CN119424606A - Application of recombinant neuropeptide OT23 in preparation of medicines for depression - Google Patents

Abstract

The invention discloses application of recombinant neuropeptide OT23 in preparation of a medicament for treating depression. The invention establishes two mouse depression models, and six behavioral analysis indexes, a T cell receptor excision ring index for evaluating thymus function and a brain-derived neurotrophic factor index for displaying the state of brain hippocampus of a depressed mouse are detected and compared after OT23 treatment. Experiments show that OT23 has therapeutic effects on both depressive mouse models. The animal behavior monitoring data of the mice in the treatment group show that the behavior of the mice is close to the normal level, the relative expression amount of T cell receptor excision loops in thymus and spleen of the mice in the treatment group is obviously increased, and the level of brain-derived neurotrophic factor in nerve tissues of the mice in the treatment group is obviously increased. It is shown that OT23 can raise the thymus generation and output capacity of mice, restore the immune function of mice, and regulate the brain hippocampal state of mice, thereby improving the depression-like behavior of mice. These suggest that recombinant neuropeptide OT23 can be used for preparing medicaments for treating depression.

Description

Application of recombinant neuropeptide OT23 in preparation of medicines for depression

Technical Field

The invention relates to the technical field of depression treatment, in particular to application of recombinant neuropeptide OT23 in preparation of a medicament for depression.

Background

Depression is a typical mental disorder condition, mainly manifested by a sustained and very marked depression, feeling anxiety and depression, so that people are more susceptible to psychological stress, and serious people may develop light behaviors. Depression is common in somatic disease patients, and has high co-disease rate with functional dyspepsia, coronary heart disease, diabetes, tuberculosis and other somatic diseases, so that positive treatment of depression has obvious benefit for improving somatic diseases.

Depression is a fairly complex disease whose pathogenesis is a variety of factors including psychological, biological, genetic, social aspects. Currently, the pathogenesis of depression is mainly focused on monoamine neurotransmitters, stress induction, neurological disorders and hypothalamic-pituitary-adrenal axis abnormalities. According to these theories, abnormal levels or functions of hypothalamic-pituitary-adrenal (HPA) axis, neurotrophic factors, sex steroids, inflammatory cytokines can lead to alterations in neurotransmitter, intracellular signaling, gene transcription, translation, and epigenetic changes, resulting in short-term and long-term imbalances in neuronal function and behavior.

There are several antidepressant therapies available, but the therapeutic effect on depressive patients is not ideal. Treatment of depression includes three principal modes, (1) antidepressants and other antidepressant-enhancing agents, (2) evidence-based psychotherapy such as Cognitive Behavioral Therapy (CBT) and human psychotherapy (IPT), and (3) somatic non-drug therapy including electroconvulsive therapy (ECT), repeated transcranial magnetic stimulation (rTMS) and Vagal Nerve Stimulation (VNS).

Currently available antidepressants are mainly classified into three categories, namely (1) monoamine oxidase inhibitors (MAOI) for preventing monoamine metabolism, (2) tricyclic drugs (TCA) for blocking monoamine reuptake, (3) Selective Serotonin Reuptake Inhibitors (SSRIs) such as Paroxetine (Paroxyetin), sertraline (SERTRALINE) and fluoxetine (Fluoxetine), and in recent decades, because SSRIs have fewer side effects and are easy to use, have replaced MAOI and TCA, and become first-line antidepressants for treating depression. It is noted that these drugs also present potential risks, such as excessive intake of the drug due to use of the drug, drug resistance, and poor patient compliance.

Several pathogenesis hypotheses are combined, and a great number of existing researches are combined to show that immune inflammation, hypofunction and disorder of immune function and the degeneration of central immune organs, namely thymus, play an important role in the occurrence and development of depression. For example, most depressed patients show a significant decrease in immune function, a significant decrease in the percentage of helper T lymphocytes monitored, a significant decrease in the response of lymphocytes to mitogens PHA and PWM in depressed and psychotic depressed patients compared to those with mild depression, a number of in vivo studies indicating a decrease in cellular immune function in depressed patients, significant increases in the expression of inflammatory factors IL-1, IL-6, IL-8, IL-18, IL-33 and IL-37 in depressed patients, particularly in the pathogenesis of depression, two pro-inflammatory cytokines IL-1 and IL-6, which have an important role in the pathogenesis of depression, pressure-related physiological responses in humans characterized by childhood abuse and adulthood by age are due at least in part to acute and lifelong immune disorders, traumatic stress and post-traumatic stress disorders are also associated with abnormal blood cell composition, immune system aging with an accelerated immune cell proportion imbalance, recent studies in Carlezon Jr. Et al indicate that exposure to chronic social stress (CSDS) can cause a significant decrease in the levels of the thymus, adrenal gland degeneration and hypertrophy in the blood glands, and the presence of negative immune markers in humans with reduced blood levels of the negative effects of the thymus, etc.

The research results show that future research will discuss the relation of different neuroendocrine changes of the immune system and depression, research the comprehensive effect of inflammatory cytokines, different immune cell subsets and neuroinflammatory factors in depression, further understand the characteristics of new biochemical pathways and neurobiological targets related to depression and the correlation of the new biochemical pathways and neurobiological targets with disease fates, and enable personalized treatment of depression. Whereas depression can be treated by taking "immune function reconstitution" and "immune inflammation modulation pathway" as main targets.

Recombinant neuropeptide OT23, also called bidirectional agonist OT23, the subject group fuses short peptide (13 peptide) consisting of 13 highly homologous amino acids of the N end of OXM and PACAP with 10 amino acids of the C end of PTD (TAT) sequence to construct new artificial polypeptide consisting of 23 amino acids, and Chinese patent (sequence HSQGTFTSDYSKYGRKKRRQRRR) has been obtained, the invention is named as bidirectional agonist OT23 and application of PAC1-R and GLP-1R, and the patent number is ZL201510996270.7. The OT23 can inhibit the apoptosis of thymus cells, inhibit thymus atrophy, has the function of regulating the immune function of the organism, and can treat inflammation and reduce inflammatory factors, so that the medicine has the characteristic of multiple effects. These suggest that OT23 can seek a new direction and thought and a new therapeutic agent for immunotherapy of depression.

Disclosure of Invention

The invention aims to overcome the defects of the existing depression treatment technology and provides application of recombinant neuropeptide OT23 in preparation of medicaments for treating depression.

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

The invention establishes two kinds of mouse depression models through establishing a mouse acquirability helplessness (LH) model and a single stress source combined modeling method, and a large number of experiments are carried out through shuttle box conditional avoidance experiments, overhead cross maze experiments, social proximity and avoidance experiments, tail suspension test experiments, open field experiments, forced swimming experiments and other mouse depression behavioral detection methods, T cell receptor excision rings (receptor excision circles, TRECs) as thymus function detection methods of biological indexes and mouse brain hippocampus state detection methods taking brain-derived neurotrophic factor (BDNF) level in nerve tissues as indexes, comprehensive experimental data find that 1, two kinds of mouse depression models are successfully modeled, and model mice have similar symptoms such as weight loss, acquirability helplessness, social avoidance, exploration desire decline, immobility time growth (presenting behavior hopeless state) and the like human depression. The method is characterized in that the weight of the model group mice is obviously reduced, the residence time and the residence times of the model group mice in a social area in a social proximity experiment are obviously reduced, the heading times and the heading times of the model group mice in an overhead cross maze experiment are obviously reduced, and the movement time of the model group mice in water is obviously reduced in a forced swimming experiment. 2. After the modeling is successful, the recombinant neuropeptide OT23 is used for treating the model mice, so that the recombinant neuropeptide OT23 has a treatment effect on two depression mice models, and animal behavior monitoring data after treatment show that the mice can be expressed at a near normal level. The effect of high concentration (10 nmol/kg) is more obvious than that of low concentration (0.1 nmol/kg), the effect of treatment is more obvious than that of one week of treatment for half a month, the BDNF level of a treatment group is obviously improved compared with that of a model group after two weeks of treatment of recombinant neuropeptide OT23, so that the BDNF level in the brain can be recovered to relieve depression-like behaviors, the visceral indexes of thymus and spleen of mice in the OT23 treatment group are both increased, the thymus index is obviously increased, and the difference has statistical significance. The relative expression level of sjTREC in thymus and spleen of mice in the OT23 treatment group is obviously increased, which indicates that the recombinant neuropeptide OT23 can increase the generation and output capacity of thymus and spleen of the mice and restore the immune function of the mice, thereby improving the depression-like behavior of the mice. These suggest that recombinant neuropeptide OT23 can be used for preparing medicaments for treating depression.

Recombinant neuropeptide OT23, also known as bi-directional agonist OT23, has been obtained in China (SEQ ID NO: HSQGTFTSDYSKYGRKKRRQRRR) entitled bi-directional agonist OT23 and application of PAC1-R and GLP-1R, ZL201510996270.7.

Compared with the prior art, the invention has the following beneficial effects that 1. The TREC is taken as a biological index for detecting the thymus function, the thymus atrophy in the depressed mice is discovered for the first time, and the related immune function degradation is initiated, so that the thymus function has close relation with the occurrence and the development of the depression. The immune treatment of the depression of the mice is carried out for the first time by taking the thymus function reconstruction and the anti-inflammatory effect of the recombinant neuropeptide OT23 as cores, and the immune treatment proves effective.

2. The single stress source can not simulate the full view of depression, and the problem that the modeling effect is unstable and the modeling effect of mice in different batches is large often occurs. The invention tries to perform combined molding by adopting two methods of acquisition helplessness (LEARNED HELPLESSNESS, LH) and Chronic constraint stress (Chronic RESTRAIN STRESS, CRS), and obtains better effect. The multidimensional combination of different models may be more helpful for inducing the animals to develop a state similar to depression, and provides opportunities for deep understanding of the general aspect of depression.

Drawings

Fig. 1 is a graph of body weight change (without sex differentiation) for each group of mice.

Figure 2 is a graph of the number of evasion failures (sex indistinguishable) of classical shock shuttle box experiments.

Figure 3 is the number of evasion failures of a classical shock shuttle box experiment.

Fig. 4 is an individual data visual trend line (male).

Fig. 5 is an individual data visual trend line (female).

Figure 6 is the time and number of arm-opening times (without sex differentiation) of the mice of the overhead plus maze experiment.

Figure 7 is the residence time of the overhead plus maze test mice at the open arm.

Figure 8 is the number of times the mice enter the open arm for the overhead plus maze experiment.

FIG. 9 is a percentage of time in the non-social zone.

FIG. 10 is a percentage of time in the non-social zone.

Figure 11 shows tail suspension immobility times (without gender discrimination).

FIG. 12 shows the tail suspension time.

Fig. 13 is an individual data visual trend line (male).

Fig. 14 is an individual data visual trend line (female).

In fig. 15, graph a shows the weight gain of mice, graph B shows the residence time of mice in social proximity experiments in social areas, graph C shows the number of times mice in the elevated plus maze have been advanced to open the arm, graph D shows the time mice were moved in water in forced swimming experiments, and P <0.05, P <0.01, P <0.005 compared to the model group.

Fig. 16 shows a graph a showing the time spent in the social proximity experiment after treatment of depressed mice, and a graph B showing the movement trace of different groups of mice in the social proximity experiment. P <0.05 compared to model group.

Fig. 17 shows a graph a total course of movement of mice with depression after treatment in open field experiments, and a graph B shows movement trajectories of mice in different groups in open field experiments. P <0.01 compared to model group.

In fig. 18, a is the number of times a depressed mouse goes to open arm after treatment in an elevated plus maze test, and B is the time the mice of different groups moved in water in a forced swimming test. P <0.05, < P <0.01 compared to model group.

FIG. 19 is a graph showing the BDNF expression level of brain tissue of mice from different groups, using western blotting experiments, and performing statistical analysis on the relative expression levels of proteins. P <0.005 compared to model group.

In FIG. 20, the A-panel shows the thymus index of the mice, and the B-panel shows the spleen index of the mice. Organ index (mg/10 g) =organ weight (mg)/(body weight×10). P <0.05, < P <0.01 compared to model group

In FIG. 21, A shows the expression level of sjTREC in thymus, B shows the expression level of sjTREC in spleen, C shows the sjTREC fragment amplification curve, D shows the RAG2 fragment amplification curve, E shows the sjTREC fragment dissolution curve, and F shows the RAG2 fragment dissolution curve. G is sjTREC fragment standard curve, and H is RAG2 fragment standard curve. P <0.05 compared to model group.

Fig. 22 is a schematic view of an overhead plus maze.

FIG. 23 is a schematic diagram of a three-chamber social box.

Detailed Description

Example 1:

1. Experimental materials physiological saline (Guangdong Kogyo Co., ltd.) OT23 solution (10 nmol/ml) 1mgOT (MW= 2899.21) powder was dissolved in 34.49ml physiological saline and mixed well. The main instrument comprises an electric shock shuttle box analysis system, a three-cavity social box, an overhead plus maze, an injector and an electronic balance. SPF-class BALB/c mice, 6 weeks old, were purchased from Guangzhou southern medical laboratory animal science and technology development Co.

2. Modeling method of mouse acquisition helplessness (LH) model

Modeling of a depression LH model was performed using classical shock shuttle box experiments, and mice were exposed to uncontrolled or unpredictable aversive stimuli (e.g., shock) when placed in an environment where shock was evaded, a phenomenon of lack of evasion behavior (evasion failure) with concomitant depression-like behavior changes. And then, analyzing modeling conditions through behavioral monitoring, including classical electric shock shuttle box escape experiments, overhead plus maze experiments, social proximity and avoidance experiments, tail suspension experiments, regular monitoring of the weight of the mice and the like.

The method comprises (1) closing the partition doors of the left and right chambers in the shuttle box. (2) The mice of the control group and the experimental group to be modeled are put into a left chamber in a box, and a metal iron grating is arranged in the left chamber as a bottom layer, so that specific current can be set through a shocker to shock. (3) Setting electric shock parameters by turning on a computer and starting a video analysis system ((current intensity zero point five milliamperes, each time lasting two seconds, average interval of eight seconds and total time interval of one hour), (4) adding an animal serial number and a test box into the system, determining an analysis range, setting an identification algorithm, performing induction training of electric shock (5) carrying out three hundred sixty times of sole electric shock every day in a continuous week, and placing mice in a blank group which is compliant with natural stay of the mice for three seconds after one electric shock in the same shuttle box left room for the same time, and not carrying out balanced grouping training on test animals with the screening condition avoidance failure times of more than or equal to 6, and continuing experiments, and other animals observe the same induction failure and are eliminated (one of the blank groups can be selected for performing the scheduling training).

Animals were grouped into (1) a blank group (10 normal mice each, 5 male and female, each, injected with physiological saline), (2) a control group (10 depressed model mice each, 5 male and female, injected with physiological saline), (3) an experimental group (5 female, 10 nmol/kgOT) and (4) an experimental group 1 (5 male, 0.1nmol/kgOT 23) and (5) an experimental group 2 (5 male, 10nmol/kgOT 23).

LH mice with successful modeling are selected, the mice are equally divided into an experimental group and a control group, and then normal mice with the same number as the control group are selected as blank groups. The mice in the experimental group are regularly injected with a proper amount of recombinant OT23, and the mice in the control group and the blank group are not injected with recombinant OT23 for normal culture. Each group of mice was tested behaviourally weekly and test data were recorded.

3. Single stress source combined modeling method for establishing mouse depression model

The single stress source can not simulate the full view of depression, and the problem that the modeling effect is unstable and the modeling effect of mice in different batches is large often occurs. We have attempted to combine molding using both the learned helplessness (LEARNED HELPLESSNESS, LH) and Chronic constraint stress (Chronic RESTRAIN STRESS, CRS) methods.

Male BALB/C mice of 3 weeks old are fed adaptively for one week with current intensity of 0.2-0.6mA, 0.1mA increased every 4 days, and one stimulation period including a stimulation period of random 5-15s and random interval, and sole electric shock is performed for 1 hour every day for three weeks. Binding for 3 hours per day for three weeks.

Animals were grouped into WT groups of five animals, and were kept normally without any treatment. Model group, five, saline injections. model+PACAP13 group, five, 10nmol/ml PACAP13 peptide was injected at a dose of 10ml/kg. model+OT23 groups, five, were injected with 10nmol/ml OT23 peptide at a dose of 10ml/kg. The mice were weighed weekly and analyzed for visual status, including comprehensive observations of hair shine, feed intake, activity status, etc.

4. Behavioural detection method for depression of mice

(1) Shuttle box conditional avoidance experiment:

After modeling, the induction effect of the experimental group was screened using a classical shuttle box condition avoidance program. The method comprises the steps of (A) closing the partition doors of the left and right chambers in the shuttle box. (B) The mice of the control group and the experimental group to be modeled are put into a left chamber in a box, and a metal iron grating is arranged in the left chamber as a bottom layer, so that specific current can be set through a shocker to shock. (C) The computer is turned on to set the shock parameters and the video analysis system is turned on (current intensity zero point five milliamperes). (D) Adding an animal serial number and a test box into the system, determining an analysis range and setting an identification algorithm. (E) The condition screening program { totally divided into ten test cycles, each cycle including a conditioning period (light, 3 s), an unconditional stimulation period (light plus electric shock, 3 s) and an intermittent period (no stimulation, 5 s) }. (F) During experiments, the doors in the shuttle box are kept normally open, and the maximum current intensity is three milliamperes at zero. The experimental mice were not effectively utilized to avoid electric shock during both conditioned and unconditioned periods, and failed to avoid.

(2) Overhead plus maze experiment:

An open arm (30 cm long, 6cm wide) and a closed arm (30 cm long, 6cm wide, 15cm high) are typically located within the overhead cross-dynasty (about 40cm from the ground). Mice were allowed to move in the closed arms due to darkness and in the open arms due to curiosity and exploratory properties. They are based on the natural aversion of rodents to the exposed environment, where unprotected or fully open elevated areas are an anxiety challenge. Following the novel stimulus, the animal also presents a desire and fear of exploration, which also creates a opposition between seeking and avoidance. In an exposed environment, unprotected or fully open elevated areas are considered to be anxiety inducing factors, the time spent in each area, and the number of travels during the test are some aspects of assessing anxiety-like behavior and exploratory activity. At present, the frequency and duration of the animal's entry into the arm are inversely related to anxiety. The lower the frequency of animals entering the open arms, but the shorter the duration, the stronger the depressed mood of the animals. The method comprises (A) placing the mouse in the middle region formed by crossing two opening arms and two closing arms, and making the head of the mouse face to the opening arms directly. (B) And the time, number of times the mice remained on the open arm was recorded over five minutes. (C) After each mouse is detected, the elevated plus maze is wiped clean by an alcohol cotton ball so as to avoid the interference of the smell and excreta of the mouse on the behavior of the next mouse. As shown in fig. 22.

(3) Social proximity and avoidance experiments

The method is carried out in a three-cavity social box. If the mouse does not have a social disorder (a model mouse with depression would exhibit a social disorder), the mouse would like to "approach" the same class, particularly behaviorally as if it were in the social area for a longer period of time than it would be in the non-social area. In contrast, if a model mouse with depression (with social disability or depression-like manifestations) is presented with social "evasion" behavior in the form of explicit behaviours, i.e. stay in the social area for a shorter period of time than not in the non-social area, and is preferred to stay in the non-social area. As shown in fig. 23. The method comprises (A) isolating a mouse on one side of a social box with a partition door, namely "social stimulation". (B) The mouse to be detected is isolated to the other end of the three-cavity social box, namely the non-social area by a pull-push door which can be pulled open. (C) the middle range of the two sliding doors is a social area. The partition board and the sliding door are transparent and are not completely sealed, so that olfactory, visual and auditory connection between two animals is ensured. (D) After allowing the test mice to stay therein for a period of time as accommodation, the sliding door is then pulled up for a total of six minutes, during which the test mice can freely enter and exit the social and non-social areas as desired by the individual, but they cannot have any chance of direct contact with the mice as "social stimuli". (E) The residence time of each mouse in the social and non-social areas, respectively, was recorded within six minutes. (F) When the mice are replaced, the social box is cleaned by alcohol cotton balls, so that the influence of the smell or excrement of the mice in the previous experiment on the mice to be tested is prevented.

(4) Tail suspension test experiment:

The tail suspension experiment is mainly used for reflecting the physical change condition and fatigue degree of the mice after modeling, shows the strong wish of trying to change uncomfortable conditions under the double emotion of fear and anger of the mice, and the immobility time can be used for evaluating the behavior destinationstate of the mice caused by the non-avoidable compression environment, is similar to the depression disease, and belongs to an effective behavioral index for judging the mental and physical double fatigue of the mice. The method comprises the steps of (A) positioning a position of about one point and five centimeters at the far end of the tail of the mouse, (B) hanging the tail of the mouse downwards from the thirty-fifth centimeters of the ground, and (C) calculating the stationary time of the mouse when the mouse is hung within three hundred sixty seconds.

(5) Forced swimming experiment

Animals are forced to swim in a confined and non-escapable space, and due to the inherent aversion of rodents to water, they struggle in the water to swim and attempt to escape from the water environment, after a period of time, the escape is not expected to stop struggling and exhibit a "behavioural despair" state (floating immobility state). The forced swimming experiment of the large and small mice uses the immobility time of swimming as a main index to detect the destinationbehavior of animals. The method comprises the steps of (A) adjusting the water temperature in a test box before an experiment, wherein the water temperature is 23-25 ℃. The water depth should be adjusted according to animal weight, and animal tail keeps a certain distance with the bottom surface of test box. An open cylindrical container (total volume 2500ml; height 20cm; diameter 14 cm), multiple animal experiments were carried out (B) as same time, with opaque baffles between every two animals, and forced swimming experiments of mice were carried out on the day of detection, the swimming time of mice was 6min, and swimming time, immobility time and climbing time were recorded for 4 min.

(6) Open field experiment

The open field experiment is a classical emotion-related behavioral detection experiment, is used for detecting the behavior of animals in strange environments, comprises spontaneous activity behaviors and exploration behaviors, and is a method for evaluating the autonomous behaviors, exploration behaviors and tenseness of the animals in new different environments. The detection index is the incubation period of the animals entering the central area for the first time, the residence time of the central area and the passing times, the 'depression-like' behaviors of the animals and the antidepressant effect of the medicines can be detected by combining a depression model, the detection index is the starting activity of the animals, the horizontal climbing times and the vertical standing times of the animals can be counted manually to serve as behavior indexes, the motion trail of the animals can be recorded by adopting automatic shooting, and the behavior indexes such as the motion distance, the motion speed, the rest time and the like can be analyzed by adopting behavioural analysis software Tracker 6. The experiment can also be used to evaluate the rate of onset of antidepressant drug action. The method comprises the steps of (A) analyzing by using a multi-unit Open Field Maze (OFM) consisting of four movable chambers. Each chamber is 50 cm (length) by 50 cm (width) by 38 cm (height) in size and is made of white high density and non-porous plastic. (B) The animal is put into the center of the test box from the same position and the same direction in each experiment, then the test is started, the test time is 5min, and the total path of the movement of the mice is calculated.

5. Mouse immune function index-thymus function detection method

(1) Determination of thymus and spleen index in mice

Thymus and spleen are immune organs of the organism, and the measurement of spleen index can reflect the immune function state of the organism. Three groups of mice are weighed respectively, the average value of the weights of the mice in each group is recorded, the mice are sacrificed by cervical dislocation, thymus and spleen are weighed, and the thymus/weight ratio and the spleen/weight ratio of the mice in each group are calculated respectively.

(2) Experimental detection of TRECs

(A) Mouse thymus and spleen cells were taken, ground, lysed and washed, and finally counted.

(B) Extracting genome DNA by adsorption column method, detecting the extracted genome DNA by agarose gel electrophoresis and ultraviolet spectrophotometry

(C) PCR amplification of target fragments

The size of the primer design and synthesis amplified fragment is 97bp.

TRECF:5‘CATTGCCTTTGAACCAAGCTG3’

RAG2F:5‘TGACGTGGTGTATAGTCGA3’

TRECR:5‘TTATGCACAGGGTGCAGGTG3’

RAG2R:5‘TCCTGAAGTTCTGGGAGA3’

The amplified fragment size was 180bp. The primer is biosynthesized by Shanghai workers. The most suitable template concentration, primer concentration, dNTP concentration and annealing temperature were searched. Different reaction combinations are set to obtain the best reaction result. Agarose gel electrophoresis detection results.

(D) Construction of Standard and Positive recombinant plasmids

PCR amplification of TREC and RAG2 fragments, agarose gel electrophoresis, cutting gel containing target fragment, recovering target PCR product, constructing plasmid of T-easy carrier+RAG2 gene fragment and T-easy carrier+TREC fragment, screening blue-white spot, extracting plasmid

(E) Dilution and preservation of standard recombinant plasmid and optimization of PCR conditions

The concentration and purity of the plasmid were measured by an ultraviolet spectrophotometer. The standard recombinant plasmid concentration was diluted and converted to copy number according to the molecular weight of the standard recombinant plasmid to form concentration gradients of 108, 107, 106, 105, 104, 103, 102 copies/. Mu.l. And primer concentration and dNTP concentration are optimized.

(F) SYBR-Green RT-PCR assay

A reaction system (overall: 25. Mu.l) was prepared in a 0.2ml PCR thin-walled tube according to SYBR GREEN QPCR Supermix UDG kit instructions and the results of ordinary PCR optimization. The reaction conditions of the system are that UDG is removed at 50 ℃ for 2min, the pre-denaturation is carried out at 95 ℃ for 3min, the reaction is carried out at 95 ℃ for 30s and at 62 ℃ for 15s, and the total reaction time is 50 cycles. In the same reaction, a standard recombinant plasmid (6 concentration gradients) reaction group, a standard plasmid negative control group, a RAG2 fragment PCR group, a RAG2 negative control group, a RAG2 positive control group, a TREC fragment PCR group, a TREC positive control group and a TREC negative control group are set, and two replicates are set in each group. The copy numbers of the TREC in 1000000 cells can be calculated according to the standard curve and the sample PCR Ct value, and the formula [ (TREC copy number 1+TREC copy number 2)/(RAG 2 copy number 1+RAG2 copy number 2) ]multipliedby 2000000.

6. Statistical analysis

The result obtained by the method adopts software data processing of SPSS10.0, the measurement result is represented by mean value +/-standard deviation, a repeated measurement and variance analysis method is adopted for different behavioural detection results, P <0.05 represents the difference extremely clearly, and P <0.01 represents the difference clearly.

7. The modeling of the depression LH (acquired helplessness) model is successful, and the recombinant neuropeptide OT23 is effective for the treatment of mice with the acquired helplessness model.

The experiment adopts a classical electric shock shuttle box experiment to model a depression LH model, and a mouse is placed in an environment capable of escaping electric shock to receive continuous uncontrollable or predicted aversive stimulation (electric shock) to show the phenomenon of lack of escaping behaviors, and meanwhile, the behavior of a depression sample is changed. And then, analyzing modeling conditions through behavioral monitoring, including classical electric shock shuttle box escape experiments, overhead plus maze experiments, social proximity and avoidance experiments, tail suspension experiments, regular monitoring of the weight of the mice and the like. Comprehensive experimental data can show that the model is successful, and the model mice have symptoms similar to human depression, such as unassisted weight loss learning, social avoidance, reduced desire to explore, increased immobility time (in a behavior desperate state) and the like. After the modeling is successful, the recombinant neuropeptide OT23 is used for treating the model mice, and analysis data shows that the recombinant neuropeptide OT23 has an effect on treating the depression LH mouse model, wherein the effect of high concentration (10 nmol/kg) is better than that of low concentration (0.1 nmol/kg), and the effect of the recombinant neuropeptide OT23 is better than that of one week of treatment for half a month. The behavioural monitoring data after treatment shows that the therapeutic effect can be near normal level.

(1) Mouse body weight and status monitoring:

As shown in fig. 1, the body weight of the model mice (control group and experimental group) after modeling has been significantly reduced after 1 week, and a two-factor analysis of variance for body weight and week number has been found to have a significant inter-group difference (P < 0.001) between the model mice and the blank mice, which is statistically significant. The experimental group had a weight gain after the recombinant neuropeptide injection treatment, and a significant difference was produced between the experimental group and the control group (P < 0.05). In addition to the change in body weight, the hair luster, feeding rate, activity state, etc. of the mice were changed. After modeling, the skin and hair of the mice are observed to be darker than before modeling, the food intake is reduced, the immobility time is increased, the mice are favored to be curled at corners, and part of the mice can be drilled into padding to hide a plurality of manifestations of the mice.

(2) Classical shock shuttle box experimental results:

As shown in fig. 2, the one-way analysis of variance for escape failure behavior in the observation of ten test periods found that there was a significant statistical significance of the apparent inter-group difference (P < 0.001) in the number of escape failures between normal mice and model mice (control group). The differences between the experimental and control groups after treatment with recombinant OT23 peptide were significant (P < 0.001) and exhibited escape behavior similar to that of the blank group, whereas there was no significant difference between the groups compared to the blank group (P > 0.05).

Specific analysis as shown in fig. 3 and 4, the number of evasion failure times of the control group after male modeling is obviously more than that of the blank group, and the single-factor analysis of variance is carried out on evasion failure behaviors in the observation of ten test periods, so that obvious inter-group differences (F=15.61 and P < 0.001) exist between the number of evasion failure times of normal mice and model mice, and the method has obvious statistical significance. After 7d treatment, the number of escape failures of the control group is still obviously more than that of the blank group, the data between the two groups have obvious differences (F=23.29, P < 0.001), the experimental group has different degrees of fallback, the experimental group 1 is not obvious compared with the experimental group 2, and the experimental group 1 and the control group have no obvious differences (P > 0.05). The escape failure times of the experimental group 2 fall back remarkably, and the depression-like performance (escape failure) of the experimental group 2 is improved to a medium to large extent. (the overall trend line equation calculates y= -0.0411x2+0.8346x +1.9798; r ² = 0.4527). After 15d treatment, experimental group 2 was almost returned to normal level, the difference between experimental group 2 and control group was significant (f=21.64, P < 0.001) and exhibited escape behavior similar to that of the blank group, and there was no significant difference between groups (P > 0.05) compared to the blank group. Significant differences (p=0.003) were also produced between experimental group 1 and control group, but were not significant compared to experimental group 2. (overall data trend line equation calculation y= -0.0508x2+0.9959x+2.2763, r ² = 0.3879)

As shown in fig. 3 and 5, the number of escape failures of the control group after female modeling is significantly greater than that of the blank, and as a result of single-factor analysis of variance on the escape failure behaviors in the observation of ten test periods, a significant statistical significance is found in that there is a significant inter-group difference in the number of escape failures between the normal mice and the control mice (f=18.83, p < 0.001). After 7d treatment, the control group had significantly more escape failures than the blank group, and the data were significantly different between the two groups (f=14.83, p < 0.001). Compared with the control group, the escape failure times of the experimental group are more remarkable, and the depression-like performance (escape failure) of the experimental group is improved to a medium extent. (overall trend line equation calculation y= -0.0628x2+1.0867x+2.4967, r ² = 0.3241). After 15d treatment, the difference between the experimental and control groups was significant (f=20.51, P < 0.001) and exhibited escape behavior similar to that of the blank group, and there was no significant difference between the groups compared to the blank group (P > 0.05). (Overall data trend line equation calculated y= -0.0549x2+0.7434x+4.4615, r ² =0.192)

(3) Results of overhead plus maze experiments:

As shown in fig. 6, there was a clear difference between groups (P < 0.001) in time and number of arm openings between model mice and normal mice after modeling, which was statistically significant. After treatment, the control group still had significantly less time and frequency to stay on arm than the blank group, the data had significant differences between the two groups (P < 0.001), the difference between the groups of the experimental group and the control group was significant (P < 0.001) and exhibited similar exploratory performance to the blank group (significantly increased time and frequency to stay on arm), and there was no significant difference between the groups of the experimental group and the blank group (P > 0.05).

Specific analysis as shown in table 1 and fig. 7 and 8, the longer open arm time than the closed arm time of the male mice in the blank group shows that the performance of the male mice in the new environment is better, the residence time of the model group (control group and experimental group) in the open arm of the elevated plus maze and the number of times of entering the open arm are obviously less than those of the blank group, and the variance analysis shows that obvious inter-group differences (time F=4.829 and P < 0.05) exist between the open arm time and the open arm time of the model mice and the normal mice (the number F=8.903 and P < 0.001) have obvious statistical significance. After one week of treatment, the control group had significantly less time and frequency to stay on arm than the blank group, the data had significant differences between the two groups (f=4.65, P < 0.05), and the experimental group 1 had no significant differences (P > 0.05) compared to the control group. After half a month of treatment, the difference between the experimental group 2 and the control group was significant (P < 0.001) and exhibited similar exploratory performance (significantly increased residence time and frequency on open arm) as the blank group, with no significant difference (P > 0.05) between the groups compared to the blank group. At the same time, the difference between the experimental group 1 and the control group (P < 0.05) was generated, but the difference was not significant compared to the experimental group 2.

As shown in table 2 and fig. 7 and 8, there was a clear difference between groups (f=8.797, p < 0.001) in time and number of arm openings between model mice and normal mice after modeling of female mice. One week after treatment, the control group still had significantly less on-arm residence time and number than the blank group, the data had significant differences between the two groups (P < 0.001), and significant inter-group differences occurred between the experimental and control groups (P < 0.05). After half a month of treatment, the difference between the experimental and control groups was significant (P < 0.001) and exhibited similar exploratory performance to the blank group (significant increase in open arm residence time and frequency), with no significant difference between the experimental and blank groups (P > 0.05).

Table 1 comparison of results of elevated plus maze experiments for each group (Male)

Note P <0.05 compared to normal blank, P <0.01 compared to model group

OT time of stay on arm OE number of times of opening arm (suffix number indicates experimental week number)

Table 2 comparison of the results of the elevated plus maze experiments of the groups (female)

Note P <0.05 compared to normal blank, P <0.01 compared to model group

OT time of stay on arm OE number of times of opening arm (suffix number indicates experimental week number)

(4) Social proximity and avoidance experimental results

As shown in fig. 9, significant differences between the model mice and the blank mice occurred after modeling (P < 0.001), while no significant differences were observed between the control group and the experimental group (P > 0.05). The data reflect social disorder depression behavior of the control group and the experimental group, and have statistical significance. After treatment, the experimental group and the control group produced significant differences between groups (P < 0.05), after half a month of treatment the experimental group and the control group were significantly different (P < 0.001), exhibited social preference behavior similar to that of the blank group, and there was no significant difference between groups compared to the blank group (P > 0.05).

Specific analysis As shown in FIG. 10, after male modeling, the control group and the experimental group have escape behaviors, namely the residence time in the social area is shorter than that in the non-social area, and when single-factor variance analysis is performed on the difference between the social area time and the non-social area time in the three-cavity social box, obvious group-to-group differences (model mouse social area: 32.06+/-2.18% and blank mouse social area: 63.89+/-3.31% and P < 0.001) are found between the model mouse and the blank mouse, and no obvious differences (P > 0.05) are found between the control group and the two experimental groups. The data reflect social disorder depression behavior of the control group and the experimental group, and have statistical significance. After one week of treatment there was a significant inter-group difference between control and blank (P < 0.05), experimental group 2 produced no significant difference (P > 0.05) from control, experimental group 1 compared to control, and after half a month of treatment experimental group 2 was almost returned to normal level, the inter-group difference between experimental group 2 and control was significant (P < 0.001), exhibited social preference performance similar to that of blank, and there was no significant difference (P > 0.05) between groups compared to blank. Significant differences (P < 0.05) were also generated between experimental group 1 and control group, but were less significant for experimental group 2 than control group.

As shown in fig. 10, significant differences between the model mice and the blank mice (P < 0.001) occurred after female modeling, while no significant differences between the control group and the experimental group (P > 0.05). The data reflect social disorder depression behavior of the control group and the experimental group, and have statistical significance. After treatment 7d, the experimental group produced significant differences between groups (P < 0.05) and the control group, after treatment 15d the experimental group showed significant differences between groups (P < 0.001) and exhibited social preference behavior similar to that of the blank group, and there was no significant difference between groups (P > 0.05) compared to the blank group.

(5) Tail suspension experimental results

As shown in fig. 11, the tail suspension struggling time of the control group and the experimental group after modeling is obviously smaller than that of the blank group, the immobility time is increased, and the single-factor variance analysis is performed on the immobility time which is abandoned after struggling in the tail suspension process, so that obvious inter-group difference (P < 0.001) exists between the immobility time of a normal mouse and a model mouse, and the model mouse has obvious statistical significance and presents a behavior hopeless state. The differences between the experimental and control groups after treatment were significant (P < 0.001) and exhibited escape behavior similar to that of the blank group, and there was no significant difference between the groups compared to the blank group (P > 0.05).

As shown in fig. 12 and 13, the tail suspension struggling time of the control group and the experimental group after male modeling is obviously smaller than that of the blank group, the immobility time is increased, and the single-factor analysis of variance is carried out on the immobility time which is abandoned after struggling in the tail suspension process, so that obvious inter-group difference (P < 0.001) exists between the immobility time of a normal mouse and a model mouse, and the model mouse has obvious statistical significance and presents a behavior destinned state. In addition, as also seen in fig. 13 based on the trend line, the immobility time of both control and both experimental groups was significantly increased (as calculated by the overall trend line equation: y=22.5x+110.5, r ² =0.305) compared to the blank group, after 7d treatment, the control immobility time was still significantly greater than the blank group, the data between the two groups had significant differences (P < 0.001), the experimental group 2 had significant differences (P < 0.001) compared to the control group, and the experimental group 1 had no significant differences (P > 0.05) compared to the control group. In addition, it is also seen from the trend line that the stationary time fall back of experimental group 2 was more remarkable, and the depression-like performance (behavior hopeless state) of experimental group 2 was improved to a medium to large extent, compared with the control group. (the overall trend line equation calculates y= -27.25x2+131.55x+30.75, r ² = 0.5148). After 15d treatment, experimental group 2 was almost returned to normal level, and the difference between experimental group 2 and control group was significant (P < 0.001) and exhibited escape behavior similar to that of the blank group, and there was no significant difference between groups (P > 0.05) compared to the blank group. Significant differences (P < 0.05) were also generated between experimental group 1 and control group, but were not particularly significant compared to differences between experimental group 2 and control group. (Overall data trend line equation calculated y= -33.75x2+165.25x-10.25, r ² = 0.6061)

As shown in fig. 12 and 14, the tail suspension struggling time of the female control group and the experimental group after modeling is obviously smaller than that of the blank group, the immobility time is increased, and the single-factor analysis of variance is carried out on the immobility time which is abandoned after struggling in the tail suspension process, so that obvious inter-group difference (P < 0.001) exists between the immobility time of a normal mouse and a model mouse, and the model mouse has obvious statistical significance and presents a behavior destinned state. In addition, as also seen in fig. 14, the immobility time of the control group and the experimental group was significantly increased (calculated by the overall trend line equation: 62.298ln (x) +115.13, r ² = 0.4499) after 7d treatment compared to the blank group, the immobility time of the control group was still significantly longer than that of the blank group, the data between the two groups had significant differences (P < 0.001), and the experimental group had significant inter-group differences (P < 0.001) compared to the control group. In addition, it is also seen from the trend line that the immobility time fall back is more remarkable in the experimental group compared with the control group, and the depression-like performance (behavior destinbed state) of the experimental group is improved to a medium to a large extent. (the overall trend line equation calculates y= -97.5x2+400.5x-190, r ² =1). After 15d treatment, the difference between the experimental and control groups was significant (P < 0.001) and exhibited escape behavior similar to that of the blank group, and there was no significant difference between the groups compared to the blank group (P > 0.05). (the overall data trend line equation calculates y= -118x2+467x-240, r ² =1).

8. The single stress source combined modeling method is proved to be capable of establishing a stable mouse depression model, and the recombinant OT23 is clear to have therapeutic effect on depression mice

8.1, After three weeks of single stress source combined modeling, multiple behavioral analysis comprehensively proves that the modeling is successful, and the combined modeling method is proved to be effective and efficient in establishing the depression mouse model.

As shown in FIG. 15, the Model mice had significantly reduced weight gain. In social proximity experiments, the time and number of stay in the social area for Model group male mice was significantly reduced after joint modeling. In the elevated plus maze experiment, the number and time of Model mice going to open arms was significantly reduced after the joint modeling. In the forced swimming experiment, the time of Model group mice moving in water is obviously reduced after combined modeling. The combined modeling method is proved to be capable of establishing a stable mouse depression model,

8.2 Multiple methods and indicators comprehensive demonstration of the efficacy of recombinant neuropeptide OT23 in combination-modeled depressive mouse treatments

As shown in fig. 16, in the social proximity experiment, male mice were exposed to the same species more than the Model group after treatment, and the number and time of reaching the social area were significantly increased, indicating a reduction in the extent of depression.

As shown in fig. 17, the total distance traveled by the mice after treatment was significantly increased over the Model group in the open field experiment, while the probability of traveling to the central area was increased, indicating improved motor status.

As shown in fig. 18A, in the elevated plus maze test, the number and time of mice entering the open arm was significantly increased, indicating that their depressed state was improved.

As shown in fig. 18B, in the forced swimming experiment, the movement time of the mice in water was significantly improved by treatment, indicating that the hopeless state was reduced.

As shown in fig. 19, OT23 can restore BDNF levels in the brains of depression model mice to reduce depression-like behavior. BDNF levels were significantly reduced in the brains of Model mice compared to WT mice. After two weeks of treatment with recombinant neuropeptide OT23, BDNF levels were significantly increased compared to the Model group, suggesting that it may alleviate depression-like behavior by restoring BDNF levels in the brain.

The thymus function detection of the mice proves that the OT23 can effectively restore thymus function, improve the immunity of the mice and improve the depression symptoms. As shown in fig. 20, after treatment, the visceral indices of thymus and spleen were elevated in the depression Model mice compared to the Model group, wherein the thymus index was elevated significantly, the difference was statistically significant, and the spleen index was not significantly different. As shown in fig. 21A, B, the relative expression level of sjTREC in thymus and spleen of mice in the group of Model mice of depression + OT23 was significantly increased by OT23, indicating that recombinant neuropeptide OT23 can increase the production and output capacity of thymus and spleen of mice, thereby improving depression-like behavior of mice.

Claims (1)

1. Application of recombinant neuropeptide OT23 in the preparation of drugs for the treatment of depression.