CN110568197A - Application of aquaporin 4 as depression drug target - Google Patents

Abstract

The invention discloses application of aquaporin 4 as a drug target for preventing and treating depression. Research results of the invention show that AQP4 has direct correlation with the morbidity and treatment of depression, the depression can block the expression of AQP4 in serum, so that the content of the AQP4 is reduced, and the content of AQP4 is increased after the treatment of medicaments such as stilbene glucoside, emodin and the like; after suffering from depression, the expression level of AQP4 in hippocampus is relatively reduced, and after being treated by medicaments such as stilbene glucoside, emodin and the like, the expression level of AQP4 is increased. AQP4 is a new therapeutic target for depression.

Description

Application of aquaporin 4 as depression drug target

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of aquaporin 4 as a depression drug target.

Background

the depression is a common disease in the psychiatric department, according to the statistics of the world health organization, the depression becomes the 4 th disease in the world, the burden of the 2 nd disease in China is predicted to be the second disease which is only second to coronary heart disease in 2020, the depression accounts for 15 percent of the global disease burden, the incidence rate of the lifetime depression is between 6 and 8 percent, and the incidence rate of the depression in the population over 60 years is up to 20 to 50 percent along with the gradual aging of the population. The depression patients are high risk groups of suicide, and the suicide risk exists from the onset to the rehabilitation period of the patients. The identification and diagnosis rate of depression is very low, and the effective cure rate is also very low. At present, the pathogenesis of the depression is not clearly analyzed, and the depression is generated by a plurality of biological hypotheses, including a monoamine neurotransmitter hypothesis, a brain reward pathway damage hypothesis, a hypothalamus-pituitary-epinephrine axis (HPA axis) dysfunction hypothesis, a neurotrophic factor hypothesis and the like, so that the selectable drug targets and effective therapeutic drugs for treating the depression are very limited.

Aquaporins (AQPs) are hydrophobic internal membrane proteins with molecular weight of 26-34 kDa, contribute to rapid and passive transport of water, and contain 250-290 amino acids. The first AQP was initially discovered in 1988 by Agre et al isolated from human erythrocytes and designated AQP from discovery to date, the number of aquaporin family members has continued to increase, and 13 AQPs, AQP0-AQP12, are now discovered in humans and rodents, with AQP4 being the most prominent aquaporin in the mammalian brain.

AQP4 was isolated from rats by Hase-gawa et al in 1994 by homologous cloning. The AQP4 gene is located at the junction between human chromosomes 18q11.2 and q 12.1. Consists of 4 exons and 3 introns, wherein the amino acid sequences coded by the 4 exons are 127, 55, 27 and 92 bits respectively, and the three introns are 0.8, 0.3 and 5.2Kb in length respectively. The basic structure of AQP4 consists of 6 transmembrane domains and 5 loops, including A, C, E3 extracellular loops and B, D2 intracellular loops. AQP4 was widely distributed in the brain and was the earliest aquaporin found in the central nervous system.

AQP4 is not expressed in excitatory cells, but is expressed in epithelial support cells such as astrocytes and ependymal cells of the central nervous system. AQP4 is distributed throughout the brain variously and includes the cerebral cortex, corpus callosum, retina, cerebellum, and magnocellular and brainstem of the hypothalamus. AQP4 exhibits a polar distribution on astrocytes and their terminal feet in direct contact with brain microvasculature. AQP4 is widely distributed in the central nervous system and plays an important role in many central nervous system diseases, such as cerebral edema, tumors, epilepsy, and neuromyelitis nervosa.

The discovery and identification of aquaporins is a milestone in human cytology, and AQP-4 is an astrocyte of the aquaporin family, distributed primarily in the brain. Recent studies have shown that AQP4 can modulate astrocyte function and play an important role in synaptic plasticity and in learning and memory.

Disclosure of Invention

the invention aims to provide application of AQP4 as a depression drug target. The research result of the invention shows that AQP4 is closely related to depression, and provides a brand-new drug target and a new treatment means and thought for the research and development of the late-stage depression treatment drug.

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

On the first hand, a plurality of stable and reliable animal models capable of accurately simulating human disease states are established, and a solid foundation is laid for subsequent researches. Finally, a Willner improvement model (CUMS) for chronic unpredictable stress stimulation is selected, and compared with a classical model, the model is modified in two aspects, namely, the intensity of a stress factor is obviously reduced, and the key to the success of the model is the measurement of anhedonia, so that the difficulty encountered in daily life of people is simulated more realistically. The reduction of the sucrose drinking amount after long-term mild stress stimulation reflects the central symptom of endogenous depression, namely anhedonia, and simultaneously simulates the symptom expression of other major depressive disorders, such as the reduction of motor ability and social interaction ability, the reduction of exploration behavior ability, the defect of invasion and attack ability, the reduction of sexual behavior ability and the like.

In the second aspect, the content change of AQP4 in mouse serum is analyzed by enzyme-linked immunosorbent assay with the anti-depression effective chemical components of prepared fleece-flower root, stilbene glucoside, rhaponticin, emodin glucoside, emodin and the like as entry points.

Finally, the change of the content of the AQP4 in the hippocampal tissues is analyzed by using a protein immunoblotting method and a PCR experiment. By comparing the expression level of aquaporin 4(AQP4), the research result indicates that AQP4 is a new therapeutic target for depression.

the research results indicate that AQP4 or the gene thereof can be used as a drug target for screening drugs for treating depression. The application of the medicine for treating various depression symptoms including endogenous depression, reactive depression, occult depression, secondary depression caused by medicines, climacteric or postpartum depression, depression induced by cerebral trauma or cerebral apoplexy and depressive neurosis and the medicine for treating diabetes and depression.

The invention has the beneficial effects that:

The invention discovers for the first time that the depression can block the expression of AQP4 in serum, so that the content of AQP4 in serum is reduced. After the treatment by medicines (the stilbene glucoside, the emodin and the like), the content of AQP4 is increased; similarly, after suffering from depression, the expression level of AQP4 in hippocampus is relatively reduced, and after being treated by medicines (stilbene glucoside, emodin and the like), the expression level of AQP4 is increased. Based on the fact, the invention firstly provides and verifies that AQP4 has direct correlation with the pathogenesis and treatment of the depression, is a new treatment target of the depression and develops a new idea for the treatment of the depression.

drawings

FIG. 1 is a graph of 1 mouse AQP4 versus absorbance value;

FIG. 2 shows the amount of AQP4 in the serum of each group administered;

FIG. 3 is an electrophoretogram of an immunoblot experiment;

FIG. 4 is a graph of the relative protein expression levels of AQP 4;

FIG. 5 is a Mus AQP4 amplification curve;

FIG. 6 is a graph of the Mus AQP4 melting curve;

FIG. 7 is a beta-actin amplification curve;

FIG. 8 is a beta-actin melting curve;

FIG. 9 shows the relative expression levels of AQP4 mRNA in each group.

Detailed Description

The technical scheme of the invention is explained in detail by animal experiments, but is not meant to limit the invention in any way.

Experimental Material

1. Experimental animals: male Kunming SPF level mice, 30 + -2 g weight, were purchased from Sichuan province laboratory medicine animal center, SCXK [ Yu ] 2018-0003. The animal experiment complies with the ethical requirements of international experimental animals, and the animal experiment can take standard feed and clean water by itself after receiving 12h of illumination/12 h of darkness every day, the illumination period is 8:00-20:00, the laboratory temperature is 20 +/-2 ℃, the humidity is 60%.

2. Reagent testing:

Stilbene glucoside: self-making;

Emodin: self-making;

Emodin glucoside: self-making;

Rhaponticin: self-making;

Preparing fleece-flower root powder: self-made

0.9% physiological saline: sichuan Koran pharmaceutical Co., Ltd.

The first embodiment is as follows: establishment of Depression model

SPF-grade KM mice, 120, were acclimatized for 1 week and randomized into normal and model groups. After molding for 2 weeks, the model groups were randomly divided into a depression model group, a stilbene glucoside group, a rhaponticin group, an emodin glucoside group, an emodin group and an injection radix polygoni multiflori powder administration group according to body weight.

Chronic mild unpredictable stimuli include 10: tail clamping for 5 minutes/time, horizontal shaking for 20 minutes/time, water deprivation (12 hours), ice water (4 ℃) swimming, fasting for 12 hours, inclination of a squirrel cage (inclination angle of 45 degrees), wet padding (can not be over wet), fasting and water deprivation, placement of foreign matters (paper scraps, orange peels and the like), and peculiar smell stimulation.

The method simulates the chronic low-intensity stress accepted in daily life of human beings, and ensures that 1 different stimuli are arranged every day, and the sequence is random and is continuous without repetition so that the animals cannot expect the stimulation. For a total of 6 weeks.

After the model is made, 16 mice in the blank group and the model group are subjected to a sugar water consumption experiment, wherein the sugar water concentration is 3%, and the sugar water consumption experiment is carried out for 2 times, and the time is four to five hours in the afternoon. The test was then performed at a fixed time, 4 times a week, monday afternoon. This operation was carried out after the mice had been fasted and deprived of water for 24 hours in the same cage, and the evaluation index was the average sugar water consumption per mouse. Sugar water consumption of mice in blank and experimental groups is shown in table 1.

TABLE 1 mouse syrup consumption (mL)

As can be seen from the table, the sugar water consumption of the mice was significantly reduced in the model group after modeling compared with the blank group; at week 5, the model group had significantly lower sugar water consumption than the blank group. The establishment of the chronic unknown depression model causes the lack of pleasure of animals, and the depression model has effectiveness and persistence, which indicates that the model building effect is good, and can be used for carrying out subsequent experiments.

Example two: injection administration and sample collection

1. Administration by injection

After the mouse model is established, the injection of the medicine is started, wherein the injection medicine powder group is prepared into a preparation by 5g/kg of powder, and the stilbene glucoside group, the rhaponticin glucoside group, the emodin glucoside group and the emodin group are prepared into a preparation by 100mg/kg of medicine. Injections were continued for 15 days.

2. Blood is collected from eyeball and tissue of Hippocampus is collected

The eyeball blood taking experiment is carried out on all mice, 1mL of blood is taken, centrifugation is carried out for 20min, and the rotating speed is 3000 r/s. Taking the supernatant, and storing in a refrigerator. Taking out brain tissue, cutting off cerebellum, separating brain along midline, placing cortex facing downwards and inner layer structure facing upwards on ice bag, carefully peeling off white matter with curved forceps, and placing Hippocampus in crescent shape in the side close to cerebellum. Taking out and storing in a refrigerator.

Example three: aquaporin 4 expression level detection

Expression level of AQP4 in serum

1. enzyme linked immunosorbent assay

(1) the experimental method comprises the following steps:

s1: firstly, the antigen is combined on the surface of the solid phase carrier, and the immunological activity of the antigen is ensured;

S2: the antigen or antibody is connected with an enzyme which can mark the antigen antibody and keep the immunocompetence, and also can keep the activity of the enzyme to form an enzyme-marked antigen or antibody;

s3: when the experiment detection is started, the marked specimen reacts with the enzyme-labeled antigen and the antigen on the solid phase carrier in the first step.

S4: washing to separate the complex formed by the reaction from other impurities. The amount of enzyme reaction in the sample is then proportional to the amount of enzyme bound to the solid phase. Since the chemical reaction takes place enzymatically after the addition of the enzymatically reacted substance and is a colored product, it is optionally possible to analyze the color by the shade of the color of the colored product. And since the effect is amplified by the action of the enzyme, the result can be clearly seen.

(2) Instruments and reagents

The apparatus and reagents used are shown in Table 2.

TABLE 2 Instrument and reagents for ELISA adsorption experiments

2. content of the experiment

The double antibody sandwich method comprises the following steps:

s1: connecting the specific antibody with a solid phase carrier, coating a microporous plate with the antibody of the purified aquaporin 4 to form the solid phase aquaporin 4 antibody, and washing to remove the antibody which is not combined and other impurities in the experimental process.

S2: adding the detected sample, making it contact with solid phase aquaporin 4 antibody and react for a period of time, making the antigen in the sample combine with aquaporin 4 antibody on the solid phase carrier, finally obtaining the solid phase antigen complex. The washing removes unbound antibodies and other impurities during the experiment.

S3: and adding an enzyme-labeled antibody, combining the antigen on the solid-phase immune complex with the enzyme-labeled antibody, and combining a Horse Radish Peroxidase (HRP) -labeled aquaporin 4 antibody to obtain the antibody-antigen-enzyme-labeled antibody complex. Unbound enzyme-labeled antibody was washed 3 times.

S4: the enzyme in the sandwich complex catalyzes the substrate to a colored product by adding the substrate Tetramethylbenzidine (TMB). The antigen is either qualitative or quantitative depending on the degree of color reaction. The substrate tetramethylbenzidine is converted into blue color under the catalysis of HRP enzyme and becomes yellow under the action of acid. (Tetramethylbenzidine TMB is the most widely used substrate in ELISA experiments. TMB is blue after reaction, turns yellow after terminating reaction by adding acid, and has the measurement wavelength of 450nm, is stable and has no carcinogenicity. the shade of color is positively correlated with aquaporin 4 in a sample.)

3. Results of the experiment

And (4) measuring the absorbance (OD value) by using an enzyme-labeling instrument, wherein the wavelength is 450nm, and obtaining a standard curve of the concentration and the absorbance value. The results are shown in FIG. 1, the degree of fitting R²0.9575, indicating that data is available.

The content of AQP4 in serum of mice in the blank and experimental groups is shown in fig. 2, from which it can be seen that: compared with a blank group, the AQP4 protein content of the depression model group is obviously reduced; compared with a depression model group, the content of AQP4 in each administration group is obviously increased. The results show that the depression can block the expression of AQP4 in serum, so that the content of the AQP4 is reduced, the effective chemical components of emodin glucoside and emodin in the prepared fleece-flower root have obvious anti-depression effect (P is less than 0.05), and the content of AQP4 is increased.

Second, AQP4 expression level in hippocampal tissue

1. Immunoblot assay

(1) The test content comprises the following steps:

S1: preparation of protein samples

Collect cells on ice, wash twice with PBS, and wash out the medium. Centrifuging at 1500r for 5min, and discarding the supernatant.

② cracking cells (lysate: RIPA lysate 100-120 microliter/dish), adding cell lysate according to the required protein concentration. And (5) uniformly mixing. The cells were lysed on ice for 20 min.

③ 14000r and 10 min. The supernatant is the required protein sample liquid.

And fourthly, measuring the protein concentration by using a Bradford protein analysis method.

Fifthly, adding loading buffer into the protein sample solution according to the proportion of adding 30ul of 6X loading buffer into 100ul of lysate.

Sixthly, performing high-temperature denaturation on the protein sample at the temperature of 95 ℃ for 5 min.

s2: polyacrylamide gel electrophoresis (SDS-PAGE)

Glue pouring:

The glass plates are enabled to be dry and clean, the glass plates are placed into a clamp for clamping after being aligned, and the glass plates are vertically clamped on a frame for glue pouring.

Secondly, preparing separation glue, adding TEMED, and pouring glue after even mixing. The glue flows down along the glass plate in the glue filling process, so that bubbles are prevented from being generated. And pouring the glue to be close to the center line of the green band.

③ using deionized water to carry out liquid sealing, the gelling speed can be faster (the speed is slow when adding water, and the glue is prevented from being punched and deformed)

fourthly, when a refracted ray exists between the water and the glue, the glue is solidified. At this time, water in the upper layer of the gel may be completely removed.

Fifthly, preparing the concentrated glue, adding TEMED, and filling the residual space with the concentrated glue after uniformly mixing. The comb is then inserted. Carefully pulling out the comb after the concentrated gel is solidified.

Sixthly, washing a lower plastic plate by water, and placing the lower plastic plate into an electrophoresis tank (the small glass plate faces inwards, the large glass plate faces outwards; if only one plate is run, the other side of the tank is provided with a plastic plate, and the side with characters faces outwards)

Electrophoresis:

an appropriate amount of Running Buffer was added to the cell and the protein sample was loaded at a volume converted from a standard of 50. mu.g. And performing electrophoresis at 120V or 160V until the electrophoresis runs to the bottom end to perform membrane rotation. The result of the electrophoresis is shown in FIG. 3,

S3: rotary film

The membrane was transferred using a nitrocellulose membrane (NC membrane). The process of assembling the Transfer film sandwich is carried out in a Transfer buffer, wherein the black side is arranged below, the filter paper, the gel, the NC film and the filter paper are sequentially arranged on the black side, and then the sandwich is closed. The whole process must be guaranteed to be bubble free. Then the transfer film sandwich is put into a transfer film groove, and the anode and the cathode are placed correctly. The appropriate amount of Transfer buffer was poured in. The film transfer was performed in an ice bath. Film transferring conditions: 120V, 1.5 h.

S4: sealing of

After the membrane transfer is finished, the NC membrane is taken out and cut according to the position of the required protein. The solution was soaked in confining liquid and shaken slowly for 1 h.

S5: one against hybridization

after sealing, the NC membrane is put into a plastic bag, a proper amount of primary antibody solution (primary antibody: sealing solution is 1:1000) is added according to the size of the membrane, sealing is carried out, and the membrane is incubated for 4 hours by slowly shaking on a shaking table.

S6: hybridization of the second antibody

And after the primary antibody incubation is finished, taking out the NC membrane, alternately adding TBST and TBS to wash the membrane, and washing for three times, wherein each time lasts for 7-10 min. Then, the NC membrane was placed in a plastic bag, and an enzyme-labeled secondary antibody (primary antibody, containing horseradish peroxidase HRP) (secondary antibody: blocking solution ═ 1:1000) was added thereto, sealed, and incubated on a shaker for 45min with gentle shaking. Finally the membrane was washed again (same as above).

S7: and (6) exposing.

(2) results of the experiment

The content of AQP4 in hippocampal tissues of mice in the blank and experimental groups is shown in fig. 4, wherein # indicates the comparative difference between the depression model group and the blank group, the administration group and the depression model group, one symbol indicates that P < 0.05 is statistically different, and two symbols indicate that P < 0.01 is statistically different; as can be seen from the figure: compared with a depression model group and a blank group, the AQP4 expression level is relatively reduced, and the statistical difference is realized (P is less than 0.05); compared with the administration group and the depression model group, the expression level of AQP4 in the emodin glucoside group and the injection drug group is increased, and the statistical difference is significant (P is less than 0.01). The results show that the expression of AQP4 is reduced in hippocampal tissues of depression mice, and the expression level of aquaporin 4 is obviously increased after administration (P is less than 0.01).

PCR experiment

(1) Instruments and reagents

The equipment used for the PCR experiments is shown in Table 3.

TABLE 3 instruments for PCR experiments

The reagents used in the PCR experiments are shown in Table 4.

TABLE 4 reagents used in PCR experiments

(2) Content of the experiment

Real-time fluorescent quantitative PCR detection of mRNA. The method for extracting RNA by using Trizol comprises the following steps:

S1: pretreatment of samples

Fresh frozen tissue, which had been stored in a freezer at-80 ℃ and weighed approximately 100mg, was first removed, 1mL of Trizol reagent was added, and then ground into a slurry using a homogenizer and transferred to a 1.5mL EP tube, which had to be kept free of RNase. Finally, the cells were lysed for 10 minutes.

S2: 200 μ L of chloroform was added, mixed vigorously by inversion several times, and left at room temperature for 5 minutes.

S3: centrifugation at 12000rpm for 15 minutes at 4 ℃ was observed to separate into upper (RNA) and lower (DNA) triphase.

s4: the upper aqueous phase (about 400. mu.L) was transferred to another new 1.5mL EP tube, 400. mu.L isopropanol was added, mixed well and allowed to stand at room temperature for 10 minutes.

S5: after centrifugation at 12000rpm for 10 minutes at 4 ℃ a white RNA precipitate was observed at the bottom of the tube.

S6: the supernatant was discarded, 1mL of RNase-free 75% ethanol was added, and the mixture was vortexed, and then centrifuged at 4 ℃ and 10000rpm for 5 minutes.

S7: repeat step 6 once.

S8: the supernatant was discarded, the RNA pellet was air dried for 5-10 minutes and the pellet was dissolved in 20. mu.L of EPC water.

s9: taking 2 microliter of dissolved RNA, measuring OD260, OD280 and OD260/OD280 values by using a micro spectrophotometer, and finally calculating the concentration and purity of the RNA.

Estimating the RNA quality according to the ratio of OD260/OD280, wherein the ratio is between 1.8 and 2.0And (5) testing requirements. The concentration of the sample RNA was calculated from the absorbance values according to the following formula: total RNA concentration (μ g/μ L) ═ OD260 × 40 × 10^-3。

The total RNA was stored in a freezer at-80 ℃ for future use.

② reverse transcription into cDNA

The reverse transcription reaction system solution is shown in Table 5.

TABLE 5 reverse transcription reaction System

The reverse transcription reaction conditions are as follows: 5 minutes at 25 ℃, 15 minutes at 50 ℃, 5 minutes at 85 ℃ and 10 minutes at 4 ℃.

③ real-time fluorescent quantitative PCR detection

The cDNA was diluted 10-fold and then subjected to PCR detection. The PCR detection reaction system is shown in Table 6.

TABLE 6 real-time fluorescent quantitative PCR reaction system

The PCR detection reaction conditions are as follows: 2 minutes at 50 ℃ and 10 minutes at 95 ℃; 95 ℃ 30sec, 60 ℃ 30sec, 40 cycles.

The primers used for PCR detection are shown in Table 6.

TABLE 6 primer sequence Listing

(3) Results of the experiment

FIG. 5 is a graph of the amplification of Mus AQP4, from which it can be seen that Mus AQP4 enters the amplification exponential phase at about the 20 th cycle. The fluorescence threshold was 0.104264, at which time the Ct value (amplification cycle number) was about 23. FIG. 6 is a Mus AQP4 melting curve showing a single peak indicating a strong specificity of primer design. The Tm, i.e., the temperature at which 50% of the DNA double strand is melted, is about 84 ℃. FIG. 7 is a beta-actin amplification curve showing that approximately cycle 15 of beta-actin enters the exponential amplification phase, with a fluorescence threshold of 0.164925, at which time the Ct value (number of amplification cycles) is approximately 20. FIG. 8 is a beta-actin melting curve, which shows a single peak indicating a strong specificity of primer design. The Tm, i.e., the temperature at which 50% of the DNA double strand is melted, is about 84 ℃.

fig. 9 is the relative expression of AQP4 mRNA in each group, with # indicating the difference between the depression model group and the blank group, the difference between the administration group and the depression model group, indicating a statistical difference (P < 0.05), the difference indicating a significant statistical difference (P < 0.01), and the difference indicating a very significant statistical difference (P < 0.001). Compared with a blank group, the AQP4 mRNA expression quantity is reduced, and the statistical difference is very significant (p is less than 0.001); compared with a depression model group, the AQP4 mRNA expression level of the administration group is increased, wherein compared with the depression model group, the AQP4 mRNA expression level of the stilbene glucoside group is increased, and statistical difference exists (P is less than 0.05); compared with the depression model group, the emodin glucoside group has the advantages that the AQP4 mRNA expression quantity is increased, and the statistical difference is very significant (P is less than 0.001); compared with the depression model group, the AQP4 mRNA expression level is increased in the drug injection group, and the statistical difference is generated (P is less than 0.05).

Through the analysis, AQP4 has direct correlation with the pathogenesis and treatment of depression. The depression can block the expression of AQP4 in serum to reduce the content of AQP4, and the content of AQP4 is increased after the treatment of medicaments (such as stilbene glucoside and emodin); after suffering from depression, the expression level of AQP4 in hippocampus is relatively reduced, and after being treated by medicaments (stilbene glucoside, emodin and the like), the expression level of AQP4 is increased. AQP4 is a new therapeutic target for depression.

While the present invention has been described in detail with reference to the embodiments, it should not be construed as limited to the scope of the patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.

Claims (6)

1. Application of aquaporin 4 in rapid detection of depression.

2. Application of aquaporin 4 as a drug target in screening drugs for treating depression.

3. The application of the gene of the aquaporin 4 as a drug target in screening drugs for treating depression.

4. use according to claim 2 or 3, characterized in that: the drug is a drug that increases the expression level of aquaporin 4.

5. Use according to claim 2 or 3, characterized in that: the drug is a drug that inhibits the level of aquaporin 4 expression.

6. Use according to any one of claims 1 to 3, characterized in that: the depression comprises endogenous depression, reactive depression, occult depression, drug-induced secondary depression, climacteric depression, postnatal depression, depression induced by cerebral trauma, depression induced by cerebral apoplexy and/or depressive neurosis.