CN105687168A - Application of resveratrol in preparation of antidepressant - Google Patents

Abstract

The invention relates to the application of resveratrol in the preparation of antidepressant drugs, which belongs to the field of medicine; the invention establishes a mouse depression model through chronic mild stress stimulation, and gives mice resveratrol by force feeding, and through the opening activity experiment, Elevated plus maze test, social interference test, forced swimming test and tail suspension test were used to evaluate its antidepressant activity, and its possible mechanism was detected by molecular biology and electron microscopy. The results show that resveratrol can reverse the behavioral manifestations of depression, such as fatigue, depression, and social decline. Integrity improvement, depression-induced damage to the structural integrity of the hippocampal myelin membrane disappears, and resveratrol may activate the SIRT1/PGC-1α/PPARr signaling pathway, increase the expression of antioxidant enzymes, and protect the hippocampal myelin membrane Structural integrity reduces stress-induced depression.

Description

Application of resveratrol in the preparation of antidepressant drugs

technical field

The invention relates to the application of resveratrol in the preparation of antidepressants and belongs to the field of medicine.

Background technique

Resveratrol (2,3,4'-trihydroxystilbene) (Resveratrol, RSV), as a natural polyphenol compound, mainly comes from plants such as grapes, knotweed, peanuts, mulberries, etc. Resveratrol was first obtained by scientists The widespread attention of wine stems from the antioxidant, anti-tumor and cardiovascular protective effects of wine; in 2003, researchers identified 21 different SIRT1 activating molecules in the screening of SIRT1 small molecule activators, among which resveratrol was Its strong antioxidant properties are widely recognized.

It has been reported in the literature that resveratrol increases the life cycle of Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster by activating SIRT1, improves the metabolic and cardiovascular functions of rodents, and reduces liver fatty degeneration , reduce inflammatory response, improve endurance (BargerJL, KayoT, PughTD, ProllaTA, WeindruchR. (2008) Short-termconsumptionofaresveratrol-containingnuraceuticalmixturemimicsgeneexpressionoflong-termcaloricrestrictioninmouseheart.ExpGerontol.43:859-866.); studies have shown that resveratrol and many neuropsychiatric Diseases are closely related, by promoting the proliferation of mitochondria, activating SIRT1 and then inhibiting the occurrence of Alzheimer's disease (PasinettiGM, WangJ, HoL, ZhaoW, DubnerL. (2015) Roles of res veratroland other grape-derived polyphenols in Alzheimer's disease prevention and treatment. BiochimBiophysActa.1852:1202-1208.) There are also reports that resveratrol plays an important role in the survival, differentiation, growth and development of neurons (DexiangL, KaiX, XudongY, JianhuaG, LiG, et al. (2014) Resveratrol reverses the effects of chronic unpredictable mild stresson behavior, serum corticosterone levels and BDNF expression in rats. Behav Brain Res268: 1-7), Inhibit the HPA axis, thereby promoting the regeneration of damaged neurons. However, whether resveratrol can activate the SIRT1 signaling pathway, regulate the activation and proliferation of mitochondria and the expression of genes related to oxygen metabolism in mitochondria, and exert antidepressant activity is rarely reported; the present invention studies the antidepressant effect of resveratrol Activity and its molecular biological mechanism.

Contents of the invention

The purpose of the invention is to overcome the vacancy in the antidepressant application of resveratrol in the prior art, contribute to the new application value of resveratrol, and provide an experimental basis for the industrialization development of resveratrol.

The invention provides the application of resveratrol in the preparation of antidepressants.

Wherein, the antidepressant-like behaviors are fatigue, anxiety, depression and decline in social interaction ability.

The present invention first establishes a mouse depression model through chronic mild stress stimulation, and gives resveratrol for three weeks by force feeding, and evaluates its antidepressant effect through open field experiment, elevated plus maze experiment, social interference experiment, forced swimming experiment and tail suspension experiment The protein expression of SIRT1 and PGC-1α in the hippocampus and the changes in the structure of the myelin sheath membrane in the hippocampus were detected by immunofluorescence and Western Blotting techniques to explore their mechanism of action. The results showed that resveratrol significantly increased the body weight of depressed mice, and reduced the immobilization time of mice forced swimming test and tail suspension test. Resveratrol has antidepressant activity by reducing the attack time and frequency of social interference experiment and increasing the interference time and frequency. Resveratrol can induce the expression of SIRT1 and PGC-1α protein in mouse hippocampus tissue and improve the ultrastructure of hippocampal myelin sheath, and the damage of hippocampal myelin sheath structure induced by depression disappears. The above results indicate that resveratrol It may be through the activation of SIRT1/PGC-1α/PPARr signaling pathway, which reduces the damage of stress-induced hippocampal myelin structure and plays an antidepressant role.

The present invention also provides an antidepressant drug, which contains resveratrol or may consist of resveratrol and a pharmaceutically acceptable carrier.

The medicament is that resveratrol is a single effective drug component or a compound form composed of other drug components with similar effects.

Beneficial effects of the present invention:

The present invention proves that resveratrol has antidepressant activity through a series of experiments; its biological activity is mainly through up-regulating the expression of SIRT1 and PGC-1α protein, activating the SIRT1/PGC-1α/PPARr signaling pathway, and then promoting the survival of neurons , morphogenesis, synaptic function and increase neuronal plasticity, play an antidepressant role.

Although the behavioral appearance of depression is difficult to evaluate, some classic behavioral experiments such as forced swimming test and tail suspension test have been proved to be an effective way to evaluate depression. Resveratrol can significantly reduce social interference in mice The time rate of invasion and the frequency of invasion in the experiment significantly increased the time and frequency of social interference experiments in mice, that is, resveratrol significantly increased social interaction ability and reduced social aggressive behavior at the same time.

In addition, resveratrol can reduce the fixed time of mice forced swimming test and increase their swimming time, suggesting that resveratrol can significantly reduce depressive symptoms; The change of the distance shows that the effect of resveratrol on the results of the above behavioral experiments is not due to the overall change of the behavioral baseline of the mice and the depression of the mice.

At present, the vast majority of antidepressant drugs are chemically synthesized, and long-term application will bring many toxic and side effects to the body. Therefore, it is urgent to find natural products with antidepressant functions and no toxic side effects. Resveratrol has been proven It can regulate the hypothalamus-pituitary-adrenal axis and the monoaminergic system to antagonize cognitive and anxiety dysfunction. Therefore, the natural product-resveratrol will have broad application prospects in antagonizing depression.

Description of drawings

Figure 1: Effect of resveratrol on body weight of depressed mice; in the figure, the inset is the average body weight of four groups of mice for 6 weeks.

Figure 2: The effect of resveratrol on the opening behavior of depressed mice; in the figure, the left picture shows the effect of exercise distance, and the right picture shows the effect of standing frequency;

Figure 3: Effects of resveratrol on social interference experiments in depressed mice: interaction time (A), interaction times (B), attack time (C) and attack times (D);

Figure 4: The effect of resveratrol on the fixed time of the forced swimming test in depressed mice;

Figure 5: The effect of resveratrol on the fixed time of the tail suspension test in depressed mice;

Figure 6: Western Blot detection of the effect of resveratrol on the expression of PGC-1α protein in depression mice; in the figure, the upper panel is the result of Western Blotting experiment; the lower panel is the result of mouse PGC-1α protein expression.

Figure 7: Immunofluorescence detection of the effect of resveratrol on the expression of SIRT1 protein in depression mice; in the figure, A. control group, B. resveratrol group, C. chronic mild stress group, D. chronic mild stress Stimulation + resveratrol group; 1 represents the nucleus, 2 represents the detected protein, and 3 represents the merged picture, and the size of the scale bar in the figure is 50 μm.

Figure 8: Immunofluorescence detection of the effect of resveratrol on PGC-1α protein expression in depression mice; in the figure, A. control group, B. resveratrol group, C. chronic mild stress group, D. chronic Mild stress + resveratrol group; 1 is the nucleus, 2 is the detected protein, 3 is the merged picture, and the size of the scale bar in the figure is 50 μm.

Figure 9: The effect of resveratrol on the structure of myelin membrane in the hippocampus of depressed mice; in the figure, A. Control group, B. Chronic mild stress group, C. Resveratrol group, D. Chronic mild stress +Resveratrol group, the size of the scale bar in the figure is 500nm.

detailed description

Embodiment 1: the establishment of depression model

Chronic mild stress stimulation (Chronicmildstress, CMS)) three weeks to establish mouse depression model, chronic mild stress stimulation followed the following experimental methods: mice were exposed to different stressful stimuli: food deprivation, water deprivation, Cage tilting, wet bedding, food restriction, restraint stress, these stress stimuli were given once a day, two random ones, for three consecutive weeks, and the control group did not receive any stress stimuli.

Embodiment 2: Behavioral experiment

C57BL/6J male mice were purchased from the Experimental Animal Center of Jiangsu University, 10-12 weeks old, and were fed alone at temperature (22±1°C), humidity (55±5%), 12h light/12h night (light time 07:30AM) -19:30PM) room, with sufficient water and food supply, the experiment was divided into four groups, 8 rats in each group: ① control group (normal saline): no exposure to chronic mild stress + normal saline treatment group; ② white quinoa Retrol group: no exposure to chronic mild stress + RSV treatment group; ③ model group (chronic mild stress group): exposure to chronic mild stress + normal saline treatment group; ④ model + resveratrol group (chronic mild stress Stress + resveratrol group): group exposed to chronic mild stress + RSV treatment. Resveratrol (purchased from Aladdin Reagent, Shanghai, China) was administered at a dose of 200 mg/kg, with a volume of 10 mL/kg, and with a normal saline volume of 10 mL/kg; administered once a day (08:00AM), continuously The drug was administered for 3 weeks, and the behavioral experiment was started on the 22nd day of administration (1h after administration).

Behavioral tests were carried out in the following order: opening activity test, elevated plus maze test, social interference test, forced swimming test and tail suspension test.

(1) Weight

The body weight of the mice was monitored once a day, with the week as the abscissa and the average body weight within a week as the ordinate. The results showed that the weight of the mice in the chronic mild stress-induced depression model group decreased significantly, and the resveratrol The body weight of depressed mice increased significantly (see Figure 1).

(2) Opening experiment

Mice were housed in a 50 cm x 25 cm x 20 cm transparent automatic activity monitor (OptoM3, Columbus Instruments, Columbus, OH). Infrared monitoring was recorded once every 10 minutes, and the opening activities of the mice were recorded continuously for 6 times for a total of 60 minutes. After the experiment was completed, the mice were sent back to the breeding cage, and the monitor was wiped clean with 10% alcohol. The results of the open-field autonomous activity experiment are shown in Figure 2. Resveratrol did not affect the time, speed and distance of the mice's autonomous activities, that is, resveratrol did not cause changes in the behavioral baseline of the mice.

(3) Elevated plus maze test (EPM)

The elevated plus maze experiment was carried out according to the routine method of our laboratory (ZhangW.N., BastT, XuY, FeldonJ. (2014) Temporaryinhibitionofdorsalorventralhippocampusbymuscimol: Distincteffectsonmeasuresoffinnateanxietyontheelevatedplusmaze, butsimilardisruptionofcontextualfearconditioning.Behav4BrainRes7-562). The elevated plus maze is a wooden cross-shaped maze 60cm above the ground, consisting of four arms, including two closed arms (24cm) and two open arms (1cm). Each arm is composed of a 30cm x 5cm area and a 5cm x 5cm central area. The mice were first placed in the central area of the maze, and their behavioral activities were recorded by the camera above the maze for 5 minutes, and then the time and times of the mice in the open arm and the closed arm were manually counted according to the camera records. was recorded. The time and times of entering the arm will be used for statistical analysis of time ratio and frequency ratio, where time ratio = open arm time / (open arm time + closed arm time); frequency ratio = open arm times / (open arm times + closed arm time) arm times). After the experiment, each mouse was returned to its home cage, and the maze apparatus was wiped clean with 10% alcohol. The time and frequency of the mice entering the open arm in the elevated plus maze experiment can be used to evaluate the anxiety-like behavior of the mice and the pharmacological activity of the drug against anxiety. The experimental results show that,

Resveratrol did not affect the time and distance that the mice stayed in the open arm and the closed arm of the elevated plus maze, that is, resveratrol did not cause changes in the anxiety-like behavior of the mice.

Table 1. Effects of resveratrol on elevated plus maze test in depressive mice

(4) Social interference experiment

Mice were transferred from their breeding cages to an empty, unfamiliar test cage for 10 minutes. The paired mice should be in the same treatment group or saline group. The behavior of the paired mice was continuously recorded through the camera above the test cage. After 10 minutes of activity, the attack time and social interference time of each mouse were manually counted according to the video recording. Attack time was defined as attempted bites, actual bites, punches, kicks, etc. Social disturbance time was defined as another form of physical contact, including sniffing, licking, or touching. Mice without mutual contact will not be recorded. The invasion ratio was defined as invasion time/(invasion time + social disturbance time). After the experiment, the mice were returned to their home cages.

The experimental results showed that compared with the control group, the attack time and times of the social interference experiment of the depressed mice were significantly increased, and the interference time and times were significantly reduced; the administration of resveratrol significantly reduced the social attack time, time and frequency of the depressed mice. The number of times increased the time and times of social interference, that is, resveratrol has the behavior of increasing the social interaction ability of depressed mice.

(5) Forced swimming test

The experimental process of the forced swimming experiment was carried out according to the contents of the reference (PorsoltR.D., BertinA., JalfreM., et al (1978). "Behavioral despair" in ratsandmice: strain differences and the effects of fimipramine. EurJPharmacol51 (3): 291-294.). Mice were placed in a plastic cylinder of 25 cm (height) × 10 cm (diameter) filled with water. The cylinder was filled with water at 23-25 °C and 10 cm in height. The mice were forced to stay in the plastic cylinder for 6 minutes. Rats that cease struggling in the plastic cylinder and remain afloat (with only minor movements to keep the head above water) are considered immobilized. The total test time lasted 6 minutes, the first 2 minutes were used as the adaptation period, and the swimming time and immobility time were recorded by the Enthovision8.5 automatic analysis software for the last 4 minutes. After each mouse swims, the water in the cylinder needs to be changed. The results showed that compared with the control group, the immobilization time of the forced swimming test of the depressed mice was significantly increased, and the immobilization time of the depressed mice was significantly reduced after the administration of resveratrol, that is, resveratrol had the effect of reducing the depression-like Behavior.

(6) Tail suspension experiment

The tail of the experimental animal was fixed and the head was down. The animal struggled in this environment, trying to get out of the predicament. After trying hard but still unable to get rid of the predicament, the animal appeared intermittently immobile, showing a state of "behavioral despair". This model of behavioral hopelessness is similar to depression. The rear 1/3 of the tail of the mouse was fixed with adhesive tape, and the head was hung down on the bracket. The head was 15cm away from the table, and the camera was taken. The background of the camera was in obvious contrast with the color of the mouse’s coat, and the black mouse used a white background. The timing was stopped after 6 minutes, and the immobility time of the mice for the last four minutes (3-6 minutes) was counted using Enthovision8.5 software. Compared with the control group, the fixation time of the tail suspension test of the depressed mice was significantly increased, and the fixation time of the depressed mice was significantly reduced after administration of resveratrol, that is, resveratrol can reduce the depression-like behavior of the mice.

Example 3: Mechanism studies

(1) Brain tissue preparation

Mice were sacrificed 1 hour after administration by CO ₂ anesthesia, and their hippocampus and brain tissues were placed in liquid nitrogen and transferred to a -80°C refrigerator.

Homogenate: Add 9 times the volume of 50mM KPO ₄ buffer, 0.5% Triton-X-100 pH7.2 and 1 times EDTA-free protease inhibitor to the brain tissue sample, use a Barnant homogenizer to homogenize, brain homogenate The mixture was centrifuged at 13,000 g for 15 min at 4°C, and the supernatant was taken as PGC-1α, which was stored in a -80°C refrigerator, and its protein concentration was detected using a BCA protein assay kit.

(2) Western Blotting experiment

The loading volume of PGC-1α was 25 μL/well, loaded into 10% gel, electrophoresed on SDS-PAGE (100V, 1h), transferred to membrane (350mA, constant current) for 2h, blocked with 5% BSA for 1h; rabbit anti-PGC-1α 1α polyclonal primary antibody (ab54481, Abcamcompany, 1:1000), incubated overnight at 4°C, goat anti-rabbit IgG secondary antibody (A0208, Beyotime Biotechnology, 1:1000) for 1h; mouse anti-Beta-catenin primary antibody (L002, EpitopeBiotechInc, 1:1000) at 4°C overnight, and goat anti-mouse IgG secondary antibody (A0216, Beyotime Biotechnology, 1:1000) was incubated for 1 hour. Protein expression was counted using Bio-RadQualityOne analysis software. The sample test results were corrected by the control sample (the same control sample was run in each gel), and finally corrected by Beta-catenin to ensure the consistency of the loading amount. The results showed that: compared with the control group, the expression level of PGC-1α protein in the depressed mice was significantly reduced; after administration of resveratrol, the expression level of PGC-1α protein in the depressed mice was significantly increased.

(3) Immunofluorescence

The hippocampal tissue was prepared for immunofluorescence histochemistry, placed in a mixture of 3% glutaraldehyde/4% paraformaldehyde/0.15moL/LPBS, and stored overnight at 4°C. On the second day, they were placed in 20% and 30% sucrose solutions prepared by 0.15moL/LPBS solution, placed in a Leica cryostat, embedded in OCT embedding agent, and coronal sections were made at 12 μm. Add 10mM citrate buffer (pH6.0) to microwave antigen retrieval for 5min, and cool naturally. The volume fraction was 5% BSA and incubated for 1h (37°C), SIRT1 rabbit anti-mouse polyclonal antibody (ab12193, Abcamcompany, 1:250), PGC-1α rabbit anti-mouse polyclonal antibody (ab54481, Abcamcompany, 1:250) incubated overnight (4°C). Rinse with PBS, and incubate with Cy3-labeled anti-rabbit IgG (P0183, Beyotime Biotechnology, 1:500) in the dark for 1 h. Rinse with PBS and counterstain with fluorescent dye (DAPI) for 5 min. Rinse, mount, photograph.

The results of immunofluorescence showed that: as shown in Figure 7, compared with the control group, the expression level of SIRT1 protein in depression mice was significantly reduced; the administration of resveratrol significantly increased the expression level of SIRT1 protein in depression mice, that is, white Veratrol may play an antidepressant role by up-regulating the expression of SIRT1 protein.

Compared with the control group, as shown in Figure 8, the expression level of PGC-1α protein in depression mice was significantly reduced; after administration of resveratrol, the expression level of PGC-1α protein in depression mice was significantly increased, that is, resveratrol Retrol may play an antidepressant role by up-regulating the expression of PGC-1 protein.

(4) Observation of the ultrastructure of the hippocampus

Take out the mouse hippocampus stored in the low-temperature refrigerator, wash it with normal saline, and process it according to the following steps:

①Cut: Trim the tissue into a size of ^1mm3 ;

②Fixation: pre-fix with 3% glutaraldehyde (0.2M, PBS, pH7.4) at 4°C for more than 2 hours, and post-fix with 1% osmic acid for 2 hours;

③Dehydration: step by step dehydration with acetone;

④ Embedding: 618 epoxy resin embedding;

⑤ Slicing: Semi-thin section positioning, sliced by LKB-5 ultra-thin microtome, thickness 700nm;

⑥Staining: double staining with uranyl acetate and lead citrate;

⑦Microscopic examination: Under a transmission electron microscope (TEM-1200EX/s), observe the changes in the ultrastructure of the hippocampus, as shown in Figure 9.

The results showed that resveratrol significantly reduced the damage of myelin sheath in the hippocampus of depressed mice, increased the integrity of myelin sheath and protected the integrity of synapses.

Claims (8)

1. resveratrol application in preparing antidepressant drug。

2. the resveratrol according to claim 1 application in antidepressant sample medicine, it is characterised in that described resveratrol can increase hippocampal tissue SIRT1 and the expression of PGC-1 α albumen。

3. the resveratrol according to claim 2 application in antidepressant drug, it is characterised in that described resveratrol can increase the integrity of Hippocampus myelin membrane structure。

4. the application in preparing antidepressant drug of the resveratrol according to claim 1-3 any one, it is characterised in that described antidepressant sample behavior includes degradation under fatigue, anxiety, depression and social contact ability。

5. an antidepressant medicine, it is characterised in that described pharmaceutical pack is containing resveratrol。

6. medicine according to claim 5, it is characterised in that described medicine is made up of resveratrol and pharmaceutically acceptable carrier。

7. medicine according to claim 5, it is characterised in that described medicine is resveratrol is single active drug composition。

8. medicine according to claim 5, it is characterised in that described medicine is the compound recipe form that resveratrol forms with other ingredients with similar effect。