CN113134003A - Application of lamivudine in preparation of medicine for treating Alzheimer's disease - Google Patents

Abstract

The invention belongs to the field of novel medicaments, relates to application of lamivudine in preparation of medicaments for treating Alzheimer disease, and simultaneously provides a medicament for treating Alzheimer disease, wherein the main active component of the medicament is lamivudine; provides a brand new idea for treating the Alzheimer disease, widens the selection field for treating the Alzheimer disease, and also makes a contribution to the further development of lamivudine.

Description

Application of lamivudine in preparation of medicine for treating Alzheimer's disease

Technical Field

The invention belongs to the field of novel medicines, relates to application of lamivudine in preparation of a medicine for treating Alzheimer disease, and simultaneously provides a medicine for treating Alzheimer disease.

Background

Alzheimer's Disease (AD) is a neurodegenerative disease, which is common in older people over 65 years of age. Irreversible damage to the brain, memory and physical function impairment occur in brain regions of part of the brain of AD patients. By 2020, more than 1000 million AD patients exist in China. The onset of AD is latent, the course of disease is long, and once the disease occurs, the disease is extremely difficult to reverse. The pathogenesis of AD is complex, and studies on its pathogenesis and prevention mechanism are actively conducted in the medical field, but the actual cause of the disease is still unclear. Unfortunately, advances in clinical trials for the treatment of AD have been limited, whether as new drug candidates or as yet marketed other disease treatment drugs.

Lamivudine (Lamivudine), also known as 3TC, is widely accepted as a nucleoside analog, is the most representative nucleoside analog with the best curative effect in clinical application at present, and is generally used for treating hepatitis b. The past research is only limited to the pharmacological action of resisting virus or resisting aging, and the research report of the action of 3TC in preparing the medicament for treating the Alzheimer disease is not available at present.

It is clear that finding new drug therapeutic targets and new therapeutic approaches for treating alzheimer's disease is a very urgent task.

Disclosure of Invention

The invention mainly relates to application of lamivudine in preparing a medicament for treating Alzheimer disease, and simultaneously provides a medicament for treating Alzheimer disease, wherein the main active component of the medicament is lamivudine; provides a brand new idea for treating the Alzheimer disease, widens the selection field for treating the Alzheimer disease, and also makes a contribution to the further development of lamivudine.

The inventor proves through experiments that:

after the administration of 3TC through gastric gavage on SAMP8 of an AD model mouse, the memory and learning capacity of an SAMP8 mouse are improved, and the inventor finds that 3TC can reduce the number of dead neurons in the hippocampal brain region of the SAMP8 mouse and can reverse the tendency of excessive rise of transposon LINE-1 and type 1 interferon reaction, aging-related secretion phenotype and AD-related pathogenic gene mRNA level in the hippocampal brain and cortical tissues of the SAMP8 mouse.

Results of network pharmacological analysis and in vitro experimental validation against 3TC indicate that 3TC can act by activating estrogen, PI3K/Akt and neuroactive ligand-receptor interaction signaling pathways. The results show that the 3TC can obviously improve AD symptoms and brain pathological injury through inhibiting inflammation, apoptosis and other ways, can be developed as a new anti-AD medicament, and provides a new means and way for treating cognitive decline and neuron injury caused by AD.

The invention has the beneficial effects that: 3TC can alleviate memory and learning decline and neuronal damage caused by AD. The invention provides a new target for preparing the medicament for treating AD, and applies 3TC to the development process of the medicament related to AD so as to prepare better treatment medicaments. The invention provides a brand new thought for the existing medicine for treating the neuron damage caused by AD, widens the selection field for treating AD, and also makes a contribution to the development of the technical field.

Drawings

FIG. 1 is a graph showing the results of improving the cognitive performance of SAMP8 mice by 3TC, using the Morris water maze test as an evaluation measure, and the Escape latency(s), the Time ratio in the target quadrant (Time in the target quadrant,%), the Number of times of crossing the platform (Number of crossing the platform), and the Time to reach the target(s) as evaluation indicators, as follows:

in the figure, A is a locus diagram of the platform found after the positioning navigation test of the 5 th day and the water enters from the second quadrant of each group of mice, the 3TC treatment group finds the platform in a shorter route than that of the untreated group,

b is a quantitative graph of the escape latency of each group of mice in a positioning navigation test every day, the escape latency of the 3 TC-treated group of mice is shorter than that of the untreated group of mice,

c is a quantitative graph of swimming speed of each group of mice in the first five days of training,

d is a trace plot of mice in a space exploration experiment, 3TC treated mice were more inclined to swim in the target quadrant than untreated mice,

e is the percentage of the total time of each group in the target quadrant of the space exploration experiment, the time of the 3TC treated mice in the target quadrant is longer than that of untreated mice,

f is the frequency of each group of mice walking through the platform in the space exploration experiment, the frequency of the 3TC treatment group of mice crossing the target platform is more than that of the untreated mice,

g is the time to target for each group of mice, the 3TC treated group of mice reached the target in a shorter time than untreated mice;

fig. 2 is a schematic diagram of the 3TC experimental results related to the reduction of the neuronal damage in the hippocampal region of the brain of the SAMP8 mouse, the tissue staining (HE staining, niemann staining and TUNEL staining) of the paraffin section of the brain is used as an evaluation means, the neuronal cell morphology in the hippocampal region, the number of healthy cells positive to niemann staining and the number of apoptotic cells positive to TUNEL staining are used as evaluation indexes, and the results are as follows:

in the figure, A is an image of HE staining of hippocampal areas of brains of all groups of mice, and the condition of disordered arrangement and loss of neuron cells of hippocampal areas of the mice after 3TC treatment is improved;

b and C are images and quantitative images of Neisseria staining of hippocampal brain of each group of mice, and healthy neuron cytosis of hippocampal region of mice after 3TC treatment;

d and E are images and quantitative graphs of TUNEL staining of hippocampal regions of brains of each group of mice, and apoptotic cells of hippocampal regions of mice are reduced after 3TC treatment;

FIG. 3 is a diagram showing mRNA levels of transposon LINE-1(L1-ORF1, L1-ORF2), type 1 interferon response (Ifna, Irf7, Oas1), senescence-associated secretory phenotype (Il6, Mmp3, Pai1) and AD-associated pathogenic gene (PS-1, APP) in the brain and hippocampal tissues of each group of mice, in which real-time fluorescent quantitative PCR was used as an evaluation means and the mRNA level of the relevant gene was used as an evaluation index;

as can be seen, 3TC reduces the mRNA levels of related markers in hippocampus (A) and cortex (B) of SAMP8 mice;

FIG. 4 is a network diagram of protein interaction nodes of 3TC action targets collected based on a public database, and 32 core targets of 3TC are screened out according to node Degree (hierarchy analysis of nodes) to serve as the most potential target proteins;

a is a network diagram of interaction of all target proteins based on data mining, and the left side is a screened core gene;

b is a protein interaction graph of the core gene, and the interaction relation among all targets of the 3TC can be known from the graph;

FIG. 5A is a Gene Ontology (GO) enrichment analysis diagram of a 3TC core target; b is KEGG (Kyoto Encyclopedia of Genes and genomes) pathway analysis diagram; c, screening out marker proteins related to Estrogen (EGFR), PI3K/Akt (pAKT1) and a neuroactive ligand-receptor interaction (ADRB2) signal channel from the protein, and carrying out 3 TC-target molecule docking simulation diagram;

FIG. 6 is a graph showing the results of in vitro experimental validation of the pharmacological analysis of the network, Formaldehyde (FA) induced damage of the neuronal cell LINE HT22, wherein A is immunofluorescence detecting the expression of LINE-1ORF1p in the control, FA and FA +3TC groups, B is the quantification of immunofluorescence analysis, C is the quantification of viable neurons per field of view for each group, D is Western blot detecting EGFR, p-AKT1 and ADRB2, E is Western blot quantification analysis, and 3TC is shown to reduce the number of Formaldehyde (FA) -induced HT22 neuronal cell death by inhibiting LINE-1 and activating the marker proteins of the Estrogen (EGFR), PI3K/Akt (pAKT1) and the neuroactive ligand-receptor interaction (ADRB2) signaling pathway.

Detailed Description

The following further description of the present disclosure is provided in conjunction with the specific drawings and the specific examples, which should not be construed as limiting the present disclosure. The animals, cells, reagents, methods and apparatus used in the examples described below are conventional reagents, methods and apparatus in the art and are commercially available, unless otherwise specified.

Example 1

3TC treatment of SAMP8 in AD model mice

(1) Experimental groups, 44-week-old normal SAMR1 mice as healthy controls, untreated SAMP8 mice as AD model group, and 3TC gavage SAMP8 mice as 3TC treatment group.

(2) Drug treatment, healthy control group (SAMR1+ Vehicle) and AD model group (SAMP8+ Vehicle) were subjected to drinking water gavage daily, and treatment group (SAMP8+3TC) was subjected to 3TC (dissolved in drinking water) gavage at a dose of 100mg/kg for 4 weeks.

Example 2 three groups of mice were simultaneously subjected to the Morris water maze test under the same conditions

(1) And (5) performing directional navigation experiments. The water maze is a circular pool (120 cm diameter with 10 cm diameter circular platform) with water temperature 25 + -1 deg.C. Mice were trained to find a platform below the water surface in one minute. If the mouse does not find a platform within 60s, it is guided to the platform and allowed to stay for 20 s. Each mouse was trained five times a day, with each mouse experiment being 15 minutes apart. In each experiment, the mice had entry initiation sites randomly distributed within four quadrants. The time from entry to landing of the platform for each training was recorded for each mouse as the escape latency.

(2) And (4) carrying out space exploration experiments. On the sixth day of the test, the platform was removed and the mouse entered the water maze from a point opposite the platform. The time for the mouse to explore the maze was set to 60s, and the experiment was terminated after 60 s. The mouse swimming path, speed and time spent in the maze search were recorded by a video camera and analyzed using Ethovision XT (Noldus software).

The results show that:

the escape latency of SAMR1 mice was significantly lower than that of SAMP8 mice (P <0.05) (fig. 1A-B). However, on day 3 and day 5, the escape latency of 3TC treated mice was significantly shorter than that of AD model group (P <0.05), but the difference was not statistically significant compared to healthy control group (P > 0.05). While the mice with 3 TC-dosed SAMP8 swim significantly faster on the first day than the other two groups, no significant difference was detected between the three experimental groups in the next four-day experiment (fig. 1C). The results at day 6 showed that both healthy control and 3TC treated mice stayed more in the target zone than AD model (P <0.05, fig. 1D-E), indicating that both healthy control and 3TC treated mice stayed more in the target quadrant than AD model (P <0.05, fig. 1D-E). In addition, the crossing times of healthy control mice and 3TC treated SAMP8 group mice were also significantly greater than those of AD model group (P <0.05, fig. 1F). Mice in the 3TC treated group had the longest latency to find the platform underwater, and the difference was statistically significant compared to the untreated group (P <0.05, fig. 1G). Data were expressed as mean ± standard error (mean ± SE) using Student's t-test, with 10 samples per group. P <0.05 is statistically different: p <0.001, P <0.01, P < 0.05.

In summary, 3TC treatment can improve the learning and memory function of SAMP8 mice.

Example 3

Example 1 post-experiment mouse brain Paraffin sectioning and tissue staining

(1) And (5) making a paraffin section of the brain. After treatment, mice were anesthetized with 5% diethyl ether and brains were collected after cardiac perfusion with Phosphate Buffered Saline (PBS) and 4% paraformaldehyde solution. Brain tissue was removed and fixed in paraformaldehyde for more than 2 days. The fixed brain was embedded in paraffin. After paraffin is fixed, the paraffin is sliced, and the paraffin mass is sliced into 4-5 mu m paraffin sections.

(2) Hematoxylin-eosin (HE) staining analyzed brain morphological changes. Taking paraffin sections of hippocampal tissues, and performing gradient dewaxing dehydration by using ethanol with a series of concentrations. HE staining was performed using HE staining kit (Solarbio, beijing, china) according to the manufacturer's instructions. After soaking in ethanol and xylene, the sections were fixed with resin and observed under an optical microscope (olympus, tokyo, japan).

(3) And detecting the neuron damage condition on the hippocampal tissue section by adopting a Niger staining method. The sections were incubated with Nisshin staining solution (Hangzhou, Biyuntian) for 30min and washed with 95% ethanol. Stained cells were counted under an optical microscope (olympus, tokyo, japan) and photographed. At random 3 non-overlapping fields were selected on each slide of hippocampal tissue for cell counting, healthy neuron index ═ normal neuron number/total neuron number of nissl bodies.

(4) TUNEL staining. Hippocampal tissue sections were incubated with proteinase K solution for 15min, followed by TUNEL reaction mixture for 30min (Beyotime, China). After three PBS washes, sections were treated with blocking solution containing the nuclear dye DAPI, and TUNEL staining positive (apoptotic) cells were observed and recorded using confocal laser fluorescence microscopy.

The results show that:

compared with a healthy control group, the mice in the AD model group have increased loss of hippocampal neurons, mild disorder of cell arrangement, partial neuron nucleus concentration and mild hippocampal edema. These lesions were improved in the 3TC treated group (fig. 2A). Nissel staining revealed loss of Nissl-positive nerve cells in the AD model group, and partial reversal of the above changes in the 3TC group (FIGS. 2B-C). TUNEL staining showed significantly less hippocampal apoptosis in 3TC treated mice than AD model (P <0.001, fig. 2D-E). Data were expressed as mean ± standard error (mean ± SE) using Student's t-test, with 5 samples per group. P <0.05 is statistically different: p < 0.001.

In summary, 3TC treatment can reduce neuronal damage in the hippocampal region of the brain of SAMP8 mice.

Example 4 mouse brain Hippocampus and cortical tissue RNA extraction and Real-time PCR

After the treatment, the mouse brain hippocampus and cortex tissue RNA was extracted by Trizol (Life technologies) method, and the specific operation was performed according to the instruction. Mu.g of total RNA was added with 2. mu.l of 5 XPrimeScript RT Master Mix (Takara), and the amount of the mixture was adjusted to 20. mu.l with deionized water for reverse transcription to synthesize cDNA; real-time PCR was performed using the set-up program with the primers listed in Table 1.

TABLE 1 Real-time PCR primer sequences

Example 5 network pharmacological analysis to find potential targets for 3TC

(1) And constructing a 3TC target data base. First, the potential target of 3TC was determined using a reverse pharmacophore profile based on the Pharmmapper database. All chemical components were converted to ". mol 2" format by chembidraw and uploaded to the pharmmmapper database. Molecules with normalized fit scores greater than 4.0 were identified as potential targets for 3 TC. Second, the CHEMBL database is used to query the biological activity of the target or compound. Proteins that were shown to be derived from homo sapiens were identified as potential targets for 3 TC.

(2)3TC target protein interaction analysis and core target screening. The selected target protein was analyzed for protein-protein interaction by String9.1 to generate a protein network interaction map. After eliminating duplicates, proteins with confidence scores >0.4 were selected in the designed set for interaction analysis. To fully study the molecular mechanism of 3TC, PPI networks were constructed using Cytoscape software version 3.6.0. Node Degree analysis was performed using centroscope 2.2 to screen the core genes in the network, with the proximity center value (Closeness center value), Betweenness center value (Betweenness center value), and Degree center value (Degree center value) set to 0.2, 450, and 50, respectively.

(3) GO and KEGG analysis. Gene Ontology (GO) enrichment analysis was performed based on the DAVID bioinformatics database using the molecular functions of biological processes, cellular components and potential targets for biological function annotation. The KEGG metabolic pathways were annotated using an online KEGG automated annotation server (http:// www.genome.jp/KEGG /) according to the Gene and genome encyclopedia (KEGG) database of the Kyoto protocol.

(4)3 TC-target molecule docking model construction. Retrieving and obtaining an SDF structure file of a compound 3TC through a pubchem website, converting the SDF file into a PDB file by utilizing Open Babel 2.3.2 software, retrieving and obtaining receptor proteins ADRB2(PDBID:3KJ6), AKT1(PDBID:4EKL) and EGFR (PDBID:6S9C) from a Protein Data Bank (http:// www.rcsb.org/PDB) database, performing operations such as dehydration and ligand removal on the receptor proteins by utilizing PYMOL 2.3.4 software, performing modification such as hydrogenation, charge balance and the like on the four receptor proteins by utilizing AutoDocktools, opening a Grid operation tool by utilizing a Grid Box command under a Grid program, determining the size of a ligand binding pocket by the number and the Grid point interval in each direction, adjusting the Grid point number in each direction of each Protein, the center of the binding pocket and the interval of the Grid point, setting the interval as1, adjusting the interval of the Grid point in each direction, and enabling the volume of the binding pocket to rotate in a state of the joint in which molecules can be in a stretching state, the pocket center is set as the center of the binding site, the receptor protein and the ligand small molecule are respectively converted into pdbqt format, and the three receptor proteins and the 3TC ligand small molecule are respectively subjected to molecular docking by utilizing AutoDock Vina 1.1.2.

The results of examples 4 and 5 show that:

in the hippocampus (fig. 3A), compared to the healthy control group, the transposons LINE-1-associated gene (L1-ORF1, P <0.05), three interferon-responsive genes (IFNA, IRF7 and Oas1, P <0.05), two AD-associated genes (PS-1 and APP, P <0.01), and three representative senescence-associated secretory phenotype genes (IL6, Mmp3, and PAI1, P <0.01) were significantly up-regulated in the AD model group, while the 3TC treatment group significantly down-regulated included one transposon-associated gene (L1-ORF2, P <0.001), two interferon-responsive genes (IFNA and Oas1, P <0.001), and three representative senescence-associated secretory phenotype-associated genes (IL6, Mmp3, PAI1, P < 0.01). Similar results were obtained with experiments on cortical tissues (fig. 3B).

Results of cyber pharmacological analysis show that a total of 269 proteins are predicted targets of 3TC, 164 proteins are identified in the Pharmmapper database, and 105 proteins are identified in the ChEMBL database. Based on the target gene of 3TC, PPI network was constructed using String database, and there are 32 core nodes in the network, which were identified as key targets of 3TC in SAMP8 mouse (fig. 4A). We used String9.1 to mimic the interaction between 3TC key target proteins, thereby identifying three groups of functionally similar proteins (fig. 4B).

GO enrichment analysis of target genes identified under 3TC treatment with STRING revealed a total of 550 biological processes (P <0.05, fig. 5A), such as reactions to acidic chemicals, reactions to reactive oxygen species, and negative control of apoptotic processes. The 3TC target network metabolic pathway KEGG analysis found that three pathways associated with neuroinflammation, cell death and neuronal signal transduction include the estrogen signaling pathway (P <0.0001), the phosphatidylinositol 3-kinase and protein kinase B (PI3K/Akt) signaling pathway (P <0.0001) and the neuroactive ligand-receptor interaction signaling pathway (P <0.001) (fig. 5B).

The 3TC was subjected to molecular docking simulation studies with three selected potential targets, namely EGFR for the estrogen signaling pathway, AKT1 for the P13K/AKT signaling pathway, ADRB2 for the neuroactive ligand-receptor interaction signaling pathway (fig. 5C). The binding mode between the receptor protein ADRB2 and the 3TC ligand small molecule is that amino acid residues Asp331, Phe332 and 3TC form hydrogen bond interaction, and amino acid residues Phe336, Ala335, Val54, Ala57, Ile58, Leu64, Asn69, Ile72, Pro330 and 3TC form hydrophobic interaction; the binding mode between the receptor protein AKT1 and the 3TC ligand small molecule is that amino acid residues Asp292, Glu191, His194, Glu198 and Thr195 and 3TC form hydrogen bond interaction, and amino acid residues Phe179, Leu295, Glu198 and Lys179 and 3TC form hydrophobic interaction; the binding pattern between the receptor protein EGFR and the 3TC ligand small molecule is that amino acid residues Thr854, Asn842, Arg841, Glu762 form hydrogen bond interactions with 3TC, and amino acid residues Lys55, Val40, Ala53, Met111, Val158, Asn114, Leu168, Ser155, Asp169, Lys153, Asn156, Gly35 form hydrophobic interactions with 3 TC.

Example 6 in vitro experiments to verify 3TC action targets

(1) And (3) constructing an in vitro AD-neuron damaged cell model. Mouse hippocampal neural cell line HT22 was purchased from Shanghai cell Biotech. HT22 cells were cultured in DMEM supplemented with 10% Fetal Bovine Serum (FBS). Cells were seeded in 6-well plates 1 day before the experiment and cell damage was induced with Formaldehyde (FA) purchased from Sigma-Aldrich, usa. HT22 cells were divided into 3 groups, the FA group was treated with 30mol/L FA for 24h, the FA +3TC group was treated with 30mol/L FA and 50 mol/L3 TC, and the control group was untreated.

(2) Immunofluorescent staining and cell counting. The culture medium was aspirated off, and the cells were fixed with 2% paraformaldehyde solution at room temperature for 1 h. Immunofluorescence assay procedures were performed according to standard procedures. The antibodies used in this example included anti-LINE-1 ORF1p antibody (1: 1000; Sigma-Aldrich) and Alexa Fluor 594 secondary antibody (1: 1000; Invitgen). Nuclei were labeled by incubation with DAPI (1: 500 dilution; Thermo Fisher Science) for 1 min. After adding the anti-fluorescence quenching mounting agent, the cells were observed and counted with a confocal microscope (nikon, tokyo, japan).

(3) Western blot was used to detect the expression of EGFR, p-AKT1 (active Akt 1: S473 phosphorylates) and ADRB 2. Total protein was collected from the cells after various treatments. Western blot experimental procedures were performed according to standard procedures. The antibodies used in this example include anti-EGFR antibody (1: 500; Sigma-Aldrich), anti-pAKT 1 antibody (1: 1000; Sigma-Aldrich), anti-ADRB 2 antibody (1: 200; Sigma-Aldrich), and anti-rabbit secondary antibody (1: 1000; Invitgen). After washing, the cells were visualized using an ECL Western blotting detection System (amersham, Aylesbury, UK).

The results showed that LINE-1 was activated in damaged neurons and that 3TC reversed this trend (fig. 6A-B) and furthermore, 3TC antagonized FA-induced cell death (fig. 6C). After 24h of FA stimulation of TH22 cells, the expression of EGFR, P-AKT1 and ADRB2 was significantly reduced (P <0.001), whereas after 3TC treatment, the expression of EGFR, P-AKT1 and ADRB2 was significantly increased (P < 0.001; FIG. 6D, E). Data were expressed as mean ± standard error (mean ± SE) using Student's t-test, with 5 samples per group. P <0.05 is statistically different. Black asterisks compared to control, red asterisks compared to FA group: p < 0.001.

The verification results of the above embodiments can be combined to prove that: after the administration of 3TC through gastric gavage on SAMP8 of an AD model mouse, the memory and learning capacity of an SAMP8 mouse are improved, and the inventor finds that 3TC can reduce the number of dead neurons in the hippocampal brain region of the SAMP8 mouse and can reverse the tendency of excessive rise of transposon LINE-1 and type 1 interferon reaction, aging-related secretion phenotype and AD-related pathogenic gene mRNA level in the hippocampal brain and cortical tissues of the SAMP8 mouse. Results of network pharmacological analysis and in vitro experimental validation against 3TC indicate that 3TC can act by activating estrogen, PI3K/Akt and neuroactive ligand-receptor interaction signaling pathways. The results show that the 3TC can obviously improve AD symptoms and brain pathological injury through inhibiting inflammation, apoptosis and other ways, can be developed as a new anti-AD medicament, and provides a new means and way for treating cognitive decline and neuron injury caused by AD.

Claims (2)

1. Application of lamivudine in preparing medicament for treating Alzheimer disease is provided.

2. A medicament for the treatment of alzheimer's disease characterized by: the main active component is lamivudine.

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