CN116159127A

CN116159127A - Use of acidic ribosomal protein P2 in the treatment of anxiety disorders

Info

Publication number: CN116159127A
Application number: CN202310059653.6A
Authority: CN
Inventors: 李燕; 罗丹蕾; 武薇; 景临林
Original assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Current assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Priority date: 2023-01-17
Filing date: 2023-01-17
Publication date: 2023-05-26
Anticipated expiration: 2043-01-17
Also published as: CN116159127B

Abstract

The invention relates to the technical field of proteins, and discloses application of acidic ribosomal protein P2 in treatment of anxiety disorder. The inventor finds that the movement induction protein generated by movement exercise, namely the acidic ribosomal protein P2 has better effect on improving anxiety and can be applied to treating anxiety. The acidic ribosomal protein P2 can improve anxiety and cognitive functions of anxiety patients, has a good effect of improving anxiety, and can be used for treating anxiety; the acidic ribosomal protein P2 can be used for preparing medicines for treating anxiety disorder and improving anxiety emotion and cognitive function of anxiety disorder patients.

Description

Use of acidic ribosomal protein P2 in the treatment of anxiety disorders

Technical Field

The invention relates to the technical field of proteins, in particular to application of acidic ribosomal protein P2 in treatment of anxiety disorder.

Background

Mental disorders are currently one of the major causes of global disease burden, with anxiety disorders having become the most common mental health problem worldwide. Anxiety disorder is one of the very disabling mental disorders, has the characteristics of high prevalence, chronicity, complications and the like, is more susceptible to young people, and is unfavorable for the healthy development of the body and the mind.

Anxiety refers to an unpleasant, complex emotional state of an individual with respect to tension, anxiety, annoyance, etc., that is forthcoming, potentially dangerous, or threatening. The specific etiology of anxiety is not completely understood and is generally thought to be the result of the effects of many factors, genetic, environmental and social. Previous studies have suggested that anxiety disorders are mainly caused by alterations in the limbic system, hypothalamic-pituitary-adrenal axis dysfunction and genetic factors. The connection and coordination between the amygdala, the ventral frontal cortex and the brain areas such as the hippocampus is related to the degree of anxiety in mice, rats or humans. Studies have also demonstrated that overactivation of the amygdala brain region or hypoactivation of the ventral prefrontal cortex during childhood may increase the likelihood of the onset of anxiety.

At present, the treatment of anxiety disorder and anxiety emotion in clinical treatment is mainly through medicine and psychological treatment. Among the most widely used are antidepressants, including selective 5-hydroxytryptamine reuptake inhibitors and 5-hydroxytryptamine-norepinephrine reuptake inhibitors, but the resistance to frequent treatments and side effects that can lead to exacerbation of gastrointestinal distress, diarrhea, nervousness, insomnia and headache, etc. have restricted the choice of clinical anxiolytic drugs. Psychological therapy is mainly cognition-behavior therapy, but the curative effect is not exact, and satisfactory effect is difficult to achieve. Thus, there is a need to find key molecules in the development of anxiety disorders from a new perspective and develop new therapeutic strategies based thereon.

There is increasing evidence that different degrees of physical exercise have many beneficial effects on brain health and cognitive function. Exercise can reduce the cognitive decline rate in patients with neurodegenerative diseases and healthy people of various ages, and has positive effects on stress, anxiety and depressed mood. Although many studies have found that movement has a better improving effect on anxiety and cognitive function, most of the current studies mainly surround the influence of movement on improving memory level and improving cognitive dysfunction, and the molecular mechanisms related to movement improving anxiety are not clear. In recent years, with the continuous development of proteomics research, the technical difficulty is overcome, and the mystery of sports is gradually revealed. Among them, proteomics studies on plasma or serum suggest a number of different proteins that play an important role in the course of exercise. Research on molecular mechanisms of motor improving anxiety is of great research value to target motor related biomarkers with direct or indirect impact on brain anxiety mood.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide an application of acidic ribosomal protein P2 in treating anxiety disorder. The inventor finds that the motion-induced protein generated by exercise, namely the acidic ribosomal protein P2, has better improving effect on focusing and can be applied to treating anxiety.

In order to achieve the above purpose, the present invention is realized by the following technical scheme.

Use of acidic ribosomal protein P2 for the treatment of anxiety disorders.

Preferably, the amino acid sequence of acidic ribosomal protein P2 is:

MRYVASYLLAALGGNSSPSAKDIKKILDSVGIEADDDRLNKVISELNG KNIEDVIAQGIGKLASVPAGGAVAVSAAPGSAAPAAGSAPAAAEEKKDEK KEESEESDDDMGFGLFD。

use of acidic ribosomal protein P2 in a medicament for the treatment of anxiety disorders.

Compared with the prior art, the invention has the beneficial effects that:

the acidic ribosomal protein P2 can improve anxiety and cognitive functions of anxiety patients, has a good effect of improving anxiety, and can be used for treating anxiety;

the acidic ribosomal protein P2 can be used for preparing medicines for treating anxiety disorder and improving anxiety emotion and cognitive function of anxiety disorder patients.

Drawings

The invention will now be described in further detail with reference to the drawings and to specific examples.

FIG. 1 is a Western immunoblot of a monoclonal bacterial fluid sample expressing acidic ribosomal protein P2; in fig. 1, 1 is a molecular weight standard, 2 is an uninduced control bacterial liquid sample, and 3, 4 and 5 are 3 monoclonal bacterial liquid samples after isopropyl-beta-D-thiogalactoside IPTG induction;

FIG. 2 is a Western immunoblot of cell lysates and supernatants; in FIG. 2, 1 is a molecular weight standard, 2 is a whole cell lysate at 22 ℃,3 is a centrifugation supernatant at 22 ℃,4 is a whole cell lysate at 37 ℃, and 5 is a centrifugation supernatant at 37 ℃;

FIG. 3 is a Western immunoblot of purified acidic ribosomal protein P2; in FIG. 3, 1 is whole cell lysate, 2 is centrifugated supernatant, 3 is penetrating fluid, 4 is 2mmol/L imidazole eluent, 5 is 20mmol/L imidazole eluent, 6 is 50mmol/L imidazole eluent, 7 is 250mmol/L imidazole eluent, and 8 is molecular weight standard;

FIG. 4 is a Western immunoblot of acidic ribosomal protein P2 after elution; in FIG. 4,1 is an enzyme-digested product, 2 is a penetrating fluid, 3 is 20mmol of imidazole eluent, 4 is 250mmol of imidazole eluent, and 5 is a molecular weight standard;

FIG. 5 is a diagram showing immunofluorescence of frozen sections of mouse brain tissue;

FIG. 6 is a graph of open arm residence time for acidic ribosomal protein P2 in the treatment of anxiety in mice;

FIG. 7 is a graph showing the number of open arm entries in the treatment of anxiety in mice with acidic ribosomal protein P2;

FIG. 8 is a graph showing the open box residence time of acidic ribosomal protein P2 in the treatment of anxiety in mice.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

According to the invention, the acidic ribosomal protein P2 is firstly extracted, the penetrability of the extracted acidic ribosomal protein P2 to the blood brain barrier is detected, then the acidic ribosomal protein P2 is injected into mice, and whether the anxiety condition of the anxiety mice is obviously improved after the exogenous acidic ribosomal protein P2 is injected is detected through behavioural detection.

The first fraction, acidic ribosomal protein P2 is extracted

Step 1, transformation and Small-Scale inducible expression of plasmids

Transferring the recombinant plasmid containing the target gene into escherichia coli to express the target protein; verifying whether the escherichia coli transferred into the plasmid can express the target protein or not through small-scale induced expression; the target protein is acidic ribosomal protein P2.

1.1, 10ng of plasmid was added to 100. Mu.l of BL21 (DE 3) E.coli competent cells and placed on ice for 20min to give a first intermediate;

1.2, rapidly placing the first intermediate product in ice for 2min at 42 ℃ for heat shock for 90s, then adding the first intermediate product into 500 mu lLB culture solution, and carrying out shaking culture at 37 ℃ and 220rpm for 30min to obtain a second intermediate product;

1.3, taking 150 μl of the second intermediate product, uniformly coating on LB plates containing 50 μg/ml kanamycin, and culturing for 24 hours at 37 ℃ in an inverted manner to enable the monoclone on the plates to grow to a proper size;

1.4, selecting a monoclonal, inoculating the monoclonal into 2ml of LB culture solution containing 50 mug/ml kanamycin, and culturing at 37 ℃ and 220rpm with shaking for 5-7 hours to obtain a third intermediate product; the absorbance OD600 of the third intermediate at 600nm should be 0.6-0.8; taking out part of the third intermediate product, adding sterilized glycerol to a final concentration of 15%, and preparing glycerinum;

1.5, taking 500 μl of the third intermediate product, adding inducer isopropyl-beta-D-thiogalactoside IPTG to the concentration of the inducer of 0.1mmol, shaking and culturing at 37 ℃ and 220rpm for 4 hours, and inducing the expression of target protein to obtain an induced bacterial liquid;

1.6, centrifuging 100 μl of the induced bacterial liquid at 10000rpm for 2min, centrifuging to remove supernatant, collecting bacterial cells, adding 50 μl of tris Buffer saline TBS to resuspend bacterial cells, adding SDS electrophoresis Loading Buffer, decocting in a metal bath at 100deg.C for 3min, centrifuging to obtain supernatant, and detecting protein expression by electrophoresis, wherein the expression result is shown in figure 1.

Step 2, verifying whether the expressed target protein is soluble;

2.1, respectively inoculating glycerol bacteria into 2 bottles of 600ml LB culture solution containing 50 mug/ml kanamycin, wherein the volume ratio of the glycerol bacteria to the culture solution is 1:1000; shaking at 37 ℃ and 220rpm until the absorbance reading OD600 of the bacterial liquid at 600nm is between 0.8 and 1.0, so as to obtain a first bacterial liquid;

2.2, respectively adding an inducer isopropyl-beta-D-thiogalactoside IPTG to the two bottles of first bacterial liquid until the concentration of the inducer is 0.1mmol, respectively placing the two bottles of first bacterial liquid in a temperature-control shaking table at 22 ℃ and 37 ℃ and continuously culturing the two bottles of first bacterial liquid for 4 hours at a rotating speed of 220rpm to obtain a second bacterial liquid at 22 ℃ and a second bacterial liquid at 37 ℃;

2.3, respectively centrifuging the second bacterial liquid at 22 ℃ and the second bacterial liquid at 37 ℃ at 4 ℃ and 5000rpm for 10min, discarding the supernatant to collect bacterial cells, and preserving at-20 ℃;

2.4, adding 10 milliliters of bacteria breaking buffer solution into each gram of bacteria according to the wet weight of the two bacteria, and respectively carrying out ultrasonic breaking on the bacteria. Crushing conditions: 150W, 1s of disruption, 1s of interval, total 15min, buffer of disruption: PBS, ph7.4; protein expression was detected by electrophoresis of the second bacterial liquid at 22℃and the second bacterial liquid at 37℃and the supernatant at 22℃and 37℃respectively, and the results are shown in FIG. 2.

Step 3, purifying the target protein;

3.1, centrifuging the crushed thalli at 12000rpm for 10min at 4 ℃, collecting supernatant, and purifying by a nickel column, wherein the purification result is shown in figure 3; wherein, the filler for nickel column purification is: the everstate world man and biotechnology super-tolerates nickel affinity filler Smart-NI6FF; the balancing liquid is as follows: phosphate buffer PBS, pH7.4; the eluent is as follows: phosphate buffer PBS containing imidazole, pH7.4, eluting the eluent according to the gradient of imidazole concentration of 2mmol/L, 20mmol/L, 50mmol/L and 250mmol/L respectively;

3.2, endotoxin removal: in the purification process of step 3.1, after sample loading is finished, target proteins are combined on the filler, and the filler is washed by endotoxin removal liquid with the volume of 20 times of column volume, so that endotoxin is washed off; eluting with 10 times of column volume of balance solution, and gradient eluting with eluent.

Wherein, endotoxin removal liquid is: phosphate buffer PBS, pH7.4,1% of surfactant TritonX-100.

3.3, enzyme cutting and dialyzing to remove labels:

taking eluted fusion protein, adding protease ULP of a protein fusion tag SUMO, wherein the ratio of target protein to protease ULP is 500:1, performing enzyme digestion overnight in a dialysis bag, taking supernatant by centrifugation with PBS as dialysis buffer, and purifying by a nickel column, wherein the SUMO fusion tag with His6-tag is adsorbed on the nickel column, and crude and pure acidic ribosomal protein P2 is in a flow-through solution;

the nickel column purification removal of the tag was similar to step 3.1. Wherein, the filler for nickel column purification is: the everstate world man and biotechnology super-tolerates nickel affinity filler Smart-NI6FF; the balancing liquid is as follows: phosphate buffer PBS, pH7.4; the eluent is as follows: phosphate buffer PBS containing imidazole, ph7.4; eluting the eluent according to gradients of 20mmol/L and 250mmol/L of imidazole concentration respectively; and (3) carrying out electrophoresis on the collected penetrating fluid and eluted proteins to detect the purity of the proteins, wherein the penetrating fluid contains target proteins, and the eluted proteins are tag proteins. The expression results are shown in FIG. 4.

And (3) dialyzing the penetrating fluid containing the target protein in phosphate buffer PBS overnight, centrifuging to obtain supernatant, filtering and sterilizing with a 0.22 mu m filter membrane to obtain the target protein, and carrying out ultrafiltration concentration on the protein as required.

In fig. 1, 1 is a molecular weight standard; 2 is an uninduced control bacterial liquid sample; 3. 4 and 5 are 3 monoclonal bacteria liquid samples after IPTG induction; as can be seen from FIG. 1, all 3 of the selected monoclonal strains expressed acidic ribosomal protein P2 and had the correct molecular weight.

In fig. 2, 1 is a molecular weight standard; 2 is whole cell lysate at 22 ℃;3 is a centrifugal supernatant at 22 ℃;4 is whole cell lysate at 37 ℃;5 is the centrifugation supernatant at 37 ℃. As can be seen from FIG. 2, the induction at 22℃and 37℃both had the expression of the target protein, and both had some proteins soluble.

In FIG. 3, 1 is whole cell lysate; 2 is centrifugal supernatant; 3 is a penetrating fluid; 4 is 2mmol imidazole eluent; 5 is 20mmol of imidazole eluent; 6 is 50mmol imidazole eluent; 7 is 250mmol imidazole eluent; 8 is a molecular weight standard; as can be seen from FIG. 3, the target protein can be bound to a nickel column, and the target protein is eluted with higher purity but little degradation.

In FIG. 4,1 is the enzyme-digested product and 2 is the permeate; 3 is 20mmol of imidazole eluent; 4 is 250mmol imidazole eluent; 5 is a molecular weight standard; as shown in fig. 4, the target protein flows through, the tag protein is combined with a nickel column, and the tag protein is separated to obtain the target protein.

The amino acid sequence of the acidic ribosomal protein P2 is:

MRYVASYLLAALGGNSSPSAKDIKKILDSVGIEADDDRLNKVISELNGKNIEDVIAQGIGKLASVPAGGAVAVSAAPGSAAPAAGSAPAAAEEKKDEKKEESEESDDDMGFGLFD。

the second part, detect the penetrability of the extracted acidic ribosomal protein P2 to the blood brain barrier

The tail of the mouse is intravenously injected with exogenous acidic ribosomal protein P2 with a Cy5.5NHS ester label, wherein the Cy5.5NHS ester is a dye which can display red fluorescence under the excitation of 648nm excitation light, and is one of the most common red fluorophores. The mouse brain tissue frozen section is observed to be subjected to immunofluorescence staining to detect red fluorescence. The results are shown in FIG. 5.

In fig. 5, green is a neuronal cell, blue is a cell nucleus, and red is an exogenous acidic ribosomal protein P2 with a cy5.5 tag. As can be seen from fig. 5, there are many red spots in the brain, and many red spots overlap with green or blue, indicating that exogenously synthesized acidic ribosomal protein P2 is able to cross the blood brain barrier into the brain parenchyma and be available to nerve cells.

Third part, use of acidic ribosomal protein P2 in the treatment of anxiety in mice

Intravenous injection of acidic ribosomal protein P2 into the tail of mice, and whether anxiety conditions of anxiety mice were significantly improved after the injection of exogenous acidic ribosomal protein P2 was detected by behavioural.

The behavioural experiment relates to two tests about anxiety emotion, namely an elevated plus maze and a light and dark box experiment, which are widely applied to a plurality of disciplines such as new drug development/screening/evaluation, pharmacology, toxicology, preventive medicine, neurobiology, animal psychology, behavioural biology and the like, and are classical experiments for developing behavioural researches, especially anxiety and depression researches.

The elevated plus maze Elevated plus maze has a pair of open arms and a pair of closed arms in which rodents would tend to move due to darkness, but would move again for curiosity and exploratory reasons. Light/dark box experiments light/dark box has a bright light box and a dark box which is closed, rodents like to move in the dark box, but the exploration habit of animals promotes the animals to try to explore the light box, however, the light stimulation of the light box inhibits the exploration activity of the animals in the light box. When the mice are placed in both devices, the mice are presented with a novel stimulus, which simultaneously produces exploratory impulse and fear, which results in exploratory and avoidance conflicting behaviors, thereby producing anxiety. The anxiolytic can obviously increase the times and time of entering the open arms of the overhead plus maze, and increase the residence time of the open boxes in the bright and dark boxes.

In the experiment, anxiety mice were modeled by a continuous 10-day constrained stress modeling method, and compared with mice in a control group without any intervention, the constrained mice were found to have significantly reduced residence time and entry times in the open arms of the elevated plus maze, and significantly reduced open box residence time in the bright and dark boxes, and the modeling method was determined to be able to obtain a stable anxiety mice model.

Subsequently, an acidic ribosomal protein P2 intervention was performed to improve anxiety experiments, which were divided into three groups: the control mice were normal saline-injected mice without restraint intervention, the anxiety group was normal saline-injected mice with restraint anxiety modeling, and the anxiety-injected protein group was mice with restraint anxiety modeling and with acid ribosomal protein P2.

Mice in anxiety-injected protein groups were injected with acidic ribosomal protein P2 once every three days at a dose of 1mg/kg, and mice in control groups and anxiety groups were simultaneously injected with equal volumes of normal saline for 10 consecutive injections, and it was found that the residence time and the number of entries in the open arms of the elevated plus maze were significantly increased, and the residence time in the open boxes of the bright and dark boxes was significantly increased, as shown in fig. 6, 7 and 8, indicating that the injection of acidic ribosomal protein P2 effectively eases anxiety mood caused by binding stress.

While the invention has been described in detail in this specification with reference to the general description and the specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. Use of acidic ribosomal protein P2 for the treatment of anxiety disorders.

2. The use according to claim 1, wherein the amino acid sequence of the acidic ribosomal protein P2 is:

3. use of acidic ribosomal protein P2 in a medicament for the treatment of anxiety disorders.