CN119868361A - Application of liensinine in preparation of anti-Alzheimer disease drugs - Google Patents

Abstract

The invention belongs to the technical field of medicines, relates to application of liensinine, and in particular relates to application of liensinine in preparation of anti-Alzheimer disease medicines. According to the invention, the study shows that the lotus plumule can obviously improve the learning and memory capacity of the mice with the Alzheimer disease induced by the combination of D-galactose and AlCl ₃, and has statistical significance. And the plumula Nelumbinis can play a role in resisting Alzheimer's disease by reducing the expression of Abeta _1‑42 protein in the hippocampal tissue of the Alzheimer's disease and inhibiting the hyperphosphorylation of Tau protein, and can be used for preparing medicines for preventing or treating Alzheimer's disease. The invention provides a new therapeutic drug selection for the treatment of Alzheimer's disease, and simultaneously digs new medicinal value of liensinine.

Description

Application of liensinine in preparation of anti-Alzheimer disease drugs

Technical Field

The invention belongs to the technical field of medicines, relates to application of liensinine, and in particular relates to application of liensinine in preparation of anti-Alzheimer disease medicines.

Background

Alzheimer's Disease (AD) is a neurodegenerative disease that severely threatens the health of the elderly. Clinical manifestations are comprehensive and persistent decline in intelligence, including decline in memory, thinking ability, emotional, behavioral abnormalities, and disabilities of work and independent life. Currently, the pathogenesis of AD is mainly based on the hypothesis of β -amyloid (aβ) cascade, tau protein hyperphosphorylation, mitochondrial dysfunction, and neuroinflammation. The clinical medicine for treating AD has the advantages of limited quantity, single acting target point, only delay of disease development, incapability of radical treatment and certain side effect. Therefore, the development of effective therapeutic drugs for AD is urgent, and is a hot spot and a difficult point of research.

The etiology of AD is complex and diverse, and many studies suggest that β -amyloid plays a critical role in the development and progression of AD. It has been found that overproduction of aβ is a major cause of amyloid plaque formation in the brains of AD patients, with a fragment of aβ _1-42 protein being the predominant neurotoxic form present. Excessive deposition of aβ initiates a series of downstream events including Tau protein hyperphosphorylation. Phosphorylated Tau proteins aggregate to form neurofibrillary tangles (NFTs), one of the classical lesions of AD, thereby enhancing the toxic response of nerve cells, leading to decline of cognitive function. Therefore, reducing the expression of aβ protein and inhibiting the hyperphosphorylation of Tau protein are advantageous for improving cognitive impairment caused by AD, and play an important role in anti-AD.

Plumula Nelumbinis alkali (English name: LIENSININE; abbreviation: lien) is a dibenzyl isoquinoline alkaloid derived from plumula Nelumbinis, and is one of main active ingredients in plumula Nelumbinis. Numerous studies have shown that liensinine has a broad pharmacological effect, such as antioxidant stress, antiarrhythmic, antihypertensive, anticancer, antiinflammatory, neuroprotection, etc. However, the therapeutic effect and mechanism of liensinine on AD are not yet known.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention aims to provide the application of the liensinine in preparing the anti-Alzheimer disease medicine.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

application of liensinine in preparing medicine for treating Alzheimer disease is provided.

Further, in the application, the structural formula of the plumula Nelumbinis alkali is shown as (I):

Further, the anti-Alzheimer's disease manifestations include improving learning and memory in AD mice, reducing Abeta _1-42 protein expression in hippocampal tissues, and inhibiting Tau protein hyperphosphorylation.

Further, for the above application, the dosage form of the medicament is any pharmaceutically acceptable dosage form.

Further, for the above-mentioned applications, the dosage of the drug is any pharmaceutically acceptable dosage.

Compared with the prior art, the invention has the following beneficial effects:

1. The invention digs out the new medicinal value of the liensinine, and the application of the liensinine in preparing the anti-Alzheimer disease medicine provides a new medicine selection for preventing and treating the Alzheimer disease.

2. According to the invention, the study shows that the liensinine can significantly improve the learning and memory capacity of the AD mice induced by the combination of the D-galactose and the AlCl ₃, and has statistical significance. And the plumula Nelumbinis can play an anti-AD role by reducing the expression of Abeta _1-42 protein in the brain of the AD mice and inhibiting the hyperphosphorylation of Tau protein, and can be used for preparing medicines for preventing or treating AD.

Drawings

Fig. 1 is the effect of liensinine on escape latency of AD mice, ^**P<0.01,^*** P <0.001, n=10 compared to model group.

Fig. 2 is the effect of plumlins on the number of AD mice crossing the plateau and target quadrant residence time, (a) number of crossing the plateau, (b) target quadrant residence time, ^nsP≥0.05,^*P<0.05,^**P<0.01,^*** P <0.001 (n=10) compared to model group.

Fig. 3 is the effect of liensinine on spontaneous alternation response rate in AD mice, ^nsP≥0.05,^**P<0.01,^*** P <0.001 (n=10) compared to model group.

Fig. 4 is the effect of liensinine on AD mice exploring new forearm time percentages, ^nsP≥0.05,^**P<0.01,^*** P <0.001 (n=10) compared to model group.

Fig. 5 is the effect of liensinine on aβ _1-42 protein expression in hippocampal tissue of AD mice, ^nsP≥0.05,^**P<0.01,^*** P <0.001 (n=3) compared to model group.

Fig. 6 is the effect of liensinine on Tau protein phosphorylation in hippocampal tissue of AD mice, ^**P<0.01,^*** P <0.001 (n=3) compared to model group.

Detailed Description

The technical scheme and technical effects of the present invention are described in detail below with reference to specific embodiments and drawings. The following examples are given for illustrative purposes only and are not intended to limit the invention to the particular embodiments disclosed, since various modifications, equivalent substitutions, improvements, etc. will be within the spirit and principles of the invention, as will occur to those skilled in the art, without departing from the spirit and scope of the invention.

Example 1

The plumula Nelumbinis alkali used in the examples (LIENSININE, lien, purity > 98%) was purchased from the company of tsukhencheng remote biotechnology, ltd, of the formula:

Experimental methods

1. Experimental grouping

60 Kunming (KM) male mice were randomly divided into 6 groups of 10 animals each, which were a blank group (Con), a model group, a Lien (1 mg/kg) group, a Lien (3 mg/kg) group, a Lien (10 mg/kg) group, and a Don positive drug (3 mg/kg) group, respectively. The mice are adaptively fed for one week, so that the mice are adapted to living environment, and the stress response in the experiment is reduced.

2. Establishment and administration of AD mouse model

Mice of the model group, the Lien (1 mg/kg) group, the Lien (3 mg/kg) group, the Lien (10 mg/kg) group, and the Don (3 mg/kg) group were intraperitoneally administered with D-galactose (D-gal, 120 mg/kg) and AlCl ₃ (20 mg/kg) by gavage every day, and molding was performed for 90 days. Meanwhile, the mice of Con group were intraperitoneally injected and lavaged with the same volume of physiological saline. Starting on day 31, mice in the dosing group were intraperitoneally injected with Lien (1, 3 or 10 mg/kg) and Don (3 mg/kg) for 60 days, respectively, in each of the different groups every day afternoon. Meanwhile, mice of the Con group and the model group were intraperitoneally injected with an equal volume of physiological saline. Behavioural experiments were performed at the last week of dosing.

3. Behavioural experiments

(1) Morris water maze experiment

① Positioning navigation experiments were performed continuously for 5 days, the mice were placed in water in four quadrants of the water maze device one after the other every day, and were allowed to swim for 90s, and their escape latency (time taken for the mice to find the platform) was recorded. If the mouse does not land on the platform within 90s, it is guided to the platform and allowed to rest on the platform for 30s, at which point the escape latency is recorded as 90s.

② Space search experiments were performed the next day after the end of the navigation experiments, the platform in the water was removed before the start of the experiments, the mice were placed in the water from the opposite quadrant to the quadrant in which the platform was located (target quadrant), and the number of times the mice traversed the original platform location and the residence time in the target quadrant were recorded.

(2) Y maze

① The spontaneous alternation reaction experiment shows that the three arms of the device are respectively an initial arm A, a new different arm B and other arms C. Each mouse was placed in arm a during the experiment, allowed to explore freely, the sequence of the mice into the three arms within 8min was recorded, and the spontaneous alternation response rate was calculated.

② Discrimination exploration experiment, which is to divide the experiment into two stages. During the training period, mice were placed in arm from the starting arm a (when the new heteroarm baffle was put down) and allowed to explore freely for 5min. After 3 hours, a test period was started, the mice were again placed from the starting arm a facing the wall (the new alien arm partition was opened) so that they were free to move in the three arms for 5min, the time for the mice to enter the new alien arm B and the total arm entry time were recorded, and the percentage of time to enter the new alien arm B was calculated.

4. Immunoblot analysis

The hippocampal tissue of the experimental mice was taken, added with RIPA lysate, homogenized for 5min in a tissue disrupter, fully lysed for 30min at 4℃and centrifuged (15000 rpm, 4℃for 15 min) and the supernatant was collected. After the BCA method determines the protein content of the sample, the protein sample is separated in an SDS-PAGE gel and transferred to a PVDF membrane. PVDF membranes were blocked with 5% nonfat dry milk at room temperature for 2h. PVDF membrane was then placed in a specific primary antibody (Abeta _1-42, 1:500; p-Tau,1:1000; beta-actin, 1:500), and incubated overnight at 4 ℃. After TBST washing PVDF membrane to remove unbound primary antibody, HRP-labeled secondary antibody (HRP-coat anti-rabbit IgG, 1:10000) was added and incubated for 2h at room temperature. After washing the PVDF membrane with TBST to remove unbound secondary antibody, 1mL of ECL solution was added in the dark, and the strip was repeatedly blown for 15s to allow the reaction to be complete. PVDF membranes were exposed in a full-automatic chemiluminescence/fluorescence Image analysis system until the protein bands were developed and the images were analyzed using Image J software.

(II) results of experiments

1. Effect of liensinine on escape latency of AD mice

In Morris water maze positioning navigation experiments, escape latency reflects the spatial memory capacity of mice. As shown in fig. 1, con group mice showed a significant decrease in escape latency from day 2 (P < 0.001) compared to the model group. After Lien (3 and 10 mg/kg) and Don (3 mg/kg) administration treatments, the escape latency of mice was significantly shortened (P < 0.001) with prolonged training time, indicating that the spatial memory capacity of AD mice was significantly enhanced after liensinine administration.

2. Influence of liensinine on the number of times of AD mice crossing the platform and target quadrant residence time

In the space exploration test, the number of times of crossing the platform and the target quadrant residence time reflect the learning and memory capacity of the mice. Experiments have found (fig. 2a and b) that model group mice are unable to pinpoint the original platform position, and AD mice pass through the platform less frequently and target quadrant residence time (P < 0.001) than Con group. Through Lien (3 and 10 mg/kg), the times of mice crossing the platform and the target quadrant residence time are both obviously increased, which shows that the treatment of the liensinine improves the learning and memory capacity of AD mice.

3. Influence of liensinine on spontaneous alternative response rate of AD mice

In the Y maze experiment, spontaneous alternation response rate can be used to evaluate spatial working memory of mice. As shown in fig. 3, the spontaneous alternation response rate of model mice was significantly reduced (P < 0.001) compared to Con mice, and it was not possible to better alternation into the three arms, indicating that they had weaker spatial memory in the Y maze. After the drug treatment, the phenomenon is improved, and the spontaneous alternation reaction rate is obviously improved (Lien 3mg/kg: P <0.001; lien 10mg/kg: P < 0.01), which indicates that the spatial memory capacity of the AD mice can be obviously improved after the administration of the liensinine.

4. Effect of liensinine on the percentage of time AD mice explored new heteroarm

In the discriminatory exploration experiment, the ratio of mice to the exploration time of the new heteroarm reflects the spatial reference memory capacity of the mice. As shown in fig. 4, the percentage of time AD mice entered the new xenograft was significantly reduced compared to the Con group (P < 0.001). After Lien (3 and 10 mg/kg) treatment, the time for mice to explore new heteroarms was significantly increased (P < 0.001), indicating that liensinine improved spatial reference memory in AD mice.

5. Effect of liensinine on Abeta _1-42 protein expression in hippocampal tissue of AD mice

As can be seen from fig. 5, the model group mice showed significantly higher expression of aβ _1-42 protein in the hippocampus than in the Con group (P < 0.001). 3 and 10mg/kg of Lien treatment showed a significant decrease in the expression of Abeta _1-42 protein in the brain of mice (Lien 3mg/kg: P <0.01; lien 10mg/kg: P < 0.001), indicating that administration of liensinine can inhibit the expression of Abeta _1-42 protein in the hippocampal tissue of AD mice.

6. Effect of liensinine on Tau protein phosphorylation in hippocampal tissue of AD mice

As shown in fig. 6, the hippocampal tissue Tau protein phosphorylation was abnormally increased in the model group mice compared to the Con group (P < 0.001). After Lien (1, 3 and 10 mg/kg) treatment, the phosphorylation level of Tau protein in the brain of mice was significantly reduced (Lien 1mg/kg: P <0.01; lien 3 and 10mg/kg: P < 0.001), indicating that liensinine inhibited abnormal phosphorylation of Tau protein in hippocampal tissues of AD mice.

Claims (5)

1. Application of liensinine in preparing medicine for treating Alzheimer disease is provided.

2. The use according to claim 1, wherein the plumula Nelumbinis alkali has the structural formula shown in (I):

3. The use according to claim 1, wherein said anti-alzheimer's disease manifestations include improving learning and memory, reducing aβ _1-42 protein expression in hippocampal tissue and inhibiting Tau protein hyperphosphorylation.

4. The use according to claim 1, wherein the pharmaceutical dosage form is any pharmaceutically acceptable dosage form.

5. The use according to claim 1, wherein the dose of the medicament is any pharmaceutically acceptable dose.