Improvement effect of metformin on post-traumatic stress disorder

Abstract

The invention provides an improvement effect of metformin on post-traumatic stress disorder, and belongs to the technical field of biological medicine. Metformin is used for relieving unbalance of mitochondrial homeostasis in amygdala nucleus on the outer side of a post-traumatic stress disorder model rat by promoting mitochondrial biosynthesis, so that synaptic and dendritic spine abnormity is improved, finally fear memory regression of the post-traumatic stress disorder model rat is promoted, and the post-traumatic stress disorder is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a new use of metformin, and particularly to the improving effect of metformin on post-traumatic stress disorder. Background Art

[0002] Metformin was first used to treat type 2 diabetes in the late 1950s and will remain the drug of choice for approximately 150 million people daily in 2024. In recent years, epidemiological and preclinical studies have clearly demonstrated that metformin has other beneficial effects beyond its effects on blood sugar. Studies have shown that metformin, as an agonist of the AMP-activated protein kinase (AMPK) pathway, also has broad protective functions in psychiatric and neurological diseases. Preclinical and clinical data suggest that metformin may protect against neurodegenerative diseases by inhibiting proinflammatory pathways, protecting mitochondrial and vascular function, and acting directly on neural stem cells.

[0003] Post-traumatic stress disorder (PTSD) is a stress-related psychiatric disorder that develops after witnessing or experiencing a life-threatening event (such as war, natural disasters, or major accidents). Abnormal fear memories and anxious or depressive behaviors are hallmarks of PTSD. Currently, the pathophysiology of this disorder is not fully understood, resulting in limited medication options for its treatment. Selective serotonin reuptake inhibitors (SSRIs), such as sertraline and parotide, are available, but they are expensive, have numerous side effects, and suffer from poor patient compliance. Therefore, a deeper understanding of the pathogenesis of PTSD and the identification of effective treatments are of great clinical significance. Growing evidence suggests that PTSD is closely associated with metabolic imbalances. Summary of the Invention

[0004] The purpose of the present invention is to address the above-mentioned problems existing in the existing technology and provide a new use of metformin, specifically a new use of metformin in mental and neurological diseases.

[0005] The present invention provides the use of metformin in preparing a product for preventing or treating post-traumatic stress disorder.

[0006] Metformin as a drug for the prevention and / or treatment of post-traumatic stress disorder.

[0007] Metformin as an add-on to pharmaceutical products for the prevention and / or treatment of post-traumatic stress disorder.

[0008] Experiments conducted in this study demonstrate that metformin alleviates mitochondrial homeostasis imbalances in the lateral amygdala of rats with post-traumatic stress disorder (PTSD) by promoting mitochondrial biogenesis, thereby improving synaptic and dendritic spine abnormalities. This ultimately promotes fear memory extinction and alleviates anxiety and depressive behaviors in these rats. This study lays the foundation for the preparation of metformin as a drug for the treatment of PTSD, and it can be used to prevent and treat PTSD. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The effect of metformin on fear behavior in rats with post-traumatic stress disorder model. Figure 1 Figure a shows the number of stiffnesses in each group on the first day of conditioning training; Figure 1 Figure b shows the number of freezing times of rats in each group on the third day of conditioning training after training; Figure 1 Figure c shows the number of freezing events in rats of each group during the seventh day of conditioning training.

[0010] Figure 2 The effect of metformin on anxiety and depression in rats with post-traumatic stress disorder model. Figure 2 Figure a is the movement heat map of rats in each group in the open field test;

[0011] Figure 2 Figure b shows the time that rats in each group stayed in the central area in the open field test; Figure 2 Figure c shows the immobility time of rats in each group in the forced swimming test.

[0012] Figure 3 The effect of metformin on synapses and dendritic spines in the lateral amygdala of rats with post-traumatic stress disorder model. Figure 3 Figure a is a transmission electron microscope image. The first row is the image of the synapses in the lateral amygdala of rats in each group (scale bar: 1 μm), and the second row is the magnified image of the synapses (arrows indicate synapses). Figure 3 Figure b is a statistical diagram of the length of the synaptic active zone; Figure 3 Figure c is a statistical diagram of the thickness of the postsynaptic density; Figure 3 Figure d shows the images of dendritic spines in the lateral amygdala of rats in each group using Golgi staining.

[0013] Figure 4 The effect of metformin on mitochondrial biogenesis in the lateral amygdala of rats with post-traumatic stress disorder model. Figure 4 Figure a shows the PGC-1α protein banding diagram and statistical analysis results in the lateral amygdala of rats in each group; Figure 4 Figure b shows the NRF2 protein banding diagram and statistical analysis results in the lateral amygdala of rats in each group; Figure 4Figure b shows the TFAM protein band diagram and statistical analysis results in the lateral amygdala of rats in each group. DETAILED DESCRIPTION

[0014] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0015] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0016] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0017] Example 1: Verification that metformin can inhibit post-traumatic stress disorder behaviors in rats induced by a single long-term stress

[0018] (1) Establishment of rat model

[0019] ① Rearing conditions

[0020] Thirty-two male SD rats weighing 200 ± 20 g and aged 7–8 weeks were selected and provided by Hubei Baient Biotechnology Co., Ltd. SCXK (E) 2021-0027. All rats were purchased and housed in an SPF-grade animal room at the Institute of Medicine of Hubei University of Science and Technology, with 2–3 rats per cage. Rats had free access to food and water, and the ambient temperature was maintained at 20–25°C and the humidity at 50%–70%.

[0021] ②Animal experimental drugs

[0022] The water-soluble drug metformin was prepared with normal saline to a concentration of 26.775 mg / mL metformin solution and stored at 4°C. The control group was given an equal amount of normal saline as a blank control.

[0023] ③Experimental plan

[0024] A) Grouping: The rats were randomly divided into 4 groups, with 8 rats in each group, namely normal group, metformin group, model group and model + metformin group.

[0025] B) Modeling Method: A single long-term stress test was used to induce a post-traumatic stress disorder model in rats. The specific steps of the single long-term stress test were as follows: The rats were immobilized for 2 hours using a transparent plastic rat restrainer. After confinement, the rats were placed in a transparent rectangular water tank (50 cm long, 35 cm wide, and 35 cm high) filled two-thirds with water at 25°C and forced to swim for 20 minutes. After the swim, the rats rested for 15 minutes. Finally, the rats were anesthetized with ether gas until unconscious and then returned to a clean cage for a week of rest with free access to food and water. No other disturbances were observed during this period.

[0026] C) Dosage regimen: Every morning, the rats in the metformin group and the model + metformin group were gavaged with 2 mL of metformin solution; the rats in the normal group and the model group were gavaged with 2 mL of normal saline for 42 days.

[0027] (2) Animal behavior detection

[0028] ① Conditioned fear experiment

[0029] A) Specific method: Each group of rats was first placed in a conditioned fear box to adapt for 2 minutes, and then allowed to explore freely. After adaptation, a sound signal of a specific frequency was given, with a duration of 18 seconds, a frequency of 2500HZ, and an intensity of 90-95db. After sound adaptation, an electric shock stimulus was given with an intensity of 1.2mA and a duration of 2s, and the rat's response was recorded at the same time. After waiting for 30 seconds after the stimulus, the same sound signal and electric shock stimulus were given again, and this process was repeated 3 times. During the experiment, the computer automatically recorded the number of stiffnesses that occurred in the rat. The experiment was repeated after 3h and 24h to evaluate its short-term memory performance. On the 7th day after the fear subsided, the rats that had established conditioned fear were placed in the conditioned fear box again and only given sound signals of the same frequency, with an interval of 1min each time, for a total of 5min, to evaluate their long-term memory performance.

[0030] B) Test results: The results are as follows Figure 1 As shown in Figure 2, there was no significant difference in the number of rigidity episodes in the model group compared with the normal group on days 1 and 2 ( Figure 1 a, b), but on the 7th day, the number of stiffness in the model group rats increased significantly ( Figure 1 c) Compared with the model group, the number of freezing episodes in the model + metformin group on day 7 was significantly reduced (***P < 0.001 model group vs normal group; #P < 0.05 model + metformin group vs model group). This result suggests that metformin can promote the extinction of fear memory in rats with post-traumatic stress disorder.

[0031] ②Open field experiment

[0032] A) Specific Methods: Experimental rats were placed in the center of an open-field reaction box measuring 100 cm x 100 cm and 40 cm high. The box's bottom was evenly divided into 125 4 cm squares. The rats' movements were observed and recorded for 5 minutes, and the time spent in the center of the open field was recorded. One rat was placed in the open-field reaction box at a time. After the experiment, the box was cleaned of feces and odors using 75% medical alcohol to maintain a clean environment.

[0033] B) Test results: The results are as follows Figure 2Figures a and b in the middle show that compared with the control group, the color of the central area in the model group rats was lighter and the time spent in the central area of ​​the open field was significantly reduced. Compared with the model group, the color of the central area in the model + metformin group rats was significantly darker and the time spent in the central area was also significantly increased (**P < 0.01 model group vs normal group; #P < 0.05 model + metformin group vs model group). These results suggest that metformin can alleviate anxiety-like behaviors in rats with post-traumatic stress disorder.

[0034] ③Forced swimming test

[0035] A) Specific Methods: The apparatus used in this test is an acrylic plastic pool (width, depth, and length (cm): 90×45×45). The pool is filled with water to a depth of 38 cm. The water temperature is maintained at 34±1°C. Before the forced swim test, rats were placed in the pool and subjected to a 15-min continuous forced swim test. Forced swimming is defined as the time from the time the mouse is placed in the pool to the time the animal remains in the water for 7 seconds without moving or rising to the surface to breathe. On the second day, the time the rat remains motionless in the pool is recorded, and the test lasts for 5 minutes.

[0036] B) Test results: The results are as follows Figure 2 Figure c shows that compared with the normal group, the model group rats spent significantly more time immobile in the water; compared with the model group, the model + metformin group rats spent significantly less time immobile in the water (*P < 0.05 model group vs normal group; ##P < 0.01 model + metformin group vs model group). This result suggests that metformin can alleviate depressive-like behaviors in rats with post-traumatic stress disorder.

[0037] Example 2: Using transmission electron microscopy and Golgi staining to study the effect of metformin on synaptic plasticity in key brain regions associated with post-traumatic stress disorder

[0038] (1) Transmission electron microscopy imaging

[0039] ① Specific methods: Three rats were randomly selected from each group and anesthetized and perfused transcardially with normal saline, 2.5% glutaraldehyde, and 4% paraformaldehyde in 0.1M PBS. After dissection of the rat brain tissue, a portion of the amygdala region tissue was immediately cut into 1mm cubes and immersed in 2.5% glutaraldehyde at 4°C overnight. The tissue was post-fixed with 1% osmium tetroxide solution in 0.2M acetate buffer for 2 hours. The tissue blocks were dehydrated with a gradient of ethanol and acetone and then embedded in epoxy resin. The tissue was stained with 3% uranyl acetate and 0.5% lead citrate for 30 minutes and 15 minutes, respectively. The ultrastructure of neurons in the amygdala region was observed using transmission electron microscopy at an accelerating voltage of 80kV.

[0040] ②Test results: Transmission electron microscopy images of synapses show ( Figure 3 (Figures a, b, and c): Compared with the normal group, the length of the presynaptic active zone in the lateral amygdala of rats in the model group was significantly shortened, and the thickness of the postsynaptic density was significantly reduced. Compared with the model group, the length of the presynaptic active zone in the lateral amygdala of rats in the model + metformin group was significantly increased, and the thickness of the postsynaptic density was also significantly increased (***P < 0.001 model group vs normal group; ##P < 0.01 or ###P < 0.001 model + metformin group vs model group). These results suggest that metformin can improve synaptic abnormalities in the lateral amygdala of rats with post-traumatic stress disorder.

[0041] (2) Golgi staining

[0042] ① Specific Methods: The remaining amygdala region brain tissue was quickly placed in Golgi solution (2.5% K₂Cr₂Oₐ; 2.5% HgCl₂; 2% K₂CrO₄) and stored in the dark for 14 days, with fresh Golgi solution replaced every two days. The tissue was then sectioned and stained by Wuhan Sevier Biotechnology Co., Ltd. Images were collected using a laser confocal microscope (OLYMPUS FV3000, Japan) equipped with a 40× objective lens.

[0043] ②Test results: Figure 3 Golgi staining results in Figure d show that compared with the normal group, the density of dendritic spines in the lateral amygdala of rats in the model group was significantly reduced; compared with the model group, the density of dendritic spines in the lateral amygdala of rats in the model + metformin group was significantly increased. This result suggests that metformin can improve the abnormalities of dendritic spines in the lateral amygdala of rats with post-traumatic stress disorder.

[0044] The results of Example 2 suggest that metformin can improve synaptic and dendritic spine abnormalities to regulate synaptic plasticity damage in the lateral amygdala of rats with post-traumatic stress disorder.

[0045] Example 3: Western blotting to investigate the effects of metformin on mitochondrial homeostasis in neurons in key brain regions involved in post-traumatic stress disorder

[0046] (1) Collection of brain tissue samples

[0047] One day before the end of the experiment, the rats were fasted but not watered, anesthetized with an overdose of sodium pentobarbital, and then the brain tissue was dissected and the lateral amygdala tissue was removed and placed in a 2 mL round-bottom centrifuge tube.

[0048] (2) Sample preparation

[0049] Lysate was added at a ratio of 1 mg to 3 uL of lateral amygdala tissue mass and lysate volume, and two steel balls were added. The mixture was placed in a homogenizer at 4°C and ground until no obvious tissue was left. The mixture was then centrifuged in an ultracentrifuge at 4°C and 12,000 rpm for 15 minutes. After centrifugation, the supernatant was aspirated and the protein concentration was detected using the BCA method. The concentration was balanced and the protein was denatured for detection.

[0050] (3) Detection

[0051] The specific method is as follows:

[0052] After heating the proteins to 95°C for 10 minutes, they were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were then transferred to a PVDF membrane using the Trans-Blot wet transfer technique. After sealing with 5% skim milk, the membrane was incubated with a specific primary antibody overnight at 4°C. A secondary antibody conjugated to horseradish peroxidase was then applied to the membrane for 2 hours at room temperature. Protein-specific signals were detected using ECL on a Bio-Rad fluorescence imaging system. The intensity of the target band was normalized to that of the corresponding internal control protein using ImageJ analysis software.

[0053] The results are as follows Figure 4 As shown, compared with the normal group, the expression levels of PGC-1α, NRF2, and TFAM proteins in the lateral amygdala of rats in the model group were significantly downregulated. Compared with the model group, the expression levels of PGC-1α, NRF2, and TFAM proteins in the lateral amygdala of rats in the model + metformin group were significantly upregulated (*P < 0.05 or **P < 0.001 or ***P < 0.001 model group vs normal group; #P < 0.05 model + metformin group vs model group). The PGC-1α signaling pathway is a key pathway in mitochondrial biogenesis. These results suggest that metformin can promote mitochondrial biogenesis and regulate the imbalance of mitochondrial homeostasis in the lateral amygdala of rats with post-traumatic stress disorder.

[0054] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described specific embodiments without departing from the spirit of the present invention or exceeding the scope of the appended claims.

Claims

1. The improving effect of metformin on post-traumatic stress disorder is characterized by: Metformin alleviates the imbalance of mitochondrial homeostasis in the lateral amygdala of rats with post-traumatic stress disorder by promoting mitochondrial biogenesis, thereby improving synaptic and dendritic spine abnormalities and having an ameliorative effect on post-traumatic stress disorder.

2. The improving effect of metformin on post-traumatic stress disorder according to claim 1, characterized in that: Metformin as a drug for the prevention and / or treatment of post-traumatic stress disorder.

3. The improving effect of metformin on post-traumatic stress disorder according to claim 1, characterized in that: Metformin as an add-on to pharmaceutical products for the prevention and / or treatment of post-traumatic stress disorder.

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