CN111588727A

CN111588727A - Medical application of balamin B in resisting Alzheimer disease

Info

Publication number: CN111588727A
Application number: CN202010570664.7A
Authority: CN
Inventors: 徐晓军; 钱程; 杨春杰
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-06-19
Filing date: 2020-06-19
Publication date: 2020-08-28

Abstract

The invention relates to medical application of balamin B in resisting Alzheimer disease. The balamin B is a Parishin derivative separated from gastrodia elata. The CL4176, CL2241 and CL2241 strains of nematodes are used for respectively inspecting the paralysis rate, chemotactic capacity and Abeta 42 plaque accumulation of the nematodes after the action of the balaneb B. And an AD model is acted by positioning and injecting Abeta 42 protein in the hippocampus of the mouse, and the behaviours of the AD model is investigated. The results show that the balaneboside B can delay the paralytic phenomenon caused by the accumulation of the nematode Abeta 42 protein, relieve the impaired learning and memory ability caused by the accumulation of the Abeta 42 protein, obviously reduce the accumulation of Abeta plaques and improve the memory retention ability of dysmnesia mice. Therefore, the barbaloside B has important application prospect in preparing the medicine for treating the Alzheimer disease.

Description

Medical application of balamin B in resisting Alzheimer disease

Technical Field

The invention relates to the field of natural medicines, in particular to medical application of Balisenside B in resisting Alzheimer disease.

Background

Alzheimer's Disease (AD) is a form of dementia that is prevalent in people of the elderly over 60 years of age, and its major pathological manifestations are progressive deterioration of learning memory and irreversible neuronal loss. One of the most prominent pathological features of AD is the appearance of senile plaques in the cerebral cortex and hippocampus, which are formed by extracellular accumulation of amyloid-beta. Furthermore, numerous studies have shown that: imbalance of A beta 42 protein metabolism causes neuroinflammation, oxidative damage, tau protein abnormal phosphorylation and finally causes irreversible damage to neurons, and is the fundamental link leading to AD. Clinical studies show that the A beta protein level begins to abnormally increase 15-20 years before obvious AD symptoms appear, the A beta protein in the brain is disturbed in metabolic balance, the clearance ability is reduced, and the activity of related secretase is abnormally increased, which can be a key factor for causing the generation and the development of AD. Therefore, the regulation of the brain A beta 42 level becomes a key link of AD treatment, and the promotion of the A beta 42 degradation is a potential AD treatment means.

The gastrodia elata (Gastrodia elata) is a dry tuber of Gastrodia elata Bl. of the family Orchidaceae and has the effects of calming wind and relieving spasm, calming liver yang, dispelling wind and dredging collaterals₃₂H₄₀O₁₉Molecular weight 728.6486, white crystalline powder, soluble in organic solvents such as methanol, ethanol, DMSO, etc., and has the following chemical formula:

disclosure of Invention

The invention discloses an anti-Alzheimer disease effect of the barbaloside B, namely the barbanoside B can be used for preparing a medicament for treating Alzheimer disease and has an excellent effect.

The inventor finds that the barbaison B can effectively reduce the level of Abeta 42 and improve the dysmnesia of AD model mice injected with Abeta 42 protein in hippocampus in a pharmacological activity research of the barbaison B.

The anti-alzheimer activity of the barbaloin B is further illustrated below with reference to the examples.

Drawings

FIG. 1 shows the results of the paralysis rate test of Balisin B

FIG. 2 shows the chemotaxis experiment results of Balisin B

FIG. 3 shows the result of the action of Balsamine B on the A beta 42 plaque accumulation of nematodes of the CL2331 line

FIG. 4 shows the treatment of SHSY5Y with Balison B_SWCells that reduce extracellular A β 42 protein levels

FIG. 5 shows treatment of SHSY5Y with Balison B_SWCells that reduce intracellular A β 42 protein levels

Fig. 6 shows the escape latency (n 10) for each group of mice in the pilot experiment.

FIG. 7 shows the number of platforms crossed by each group of mice in the space exploration experiment (n ═ 10)

Figure 8 is the percentage of time each group of mice stayed in the target quadrant in the space exploration experiment (n-10).

Fig. 9 shows the percentage of distance traveled by each group of mice in the target quadrant in the space exploration experiment (n-10).

FIG. 10 shows the swimming trajectories of groups of mice in a space exploration experiment.

Wherein the sham in the figure is a sham group; model is a Model group; DPZ is a positive drug group, namely donepezil; ParB is the group administered, Balison B.

Detailed Description

Example 1 Balisin B delays muscle paralysis of Exophiala Alzheimer's disease

1. Biological material

(1) CL4176, the CL802 line transgenosis nematode is given by ChristopherD. Link, the CL4176 line nematode is a temperature-sensitive muscle cell specificity expression A beta 42 protein nematode line, the transcription expression of A beta 42 is started when the environmental temperature is increased from 16 ℃ to 25 ℃, the paralysis phenomenon is finally caused along with the accumulation of A beta 42 protein, and the C.elegans CL4176 line is taken as a pharmacological model for screening the anti-AD drugs.

(2) Coli OP50 uracil leak-deficient strains were purchased from the university of Nanjing model animal research center as food for C.elegans nematodes.

2. Reagent

(1) Liquid LB culture medium preparation

(2) Preparing a solid LB culture medium:

(3) OP50 strain culture: a small amount of the bacterial liquid is dipped by using an inoculating loop, and the streaked liquid is inoculated on a solid LB culture medium and cultured overnight at 37 ℃. A single clone on a solid medium was picked, inoculated into a liquid LB medium, and shaken overnight. The bacterial solution can be stored at 4 ℃ for later use. Care was taken in the above steps to maintain sterile operation. The bacterium liquid preserved at 4 ℃ is reused for obtaining the monoclonal by using a plate marking method every 7 days so as to ensure the activity of the bacteria.

(4) Preparation of Standard nematode growth plates (NGM)

When the temperature of the sterilization solution is reduced to about 55 ℃, placing the conical flask on a heating magnetic stirrer, and sequentially adding:

composition (I)	Content (wt.)
		1M calcium chloride solution	0.5mL
5mg/mL Cholesterol solution	1mL
		1M magnesium sulfate solution	1mL
Potassium phosphate buffer	25mL

And adding the prepared culture medium liquid into a corresponding plate or a corresponding hole plate respectively by using a peristaltic pump or a pipette, and cooling and solidifying the culture medium liquid overnight.

(5) M9 buffer preparation:

(6) preparing a lysis solution: 1.2mL of NaCl solution, 2mL of 8M NaOH solution and 6.8mL of water are mixed uniformly for later use. It is prepared fresh before use.

3. Preparing NGM flat plate containing Balison B

And (3) dropwise adding a proper amount of appropriate dilution of the barbaloin B into the NGM culture center, transferring the plate into a 37 ℃ incubator after the bacterial liquid is dried, and culturing overnight to form a circle of lawn with uniform thickness in the center of the NGM plate. The prepared food-containing NGM panels were sealed with a sealing film and stored at 4 ℃ before use, the panels were allowed to return to room temperature. The above operations all use sterile consumables and are carried out in a super clean bench, so that pollution is avoided.

4. Carrying out the step

(1) Nematode culture: inoculating the nematodes on a solid NGM flat plate containing OP50 bacterial liquid, culturing the nematodes with a constant-temperature incubator at 16 ℃ until the nematodes become adults, and performing synchronization treatment

(2) Nematode synchronization: gently washing the nematodes in the culture plate by using deionized water, collecting the nematodes in a 1.5mL sterile centrifuge tube, centrifuging and removing supernatant; adding a proper amount of lysate, performing vortex oscillation, and centrifuging again; removing supernatant, washing precipitate with M9 solution, centrifuging to obtain ovum and broken insect body; adding appropriate amount of M9 solution, blowing off the precipitate, inoculating into fresh NGM plate or medicated plate, and culturing. And continuously culturing at the corresponding culture temperature to obtain the synchronized nematodes.

(3) Paralysis induction and paralysis rate detection: paralysis induction experiments were performed using the CL4176 line nematode, using the CL802 line nematode as a negative control. Transferring the culture plate to a condition of 25 ℃ to induce the A beta gene to start transcription expression 48 hours after the synchronized egg inoculation, namely when the nematode grows to the L3 stage; the plates were transferred to 25 ℃ culture conditions and after 24h the number of paralysed nematodes was recorded, once every two hours and the paralysed individuals were transferred to the periphery of the plates to avoid repeated counts.

(4) And drawing a paralysis rate curve. And drawing a paralysis rate curve by taking the time as an abscissa and the percentage of the non-paralyzed nematodes in each time point as an ordinate. The ability of the target active ingredient to resist a β 42 toxicity is assessed by comparing the rate of paralysis of the various groups of nematodes.

The experimental results are as follows: the detection of paralysis rate finds that: the balaneb B can remarkably delay the paralysis phenomenon caused by accumulation of nematode Abeta 42 protein (figure 1).

Example 2, the balaneboside B can significantly alleviate cognitive impairment caused by accumulation of Abeta 42 protein

1. Biological material

Nematode of CL2241 line (nematode line of neuron intrinsic expression Abeta 42 protein)

2. Reagent

Diacetyl, a classical nematode chemotactic agent, purchased from Merck, tended to migrate and accumulate to the diacetyl odor source under normal physiological conditions, whereas during the induction of hunger, both hunger and treatment with the chemotactic agent were given to cause the nematode to remember its association with the environment of the chemotactic agent, so that the nematode did not significantly accumulate to the diacetyl odor source in the test plate.

Other reagents were prepared as in example 2

3. Carrying out the step

Nematode culture and synchronization (same procedure as in example 1)

After the nematodes are synchronized and grow for 36h at 16 ℃, the culture temperature is raised to 23 ℃ for further treatment for 36h, and then behavioral investigation is carried out: pick each group of nematodes to no food culture plate, drop diacetyl on a plate cover, culture for 2h in a reversed buckle way, and pick the nematode colony which is just cultured to detect in a detection plate: dripping 1 drop of diacetyl or ethanol at two ends of the diameter of the culture plate respectively to enable the culture plate to move freely for 1h, selecting whether to move towards the diacetyl direction according to the strength of the food-smell correlation, recording the number of the surrounding nematodes at diacetyl and ethanol marking points to calculate the chemotactic index, and evaluating whether the target active ingredient can enhance the learning and memory ability.

The experimental results are as follows: by utilizing a CL2241 line nematode (namely a neuron intrinsic expression A beta 42 protein nematode line), chemotactic experiments show that the balisonin B can obviously relieve the learning and memory capacity damage caused by the accumulation of A beta 42 protein, and the administered group nematodes successfully generate the associative memory of hunger and a chemotactic agent environment, and the group chemotactic factors are obviously lower than those of a control group (figure 2).

Example 3 effect of balisonide B on Α β 42 plaque accumulation in nematode of CL2331 strain

The influence of the target active ingredient on the accumulation of the A beta plaque is examined by an A beta plaque fluorescence counting method: a CL2331 line which inherently expresses the Abeta 42-GFP fusion protein in muscle cells is used as a research model, an insoluble Abeta plaque can be formed in the muscle cells and among the cells in the line nematode model, and the accumulation condition of the Abeta plaque in vivo is evaluated by observing a GFP signal by using a fluorescence microscope.

The cultured CL2331 line nematodes are synchronized, eggs obtained by the synchronization (the step is the same as the step in the embodiment 1) are inoculated into NGM plates containing 1% DMSO and 50 mu M of balamin B, the NGM plates are treated for 80 hours under the culture condition of 16 ℃, then various groups of nematode individuals are collected and fixed for photographing, and the influence of the target active ingredient on the accumulation of Abeta is evaluated by observing the quantity of Abeta plaques among muscles of various groups of nematode individuals. The results are shown in FIG. 3: the balaneb B was able to significantly reduce the accumulation of nematode Α β plaques in the CL2331 line compared to the control group.

Example 4 treatment of SHSY5Y with Balison B_SWEffect of cellular A β 42 protein levels

SHSY5Y treated with 50 μ M of the Balison B Compound_SWCells were incubated overnight with 1% DMSO as a normal control, and then cell culture supernatants and intracellular soluble A β 42 levels were examined using the MSD method, respectively, the results are shown in FIGS. 4 and 5, with 50 μ M of Barlin B treated SHSY5Y_SWThe level of a β 42 protein was decreased both extracellularly (figure 4) and intracellularly (figure 5) compared to normal controls (1% DMSO-treated cells).

Example 5 mouse hippocampal localization injection Abeta 42 protein AD model establishment, grouping and administration mode

1. Establishing AD injection model mouse

Sterilizing the surgical instruments at high temperature, and naturally cooling for later use. The surface of the surgical area was wiped with 75% medical alcohol. Mice were collected and anesthetized by intraperitoneal injection (i.p.) of pentobarbital sodium solution. The mice successfully anesthetized are fixed on a brain stereotaxic apparatus, hairs in an operation area are removed, and iodophor is wiped for disinfection. The skin above the skull is cut open, and a small amount of 3% hydrogen peroxide is dipped to quickly destroy the meninges and expose bregma. According to the stereotactic map of the mouse brain, with bregma as a reference point, the microinjector is moved as follows: 1.7mm behind the fontanel and 1.8mm away from the left and right sides respectively; marking a locating point on the skull, drilling a hole at the locating point by using a handheld electric drill, and carefully controlling the strength to ensure that the drilled hole just penetrates the skull without damaging brain tissues.

A microinjector is used for positioning and injecting a specified solution into the hippocampus, the needle insertion depth of the microinjector is 2mm, 410pmol (2.5 mu L) of A beta solution is injected into each side of the model group, the injection time of each side is controlled to be 5min by using a constant flow pump, and the needle is left for 5 min. The control group was injected with an equal amount of physiological saline in the same manner.

After the injection, the incision was sutured and covered with chlortetracycline ointment, and the mice were returned to their home cages for natural recovery. All mice subjected to surgery were given an intramuscular injection of penicillin 20000 units/day for 3 consecutive days after surgery.

2. Animal grouping and administration

After 3 days of recovery of 50 model-making mice and 10 sham-operated mice, 50 of the model-making mice were randomly divided into model groups, a positive medicine gastrodin low-dose group, a medium-dose group and a high-dose group, and each group contains 10 mice. The administration is carried out by gavage at nine am every day, and the continuous administration is carried out, wherein the gavage volume is 0.01 ml/g. The gavage situation is shown in the following table:

group of	Administration by intragastric administration	Dosage (mg/kg)
			Artificial operation group	Physiological saline	--
Model set	Physiological saline	--
			Positive drug group	Donepezil	1.0
Low dose group	Balisin B					10
			Middle dose group	Balisin B			20
High dose group	Balisin B					40

After 10 days of administration, a new object identification experiment is carried out, and the test time lasts for 3 days from 9 am to 3 pm every day; on days 14-19, water maze experiments were performed with test times ranging from 9 am to 5 pm each day and 30min before the experiments, and the administration was by gavage.

Example 6 Morris Water maze method for testing learning and memory abilities of mice

1. Device for measuring the position of a moving object

The Morris water maze consists of a pool, a platform, a curtain and a tracking camera system. The pool is virtually divided into four quadrants, a cylindrical platform is fixed at the center of the fourth quadrant, the diameter of the platform is 8cm, and colorless and transparent materials are adopted. And (5) injecting water into the pool to enable the final water level to be 0.5cm higher than the platform. Adding edible titanium dioxide into water, and stirring uniformly to make the water in the pool be opaque, uniform and milky white. The water in the pool was warmed to 22 ℃ before use. The curtain is white, four images are hung at the corresponding quadrant positions and used as a clue for the mouse to find the platform. The water maze system is placed in a single and quiet room to avoid direct light.

2. Acquired training

When training begins, the platform is placed in the fourth quadrant, the mouse is slightly released into water at the horizontal plane facing the direction of the pool wall at a preset position, and the mouse can be freely searched in the pool. The time was started at the moment the mouse entered the water. When the mouse is searched and stays on the platform, the timing is ended, and the corresponding time and the movement route are recorded. If the mouse fails to find the hidden platform within 90s, the mouse is guided onto the platform and allowed to stay on the platform for 30 s. After one training, the mouse is wiped and then placed in a feeding cage paved with a dry padding, and if necessary, the mouse can be irradiated under an infrared lamp to accelerate drying. Each mouse was trained four times a day (i.e., placed in water from four different quadrants), with 30min intervals. The acquired training was continued for 5 days.

3. Space exploration training

And performing space search training the next day after the acquired training is finished, namely the total sixth day. The platform is removed. The mice were released into the water from the second quadrant and allowed to freely search the pool for 90 s. And recording the movement route of the mouse, and analyzing the data of the mouse such as the residence time, the traveling distance, the number of times of crossing the original platform and the like in the fourth quadrant.

And (3) analyzing the result of the positioning navigation of the water maze, wherein the escape latency is an important index of the positioning navigation stage of the Morris water maze, and is the time required for successfully finding the platform for the first time after the animal enters water each time. The length of the compound represents the quality of the spatial learning ability of the animal, the incubation period is short, and the compound indicates that the learning ability of the animal is good. The spatial memory ability of the mice was examined by the Morris water maze behavioural test method. In the acquired training stage, the time (namely the latency) for successfully finding the platform by the Sham group mice is rapidly reduced, and the time for finding the hidden platform by the model group mice is longer, which indicates that the learning and memory ability of the mice is damaged by intracerebral injection of the Abeta protein; after the treatment of the treatment with the balamin B, the incubation period of the molding mice can be reduced in a dose-dependent manner (figure 6); in the space exploration experiment, the residence time, the walking distance and the platform crossing times of the mice in the target quadrant are obviously lower than those of the mice in the Sham group, and compared with the mice in the model group, the times of crossing the platform, the time percentage of residence in the target quadrant and the distance percentage of residence in the target quadrant of the mice in the model group are increased in a dose-dependent manner by giving treatment of the Balsinoside B (FIGS. 7, 8 and 9)

The swimming trajectory of the mice is shown in fig. 10, and the normal group is mainly trending and shows purposeful search; the model group mainly takes edge type as main and represents purposeless search; the administration group improved the swimming pattern of mice, especially in the high dose group, with an increased tendency to track. The result shows that the intervention of the balaneboside B can improve the memory retention capacity of the dysmnesia mice.

Claims

1. Use of barban B for the manufacture of a medicament for the treatment of Alzheimer's disease.