CN111249262A

CN111249262A - Application of alkyl resorcinol compound in preparing medicine for preventing or treating Alzheimer's disease

Info

Publication number: CN111249262A
Application number: CN202010082788.0A
Authority: CN
Inventors: 王静; 刘洁; 王子元; 王玉
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2020-02-07
Filing date: 2020-02-07
Publication date: 2020-06-09
Anticipated expiration: 2040-02-07
Also published as: CN111249262B; WO2021155633A1

Abstract

The invention provides an application of an alkyl resorcinol compound in preparation of a medicine for preventing or treating Alzheimer's disease, and belongs to the field of biological medicines. The structure of the alkyl resorcinol compound is shown as a general formula (I), wherein R1 is any one of C5-C50 straight-chain or branched-chain alkyl. Experiments prove that the alkyl resorcinol compound can realize the prevention or treatment of the Alzheimer's disease by changing the expression of various pathogenic related proteins and pathway proteins in the brain and regulating the composition of intestinal flora and the abundance of characteristic flora.

Description

Application of alkyl resorcinol compound in preparing medicine for preventing or treating Alzheimer's disease

Technical Field

The invention relates to the field of biological medicines, in particular to application of an alkyl resorcinol compound in preparing a medicine for preventing or treating Alzheimer's disease.

Background

The main pathological features of AD are the deposition of extracellular amyloid plaques of the brain (a β), the accumulation of Tau tubulin, abnormal manifestations of neurofibrillary tangles and neuronal, synaptic loss, which in turn manifest as cognitive impairment, behavioral abnormalities and even death.

Epidemiological investigations have shown that consumption of whole grain products containing wheat bran can act to resist oxidative damage, reduce the incidence of obesity, diabetes and some cancers. The testa Tritici fat-soluble part can exert its physiological effect more effectively than water-soluble part. Further research shows that the fat-soluble active substance, namely Alkylresorcinols (ARs), in the wheat bran has good biological activity of resisting oxidation, reducing blood sugar and resisting tumors. The wheat alkyl resorcinol is a1, 3-dihydroxybenzene derivative, an odd-carbon alkyl side chain is connected to the 5 th carbon of a benzene ring, and the side chain is mainly composed of C15: 0. c17: 0. c19: 0. c21: 0. c23: 0 and C25: 0, the wheat bran is mixed with the mixture of C19: 0 and C21: the highest content of 0. Other researches show that the alkylresorcinol can play a role in protecting against liver cancer by inducing autophagy and apoptosis of HepG2 cells; the alkyl resorcinol can also play a role in protecting cells from oxidative stress by regulating an Nrf2/HO-1 pathway, and the like, but the action and mechanism of the alkyl resorcinol which is particularly applied to the aspect of the antioxidant stress of the nervous system are not reported yet and are still yet to be explored and researched.

Chinese patent application 201910981462.9 discloses the use of alkylresorcinols in the preparation of products for the prevention or treatment of obesity related diseases. The product comprises an active ingredient of alkylresorcinol, wherein the alkylresorcinol consists of the following monomers in percentage by weight: 1-10% of heptadecyl resorcinol; nonadecyl resorcinol 20-25%; 50-55% of heneicosyl resorcinol; 5-10% of eicosatriylresorcinol; 5-12% of pentacosyl resorcinol. The invention defines the proper proportion of the corresponding monomers when the alkylresorcinol plays a role in preventing or treating obesity by improving the energy metabolism or the heat production of an organism.

Chinese patent application 201910593007.1 discloses a composition comprising an amide derivative and its use in cosmetics, said composition comprising: a. at least one amide derivative (A) represented by the formula (1); b. at least one resorcinol derivative (B) represented by the formula (2); and at least one cosmetically acceptable component (C). The amide derivatives contained in the compositions described herein have a positive enhancing effect on the transdermal delivery of the said compositions, favouring the active substances of the compositions to reach the site of action through the layers of skin and generating a preventive and/or inhibitory effect on the visible discontinuous appearance of the skin caused by the pigmentation of the skin, by the ageing of the skin or by other conditions, treating, regulating or preventing the oxidative stress or the processes of deterioration of the skin, inhibiting the formation and precipitation of melanin, reducing the darkening of the skin, increasing the volume of the skin and keeping the skin shiny and fair.

However, the above patent applications do not mention the specific application of alkylresorcinol compounds in the anti-oxidative stress of nervous system, and no report on the use of alkylresorcinol compounds for preparing drugs for preventing or treating alzheimer's disease has been found so far. In view of the above, the invention provides an application of an alkylresorcinol compound in preparing a medicament for preventing or treating alzheimer's disease, which provides a new idea for preventing and treating alzheimer's disease and has important significance.

Disclosure of Invention

The invention aims to provide application of alkyl resorcinol compounds in preparing medicaments for preventing or treating Alzheimer's disease. The applicant researches the expression of various pathogenic related proteins and pathway proteins in the brain and regulates the composition of intestinal flora and the abundance of the flora to change through animal experiments and cell experiments so as to clarify the mechanism of the action of the alkyl resorcinol compounds on the Alzheimer's disease and the prevention or treatment effect.

In order to achieve the above object, the present invention provides the following technical solutions:

the application of the alkyl resorcinol compound in preparing the medicine for preventing or treating the Alzheimer disease.

Wherein the alkyl resorcinol compound is shown as the following general formula (I).

In the formula, R1 is C5-C50 straight chain or branched chain alkyl; preferably, R1 is a C9-C35 linear or branched alkyl group, more preferably a C15-C30 linear or branched alkyl group; still more preferably a C15 linear or branched alkyl group, a C17 linear or branched alkyl group, a C19 linear or branched alkyl group, a C21 linear or branched alkyl group, a C23 linear or branched alkyl group, a C25 linear or branched alkyl group, a C27 linear or branched alkyl group, or a C29 linear or branched alkyl group; still further, C17 straight or branched chain alkyl group, i.e., heptadecyresorcin (AR-C17), is preferred, as shown in formula (II). The alkyl resorcinol compounds can change the expression of various pathogenic related proteins and pathway proteins in the brain, thereby playing a role in preventing or treating the Alzheimer disease.

The medicine can be prepared into any one of tablets, powder, paste, aqueous solution, injection or infusion solution.

The dosage of the medicine is 150 mg/kg/day.

The preparation of the medicine for preventing or treating the Alzheimer disease by the alkyl resorcinol compound is realized by changing the expression of various pathogenic related proteins and pathway proteins in the brain and regulating the flora composition and the abundance of characteristic flora.

Specifically, the preparation of the medicine for preventing or treating the Alzheimer disease by the alkyl resorcinol compounds is realized by reducing the accumulation of A β plaques in the brain, reducing the activation accumulation and inflammation expression of microglia, reducing the expression of β -amyloid protein and Tau protein in the brain of a mouse, obviously reducing the expression of ADAM10 and synapsin so as to improve the intervention of AR-C17, activating a SIRT3 signal channel and a downstream effector SOD2 thereof and improving the abundance of flora with a protective effect on AD.

The research method mainly comprises the following steps:

1. animal feeding:

all mice were housed in a single cage under standard SPF-level conditions (20-25 ℃, 55% humidity, 12 hours light and dark cycle, free access to food and water). A three-month-old male APP/PS1 double-transgenic knockout mouse B6C3-Tg [ APPSwe, PSEN1dE9] is selected, the body weight is about 30g, the drug resorcinol (AR) is uniformly suspended in 0.5% food-grade sodium carboxymethylcellulose (CMC-Na) solution, and the drug is administrated in a gastric perfusion mode. AD model animal (APP/PS1) mice were randomly divided into two groups: AD control group (APP/PS1+ CMC-Na) and dosing AR group (APP/PS1+ AR), wherein each group contains 12 animals, and the administration dose concentration is 150 mg/kg/day. 12 non-transgenic mice were additionally set as a background group (C57/BL6J) and given an equivalent amount of CMC-NA (0.5%) per day as a control. The administration was performed daily by gavage for 5 months.

2. Improvement of cognitive behaviour in APP/PS1 mice by AR:

adopting an internationally accepted animal behavioural detection method: and in the Morris water maze experiment, the spatial memory ability, the new memory forming ability and the cognitive ability of the mouse are subjected to apparent evaluation, and the continuous administration or the death is determined according to the evaluation result. The Morris water maze test was performed 1 week before sacrifice. The Morris water maze is a circular water tank with the diameter of 120cm multiplied by 50cm, is filled with water (22 +/-1 ℃), is provided with a platform with the diameter of 10cm in the third quadrant, and is submerged at the position 0.5cm below the water surface. In the 5-day trial, the hidden platform was fixed in the same place. 4 days before the last probe test, each mouse was allowed to freely search for a platform hidden under the water surface within 120s, and mice that could not find a hidden platform within 120s would be guided onto the platform, staying for 20 seconds to remember the spatial location. The last probe test was performed on day 5, the hidden platform was removed and each mouse was free to look for 120 seconds. All data, such as swimming trajectories, target quadrant crossing times, time to first reach the target area, etc., are automatically recorded and collected and statistically analyzed using SPSS software.

3. Improvement of the pathological changes of brain tissue of APP/PS1 mice by AR:

2 days after the behavior test, 4 mice (n is 4) are selected for each group, pentobarbital sodium (50mg/kg) is injected into the abdominal cavity, then normal saline and 4% paraformaldehyde solution are used for cardiac perfusion, after about 10min of fixation, the whole brain is soaked in 4% paraformaldehyde for 2h, after 2h, the brain is soaked in sucrose solution for dehydration, and the brain is placed in a refrigerator at 4 ℃ for storage until being sliced.

The analysis method comprises the following steps:

iba1/Nissl double staining experiment: frozen brain tissue sections were removed, blocked with blocking solution (3% donkey serum, 10% Triton in 0.1M PBS) for 30min, and incubated overnight at 4 ℃ with primary antibody (anti-Iba 1). After 3 washes in PBS buffer, brain sections were placed in secondary antibodies containing target different excitation wavelength secondary antibodies (Donkey anti-Ms594, Nissl 500/525) and incubated for 1.5h in the dark. The sections were then washed 3 times in PBS buffer, mounted, and the mounted sections were imaged using an Olympus FV1200 confocal microscope.

A β/GFAP/Nissl triple stain experiment frozen brain tissue sections were removed and blocked with blocking solution (3% Donkey serum, 10% Triton in 0.1M PBS) for 30min, incubated overnight at 4 ℃ with primary antibodies (anti-A β, anti-GFAP) and washed 3 times in PBS buffer, then the brain sections were incubated in dark for 1.5h in secondary antibodies containing target different excitation wavelength secondary antibodies (Donkey anti-Rb 488, Donkey anti-Ms594, Nissl 435/455) and then washed 3 times in PBS buffer, mounted and the mounted sections were imaged using an Olympus FV1200 confocal microscope.

4. The improvement effect of AR on the change of the brain tissue biochemical indexes of APP/PS1 mice:

the perfused mice were removed, the remaining mice were sacrificed by decapitation, and cerebral cortex and hippocampus were separated and stored at-80 ℃ for biochemical index analysis. Pathological related proteins, nerve health related factors, pathway related proteins and the like are analyzed by a western blotting method. The analysis and research on biochemical mechanism are carried out on how AR improves AD pathological change.

The analysis method comprises the following steps:

the method adopts a Western-blot Western blotting method to carry out quantitative analysis on the related phenotypic proteins, and comprises the following specific operation steps: tissue lysates were prepared by RIPA and total protein was determined by BCA. After gel electrophoresis with 12% separation gel, the protein isolate was transferred to PVDF membrane, washed and blocked with skim milk powder antigen, and the antibody to be detected was incubated overnight at 4 ℃. The membrane was then washed with TBST for 5 minutes and then incubated with goat anti-rabbit IgG H & l (hrp) for 1 hour at room temperature. The blot was washed again with TBST for 5 min and finally detected on a gel imager using chemiluminescent reagents.

5. Improvement of intestinal flora abundance by AR in APP/PS1 mice:

mouse treatment method: feces are collected from all mice one day before the behavioral experiments, and are frozen in a refrigerator at the temperature of minus 80 ℃ until intestinal bacteria are detected.

And (3) measuring the abundance of the intestinal flora: fecal samples from each group were analyzed for microflora. Extracting the total DNA of the frozen stool sample, and detecting the concentration of the separated DNA. PCR amplification and purification are carried out on the V3-V4 region of 16S rRNA, sequencing is carried out by using QIIME software package and Illumina MiSeq platform, and bioinformatics analysis is carried out.

In the present invention, AR means alkylresorcinol compounds.

The invention has the following beneficial effects:

the alkyl resorcinol compound is used as a new tool and applied to the preparation of a pharmaceutical composition or a preparation for preventing or treating the Alzheimer disease, can obviously improve the spatial memory capacity and the cognitive ability, delays the AD generation and plays a role in protection. In addition, the present invention demonstrates that AR is able to activate SIRT3 signaling pathway and its downstream effector SOD 2.

Drawings

FIG. 1 is a graph of the escape time (A) of the first 4 days, the time (B) for finding a target area on the last day, the number of times of target crossing (C) and the swimming trajectory (D) of three groups of mice in a water maze experiment, wherein WT refers to a background group of mice (C57BL/6J), AD refers to an AD control group of mice (APP/PS1 double transgenic mice), AR-C17 refers to a medicated APP/PS1 transgenic mouse (APP/PS1+150 mg/kg/day AR-C17);

FIG. 2 is a hippocampal pathology plot of three groups of mice, A β/GFAP/Nissl triple-staining immunofluorescence plot, wherein WT refers to a background group of mice (C57BL/6J), AD refers to an AD control group of mice (APP/PS1 double transgenic mice), AR-C17 refers to a medicated APP/PS1 transgenic mouse (APP/PS1+150 mg/kg/day AR-C17);

fig. 3 is a hippocampal pathology plot for three groups of mice: iba1/Nissl double-staining immunofluorescence map, wherein, WT refers to a background group mouse (C57BL/6J), AD refers to an AD control group mouse (APP/PS1 double-transgenic mouse), AR-C17 refers to a medicated APP/PS1 transgenic mouse (APP/PS1+150 mg/kg/day AR-C17);

FIG. 4 is a diagram of pathological protein expression and neuroinflammation intervention related to AD in hippocampal and cortical tissues of three groups of mice, wherein WT refers to a background group of mice (C57BL/6J), AD refers to an AD control group of mice (APP/PS1 double transgenic mice), AR-C17 refers to a medicated APP/PS1 transgenic mouse (APP/PS1+150 mg/kg/day AR-C17);

FIG. 5 is a graph showing the effect of AR-C17 on SIRT3/SOD2 signal pathway activation, wherein WT refers to a background group mouse (C57BL/6J), AD refers to an AD control group mouse (APP/PS1 double transgenic mouse), AR-C17 refers to a medicated APP/PS1 transgenic mouse (APP/PS1+150 mg/kg/day AR-C17);

FIG. 6 is a graph showing the abundance of intestinal flora in three mice, wherein WT refers to a background group of mice (C57BL/6J), AD refers to an AD control group of mice (APP/PS1 double transgenic mice), and AR-C17 refers to a medicated APP/PS1 transgenic mouse (APP/PS1+150 mg/kg/day AR-C17).

Detailed Description

The present invention will be further explained with reference to specific examples in order to make the technical means, the technical features, the technical objectives and the effects of the present invention easier to understand, but the following examples are only preferred embodiments of the present invention, and not all embodiments of the present invention. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The heptadecafluororesorcinol of the following examples was prepared by the following method:

selecting dried wheat bran as a raw material, selecting, finishing and grinding the raw material into powder, adding ethyl acetate to perform ultrasonic extraction, centrifuging to obtain a supernatant, and performing rotary evaporation to obtain a concentrated solution. And then, carrying out silica gel column chromatography for separation and purification by utilizing solid phase extraction or silica gel mixing samples, collecting eluent for further concentration to obtain mixed liquor of different homologues of the alkylresorcinol, and collecting heptadecyltrenol samples according to different peak times, wherein the purity is more than 97%.

Example 1 animal feeding

All mice were housed in a single cage under standard SPF-level conditions (20-25 ℃, 55% humidity, 12 hours light and dark cycle, free access to food and water). A three-month-old male APP/PS1 double-transgenic knockout mouse B6C3-Tg [ APPSwe, PSEN1dE9] is selected, the weight of the mouse is about 30g, and a medicament of heptadecyl resorcinol (AR-C17) is uniformly suspended in a 0.5% food-grade sodium carboxymethylcellulose (CMC-Na) solution and is administrated in a gastric perfusion mode. AD model animal (APP/PS1) mice were randomly divided into two groups: an AD control group (APP/PS1+ CMC-Na) and a dosing AR-C17 group (APP/PS1+ AR-C17), wherein each group contains 12 animals, and the administration dose concentration is 150 mg/kg/day. 12 non-transgenic mice were additionally set as a background group (C57/BL6J) and given an equivalent amount of CMC-NA (0.5%) per day as a control. The administration was performed daily by gavage for 5 months.

Example 2 Water maze experiment

Adopting an internationally accepted animal behavioural detection method: and in the Morris water maze experiment, the spatial memory ability, the new memory forming ability and the cognitive ability of the mouse are subjected to apparent evaluation, and the continuous administration or the death is determined according to the evaluation result. The Morris water maze test was performed 1 week before sacrifice. The Morris water maze is a circular water tank with the diameter of 120cm multiplied by 50cm, is filled with water (22 +/-1 ℃), is provided with a platform with the diameter of 10cm in the third quadrant, and is submerged at the position 0.5cm below the water surface. In the 5-day trial, the hidden platform was fixed in the same place. 4 days before the last probe test, each mouse was allowed to freely search for a platform hidden under the water surface within 120s, and mice that could not find a hidden platform within 120s would be guided onto the platform, staying for 20 seconds to remember the spatial location. The last probe test was performed on day 5, the hidden platform was removed and each mouse was free to look for 120 seconds. All data, such as swim trajectory, target quadrant crossing times, time to first reach target area, etc., were automatically recorded and collected and statistically analyzed using SPSS software.

The result shows that compared with the WT group, the APP/PS1 transgenic mouse has obviously disordered track, the escape time and the time for reaching the target area for the first time are obviously prolonged, and the times of crossing the target quadrant are obviously reduced; the medicine (AR-C17) intervenes in APP/PS1 mice for 5 months, and can obviously improve the spatial memory ability and cognitive ability of the mice.

EXAMPLE 3 pathological changes in brain tissue

The A β/GFAP/Nissl triple-staining fluorescence diagram shows that the brain of mice in the blank APP/PS1 group has A β plaques and astrocytes, while the brain of mice in the non-transgenic mice (C57BL/6J) and APP/PS1 mice with gavage AR-17 has no or significantly less aggregation, so that the AR can reduce the aggregation of A β plaques in the brain, thereby delaying the occurrence of AD and playing a role in protection.

Example 4 brain tissue Key Effector protein changes

The perfused mice were removed, the remaining mice were sacrificed by decapitation, and cerebral cortex and hippocampus were separated and stored at-80 ℃ for biochemical index analysis. By means of Western blotting, pathological related protein and nerve health related factor are analyzed to research how AR can improve AD pathological change in biochemical mechanism.

The experimental results show that β -amyloid protein in brains of mice in the APP/PS1 group is greatly aggregated and Tau protein is over-phosphorylated, while APP/PS1 mice intervened by Wild Type (WT) and AR-C17 are not or are remarkably reduced in brains, therefore, AR-C17 remarkably reduces β -amyloid protein and Tau protein expression in brains of mice, thereby delaying AD generation and playing a role in protection.

Example 5 AR-C17 activation of the SIRT3/SOD2 Signal pathway

Pathway-associated proteins and the like were analyzed by western blotting. Sirt3 is an important deacetylation modification enzyme in mitochondria, can regulate and control the activity of a plurality of metabolic enzymes in mitochondria, and further regulate and control the metabolism of cell mitochondria, the activation of Sirt3 is closely related to anti-aging, anti-tumor, immunity improvement and the like, and an effective way and a medicine for activating Sirt3 are not found so far. And (3) carrying out quantitative analysis on related phenotypic proteins by using a Western-blot Western blotting method, preparing a tissue lysate by using a RIPA method, and determining total protein by using a BCA method. After gel electrophoresis with 12% separation gel, the protein isolate was transferred to PVDF membrane, washed and blocked with skim milk powder antigen, and the antibody to be detected was incubated overnight at 4 ℃. The membrane was then washed with TBST for 5 minutes and then incubated with goat anti-rabbit IgG H & l (hrp) for 1 hour at room temperature. The blot was washed again with TBST for 5 min and finally detected on a gel imager using chemiluminescent reagents.

As shown in fig. 5, AR-C17 was able to activate the SIRT3 signal pathway and its downstream effector SOD 2. In cerebral cortex and hippocampus tissues, compared with the WT group, in the AD group, the SIRT3/SOD2 signal channel expression is obviously reduced, and the AR-C17 dry prognosis shows that the signal channel is obviously improved.

Example 6 Enterobacter abundance test

Mouse treatment method: feces are collected from all mice one day before the behavioral experiments, and are frozen in a refrigerator at the temperature of minus 80 ℃ until intestinal bacteria are detected. Fecal samples from each group were analyzed for microflora. Extracting the total DNA of the frozen stool sample, and detecting the concentration of the separated DNA. PCR amplification and purification are carried out on the V3-V4 region of 16S rRNA, sequencing is carried out by using QIIME software package and Illumina MiSeq platform, and bioinformatics analysis is carried out.

FIG. 6 shows that APP/PS1 model mice are significantly reduced in abundance relative to background group flora, particularly Akkermansia (muciniphilic-Ackermansia) and Lactobacillus (Lactobacillus). Both of these two bacteria have been demonstrated to have protective effects on AD in clinical and different experimental animals. The intervention group AR-C17 can obviously improve the abundance of the flora of the AD model mouse, particularly improve the abundance of the two beneficial bacteria.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. The application of the alkyl resorcinol compound in preparing the medicine for preventing or treating the Alzheimer disease is characterized in that the alkyl resorcinol compound is shown as the following general formula (I),

wherein R1 is any one of C5-C50 straight chain or branched chain alkyl.

2. The use of claim 1, wherein the medicament can be prepared into any one of tablets, powders, pastes, aqua, injections or infusion solutions.

3. The use of claim 1, wherein the medicament is present in an amount of 150 mg/kg/day.

4. The use according to claim 1, wherein the alkylresorcinol compounds are used for preparing the medicament for preventing or treating the Alzheimer's disease by changing the expression of various pathogenic related proteins and pathway proteins in the brain and regulating the flora composition and the abundance of characteristic flora.

5. The use of claim 1, wherein the alkylresorcinol compounds are used for the preparation of a medicament for preventing or treating Alzheimer's disease by reducing the accumulation of A β plaques in the brain, reducing the activated accumulation of microglia and the expression of inflammation, reducing the expression of β -amyloid protein and Tau protein in the brain of mice, significantly reducing the expression of ADAM10 and synaptosomes, activating a SIRT3 signal pathway and a downstream effector SOD2 thereof, and increasing the abundance of flora having a protective effect on Alzheimer's disease.