Method for verifying LA as peripheral marker of depression
By detecting the plasma LA content of depression patients and control groups, and combining behavioral and LA expression analysis of depressive model rats, LA is verified as an early peripheral marker of depression, solving the problem of lack of effective biomarker molecules in the prior art, and providing new diagnostic and therapeutic guidance.
Patent Information
- Application Number
- CN202411739165.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-02-25
AI Technical Summary
The lack of effective biomarker molecules to assist the diagnosis and treatment of depression in the prior art, resulting in a diagnosis that depends on the experience of clinicians and lack of early warning and personalized treatment guidelines.
By collecting plasma samples from depression patients and control groups who meet ICD-10 diagnostic criteria, LA content in peripheral plasma was detected by spectrophotometry, and combined with behavioral and LA expression analysis of rats established by CUMS method, LA was verified as an early peripheral marker of depression.
It was found that the peripheral plasma LA content of depression patients and depressive model rats was downregulated, suggesting that LA can be used as an early peripheral marker molecule to evaluate depression and has important clinical diagnosis and treatment guidance.
Smart Images
Figure CN119510739A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of mental illness research, and in particular to a method for verifying LA as a peripheral marker of depression. Background Art
[0002] Depression is one of the main causes of the global burden of mental illness. Currently, the diagnosis of depression relies more on the experience of clinicians and lacks potential biomarker molecules. In order to alleviate the burden of depression on society and reduce the suffering of patients and their families, it is urgent to discover new targets to guide the prevention, differential diagnosis, early intervention and medication of depression.
[0003] Studies at the animal level have confirmed that abnormal expression of LA (lactic acid) in depression model rats is accompanied by abnormal behavior, indicating that LA dysfunction plays a role in mental illness and suggesting that it may serve as a potential biomarker molecule for the disease.
[0004] Therefore, the applicants speculated that LA could be used as an early peripheral marker molecule for assessing depression. Summary of the invention
[0005] The purpose of the present invention is to provide a method for verifying LA as a peripheral marker of depression and a method of using the same in view of the deficiencies of the prior art. The specific scheme is as follows: A method for validating LA as a peripheral marker of depression, characterized by comprising the following steps: Step 1: Collect 11 plasma samples from adolescent depression patients who meet the ICD-10 diagnostic criteria before treatment and 11 plasma samples from healthy volunteers in the adolescent control group, and detect the peripheral plasma LA content of each group by spectrophotometry; Step 2, collecting plasma samples from 12 adult depression patients who met the ICD-10 diagnostic criteria before treatment and plasma samples from 12 healthy volunteers in the adult control group, and detecting the peripheral plasma LA content of each group by spectrophotometry; Step 3, rat preparation: 31 SPF grade 10-14 week old SD rats were selected and adapted to the new environment for 1 week; Step 4, CUMS method to establish depression model rats: The rats in step 3 were divided into 2 groups, one group of 20 rats were subjected to chronic mild unpredictable stimulation for 4-6 weeks, and different stimulations were given every day, which served as the model group; the other group of 11 rats were not subjected to stimulation as the control group; Step 5, conduct behavioral tests on the model group rats in step 4, and remove the rats that fail to form a model, resulting in 11 rats in the control group and 11 rats in the model group; Step 6: Detect the LA levels in the peripheral blood of the model group rats and the control group rats.
[0006] Based on the above, the behavioral testing time in step 5 is controlled between 8:00-20:00, and the temperature, humidity and lighting in the room are controlled appropriately.
[0007] Based on the above, in step 6, peripheral blood samples of rats in the control group and rats in the model group were collected, plasma was separated, and the peripheral plasma LA content of each rat was detected by spectrophotometry.
[0008] The present invention has outstanding substantive features and significant progress compared to the prior art. Specifically, the present invention has the following advantages: The method for verifying LA as a peripheral marker of depression provided by the present invention comprises the following steps: collecting 11 plasma samples of adolescent depression patients meeting the ICD-10 diagnostic criteria before treatment and 11 plasma samples of healthy volunteers in the adolescent control group, detecting the peripheral plasma LA content of each group by spectrophotometry, and finding that the peripheral plasma LA content of the depression group was down-regulated; collecting 12 plasma samples of adult depression patients meeting the ICD-10 diagnostic criteria before treatment and 12 plasma samples of healthy volunteers in the adult control group, detecting the peripheral plasma LA content of each group by spectrophotometry, and finding that the peripheral plasma LA content of the depression group was significantly down-regulated; establishing depression model rats by the CUMS method, combining behavioral and LA expression analysis, finding that the model rats had abnormal LA expression and abnormal behavior, detecting the peripheral plasma LA content of each group of rats by spectrophotometry, and finding that the peripheral plasma LA content of the modeling group was down-regulated; therefore, it is suggested that LA can be used as an early peripheral marker molecule for evaluating depression; the present invention is ingenious in conception and scientific in scheme, and is of great significance to human psychiatric research and drug development. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figure 1 This is a comparative analysis chart of LA content between the adolescent depression group and the control group in the present invention.
[0010] Figure 2 This is a comparative analysis chart of LA content between the adult depression group and the control group in the present invention.
[0011] Figure 3 This is a comparative analysis diagram of the sucrose preference rate between the rat model group and the control group in the present invention.
[0012] Figure 4 This is a comparative analysis chart of the total moving distance in the open field between the rat model group and the control group in the present invention.
[0013] Figure 5 This is a comparative analysis chart of the forced swimming immobility time of the rat model group and the control group in the present invention.
[0014] Figure 6 This is a comparative analysis chart of LA content between the rat model group and the control group in the present invention. DETAILED DESCRIPTION
[0015] The technical solution of the present invention is further described in detail below through specific implementation methods. Example
[0016] like Figure 1-6 As shown, the present invention provides a method for validating LA as a peripheral marker of depression, characterized by comprising the following steps: Step 1: Collect 11 plasma samples from adolescent depression patients who meet the ICD-10 diagnostic criteria before treatment and 11 plasma samples from healthy volunteers in the adolescent control group, and use spectrophotometry to detect the peripheral plasma LA content of each group; Figure 1 As shown, the LA (lactic acid) content in the adolescent facial depression group was significantly lower than that in the control group; Step 2: Collect plasma samples from 12 adult depression patients who meet the ICD-10 diagnostic criteria before treatment and plasma samples from 12 healthy volunteers in the adult control group, and detect the peripheral plasma LA content of each group by spectrophotometry; Figure 2 As shown, the LA (lactic acid) content in the adult depression group was significantly lower than that in the control group; Step 3, rat preparation: 31 SPF grade 10-14 week old SD rats were selected and adapted to the new environment for 1 week; Step 4, CUMS method to establish depression model rats: The rats in step 3 were divided into 2 groups, one group of 20 rats were subjected to chronic mild unpredictable stimulation for 4-6 weeks, and different stimulations were given every day, which served as the model group; the other group of 11 rats were not subjected to stimulation as the control group; Step 5, conduct behavioral tests on the model group rats in step 4, and remove the rats that fail to form a model, resulting in 11 rats in the control group and 11 rats in the model group; Step 6: Detect the LA levels in the peripheral blood of the model group rats and the control group rats.
[0017] In step 4, when the CUMS method was used to establish a rat model of depression, the stimulation applied to the rats mainly included: electric shock, restriction, tail clamping, fasting, water deprivation, crowding, empty bottle, ice water swimming, day and night reversal, and stroboscopic. Rats were randomly given 2-3 stimulations every day but not continuously to prevent rats from predicting the occurrence of stimulation. The modeling process lasted for 6 weeks, and the rats were kept in solitary cages during the modeling period to simulate the state of reduced social activities in patients with depression.
[0018] The specific operations of the above stimulation are: Electric shock: The rats were placed in an electric shock box, with the soles of the rats touching the conductive metal rods at the bottom of the box. The power supply was set to 0.6 mA, 10 seconds / time, 1-10 seconds interval, and lasted for 5 minutes. After the electric shock, the box was thoroughly cleaned with 30-40% alcohol to remove the odor and excrement left by the previous rat, and the next rat was shocked; Restraint: Choose a suitable restrainer and place the rat in it. Adjust the length of the restrainer to keep the rat's body fixed and able to breathe freely. Expose the rat's tail and tighten the screws of the end restraint to prevent the rat from escaping. All restraints of rats are performed in the same time period. After 6 hours, the rats are released from restraint and placed back in their respective cages. Tail clamp: Clamp the rat's tail at about 1 / 3 with an oval clamp, tighten the buckle, and release the oval clamp after 6 minutes; Fasting: No food intake for 24 hours from 8:00 am to 8:00 am the next day, free access to water. Water deprivation: No water intake for 24 hours from 8:00 am to 8:00 am the next day, free access to food; Crowding: From 8:00 am to 8:00 am the next day, 10 rats were housed in a 488x320x257 mm cage. After 24 hours, the grouped rats were de-crowded and returned to their original positions to continue solitary cage housing. Empty bottle: Place an empty drinking bottle at the drinking area in the mouse cage for 2 hours; Ice water swimming: Pour 40 cm of water into a forced swimming bucket that is 80 cm high and 20 cm in diameter, then add ice to lower the water temperature to 4°C. Place the rat in the ice water and swim for 5 minutes. After the swimming, wipe the rat dry to quickly restore body temperature. Reversal of day and night: from 8:00 a.m. to 8:00 p.m., turn off all light sources to make the rats live in a dark environment, and from 8:00 p.m. to 8:00 a.m. the next day, turn on the lights to give the rats a light source that is suitable for their living conditions; Strobe: Strobe lighting is provided from 20:00 pm to 8:00 am the next day, and the strobe is turned off at 8:00 am the next day.
[0019] The behavioral testing time in step 5 above is controlled between 8:00-20:00, and the temperature, humidity and lighting in the room are controlled appropriately. The behavioral testing in step 5 includes sucrose preference test, open field test and forced swimming test. Figure 3-5 As shown, after comprehensive comparison, 11 rats whose sucrose preference rate was significantly lower than that of the control group, whose activity level in the open field test was significantly lower than that of the control group, and whose immobility time in the forced swimming test was significantly higher than that of the control group were selected as the model group.
[0020] The sucrose preference experiment takes advantage of the rats' natural preference for sweets. It tests whether the experimental animals have changed their preference for sweets after modeling to detect whether they are in a state of reduced interest and anhedonia in patients with depression. The specific experimental steps are as follows: All rats are housed in single cages before the experiment begins. The rats need to adapt before the formal experiment. Prepare two bottles of 1% sucrose water (about 250mL) on the first day. Replace them with a bottle of ordinary drinking water and a bottle of 1% sugar water on the second day. The formal experiment begins on the third day. Place a bottle of ordinary drinking water and a bottle of 1% sugar water at the drinking water place of the mouse cage. Exchange the positions of the two bottles 6 hours after the start of the experiment to prevent the memory of the position of the drinking water bottle from affecting the experimental results. The drinking water bottle and the sugar water bottle need to be weighed before and after the experiment, and the mass of the water in the bottle at 0 hours and 12 hours is recorded. The calculation method for sucrose preference is: Sucrose preference rate = (weight before sugar water consumption - weight after sugar water consumption) / ((weight before sugar water consumption - weight after sugar water consumption + (weight before drinking water consumption - weight after drinking water consumption)) x 100%.
[0021] The day before the open field experiment, the rats were moved to the room where the open field experiment was conducted to adapt to the environment. The experimental room should be quiet and should avoid other sound interference. The light in the room was adjusted with a shadowless lamp to simulate the growth environment of rats. In order to reduce the influence of factors such as the sound and smell of the experimenters on the behavior of the rats, the participants in the experiment were in another room adjacent to the open field experiment during the experiment. During the experiment, the rats were placed in the center of a rectangular open field (100cmx100cmx40cm) each time, and the behavioral analysis recording software Smart3.0 was set up on the computer to record all the movement trajectories of the rats within 5 minutes after 30 seconds of adaptation. After the experiment of each rat, the excrement left by the previous rat should be thoroughly cleaned, and the open field should be sprayed with 30-40% alcohol to eliminate the influence of the odor. After drying, the next rat can be experimented. After the experiment, the total moving distance of each rat was counted. The total moving distance was used to evaluate the spontaneous activity, exploration and anxiety state of the rats in the new environment. When the rats are in a depressed state, the spontaneous activity decreases, which is manifested as a decrease in the total moving distance.
[0022] In the forced swimming test, rats swim in water that has been sterilized by high temperature and high pressure. Before the experiment, sterilized water with a depth of about 40 cm was poured into a transparent round swimming bucket (80 cm high and 20 cm in diameter). The water temperature was maintained at 24±1℃. Smart3.0 was used to record the swimming process of the rats within 6 minutes, and then the immobility time of the rats was analyzed. If multiple rats are carried out at the same time, opaque baffles should be used to separate the animals to ensure that the behavior of each animal will not affect other animals. In addition, the experimenters observed the rats in another room separated from the rats to prevent the rats from drowning and avoid affecting the behavior of the rats. After 6 minutes, the rats were fished out, immediately wiped dry with absorbent paper, restored their body temperature, and put back into the cage. After the experiment, the immobility time was analyzed. The immobility time refers to the state in which the rats give up struggling for survival and occasionally slap the water surface with their limbs to keep their mouths and noses out of the water to maintain breathing. It simulates the passive clinical manifestations of patients with depression. Compared with normal rats, depression model rats are more likely to enter an immobile state. The immobility time is a good indicator of whether the rats are more prone to a state of despair and helplessness.
[0023] In step 6 above, peripheral blood samples of rats in the control group and rats in the model group were collected, plasma was separated, and the LA content in peripheral plasma of each rat was detected by spectrophotometry. Figure 6 As shown, the LA (lactic acid) content of rats in the model group was significantly lower than that in the control group.
[0024] The method for verifying LA as a peripheral marker of depression provided by the present invention comprises the following steps: collecting 11 plasma samples of adolescent depression patients meeting the ICD-10 diagnostic criteria before treatment and 11 plasma samples of healthy volunteers in the adolescent control group, detecting the peripheral plasma LA content of each group by spectrophotometry, and finding that the peripheral plasma LA content of the depression group was down-regulated; collecting 12 plasma samples of adult depression patients meeting the ICD-10 diagnostic criteria before treatment and 12 plasma samples of healthy volunteers in the adult control group, detecting the peripheral plasma LA content of each group by spectrophotometry, and finding that the peripheral plasma LA content of the depression group was significantly down-regulated; establishing depression model rats by the CUMS method, combining behavioral and LA expression analysis, finding that the model rats had abnormal LA expression and abnormal behavior, detecting the peripheral plasma LA content of each group of rats by spectrophotometry, and finding that the peripheral plasma LA content of the modeling group was down-regulated; therefore, it is suggested that LA can be used as an early peripheral marker molecule for evaluating depression; the present invention is of great significance to human psychiatric research and drug development.
[0025] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.
Claims
1. A method for validating LA as a peripheral marker of depression, characterized in that The following steps are involved: Step 1: Collect 11 plasma samples from adolescent depression patients who meet the ICD-10 diagnostic criteria before treatment and 11 plasma samples from healthy volunteers in the adolescent control group, and detect the peripheral plasma LA content of each group by spectrophotometry; Step 2, collecting plasma samples from 12 adult depression patients who met the ICD-10 diagnostic criteria before treatment and plasma samples from 12 healthy volunteers in the adult control group, and detecting the peripheral plasma LA content of each group by spectrophotometry; Step 3, rat preparation: 31 SPF grade 10-14 week old SD rats were selected and adapted to the new environment for 1 week; Step 4, CUMS method to establish depression model rats: The rats in step 3 were divided into 2 groups, one group of 20 rats were subjected to chronic mild unpredictable stimulation for 4-6 weeks, and different stimulations were given every day, which served as the model group; the other group of 11 rats were not subjected to stimulation as the control group; Step 5, conduct behavioral tests on the model group rats in step 4, and remove the rats that fail to form a model, resulting in 11 rats in the control group and 11 rats in the model group; Step 6: Detect the LA levels in the peripheral blood of the model group rats and the control group rats.
2. The method for validating LA as a peripheral marker of depression according to claim 1, characterized in that: In step 5, the behavioral testing time is controlled between 8:00-20:00, and the temperature, humidity and lighting in the room are controlled to be appropriate.
3. The method for validating LA as a peripheral marker of depression according to claim 1, characterized in that: In step 6, peripheral blood samples of rats in the control group and rats in the model group were collected, plasma was separated, and the peripheral plasma LA content of each rat was detected by spectrophotometry.