, but it is known to be related to the process of neuronal protein forming Lewy bodies [Kalia LV; Lang AE, Lancet, 386(9996):896–912, 2015].

In terms of pathophysiology, due to the accumulation of α-synuclein in the form of Lewy bodies, Parkinson’s disease is regarded as a synuclein disease, which is the same as the nerve formed by the accumulation of Tau protein in Alzheimer’s disease. Fiber entanglement is different. However, synucleinopathy and Tau proteinopathy have clinical overlaps. Patients with severe Parkinson's disease often have typical symptoms of Alzheimer's disease (dementia), and they often find it in their brains. Nerve fiber entanglement [Galpern WR, Lang AE, Annals of Neurology, 59(3):449-458, 2006].

The latest research shows that Tau is an important participant in neurodegenerative diseases and is related to Parkinson's disease. The concentration of total Tau (t-Tau) in cerebrospinal fluid (CSF) and the ratio of cerebrospinal fluid/serum albumin gradually increase during the H and Y stages of Parkinson's disease. At the same time, there is a positive correlation between the t-Tau level in CSF of PD patients and the ratio of cerebrospinal fluid/serum albumin and motor dysfunction. Studies conducted in cognitively intact patients with Parkinson's disease confirmed the gradual increase in Tau protein levels and blood-brain barrier (BBB) damage in CSF and the pathological progression of PD. Since the blood-brain barrier ensures the removal of Tau protein from the brain, the dysfunction of the blood-brain barrier during the entire disease progression may lead to an increase in the level of Tau protein in the CSF in PD at the same time [Liguori Cetal, CNS Neurol Disord Drug Targets, 16(3 ):339-345,2017].

There is currently no cure for Parkinson's disease. The initial symptoms are usually treated with L-dopa. When the effect of L-dopa decreases, dopamine agonists are used together. As the course of the disease worsens, neurons will continue to be lost, so the dose of the drug must be increased accordingly, but when the dose is just increased, side effects of dyskinesia such as involuntary tics will occur. For severe patients whose drugs are not effective, deep brain stimulation surgery in neurosurgery can be considered, which uses microelectrode discharge to reduce motor symptoms. As for non-exercise-related symptoms of Parkinson's disease (such as patients with sleep disturbance or emotional problems), the treatment effect is usually poor [The National Collaborating Centre for Chronic Conditions, Symptomatic pharmacological therapy in Parkinson's disease. Parkinson's Disease.London: Royal College of Physicians. 2006: 59-100.; Kalia LV, Lang AE, Lancet, 386(9996):896-912, 2015.].

It can be seen that there is currently no particularly effective treatment for Alzheimer's disease and Parkinson's disease, and finding new Tau protein expression inhibitors that can smoothly pass through the blood-brain barrier will be a way out.

Histone post-translational modification (PTM) regulates the organization and function of vertebrate genomes, and is a key participant in epigenetic regulation of gene activity [Di Lorenzo A, Bedford MT, FEBS Letters, 585(13): 2024-2031, 2011 ]. So far, most of the modified enzymes of PTM have been studied through isolation, in vitro modification or chemical inhibition or silencing/overexpression methods. Several evidences show that post-translational modifications (PTMs) regulate the functions of Tau, including: its subcellular localization, clearance, aggregation, toxicity and pathological spread [Coughlin D, Irwin D, Current Neurology and Neuroscience Reports, 17(9): 72, 2017; Hanger DP, et al., Trends in Molecular Medicine, 15(3): 112-119, 2009]. Recent advances in semi-synthetic strategies have allowed the introduction of single or multiple different PTMs into recombinant proteins at specific sites, successfully demonstrating K acetylation, Y and S phosphorylation of the microtubule binding domain of Tau. Such a strategy provides new opportunities for investigating other types of PTM in Tau or other severely post-translationally modified proteins [Haj-Yahya M, Luxuel HA, Journal of the American Chemical Society 140:6611-6621, 2018].

The mono- and dimethylation of arginine is catalyzed by three types of protein arginine methyltransferase (PRMT). 11 PRMTs have been reported in mammals: PRMT1, 3, 4, 6 and 8 belong to type I. PRMT5 and 9 belong to type II, and PRMT7 is a type III methyltransferase. PRMT uses S-adenosyl-L-methionine (AdoMet) to catalyze the methylation of arginine to form monomethylarginine (MMA) and asymmetric (u-NG, u-NG-dimethyl- arginine or ADMA (Type I) or symmetric (u-NG, u-N0G-dimethylarginine or SDMA) (Type II) [Bedford MT, Clarke SG, Molecular Cell, 33:1-13, 2009]. PRMT participates in the regulation of chromatin structure and function through transcriptional activation, inhibition and its interaction with chromatin barrier elements [Aravind L, et al., Progress in Molecular Biology and Translational Science, 101: 105-76, 2011]. PRMT is also involved in pre-mRNA splicing, nuclear/cytoplasmic shuttling, cell cycle and DNA repair [Bezzi M, et al., Genes & Development, 27:1903-1916, 2013].

PRMT8 is a type I PRMT, which mainly exists in the neuronal area of the brain. It can asymmetrically dimethyl histone H4R3 to form H4R3me2a [Scaramuzzino C, PLoS One, 8: e61576, 2013]. The subcellular location of the enzyme is located in the plasma membrane because it can interact with membrane lipids using its N-terminal myristoylation motif. The N-terminal region also contains two proline-rich motifs, allowing it to interact with several SH3 domains and PRMT2. The enzyme activity of this enzyme exists in its N-terminal conformation, and its enzyme activity increases with the loss of this domain [Sayegh J, et al., Journal of Biological Chemistry, 282:36444-36453, 2007].

ADDA (adenosine-2', 3'-dialdehyde), 2', 3'-dialdehyde adenosine, its C ₁₀ H ₁₁ N ₅ O ₄ molecular weight is 265.23, and the structural formula is:

At present, there is no report on the application of ADDA in the preparation of inhibitors of Tau protein and in the preparation of drugs for the treatment of Alzheimer's disease and Parkinson's disease.

Summary of the invention

The purpose of the present invention is to provide ADDA for preparing inhibitors of Tau protein, and new uses in preparing drugs for treating Alzheimer's disease and Parkinson's disease.

The technical scheme of the present invention includes:

Use of the compound represented by formula (I) or its metabolites, tautomers, stereoisomers, pharmaceutically acceptable salts, hydrates and/or solvates in the preparation of Tau protein inhibitors; (I):

According to the aforementioned use, the Tau protein inhibitor is a drug that inhibits the expression of Tau protein.

According to the aforementioned application, the Tau protein inhibitor is a drug that inhibits the asymmetric dimethylation of the third arginine residue of histone H4.

According to the aforementioned use, the Tau protein inhibitor is a drug for inhibiting the formation of Aβ senile plaques in the brain.

According to the aforementioned use, the drug is a drug for treating diseases of the central nervous system.

According to the aforementioned use, the drug is a drug for treating chronic diseases of the central nervous system.

According to the aforementioned use, the drug is a drug for treating Alzheimer's disease and Parkinson's disease.

A drug for the treatment of central nervous system diseases, which is a compound represented by formula (I) or its metabolites, tautomers, stereoisomers, pharmaceutically acceptable salts, hydrates and/or solvents The compound is the active ingredient, plus pharmaceutically acceptable excipients or auxiliary ingredients to prepare a preparation; formula (I):

According to the aforementioned drugs, the auxiliary materials include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, and synergists in the pharmaceutical field. Agent.

According to the aforementioned medicine, the preparation is an injection preparation or an oral preparation.

According to the aforementioned medicine, the injection preparation is a powder injection.

The term "pharmaceutically acceptable salt" refers to a salt formed by the compound of the present invention and an acid or a base suitable for use as a medicine. Pharmaceutically acceptable salts include inorganic salts and organic salts.

The pharmaceutically acceptable excipient has certain physiological activity, but the addition of the component will not change the dominant position of the above-mentioned pharmaceutical composition in the course of disease treatment, but only exerts auxiliary functions. These auxiliary functions are only for the component The utilization of known activity is a commonly used adjuvant therapy in the medical field. If the aforementioned auxiliary components are used in combination with the pharmaceutical composition of the present invention, they should still fall within the protection scope of the present invention.

The present invention has the following beneficial effects:

1) The ADDA of the present invention can effectively inhibit the expression of Tau protein, and thus can theoretically be used to treat various central nervous system diseases caused by Tau protein, especially neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease.

2) The ADDA of the present invention can inhibit the asymmetric double methylation of the third arginine residue of histone H4.

3) The ADDA of the present invention has the ability to penetrate the blood-brain barrier and is suitable for various administration methods.

4) ADDA of the present invention can inhibit the formation of Aβ senile plaques in the brain, so that it can be used to treat AD in theory; at the same time, the experiment of the present invention also proves that ADDA can be used to treat AD.

Obviously, according to the above content of the present invention, according to common technical knowledge and conventional means in the field, various other modifications, substitutions or changes can be made without departing from the above basic technical idea of the present invention.

Hereinafter, the above-mentioned content of the present invention will be further described in detail through specific implementations in the form of examples. However, it should not be understood that the scope of the above-mentioned subject of the present invention is limited to the following examples. All technologies implemented based on the foregoing content of the present invention belong to the scope of the present invention.

Description of the drawings

Figure 1: A statistical diagram of the effects of different concentrations of ADDA on the transcription of Tau genes in human astrocytes.

Figure 2: Western blot detection of Tau protein in human astrocytes treated with different concentrations of ADDA.

Figure 3: The effect of PRMT8 expression interference on the level of histone modification H4R3me2a.

Figure 4: Detection diagram of PRMT8 and Tau gene expression.

Figure 5: HPLC detection chart of ADDA content in orbital blood (serum sample 0-4h) and brain tissue (brain tissue 0-4h).

Figure 6: A statistical diagram of the effect of ADDA on the expression of Tau mRNA in primary neurons of the mouse cerebral cortex.

Figure 7: Immunoblotting image of Tau protein in primary neurons of mouse cerebral cortex under ADDA treatment.

Figure 8: Immunoblot of H4R3me2a in primary neurons of mouse cerebral cortex under ADDA treatment.

Figure 9: The escape latency and escape route diagram of AD model mice in the water maze experiment.

Figure 10: Detection of senile plaque formation in the brain region of AD model mice.

Detailed ways

Material: ADDA (adenosine-2', 3'-dialdehyde, 2', 3'-dialdehyde adenosine) was purchased from Sigma, USA.

Example 1 Preparation of anti-Alzheimer's disease and Parkinson's disease medicine of the present invention

Take ADDA (adenosine-2’, 3’-dialdehyde, 2’, 3’-dialdehyde adenosine), add pharmaceutically acceptable excipients, and make a medicine.

The following test examples are used to verify the beneficial effects of the present invention:

Test Example 1. Real-time PCR detection of the effect of ADDA on Tau mRNA expression

Using human astrocyte SVG p12 as the research object, the cells were treated with different ADDA concentrations to explore the effect of different ADDA on the expression of Tau mRNA in SVG p12. As shown in Figure 1, in human astrocyte SVG p12, compared with the control group, ADDA can significantly inhibit Tau mRNA expression; and, the higher the concentration of ADDA, the more obvious the inhibitory effect on Tau mRNA.

The implementation steps are as follows:

Firstly, prepare 1mM ADDA stock solution with 0.04M HCl, and add the mother solution to the culture medium to make the final concentration of ADDA be 1μM, 2μM, 4μM and 8μM, respectively, 0.04M HCl as a control, re-dosing every 24h; ADDA treatment After 72 hours, the cells were trypsinized. The cells were collected by centrifugation at 1000 rpm. The cells were washed twice with 1′PBS. Then, 1 mL of Trizol was added to gently blow the cells evenly, and all the cell solutions were transferred to a 1.5 mL EP tube (all the cells were removed during the RNA extraction process). The EP tubes and Tip tips used must be treated with 0.1% DEPC water and used after damp heat sterilization), and let stand at room temperature for 5 minutes; add 200 mL of chloroform (chloroform), shake vigorously for 15 seconds, and stand at room temperature for 2 to 3 minutes. Centrifuge for 15 minutes at 12000'g, 4°C. Transfer the upper aqueous phase to a new enzyme-free EP tube, add 500 mL of isopropanol, and let it stand at room temperature for 10 minutes. Centrifuge at 12000g, 4°C for 10min. Remove the supernatant, add 1ml of 75% pre-cooled ethanol (prepared with 0.1% DEPC water), and vortex for a while. 7500'g, 4℃, centrifuge for 5min. Remove the supernatant, dry, and dissolve it in 20 mL of 0.1% DEPC water. Place it at 55°C for 10 minutes to promote dissolution. Use a spectrophotometer to determine the concentration and purity, and perform the following RNA reverse transcription procedures:

Prepare the following mixture in the RNase free EP tube:

Mix gently with a pipette, 42°C for 2 min. Then add 5×qRT SuperMix II, mix gently with a pipette, and synthesize cDNA according to the following procedure:

25℃ 10min

50℃ 30min

85℃ 5min

The product can be used in PCR reaction immediately or stored at -30°C for later use.

Real-time PCR uses the Rotor-gene 6000 quantitative PCR instrument from Gene's Corbett Research, the reagent is Roche FastStart Universal SYBR Green Master Mix, and the reaction system is as follows:

Real-time PCR reaction conditions are as follows:

The list of Tau primers is as follows:

Test Example 2. Western blot to detect the effect of ADDA on the expression of Tau protein

The results in Figure 2 show that after ADDA treatment of astrocytes SVG p12, compared with the control (0.04M HCl), the Tau protein level was significantly down-regulated, indicating that in astrocytes SVG p12, ADDA can inhibit the expression of Tau protein.

The specific implementation steps are as follows:

Using human astrocyte SVG p12 as the research object, the cells were treated with different ADDA concentrations to explore the effect of different ADDA on the expression of Tau protein in SVG p12. Before treating the cells, first prepare 1 mM ADDA stock solution with 0.04M HCl, and add the mother solution to the culture medium to make the final concentration of ADDA 2μM, 4μM and 8μM, and re-dosing every 24h; after ADDA treatment cells 72h, Trypsin digest the cells, collect the cells by centrifugation at 1000 rpm, and wash the cells twice with 1'PBS, add RIPA cell lysate (effective lysis component is 1% Triton X-100), lyse on ice for 30 minutes and centrifuge at 14000 rpm for 10 minutes to collect the protein Use the BSA method to determine the protein concentration of the supernatant and store it at -30°C for later use.

According to the molecular weight of the protein, the concentrated gel and separating gel of the appropriate concentration are prepared. Take 40-60μg protein sample and mix it with 4'SDS loading buffer 1:4. Then heat at 100°C for 5 min, and centrifuge at 12000 rpm for 5 min. Add the protein sample to the loading channel, and add 5μL of protein Marker to the left channel of the sample. After loading the sample, turn on the power and adjust the constant voltage for 90V electrophoresis. After the protein sample enters the separation gel, adjust the voltage to 120V to continue electrophoresis. When the bromophenol blue front reaches the bottom of the separation gel, stop the electrophoresis and take out the gel. . Remove the gel sandwich and take out the gel. According to the position of the band indicated by the protein Marker, cut the gel corresponding to the target band. Cut the PVDF membrane corresponding to the size of the gel, soak it in methanol for activation, and soak the transferred membrane filter paper in a semi-dry transfer buffer for 10 minutes. Assemble the transfer membrane sandwich in the membrane transfer instrument, and load it in the order of filter paper-gel-PVDF membrane-filter paper from top to bottom, and pay attention to no bubbles in the sandwich, adjust the voltage to 24V, and set the transfer membrane according to the size of the protein time. After the transfer, the PVDF membrane was placed in a PBST solution containing 5% skimmed milk powder. After blocking at room temperature for 1 hour, the blocking solution was discarded, the corresponding primary antibody and internal reference GAPDH antibody (diluted according to the antibody instruction) were added, and incubated overnight at 4°C. The next day, the PVDF membrane was taken out and washed 4 times with 1'PBST for 10 minutes each time. Add HRP-labeled anti-mouse or rabbit secondary antibody and incubate at room temperature for 1 hour. After the secondary antibody incubation, wash 4 times with 1'PBST for 10 minutes each time. Drop the ECL luminescent liquid on the PVDF film. In the dark room, cut the X film of the appropriate size, put it in the press clamp and press for 1 to 5 minutes, open the press clamp and take out the light film for development, fixation, rinse and dry , Scan the film and observe the results.

Test Example 3. Western blot detects the effect of PRMT8 expression interference on the level of histone modification H4R3me2a

In human astrocytes SVG p12, specific interfering small RNA (siRNA) was used to silence protein arginine methyltransferase PRMT8 gene expression, and to detect PRMT8's potential catalytic substrate H4R3me2a (Histone H4 third arginine residue) Base asymmetric bismethylation) level changes. The results in Figure 3 show that the H4R3me2a modification level is significantly reduced after interference with PRMT8 expression.

The implementation steps are as follows:

Using human astrocyte SVG p12 as the research object, firstly use artificially synthesized PRMT8 interference small RNA (siRNA; the interference sequence is GACAGUACAAGGACUUCAA) to transfect human astrocyte SVG p12, which will grow well the day before transfection. Cells are seeded in a six-well plate, and transfection can be performed the next day when the cell confluence reaches 60% to 70%. Gently mix PRMT8siRNA and Lipo3000 transfection reagent in two EP tubes, then gently mix the two together. After standing at room temperature for 10-15 minutes, add the transfection reagent mixture dropwise to the cell culture medium . After 72 hours of culture, the cells were trypsinized, collected by centrifugation, and washed twice with 1′PBS. The cells were divided into two parts, one part was used to extract whole cell proteins, and the other part was used to extract nuclear histones. For whole-cell protein extraction, add RIPA cell lysate (effective lysis component is 1% Triton X-100), lyse on ice for 30 minutes and centrifuge at 14000 rpm for 10 minutes, collect the protein supernatant, and determine the protein concentration by BSA method, -30℃ Save it for later use. For nuclear histone extraction, use Triton extraction solution (containing 0.5% Triton X-100, 2mmol/L PMSF and 0.02% NaN ₃ ) to resuspend the cells, lyse on ice for 10 minutes, centrifuge at 6500′g for 10 minutes, and discard The supernatant, washed the cells with half of the Triton extract in the previous step, added 0.2M HCl suspension cell mass, 4°C overnight, centrifuged at 6500'g for 10 minutes to collect the supernatant, measured the concentration and stored at -30°C for use.

Next, perform a Western blot experiment. According to the molecular weight of the protein, prepare concentrated gels and separation gels with appropriate concentrations. Take 40-60μg protein sample and mix it with 4'SDS loading buffer 1:4. Then heat at 100°C for 5 min, and centrifuge at 12000 rpm for 5 min. Add the protein sample to the loading channel, and add 5μL of protein Marker to the left channel of the sample. After loading the sample, turn on the power and adjust the constant voltage for 90V electrophoresis. After the protein sample enters the separation gel, adjust the voltage to 120V to continue electrophoresis. When the bromophenol blue front reaches the bottom of the separation gel, stop the electrophoresis and take out the gel. . Remove the gel sandwich and take out the gel. According to the position of the band indicated by the protein Marker, cut the gel corresponding to the target band. Cut the PVDF membrane corresponding to the size of the gel, soak it in methanol for activation, and soak the transferred membrane filter paper in a semi-dry transfer buffer for 10 minutes. Assemble the transfer membrane sandwich in the membrane transfer instrument, and load it in the order of filter paper-gel-PVDF membrane-filter paper from top to bottom, and pay attention to no bubbles in the sandwich, adjust the voltage to 24V, and set the transfer membrane according to the size of the protein time. After the transfer, the PVDF membrane was placed in a PBST solution containing 5% skimmed milk powder. After blocking for 1 hour at room temperature, the blocking solution was discarded, the corresponding primary antibody and internal reference antibody (diluted according to the antibody instruction) were added, and incubated overnight at 4°C. The next day, the PVDF membrane was taken out and washed 4 times with 1'PBST for 10 minutes each time. Add HRP-labeled anti-mouse or rabbit secondary antibody and incubate at room temperature for 1 hour. After the secondary antibody incubation, wash 4 times with 1'PBST for 10 minutes each time. Drop the ECL luminescent liquid on the PVDF film. In the dark room, cut the X film of the appropriate size, put it in the press clamp and press for 1 to 5 minutes, open the press clamp and take out the light film for development, fixation, rinse and dry , Scan the film and observe the results.

Test Example 4. PRMT8 regulates the expression of Tau gene

In human astrocytes SVG p12, specific interfering small RNA (siRNA) is used to silence the expression of protein arginine methyltransferase PRMT8 gene, and the expression changes of Tau mRNA are detected. The results in Fig. 4 show that the expression level of Tau mRNA that interferes with PRMT8 expression is significantly reduced compared with the control.

The specific experimental steps are as follows:

Using human astrocyte SVG p12 as the research object, firstly use artificially synthesized PRMT8 interfering small RNA (siRNA; the interference sequence is GACAGUACAAGGACUUCAA) to transfect human astrocyte SVG p 12, which will grow well one day before transfection Inoculated into a six-well plate, the next day when the cell confluence reaches 60% to 70%, transfection can be performed. Gently mix PRMT8siRNA and Lipo3000 transfection reagent in two EP tubes, then gently mix the two together. After standing at room temperature for 10-15 minutes, add the transfection reagent mixture dropwise to the cell culture medium . After culturing for 72 hours, trypsinize the cells, collect the cells by centrifugation, wash the cells twice with 1′PBS, add 1mL Trizol to gently blow the cells, and transfer all these cell solutions to a 1.5mL EP tube (used in the RNA extraction process) The EP tube and Tip must be treated with 0.1% DEPC water and used after damp heat sterilization), stand at room temperature for 5 minutes; add 200 mL of chloroform (chloroform), shake vigorously for 15 seconds, and stand at room temperature for 2 to 3 minutes. Centrifuge for 15 minutes at 12000'g, 4°C. Transfer the upper aqueous phase to a new enzyme-free EP tube, add 500 mL of isopropanol, and let it stand at room temperature for 10 minutes. Centrifuge at 12000g, 4°C for 10min. Remove the supernatant, add 1ml of 75% pre-cooled ethanol (prepared with 0.1% DEPC water), and vortex for a while. 7500'g, 4℃, centrifuge for 5min. Remove the supernatant, dry, and dissolve it in 20 mL of 0.1% DEPC water. Place it at 55°C for 10 minutes to promote dissolution. Use a spectrophotometer to determine the concentration and purity, and perform the following RNA reverse transcription procedures:

Prepare the following mixture in the RNase free EP tube:

Mix gently with a pipette, 42°C for 2 min. Then add 5×qRT SuperMix II, mix gently with a pipette, and synthesize cDNA according to the following procedure:

25℃ 10min

50℃ 30min

85℃ 5min

The product can be used in PCR reaction immediately or stored at -30°C for later use.

Real-time PCR uses the Rotor-gene 6000 quantitative PCR instrument from Gene's Corbett Research, the reagent is Roche FastStart Universal SYBR Green Master Mix, and the reaction system is as follows:

Real-time PCR reaction conditions are as follows:

Test Example 5. ADDA permeates the blood-brain barrier in mice

The ability of drugs to penetrate the blood-brain barrier is a prerequisite for the treatment of Alzheimer's disease. Use tail vein injection to inject ADDA into mice to observe and analyze whether ADDA exists in the blood and brain tissues of mice to verify whether ADDA can penetrate the blood-brain barrier of mice. As shown in Figure 5, tail vein injection of ADDA Later, we observed that ADDA was present in the blood and brain tissues of mice, and the ADDA content reached its peak 2 to 3 hours after injection.

The implementation steps are as follows:

The experimental mice were purchased from the Model Animal Research Institute of Nanjing University, females, 6-8 weeks old. The concentration of ADDA mother solution was 0.25mg/100μL, the solvent was 0.04M HCl, and 100μL of ADDA solution was injected into mice by tail vein injection. After 0, 1, 2, 3, 4, and 5 hours after injection of ADDA into the tail vein, the mice were sacrificed by cervical dislocation, blood was collected from the orbit and brain tissue was extracted. For serum samples, add 1 times the amount of normal saline to the blood, centrifuge at 10,000 rpm for 10 minutes, aspirate the supernatant, and filter with 0.22 μm for use. For brain tissue samples, wash the taken brain tissue with normal saline to remove blood stains, blot dry with filter paper, peel off meningeal blood vessels, homogenize the brain tissue: normal saline=1:1.5 (W/V), centrifuge for 10 minutes, and take it. Clear liquid, 0.22μm filter for use. Strictly filter the chromatographic pure mobile phase before sampling, and choose different filter membranes as needed. The mobile phase after suction filtration is ultrasonically degassed for 20-30 minutes. Open the chromatographic workstation, connect the mobile phase pipeline, and connect the detection system. For a new mobile phase, the pump and injection valve need to be flushed first. Flush the pump directly at the outlet of the pump and draw with a needle. Flush the injection valve. The flushing speed is not too high. Adjust the flow rate. When using a new mobile phase for the first time, try the pressure first. The greater the flow rate, the greater the pressure, generally not more than 2000psi. Choose an appropriate flow rate. Set the detector analysis parameters for different samples. When the baseline becomes stable, the sample can be injected and analyzed. The pre-processing of the sample is very important when analyzing the sample. Before the sample is subjected to high-performance liquid chromatography (HPLC), analyze the ADDA standard, explore the column and mobile phase of ADDA detection and analysis, determine the detection wavelength of the standard, and determine the time and location of the peak of the standard. Then, compare the analysis results of the samples with those of the standard products.

Test Example 6. Real-time PCR detection of the effect of ADDA on the expression of Tau mRNA in primary neurons of mouse cerebral cortex

Using mouse cerebral cortex primary neuron cells as the research object, we explored the effects of different ADDA treatment concentrations on the expression of Tau mRNA in mouse neuronal cells. It was found that as the concentration of ADDA increased, the expression of Tau mRNA decreased.

The specific implementation steps are as follows:

First, the primary neurons of the mouse cortex were extracted. The pregnant mouse (C57BL/6J) was sacrificed by cervical dissection, the abdominal cavity of the pregnant mouse was opened with dissecting scissors, and the fetal mouse (E12.5) was taken out, and the brain of the fetal mouse was immersed in pre-cooled PBS. The skin was removed, the skull was removed, and the fetal rat cortex was separated and placed in pre-cooled HBSS (Hanks balanced salt solution). Under a dissecting microscope, use ophthalmic forceps and tweezers to remove the meninges and superficial capillaries until the tissue is milky white. Cut the cortex tissue and move it to a 15mL petri dish, add 0.25% EDTA trypsin to digest at 37°C for 30 minutes, and observe the digestion under a microscope every 10 minutes. Add the same amount of Neurobasal medium containing 10% FBS to terminate the digestion. After centrifugation, the cells were resuspended in Neurobasal complete medium (containing 2% B27 and 2mM Glutamine) and seeded with Poly-D-lysine (PDL) at a density of 1.5×10 ⁵ cells/cm ² ) The coated 24-well plate is cultured. After the cells adhere to the wall, the mouse neuron cells are treated with different ADDA concentrations.

Prepare 1 mM ADDA stock solution with 0.04M HCl, and add the mother solution to the culture medium to make the final concentrations of ADDA be 2μM, 4μM, 8μM and 16μM, respectively, 0.04M HCl as a control, re-dosing every 24h; ADDA treated cells After 72 hours, trypsinize the cells, collect the cells by centrifugation at 1000 rpm, and wash the cells twice with 1′PBS. Then add 1 mL Trizol to gently blow the cells, and transfer all the cell solutions to a 1.5 mL EP tube (used in the RNA extraction process). The EP tube and Tip must be treated with 0.1% DEPC water and used after damp heat sterilization), stand at room temperature for 5 minutes; add 200 μL of chloroform (chloroform), shake vigorously for 15 seconds, and stand at room temperature for 2 to 3 minutes. Centrifuge for 15 minutes at 12000'g, 4°C. Transfer the upper aqueous phase to a new enzyme-free EP tube, add 500 μL of isopropanol, and let it stand at room temperature for 10 minutes. Centrifuge at 12000g, 4°C for 10min. Remove the supernatant, add 1ml of 75% pre-cooled ethanol (prepared with 0.1% DEPC water), and vortex for a while. 7500'g, 4℃, centrifuge for 5min. Remove the supernatant, dry, and dissolve it in 20μL of 0.1% DEPC water. Place it at 55°C for 10 minutes to promote dissolution. Use a spectrophotometer to determine the concentration and purity, and perform the following RNA reverse transcription procedures:

Prepare the following mixture in the RNase free EP tube:

Mix gently with a pipette, 42°C for 2 min. Then add 5×qRT SuperMix II, mix gently with a pipette, and synthesize cDNA according to the following procedure:

25℃ 10min

50℃ 30min

85℃ 5min

The product can be used in PCR reaction immediately or stored at -30°C for later use.

Real-time PCR uses the Rotor-gene 6000 quantitative PCR instrument from Gene's Corbett Research, the reagent is Roche FastStart Universal SYBR Green Master Mix, and the reaction system is as follows:

Real-time PCR reaction conditions are as follows:

Test Example 7. Western blot detection of the effect of ADDA on the expression of Tau protein in primary neurons of mouse cerebral cortex

After ADDA treated mouse cerebral cortex primary neuron cells, compared with the control (0.04M HCl), Western blot was used to detect the expression level of Tau protein. It was found that the Tau protein in the primary cells of the cerebral cortex of mice treated with ADDA decreased with the increase of ADDA concentration (Figure 7).

The specific implementation steps are as follows:

The primary neuron cells of the mouse cerebral cortex were extracted according to the method described in Test Example 6, and the cells were treated with different ADDA concentrations to explore the effect of different ADDA on the expression of Tau protein in neuronal cells. Before treating the cells, first prepare 1mM ADDA mother liquor with 0.04M HCl, and add the mother liquor to the culture medium to make the final concentrations of ADDA be 2μM, 4μM, 8μM and 16μM respectively, and re-dosing every 24h; ADDA treated cells for 72h After that, trypsinize the cells, collect the cells by centrifugation at 1000 rpm, wash the cells twice with 1'PBS, add RIPA cell lysate (effective lysis component is 1% Triton X-100), lyse on ice for 30 minutes and centrifuge at 14000 rpm for 10 minutes. Collect the protein supernatant, use the BSA method to determine the protein concentration, and store it at -30°C for later use.

According to the molecular weight of the protein, the concentrated gel and separating gel of the appropriate concentration are prepared. Take 40-60μg protein sample and mix it with 4'SDS loading buffer 1:4. Then heat at 100°C for 5 min, and centrifuge at 12000 rpm for 5 min. Add the protein sample to the loading channel, and add 5μL of protein Marker to the left channel of the sample. After loading the sample, turn on the power and adjust the constant voltage for 90V electrophoresis. After the protein sample enters the separation gel, adjust the voltage to 120V to continue electrophoresis. When the bromophenol blue front reaches the bottom of the separation gel, stop the electrophoresis and take out the gel. . Remove the gel sandwich and take out the gel. According to the position of the band indicated by the protein Marker, cut the gel corresponding to the target band. Cut the PVDF membrane corresponding to the size of the gel, soak it in methanol for activation, and soak the transferred membrane filter paper in a semi-dry transfer buffer for 10 minutes. Assemble the transfer membrane sandwich in the membrane transfer instrument, and load it in the order of filter paper-gel-PVDF membrane-filter paper from top to bottom, and pay attention to no bubbles in the sandwich, adjust the voltage to 24V, and set the transfer membrane according to the size of the protein time. After the transfer, the PVDF membrane was placed in a PBST solution containing 5% skimmed milk powder. After blocking for 1 hour at room temperature, the blocking solution was discarded, the corresponding primary antibody and internal reference antibody (diluted according to the antibody instruction) were added, and incubated overnight at 4°C. The next day, the PVDF membrane was taken out and washed 4 times with 1'PBST for 10 minutes each time. Add HRP-labeled anti-mouse or rabbit secondary antibody and incubate at room temperature for 1 hour. After the secondary antibody incubation, wash 4 times with 1'PBST for 10 minutes each time. Drop the ECL luminescent liquid on the PVDF film. In the dark room, cut the X film of the appropriate size, put it in the press clamp and press for 1 to 5 minutes, open the press clamp and take out the light film for development, fixation, rinse and dry , Scan the film and observe the results.

Test Example 8. Western blot detection of the effect of ADDA on the H4R3me2a level of primary neurons in the cerebral cortex of mice

Using mouse cerebral cortex primary neuron cells as the research object, to explore the effect of ADDA treatment on the level of mouse neuronal cells H4R3me2a.

The results showed that the H4R3me2a of 4μM ADDA-treated cells could not be detected (Figure 8).

The specific implementation steps are as follows:

The primary neuron cells of the mouse cerebral cortex were extracted according to the method described in Example 6, and the mouse cerebral cortex neuron cells were treated with ADDA to explore the effect of ADDA on the level of H4R3me2a in neuronal cells. Before treating the cells, first prepare 1mM ADDA mother liquor with 0.04M HCl, and add the mother liquor to the culture medium to make the final concentration of ADDA 4μM, and re-dosing every 24h; after 72h ADDA treating the cells, trypsinize the cells. The cells were collected by centrifugation at 1000 rpm and washed twice with 1×PBS. The cells were resuspended in Triton extract (containing 0.5% Triton X-100, 2mmol/L PMSF and 0.02% NaN3), and lysed on ice for 10 minutes, 6500× Centrifuge for 10 minutes at g, discard the supernatant, and add 0.2M HCl suspension cell pellet, overnight at 4°C, and centrifuge at 6500×g for 10 minutes to collect the supernatant. After determining the concentration, store at -30°C for later use.

Prepare the concentrated gel and separating gel of the appropriate concentration. Mix the protein sample with 4×SDS loading buffer 1:4. Then heat at 100°C for 5 min, and centrifuge at 12000 rpm for 5 min. Add the protein sample to the loading channel, and add 5μL of protein Marker to the left channel of the sample. After loading the sample, turn on the power and adjust the constant voltage for 90V electrophoresis. After the protein sample enters the separation gel, adjust the voltage to 120V to continue electrophoresis. When the bromophenol blue front reaches the bottom of the separation gel, stop the electrophoresis and take out the gel. . Remove the gel sandwich and take out the gel. According to the position of the band indicated by the protein Marker, cut the gel corresponding to the target band. Cut the PVDF membrane corresponding to the size of the gel, soak it in methanol for activation, and soak the transferred membrane filter paper in a semi-dry transfer buffer for 10 minutes. Assemble the transfer membrane sandwich in the membrane transfer instrument, and load it in the order of filter paper-gel-PVDF membrane-filter paper from top to bottom, and pay attention to no bubbles in the sandwich, adjust the voltage to 24V, and set the transfer membrane according to the size of the protein time. After the transfer, the PVDF membrane was placed in a PBST solution containing 5% skimmed milk powder. After blocking for 1 hour at room temperature, the blocking solution was discarded, the corresponding primary antibody and internal reference antibody (diluted according to the antibody instruction) were added, and incubated overnight at 4°C. The next day, the PVDF membrane was taken out and washed 4 times with 1×PBST for 10 minutes each time. Add HRP-labeled anti-mouse or rabbit secondary antibody and incubate at room temperature for 1 hour. After the secondary antibody incubation, wash 4 times with 1×PBST for 10 minutes each time. Drop the ECL luminescent liquid on the PVDF film. In the dark room, cut the X film of the appropriate size, put it in the press clamp and press for 1 to 5 minutes, open the press clamp and take out the light film for development, fixation, rinse and dry , Scan the film and observe the results.

Test Example 9. The effect of ADDA on spatial learning and memory in AD mice

Using APP/PS1 double transgenic Alzheimer's disease model mice (C57BL/6J) as the research object, after intraperitoneal injection, observe the effect of ADDA on the spatial learning and memory ability of APP/PS1 mice.

As shown in Figure 9, the water maze experiment shows that as the number of training days increases, the time for the mice in the control group and the administration group to find an underwater platform is significantly shortened. And, starting from the fourth day, compared with the APP/PS1 mice in the control group (0.04M HCl treatment), the time for the ADDA-administered mice to find an underwater platform was significantly shorter. It shows that ADDA treatment can improve the spatial learning and memory ability of APP/PS1 mice.

The specific implementation steps are as follows:

The experimental APP/PS1 double transgenic Alzheimer's disease model mice (C57BL/6J) were purchased from the Institute of Model Animals of Nanjing University, males, and started to be administered at 6 months of age. Use 0.04M HCl as a solvent to dissolve ADDA, the dosage is 12.5mg/kg, control mice (Control) are injected with the same volume of 0.04M HCl solvent, and 100μL of the prepared ADDA solution and control solvent are injected by intraperitoneal injection Mice were injected every two days for a duration of 2.5 months. The water maze experiment is a classic method to test the spatial learning and memory abilities of mice. It mainly includes a round game pool with a diameter of 1.5m, a detachable platform, and a video tracking system. In the experiment, the swimming pool is divided into four quadrants. The whole experiment should be kept in a quiet environment, and the water temperature should be maintained at 23±1℃. Before the experiment, let the mice adapt to the familiar environment, put all the mice in the swimming pool in turn, let them swim freely for 1 minute, and then guide the mice to stay on the platform for 20 seconds to help them become familiar with the surrounding environment. In the formal experiment, the mice were placed in different quadrants for training in sequence, the mice were faced to the pool wall, gently put into the water, and the swimming time (escape latency) of the mice in the water was recorded. The time is 1 min. If the mouse can find the hidden platform within 1 min and stay on the platform for 5 seconds, it is deemed to have successfully found the platform. If the mouse fails to find the platform within 1 min, it will be artificially guided to the platform to stay for 20 seconds, and the swimming time (escape latency) is 1 min by default. After each training, wipe off the body water in time and keep the body temperature. The training interval is no less than 30 minutes, and the training lasts for a total of 5 days. The training is performed at the same time every day. The video analysis system is used to record and analyze the mouse activity track. During the water maze experiment, the mice still kept the drug once every two days.

Test Example 10. Thioflavin-S (Thioflavin-S) and immunohistochemical staining methods were used to detect the effect of ADDA on the formation of senile plaques in AD mice

Aβ (β-amyloid) deposition is the main cause of senile plaques in the brain, and the corresponding senile plaques are called Aβ senile plaques. According to the amyloid cascade hypothesis, Aβ senile plaque formation is the main pathological manifestation of Alzheimer's disease (AD).

The senile plaque referred to in this test example is Aβ senile plaque.

APP/PS1 double transgenic Alzheimer's disease model mice (C57BL/6J) will have senile plaques in the cerebral cortex and hippocampus at the age of 4 months, and the number and size of senile plaques will increase with the increase of months. In order to study whether ADDA affects the formation of senile plaques in mice, we used Thioflavin-S (Thioflavin-S) and immunohistochemical staining to detect senile plaques in the brain cortex and hippocampus of control mice and ADDA-treated mice Formation. Experiments 10 and 9 used the same batch of mice. After all behavioral experiments, mice (9 months old) were sacrificed for senile plaque and IHC analysis.

As shown from the staining results in Fig. 10, compared with the control group, the ADDA treatment group had significantly less senile plaque formation in the brain cortex and hippocampus. It shows that ADDA can inhibit the formation of senile plaques in the brain regions of AD mice.

The specific implementation steps are as follows:

After the AD mouse model was put to death, the mouse's chest cavity was quickly opened, and 4% paraformaldehyde was infused from the left ventricle with a disposable syringe to remove blood from the brain. Subsequently, the mouse skull was peeled off with dissecting scissors, the brain was exposed, and the brain was separated and placed in formalin fixation solution to prepare paraffin sections (thickness of 4 μm) and frozen sections (thickness of 30 μm). Among them, paraffin sections were stained with immunohistochemistry, and frozen sections were stained with Thioflavin-S. For Thioflavin-S staining, after dewaxing with xylene and alcohol, the sections are bleached in 0.25% permanganic acid solution for 30 minutes, rinsed in deionized water for 5 minutes, and then rinsed in 0.25% acetic acid solution for 5 seconds; after rinsing in deionized water for 5 minutes Put it in blocking solution (BSA) and block for 30 minutes; rinse with deionized water for 5 minutes and drip with Thioflavin-S dye solution (prepared with 50% ethanol) for 5-8 minutes; rinse with 50% ethanol and deionized water for 2 times and use glycerin Mount the gel and take pictures under a fluorescence microscope. For immunohistochemical staining, the tissue sections were baked in a 60°C incubator for 2 hours before deparaffinization. Soak and dewax in xylene and gradient ethanol respectively, incubate with 0.5% Triton X-100 at room temperature for 15 minutes, rinse with PBS for 5 minutes; immerse the slices in 0.01M citrate buffer (pH 6.0), heat to boiling for 10-8 minutes, and take out Then it was naturally cooled to room temperature, washed with PBS and incubated with 3% H ₂ O ₂ at room temperature for 10 min to remove endogenous peroxidase. After washing with PBS, mount the slide with goat serum for 30 minutes, add Aβ antibody (1:200) dropwise, incubate at room temperature for 1 hour, wash 3 times at room temperature, add goat anti-mouse secondary antibody diluent and incubate for 30 minutes; after washing with PBS, add DAB dropwise to display Color solution, observe the staining under a microscope; rinse with deionized water and add hematoxylin for counterstaining for 5 min, rinse with deionized water; after the sections are dehydrated by alcohol gradient and transparent with xylene, mount and take pictures.

In summary, the ADDA of the present invention can be used to inhibit the expression of middle-aged dementia and Parkinson's disease related genes/protein Tau, easily penetrate the blood-brain barrier, and play a role in treating middle-aged dementia and Parkinson's disease. ADDA can also inhibit histone H4 methylation (H4R3me2a), which may be achieved by inhibiting PRMT8.

ADDA is colorless, tasteless, easy to dissolve, low concentration, stable, and safe. It has broad application prospects in the treatment of brain degenerative diseases.

Claims (11)

1. Use of the compound represented by formula (I) or its metabolites, tautomers, stereoisomers, pharmaceutically acceptable salts, hydrates and/or solvates in the preparation of Tau protein inhibitors; (I):

   
2. The use according to claim 1, wherein the Tau protein inhibitor is a drug that inhibits the expression of Tau protein.
3. The use according to claim 2, wherein the Tau protein inhibitor is a drug that inhibits the asymmetric dimethylation of the third arginine residue of histone H4.
4. The use according to claim 2, wherein the Tau protein inhibitor is a drug for inhibiting the formation of Aβ senile plaques in the brain.
5. The use according to claim 2, wherein the medicine is a medicine for treating diseases of the central nervous system.
6. The use according to claim 5, wherein the medicine is a medicine for treating chronic diseases of the central nervous system.
7. The use according to claim 5, wherein the medicine is a medicine for treating Alzheimer's disease and Parkinson's disease.
8. A drug for the treatment of central nervous system diseases, characterized in that: it is a compound represented by formula (I) or its metabolites, tautomers, stereoisomers, pharmaceutically acceptable salts, hydrates And/or solvate is a preparation prepared by adding pharmaceutically acceptable excipients or auxiliary ingredients as active ingredients; formula (I):

   
9. The medicament according to claim 8, wherein the auxiliary materials include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, and Adsorption carrier, lubricant, synergist.
10. The medicine according to claim 8, wherein the preparation is an injection preparation or an oral preparation.
11. The medicine according to claim 8, wherein the injection preparation is a powder injection.

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