Virus peptide of Borna disease virus type 1 and application of derivative of virus peptide in preparation of medicine for preventing and / or treating Alzheimer disease

Abstract

The invention belongs to the technical field of Alzheimer's disease, and particularly relates to application of a Borna disease virus type 1 virus peptide and a derivative of the Borna disease virus type 1 virus peptide to preparation of a medicine for preventing and / or treating Alzheimer's disease, the virus peptide is X protein, and the derivative of the virus peptide is X protein derived peptide PX3, the derivative PX3 of the virus peptide has a remarkable protection effect on SH-SY5Y cell injury induced by A beta, and a new strategy is provided for preventing and / or treating the Alzheimer disease.

Description

Application of a viral peptide of Borna disease type 1 virus and its derivatives in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease Technical Field

[0001] This invention belongs to the field of Alzheimer's disease technology, specifically relating to the use of a viral peptide of type 1 Borna disease virus and its derivatives in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive impairment. Clinically, it progresses from mild cognitive impairment to dementia. Currently, AD treatment primarily focuses on symptom relief and cannot stop or reverse disease progression; therefore, there is an urgent need to develop new treatment strategies. Summary of the Invention

[0003] To address the problems in the prior art, this invention provides the application of viral peptides and their derivatives of Borna disease type 1 virus in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease, offering a new strategy for the prevention and / or treatment of Alzheimer's disease.

[0004] The technical problem solved by this invention is achieved by the following technical solution:

[0005] The purpose of this invention is to provide the application of a viral peptide of type 1 Borna disease virus and its derivatives in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease. The viral peptide is the X protein, and the viral peptide derivative is the X protein-derived peptide PX3.

[0006] Furthermore, the amino acid sequence of the X protein-derived peptide PX3 is SEQ ID NO: 1.

[0007] Non-cytolytic replication of Borna disease virus 1 (BoDV-1) depends on the expression of the viral X protein. The X protein, composed of 87 amino acids, is the first mitochondrial localization protein discovered in RNA viruses. The X protein inhibits, rather than promotes, apoptosis, thus facilitating the persistence of the non-cytolytic virus in host cells. The X protein-derived peptide (PX3) is a short peptide composed of 29 amino acid residues at the C-terminus of the X protein.

[0008] The amino acid sequence of the virus-derived peptide PX3, SEQ ID NO: 1, is: F{d-ARG}{Cha}KF{d-ARG}{Ch a}KSRPAPEGPQEEPLHDLRPRPANRKGAAVE.

[0009] The amino acid Cha in the X protein-derived peptide PX3 is cyclohexylalanine. Except for ARG arginine, which is a D-amino acid, all others are L-amino acids.

[0010] Furthermore, the drug is prepared using viral peptides of type 1 Borna virus or their derivatives as active ingredients, along with pharmaceutically acceptable excipients or auxiliary ingredients.

[0011] "Pharmaceutical acceptable" means that a carrier, delivery substance, diluent, excipient, and / or the salt formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with the receptor.

[0012] Furthermore, the drug is in liquid form.

[0013] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0014] 1. This invention, through animal experiments, found that in AD animal models APP / PS1 mice, administration of viral peptide PX3 or control saline solution significantly improved spatial memory and learning impairment in APP / PS1 mice, and the Aβ aggregation and inflammatory response in the cortex and hippocampus were significantly lower than those in the solvent control group. In normal C57 mice, administration of viral peptide PX3 did not cause significant pathological changes in the heart, liver, spleen, lungs, kidneys, or brain, nor did it lead to abnormal changes in key liver and kidney function indicators.

[0015] 2. Through cell experiments, this invention has found that the viral peptide derivative PX3 has a significant protective effect against Aβ-induced SH-SY5Y cell damage, providing a new strategy for the prevention and / or treatment of Alzheimer's disease.

[0016] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. Furthermore, in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0017] Figure 1 shows the HPLC data of PX3 in Example 1.

[0018] Figure 2 shows the mass spectrometry data of PX3 in Example 1.

[0019] Figure 3 shows the protective effect of PX3 against Aβ-induced SH-SY5Y cell damage in Example 1.

[0020] Figure 4 shows the safety evaluation of PX3 administration to normal mice in Example 1.

[0021] Figure 5 shows the effect of PX3 administration on serum indicators of liver and kidney function in mice in each group in Example 1.

[0022] Figure 6 shows the behavioral results of the open field experiment in Example 1: there was no significant difference in the motor ability of the mice in each group.

[0023] Figure 7 shows the behavioral results of the Morris water maze in Example 1: the virus-derived peptide PX3 improved cognitive dysfunction in AD model mice.

[0024] Figure 8 shows the results of thiamine S staining of mouse brain tissue in Example 1: the virus-derived peptide PX3 reduced the aggregation of amyloid protein in the cortex and hippocampus of APP / PS1 mice.

[0025] Figure 9 shows the GFAP staining results of mouse brain tissue in Example 1: the virus-derived peptide PX3 reduced the overactivation of astrocytes in the cortex and hippocampus of APP / PS1 mice.

[0026] Figure 10 shows the IBA1 staining results of mouse brain tissue in Example 1: the virus-derived peptide PX3 reduces the overactivation of microglia in the cortex and hippocampus of APP / PS1 mice. Detailed Implementation

[0027] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0028] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing from the market or prepared by existing methods.

[0029] Example 1: The role of virus-derived peptide PX3 in the prevention and treatment of Alzheimer's disease

[0030] This study investigated the effects of intranasal administration of PX3 on the APP / PS1 mouse model of Alzheimer's disease. The APP / PS1 mice carry mutations in the human APP and PS1 genes, both originating from familial AD patients. Their disease origin is the same as the pathogenic genes in some AD patients, mimicking the molecular initiation events of AD. Simultaneously, this model can reproduce key features of AD such as Aβ amyloid plaque deposition, chronic neuroinflammation, and synaptic dysfunction, and exhibits a decline in learning and memory abilities with age, a process similar to the pathological development of human AD. The experiments in this study demonstrate the preventive and therapeutic effects of PX3 on AD.

[0031] Cell grouping and experimental methods

[0032] Aβ25-35 It is β-amyloid protein (Aβ) 1-42 or Aβ 1-40 A core fragment of GSNKGAIIGLM, whose sequence is considered to be its toxic domain, is widely used in vitro (especially in the human SH-SY5Y neuroblastoma cell line) to rapidly induce Alzheimer's disease-related cytotoxicity models due to its good water solubility, rapid aggregation, significant toxic effects, and low cost. SH-SY5Y cells were seeded into three groups: control group, Aβ... 25-35 Group and Aβ 25-35 Group +PX3, Aβ was detected by CCK8 assay. 25-35 The cytotoxic effect of PX3 on SH-SY5Y cells and its protective effect against Aβ-induced SH-SY5Y cell damage were shown in Figure 3. Experiments revealed that SH-SY5Y cells, after exposure to 10 μM Aβ... 25-35 After 24 hours of stimulation, cell viability decreased by 52.12% (p < 0.001), while PX3 and Aβ... 25-35 After co-incubation for 24 hours, the viability of damaged SH-SY5Y cells was increased by 30.69% (p<0.001).

[0033] Animal grouping and experimental methods

[0034] APP / PS1 transgenic Alzheimer's disease model mice (AD model mice) were randomly divided into three groups: the APP / PS1 group (control group) and the APP / PS1+PX3 group (treatment group). Normal mice were divided into the WT group (wild-type normal genome mice) and the WT+PX3 group (wild-type normal genome mice treatment group). All mice had normal access to water and food. Administration: The solid powdered peptide was dissolved in physiological saline to prepare a 1 mg / ml peptide solution. After brief anesthesia with isoflurane, the mice were positioned at a 45-degree angle, and the peptide solution (1 mg / ml of X) or physiological saline was added using a 10 μl pipette. The peptide was administered bilaterally via nasal drops at a dose of 0.4 μl / g / day. The APP / PS1+PX3 group and the WT+PX3 group received PX3 dissolved in physiological saline via nasal drops once daily. The APP / PS1 group and the WT group received the same volume of physiological saline once daily for a total of 4 weeks.

[0035] One week before the end of the experiment, all mice underwent behavioral tests, including the mouse open field test and the Morris water maze test. Changes in cognitive status in each group of mice were observed and recorded, and the data were analyzed. At the end of the experiment, after anesthesia, blood was collected from the eyeballs of all mice in each group, and the blood supernatant was separated for serological testing. After perfusion with physiological saline and paraformaldehyde, mouse brain tissue was fixed, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE) to examine the basic tissue and cell morphology of important organs in each group of mice. Thioflavin S fluorescence staining was used to detect the aggregation of amyloid protein in the cerebral cortex and hippocampus of each group of mice. Immunohistochemical staining for IBA1 and GFAP in the mouse cortex and hippocampus was also performed to assess the activation of microglia and astrocytes.

[0036] I. Security Assessment

[0037] As shown in Figure 4, in the brain slices of mice in the WT group, WT+PX3 group, and APP / PS1+PX3 group, the hippocampal neurons were closely arranged, with normal morphology and structure, clear demarcation between the nucleus and cytoplasm, and prominent nucleoli, and no inflammatory response was observed. In contrast, in the brain slices of mice in the APP / PS1 group, a small number of hippocampal cells showed increased staining, neurons were shrunken, and the demarcation between the nucleus and cytoplasm was unclear.

[0038] After intranasal administration of PX3 to mice, no significant pathological changes were observed in the vital organs (heart, liver, spleen, lung, kidney, and brain) of normal mice after HE staining, indicating that the polypeptide is safe and reliable and can be used for subsequent experiments.

[0039] II. Serological Tests

[0040] The following indicators were measured in serum: T-Bil (total bilirubin), ALT (alanine aminotransferase), AST (aspartate aminotransferase), TP (total protein), CREA (serum creatinine), UA (uric acid), and UREA (blood urea nitrogen). T-Bil, ALT, AST, and TP are indicators of liver function, while CREA, UA, and UREA are indicators of kidney function.

[0041] As shown in Figure 5, except for T-Bil, the differences in other indicators were not statistically significant. There was no significant difference in renal function between the APP / PS1 group mice and the littermate WT group mice. At the same time, the administration of the peptide to the APP / PS1 mice did not cause liver and kidney damage, further demonstrating the safety of the peptide.

[0042] Compared with the WT group, the T-Bil level in the APP / PS1 group mice was significantly elevated. After administration of PX3 via nasogastric tube, the T-Bil level decreased significantly, and the difference was statistically significant. This indicates that the APP / PS1 group mice had abnormal liver function, and that peptide treatment could improve the degree of liver damage.

[0043] Theoretically, the WT group mice and the APP / PS1 group mice have the same genetic background and represent a normal physiological state. The APP / PS1 group mice are only used as a transgenic model of Alzheimer's disease, and the changes in their liver function may be indirectly caused by certain secondary factors. Some studies have revealed that Alzheimer's disease is closely related to lipid metabolism.

[0044] III. Open Field Experiment

[0045] The open field test assesses the basic motor abilities of mice, providing a prerequisite for subsequent more complex behavioral tests (such as the Morris water maze test), and can serve as a rapid and effective preliminary screening tool for cognitive impairment models. The total distance traveled and speed measured in the open field test reflect the overall activity level, as shown in Figure 6.

[0046] Total Distance: Compared with the APP / PS1 group, the total distance of mice in the WT group and the APP / PS1+PX3 administration group both showed a decreasing trend, but the difference did not reach statistical significance, indicating that there was no obvious abnormality in the motor function of mice in each group.

[0047] Velocity: Compared with the APP / PS1 group, the WT group and the APP / PS1+PX3 administration group also showed reduced velocities, but there was no statistically significant difference.

[0048] The results of the mine field test showed no significant difference in the motor ability of the mice in each group, and the differences in performance in the subsequent water maze test can be ruled out as possibly due to differences in motor ability.

[0049] IV. Morris Water Maze Experiment

[0050] The Morris water maze test is a behavioral test used to assess spatial learning and memory in mice. The test lasts for six days. The first five days are the training period, during which mice are trained to remember the location of the platform. The sixth day is the testing period, during which the number of times mice traverse the platform and the time spent in the target quadrant (where the platform is located) are compared between the groups after the platform is removed.

[0051] The training period test results (see Figure 7B) showed that the latency of the four groups of mice to find the platform gradually shortened with the increase of training sessions. On day 5 of the training period (see Figure 7A), compared with WT mice, the average time for the APP / PS1 group mice to find the hidden platform was prolonged (####p<0.0001). Compared with the APP / PS1 group mice, the average time for the APP / PS1+PX3 group mice to find the hidden platform was shortened (****p<0.0001). On day 6 of the test period (see Figure 7D and E), the number of times the APP / PS1 group mice crossed the platform and the time spent crossing the platform quadrant were significantly less than those of the WT group mice (#p<0.05). Compared with the APP / PS1 group mice, the number of times the APP / PS1+PX3 group mice crossed the platform and the time spent crossing the platform quadrant were significantly increased (*p<0.05). These experimental results indicate that the spatial memory cognitive function of the APP / PS1 model mice is significantly impaired, and peptide treatment intervention can significantly improve the spatial memory function impairment of APP / PS1 mice.

[0052] V. Thioflavin S Fluorescent Staining

[0053] The results of thioflavin S fluorescence staining of mouse brain tissue (as shown in Figure 8, B is the staining of the hippocampus, and C is the staining of the cortex) are as follows:

[0054] Compared with the WT group, mice in the APP / PS1 group and the APP / PS1+PX3 group showed obvious apple green fluorescent spots in the cerebral cortex and hippocampus. Compared with the APP / PS1 group, mice in the APP / PS1+PX3 group showed a significant reduction in apple green fluorescent spots, and the difference was statistically significant (***=P<0.001, ****=P<0.0001).

[0055] This indicates that treatment with the viral peptide PX3 can reduce the abnormal accumulation of amyloid protein to a certain extent, thereby further alleviating learning and cognitive impairment in AD mice.

[0056] VI. GFAP Immunohistochemical Staining

[0057] Immunohistochemical staining results of GFAP in mouse cerebral cortex and hippocampus (as shown in Figure 9):

[0058] When the central nervous system is damaged or inflamed, astrocytes produce large amounts of GFAP, a process known as "reactive gliosis".

[0059] Immunohistochemical staining of GFAP showed that, compared with the WT group, astrocytes in the cortex and hippocampus of mice in the APP / PS1 group were widely activated and GFAP expression was significantly upregulated. After PX3 intervention, GFAP expression decreased significantly, and the difference was statistically significant.

[0060] VII. IBA1 Immunohistochemical Staining

[0061] Immunohistochemical staining results of IBA1 in mouse cerebral cortex and hippocampus (as shown in Figure 10):

[0062] Ionized calcium adaptor molecule (IBA1), also known as allogeneic inflammatory factor (AIF1), is specifically highly expressed in macrophage lineage cells. Microglia, as resident immune cells of the brain, can express large amounts of IBA1 when activated, thus serving as a marker for microglia.

[0063] IBA1 expression in the cortex and hippocampus of APP / PS1 mice was higher than that in the WT group, suggesting significant and widespread neuroinflammation in the cortex and hippocampus of AD mice, leading to the activation of a large number of microglia. Compared with APP / PS1, IBA1 expression in mice treated with peptides decreased to near-normal levels.

[0064] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0065] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. The use of a viral peptide of Borna disease type 1 virus and its derivatives in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease, characterized in that, The viral peptide is the X protein, and the derivative of the viral peptide is the X protein-derived peptide PX3.

2. The application as described in claim 1, characterized in that: The amino acid sequence of the X protein-derived peptide PX3 is SEQ ID NO:

1.

3. The application as described in claim 1, characterized in that: The drug is prepared using viral peptides or derivatives of type 1 Borna virus as the active ingredient, along with pharmaceutically acceptable excipients or auxiliary ingredients.

4. The application as described in claim 1, characterized in that: The drug is a liquid formulation.

Images