Application of liensinine in preparation of anti-Alzheimer disease drugs

Abstract

The invention belongs to the technical field of medicines, and relates to application of liensinine, in particular to application of liensinine in preparation of an anti-Alzheimer disease medicine. Research finds that the liensinine can obviously improve the learning and memory ability of a mouse suffering from the Alzheimer disease jointly induced by D-galactose and AlCl3, and has statistical significance. The liensinine can play a role in resisting the Alzheimer's disease by reducing expression of Abeta1-42 protein in hippocampal tissues of mice with the Alzheimer's disease and inhibiting excessive phosphorylation of Tau protein, and can be used for preparing the medicine for preventing or treating the Alzheimer's disease. The invention provides a new therapeutic drug choice for the treatment of Alzheimer's disease, and digs the new medicinal value of liensinine at the same time.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to the application of liesinine, and in particular to the application of liesinine in the preparation of anti-Alzheimer's disease drugs. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease that seriously threatens the health of the elderly. Clinical manifestations include comprehensive and persistent intellectual impairment, including memory and thinking impairment, emotional and behavioral abnormalities, and loss of the ability to work and live independently. At present, the main hypotheses about the pathogenesis of AD include β-amyloid (Aβ) cascade, Tau protein hyperphosphorylation, mitochondrial dysfunction and neuroinflammation. The number of drugs used in the clinic to treat AD is limited, and the target of action is single. They can only delay the development of the disease, but cannot cure it, and have certain side effects. Therefore, the development of effective therapeutic drugs for AD is very urgent, and it is also a hot spot and difficulty in research.

[0003] The causes of AD are complex and diverse. Many studies have shown that β-amyloid plays a vital role in the occurrence and development of AD. Studies have found that excessive production of Aβ is the main cause of amyloid plaque formation in the brains of AD patients. 1-42 Protein fragments are the main form of neurotoxicity. Excessive deposition of Aβ triggers a series of downstream events, including hyperphosphorylation of Tau protein. Phosphorylated Tau protein aggregates to form neurofibrillary tangles (NFTs), one of the classic lesions of AD, thereby enhancing the toxic response of nerve cells and leading to cognitive decline. Therefore, reducing the expression of Aβ protein and inhibiting the hyperphosphorylation of Tau protein are beneficial to improving cognitive impairment caused by AD and play an important role in anti-AD.

[0004] Liensinine (English name: Liensinine; abbreviation: Lien) is a dibenzylisoquinoline alkaloid derived from lotus seed core and is one of the main active ingredients in lotus seed core. Numerous studies have shown that liensinine has a wide range of pharmacological effects, such as anti-oxidative stress, anti-arrhythmia, anti-hypertension, anti-cancer, anti-inflammatory and neuroprotective effects. However, the therapeutic effect and mechanism of liensinine on AD are still unclear. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide an application of liesinine in the preparation of anti-Alzheimer's disease drugs.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] Application of liesinine in the preparation of anti-Alzheimer's disease drugs.

[0008] Further, in the above application, the structural formula of liesinine is as shown in (I):

[0009]

[0010] Furthermore, in the above application, the anti-Alzheimer's disease performance includes improving the learning and memory ability of AD mice, reducing Aβ in hippocampal tissue 1-42 The expression of Tau protein was inhibited and the hyperphosphorylation of Tau protein was inhibited.

[0011] Furthermore, in the above application, the dosage form of the drug is any dosage form acceptable in pharmacological therapy.

[0012] Furthermore, in the above application, the dosage of the drug is any therapeutically acceptable dosage.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. The present invention explores the new medicinal value of liesinine, and its application in the preparation of anti-Alzheimer's disease drugs provides a new drug option for the prevention and treatment of Alzheimer's disease.

[0015] 2. The present invention has found through research that liensinine can significantly improve the 3 The learning and memory ability of AD mice induced by the combination was statistically significant. And Liensinine can reduce the Aβ in the brain of AD mice 1-42 The protein can be expressed and the hyperphosphorylation of Tau protein can be inhibited to play an anti-AD role, and can be used to prepare drugs for preventing or treating AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of liensinine on the escape latency of AD mice was compared with the model group. ** P<0.01, *** P<0.001, n=10.

[0017] Figure 2 Figure 2 Effects of liensinine on the number of times AD mice crossed the platform and the time they stayed in the target quadrant. (a) The number of times they crossed the platform, (b) The time they stayed in the target quadrant, compared with the model group. ns P ≥ 0.05, * P<0.05, ** P<0.01, *** P<0.001(n=10).

[0018] Figure 3 is the effect of liensinine on the spontaneous alternation reaction rate of AD mice. Compared with the model group,ns P ≥ 0.05, ** P<0.01, *** P<0.001(n=10).

[0019] Figure 4 is the effect of liensinine on the percentage of time spent exploring the novel arm in AD mice. Compared with the model group, ns P ≥ 0.05, ** P<0.01, *** P<0.001(n=10).

[0020] Figure 5 Effect of liensinine on Aβ in hippocampus of AD mice 1-42 Compared with the model group, ns P ≥ 0.05, ** P<0.01, *** P<0.001(n=3).

[0021] Figure 6 The effect of liensinine on Tau protein phosphorylation in the hippocampus of AD mice. Compared with the model group, ** P<0.01, *** P<0.001(n=3). DETAILED DESCRIPTION

[0022] The technical scheme and technical effects of the present invention are described in detail below in conjunction with specific embodiments and drawings. Experimental methods without specifying specific conditions are usually based on conventional conditions, such as the conditions described in textbooks and experimental guides, or the conditions recommended by manufacturers, which are well known or easily known to ordinary technicians in the field. The following embodiments are only preferred embodiments of the present invention and do not limit the present invention. For those skilled in the art, the present invention can have various changes and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0023] Example 1

[0024] Liensinine (Lien, purity> 98%) used in the examples was purchased from Sichuan Hengcheng Zhiyuan Biotechnology Co., Ltd., and the structural formula is shown below:

[0025]

[0026] 1. Experimental methods

[0027] 1. Experimental Grouping

[0028] Sixty Kunming (KM) male mice were randomly divided into 6 groups, 10 mice in each group, namely, blank control group (Con), model group, Lien (1mg / kg) group, Lien (3mg / kg) group, Lien (10mg / kg) group and positive drug Donepezil Don (3mg / kg) group. The mice were adaptively raised for one week to adapt to the living environment and reduce the stress response during the experiment.

[0029] 2. Establishment of AD mouse model and drug administration

[0030] Every morning, mice in the model group, Lien (1 mg / kg), Lien (3 mg / kg), Lien (10 mg / kg), and Don (3 mg / kg) groups were intraperitoneally injected with D-galactose (120 mg / kg) and intragastrically administered with AlCl 3 (20mg / kg) for 90 days of modeling. At the same time, mice in the Con group were intraperitoneally injected and gavaged with the same volume of saline. Starting from the 31st day, mice in the drug-treated groups were intraperitoneally injected with Lien (1, 3 or 10mg / kg) and Don (3mg / kg) every afternoon according to different groups for 60 days. At the same time, mice in the Con group and the model group were intraperitoneally injected with an equal volume of saline. Behavioral experiments were performed in the last week of drug administration.

[0031] 3. Behavioral Experiment

[0032] (1) Morris water maze test

[0033] ① Positioning and navigation experiment: This experiment was carried out for 5 consecutive days. Every day, the mice were placed in the water of the four quadrants of the water maze device in turn and allowed to swim for 90 seconds. The escape latency (the time it took for the mice to find the platform) was recorded. If the mouse did not land on the platform within 90 seconds, it was guided to the platform and allowed to stay on the platform for 30 seconds. At this time, the escape latency was recorded as 90 seconds.

[0034] ② Spatial search experiment: It was conducted on the second day after the positioning navigation experiment. Before the experiment began, the underwater platform was removed and the mice were placed in the water from the quadrant opposite to the quadrant where the platform was located (target quadrant). The number of times the mice crossed the original platform position and the time they stayed in the target quadrant were recorded.

[0035] (2) Y-maze

[0036] ① Spontaneous alternation reaction experiment: The three arms of the device are the starting arm A, the novel arm B and the other arm C. During the experiment, each mouse was placed in arm A and allowed to explore freely. The order in which the mouse entered the three arms within 8 minutes was recorded, and the spontaneous alternation reaction rate was calculated.

[0037] ②Discriminative exploration experiment: The experiment consists of two stages. During the training phase, mice were placed from the starting arm A facing the arm (the partition of the novel arm was lowered at this time) and allowed to freely explore for 5 minutes. After 3 hours, the test phase began, and mice were placed from the starting arm A facing the wall again (the partition of the novel arm was opened), and allowed to move freely in the three arms for 5 minutes. The time when the mice entered the novel arm B and the total time they entered the arm were recorded, and the percentage of time they entered the novel arm B was calculated.

[0038] 4. Immunoblotting analysis

[0039] Take the hippocampal tissue of the experimental mouse, add RIPA lysis buffer, place it in a tissue disruptor for 5 minutes, fully lyse it at 4°C for 30 minutes, centrifuge (15000rpm, 4°C, 15min) and collect the supernatant. After the protein content in the sample is determined by the BCA method, the protein sample is separated in the SDS-PAGE gel and transferred to the PVDF membrane. Use 5% skim milk powder to block the PVDF membrane at room temperature for 2 hours. Then place the PVDF membrane in a specific primary antibody (Aβ 1-42 , 1:500; p-Tau, 1:1000; Tau, 1:1000; β-actin, 1:500), incubated at 4°C overnight. After washing the PVDF membrane with TBST to remove unbound primary antibody, HRP-labeled secondary antibody (HRP-goat anti-rabbit IgG, 1:10000) was added and incubated at room temperature for 2h. After washing the PVDF membrane with TBST to remove unbound secondary antibody, 1mL ECL solution was added in the dark, and the strips were repeatedly blown for 15s to make the reaction complete. The PVDF membrane was exposed in a fully automatic chemiluminescence / fluorescence image analysis system until the protein bands were developed, and the images were analyzed using Image J software.

[0040] (II) Experimental results

[0041] 1. Effect of Liensinine on the Escape Latency of AD Mice

[0042] In the Morris water maze navigation test, the escape latency reflects the spatial memory ability of mice. Figure 1 As shown in the figure, compared with the model group, the mice in the Con group showed a significant decrease in escape latency from the second day (P<0.001). After treatment with Lien (3 and 10 mg / kg) and Don (3 mg / kg), the escape latency of mice was significantly shortened as the training time prolonged (P<0.001), indicating that the spatial memory ability of AD mice was significantly enhanced after administration of liensinine.

[0043] 2. Effect of Liensinine on the Number of Crossing Platforms and the Target Quadrant Stay Time of AD Mice

[0044] In the spatial exploration test, the number of times the platform was crossed and the time spent in the target quadrant reflected the learning and memory ability of mice. Figure 2 a and b), the model group mice could not accurately locate the original platform position, and compared with the Con group, the number of times the AD mice crossed the platform and the time they stayed in the target quadrant were reduced (P<0.001). After administration of Lien (3 and 10 mg / kg), the number of times the mice crossed the platform and the time they stayed in the target quadrant were significantly increased, indicating that the treatment of lianxinine improved the learning and memory ability of AD mice.

[0045] 3. Effect of Liensinine on the Spontaneous Alternation Rate in AD Mice

[0046] In the Y-maze test, the spontaneous alternation response rate can be used to assess the spatial working memory of mice. Figure 3 As shown in the figure, compared with the mice in the Con group, the spontaneous alternation reaction rate of the mice in the model group was significantly reduced (P<0.001), and they could not alternately enter the three arms well, indicating that their spatial memory of the Y maze was weak. After drug treatment, this phenomenon was improved, and the spontaneous alternation reaction rate was significantly increased (Lien 3mg / kg: P<0.001; Lien 10mg / kg: P<0.01), indicating that the administration of liensinine can significantly improve the spatial memory ability of AD mice.

[0047] 4. Effect of Liensinine on the Percentage of Time of Exploring the Novel Arm in AD Mice

[0048] In the discriminative exploration experiment, the proportion of time that mice spend exploring the novel arm reflects their spatial reference memory ability. Figure 4 As shown in the figure, compared with the Con group, the percentage of time that AD mice entered the novel arm was significantly reduced (P<0.001). After treatment with Lien (3 and 10 mg / kg), the time that mice spent exploring the novel arm was significantly increased (P<0.001), indicating that Lienxinine improved the spatial reference memory of AD mice.

[0049] 5. Effect of Liensinine on Aβ in the hippocampus of AD mice 1-42 Effect of protein expression

[0050] Depend on Figure 5 It can be seen that Aβ in the hippocampus of mice in the model group 1-42 The protein expression was significantly higher in the Con group (P<0.001). After treatment with 3 and 10 mg / kg of Lien, Aβ in the brain of mice 1-42 The protein expression was significantly reduced (Lien 3mg / kg: P<0.01; Lien 10mg / kg: P<0.001), indicating that the administration of liensinine can inhibit the Aβ in the hippocampus of AD mice 1-42 Protein expression.

[0051] 6. Effect of Liensinine on Tau Protein Phosphorylation in Hippocampal Tissue of AD Mice

[0052] like Figure 6 As shown in the figure, compared with the Con group, the phosphorylation of Tau protein in the hippocampus of the model group mice was abnormally increased (P<0.001). After treatment with Lien (1, 3 and 10 mg / kg), the phosphorylation level of Tau protein in the brain of mice was significantly decreased (Lien 1 mg / kg: P<0.01; Lien 3 and 10 mg / kg: P<0.001), indicating that Lienxinine inhibited the abnormal phosphorylation of Tau protein in the hippocampus of AD mice.

Claims

1. Application of liensinine in the preparation of anti-Alzheimer's disease drugs.

2. The use according to claim 1, characterized in that: The structural formula of the liensinine is shown in (I):

3. The use according to claim 1, characterized in that: The anti-Alzheimer's disease manifestations include improving learning and memory ability, reducing Aβ in hippocampal tissue 1-42 The expression of Tau protein was inhibited and the hyperphosphorylation of Tau protein was inhibited.

4. The use according to claim 1, characterized in that: The dosage form of the drug is any pharmaceutically therapeutically acceptable dosage form.

5. The use according to claim 1, characterized in that: The dosage of the drug is any therapeutically acceptable dosage.

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