Application development of APR3 as analgesic target

Abstract

The invention discloses application and development of APR3 as an analgesic target. According to the invention, the space-time specific knockout of the gene is realized in the mouse body by using the Cre-lox system. The method comprises the following steps: firstly, injecting a complete Freund's adjuvant (CFA) into toes of a mouse by using a chronic inflammatory pain model, and comparing the expression of the gene in different brain regions of a control group and a modeling group mouse; the result shows that the expression of the APR3 is obviously increased in the pain strongly associated brain region ACC after modeling. Immunofluorescence co-staining cells show that the expression of the gene is mainly increased in astrocytes. Then, a specific Aldh1l1-Cre mouse is established, Cre recombinase in the mouse is driven by a specific promoter, the Cre recombinase can be expressed in astrocytes, and target gene knockout is successfully achieved. A pain behavioral test is carried out on the knockout mouse, the pain threshold value of the knockout mouse after modeling is obviously increased, and an electrophysiological result also shows that the current frequency and amplitude of the knockout mouse after inhibitory synapse are higher than those of a non-knockout group. Afterwards, ACC brain tissue transcriptome sequencing genes are enriched in endocannabinoid signal transduction, then q-PCR is applied to verify key genes on the pathway, and the result shows that the pathway is obviously activated. The CB1 on the surface of the astronomical gum is activated, so that the astronomical gum releases a small amount of GABA, more expression on a GABRA2 receptor membrane is induced, inhibitory neuron signal transduction is aggravated, and the result that the pain threshold value is increased is achieved. The invention researches the influence of APR3 on mouse inflammatory pain, and the research finds that the pain can be obviously inhibited by knocking out APR3. In addition, APR3 is found to participate in pain inhibition through an endocannabinoid signaling pathway.

Description

Technical Field

[0001] The present invention relates to the application of APR3 in the preparation and development of target drugs for treating chronic inflammatory pain Background Art

[0002] Chronic pain refers to pain that persists or recurs for more than 3 months and has been defined as an independent disease. In developed countries in Europe and the United States, the incidence of chronic pain is as high as 30%. In my country, there are more than 300 million chronic pain patients, and the patient population is growing rapidly and showing a trend of getting younger. Chronic pain patients often show a variety of physical and psychological problems, such as depression, anxiety, trauma, stress response, cognitive dissonance, and impaired social function. A survey in my country in 2020 showed that the overall cost of treating chronic pain may exceed 500 billion yuan per year. The 2016 Global Burden of Disease Study on Pain and Pain-Related Diseases showed that chronic low back pain and neck pain were among the top 10 healthy life years lost due to disability, among which chronic low back pain was the leading disease causing functional impairment and missed work. Chronic pain is also the most common health problem related to long-term disability among the elderly. Therefore, chronic pain has become one of the third most serious chronic diseases that seriously affect human life and quality of life after cardiovascular and cerebrovascular diseases and tumors.

[0003] Although there have been many promising results in the study of pain mechanisms, there is still a lack of effective, non-invasive, and objective examination methods in clinical practice. The study of the mechanism of chronic neuropathic pain is still underway, and treatment and management are still full of challenges. The influencing factors and impacts of chronic pain are diverse. For example, chronic low back pain is the leading disease causing disability burden and missed work days in the global population. It manifests as recurrent pain and functional impairment, and is often accompanied by psychological problems such as sleep disorders, anxiety, and depression, and even has a negative impact on family relationships and the social environment. Multiple factors such as negative coping styles, adverse psychological stimulation or depression, fear of pain or exercise, and lack of confidence in rehabilitation are related to the prognosis of chronic low back pain.

[0004] However, there is currently a lack of effective prevention and treatment methods for chronic pain, which is so harmful. Therefore, new therapies are urgently needed, such as targeted therapy, immunotherapy, and traditional Chinese medicine treatment to alleviate patients' pain. Therefore, developing new safe and effective therapeutic targets and conducting in-depth research on the regulatory mechanism of chronic pain have important scientific and social significance.

[0005] In recent years, the association between apoptosis genes and pain has gradually become a research hotspot, and APR3, as an apoptosis-related protein, has also received a lot of attention. APR3 is a new gene cloned in 1999. Its sequence is conserved among various species. The human APR3 gene is located on chromosome 2, so it is also called C2ORF28. Their research found that the addition of all-trans retinoic acid to the HL60 cell line can induce an increase in the expression of the gene and apoptosis of the cells, so it was named apoptosis-related protein 3 (APR3). Studies have found that APR3 is a single transmembrane protein with 223 amino acids, but 193 amino acids are located outside the cell. At the same time, it has been reported that APR3 is a lysosomal membrane protein that is important in the expression of cell surface receptors. However, there are only a dozen articles on the function of APR3, and almost all of them are concentrated in the field of apoptosis. Its function in the central nervous system has not been reported; so APR3 is selected as the research focus, and its regulatory role in chronic inflammatory pain is elaborated in detail.

[0006] Chronic pain is the most common health problem associated with long-term disability in the elderly, and the current methods and strategies for alleviating it are very limited. APR3 is a new gene related to apoptosis, and its regulation of chronic inflammatory pain has been less studied. ① The present invention deeply explores the mechanism of APR3's action on chronic inflammatory pain at the cellular level and in vivo level, and clarifies the mechanism of action of APR3 in regulating chronic inflammatory pain at the molecular level, laying the foundation for clinical application. ② From the perspective of drug targets, the present invention clarifies the mechanism of action of APR3 as a target gene in regulating chronic inflammatory pain, providing a new target for pain relief. Summary of the invention

[0007] The first objective of the present invention is to address the deficiencies of the prior art and provide an application of the APR3 gene as a target for alleviating chronic inflammatory pain.

[0008] The structure of the APR3 is as follows: Preferably, APR3 is involved in inhibiting chronic inflammatory pain by activating the endocannabinoid signaling pathway.

[0009] The second objective of the present invention is to provide the use of APR3 in the preparation of medicines or health products for relieving chronic inflammatory pain.

[0010] The third object of the present invention is to provide a target for treating or preventing chronic inflammatory pain.

[0011] Preferably, the gene used as a therapeutic target is a gene recognized medically.

[0012] The present invention utilizes the Cre-lox system to achieve spatiotemporal specific knockout of genes in mice. First, a chronic inflammatory pain model was used, complete Freund's adjuvant (CFA) was injected into the toes of mice, and the expression of the gene in different brain regions of the control group and the modeling group was compared. The results showed that APR3 was significantly increased in the ACC, a brain region strongly associated with pain, after modeling. And immunofluorescence co-staining cells showed that the gene was mainly increased in expression in astrocytes. Subsequently, a specific Aldh1l1-Cre mouse was established, in which the Cre recombinase was driven by a specific promoter, and the Cre recombinase could be expressed in astrocytes, successfully achieving target gene knockout.

[0013] The knockout mice were also tested for pain behavior, which showed that the pain threshold of the knockout mice was significantly increased after modeling. The electrophysiological results also showed that the frequency and amplitude of inhibitory postsynaptic currents in the knockout mice were higher than those in the non-knockout group. Subsequently, the ACC brain tissue transcriptome sequencing genes were enriched in endocannabinoid signaling, and q-PCR was then used to verify the key genes in this pathway. The results showed that the pathway was indeed significantly activated. The CB1 on the surface of the astrocytes was activated, causing the astrocytes to release a small amount of GABA, inducing more expression on the GABRA2 receptor membrane, resulting in increased inhibitory neuron signaling, resulting in an increase in the pain threshold.

[0014] Therefore, the present invention provides a new idea for the treatment of chronic inflammatory pain and can also provide a new target for the development of drugs.

[0015] Preferably, the knockout mouse is Aldh1h1-cre; APR3 f / f tool mouse.

[0016] Therefore, the present invention has the following beneficial effects: (1) APR3 was knocked out by the Cre-loxP system, the target cells were clear, and the knockout was stable; (2) Easy to operate, with high success rate and good effect; (3) It can not only provide new ideas for the treatment of chronic inflammatory pain, but also provide new targets for drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The changes in the expression of APR3 in the pain-sensitive brain area ACC after the establishment of the chronic inflammatory pain model, as well as the co-labeling of APR3 with astrocytes, oligodendrocytes, and microglia. (n=3), Student's t-test; data are mean ± standard error, *p<0.05, **p<0.01.

[0018] Figure 2Figure 1 is the construction of conditional knockout mice, B and C are the successful knockout of APR3 verified by WB and IF. D and E are pain behavioral tests, representing the thresholds of mechanical pain and thermal pain, respectively (n=5), two-way ANOVA; FK is the behavioral test results of elevated and open fields, and the statistical graphs are the center time and total distance of the mine, the time and moving distance of elevated open arms, respectively (n=5), two-way ANOVA. Data are mean ± standard error, *p<0.05.

[0019] Figure 3 Typical electrophysiological graphs and statistical graphs of knockout mice. A and B are the number of current signal spikes, CF is the frequency and amplitude of excitatory postsynaptic current, and GJ is the frequency and amplitude of inhibitory postsynaptic current. One-way ANOVA; data are mean ± standard error, and p values ​​are shown in the figure.

[0020] Figure 4 The enrichment map of gene pathways in transcriptome sequencing of ACC tissue and q-PCR validation. A is the KEGG pathway enrichment, B is the regulation of genes in the pathway, and flow cytometry is used to detect cell apoptosis; C is a heat map; D is a statistical chart of q-PCR validation results. E is the expression level of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) in ACC. Student's t-test. Data are mean ± standard error, *p<0.05, **p<0.01. Implementation

[0021] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. The following description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those familiar with the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without having to go through creative work. Therefore, the present invention is not limited to the following embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

[0022] Example 1: Changes in the expression of APR3 in chronic inflammatory pain In order to detect whether APR3 changes after CFA complete Freund's reagent modeling, Western blot (WB) and immunofluorescence IF were used to detect the changes in the expression of APR3 in the ACC brain region after CFA modeling. The specific operations are as follows: 1) Use a microsyringe to pierce the mouse toes with 10 μl CFA working solution (complete Freund's reagent: saline / 1:1), and divide the mice into two groups three days later. One group is used for Western blot, and the sample is immersed in RIPA lysis buffer, and the tissue is ultrasonically broken, with 20% power, ultrasonic for 2s and 3s, and lasts for 1 minute. Place it on ice for lysis for 15 minutes, centrifuge it at 12,000 rpm for 15 minutes, and take the supernatant. Use BCA quantitative method for quantification, add 5 x protein loading buffer and denature it at 100℃ for 15 minutes to obtain protein samples for western blot. 10% SDS-PAGE was run, and the PVDF membrane was transferred under 20% methanol conditions at 100V for 90 minutes. The membrane was blocked with 5% skim milk at room temperature for 1 hour, and the membrane was cut according to the molecular weight. The related protein APR3 was incubated at 4°C overnight, and then washed with 1 x TBST. The HRP secondary antibody conjugated to the corresponding species was incubated, and then washed with 1 x TBST. The membrane was incubated with ECL luminescent solution, and the signal was collected by chemiluminescence. The protein content was reflected according to the signal strength. Image J was used to calculate the grayscale of the bands and perform statistical analysis on the differences. The other group of mice were perfused with 1xPBS, fixed with 4% PFA for 24 h, and dehydrated by precipitation with 30% sucrose. After complete precipitation, the mice were embedded with embedding agent, sliced ​​with a freezing microtome, washed with 1xPBS three times after mounting, fixed with 4% PFA for 15 minutes, permeabilized with 1.5% Triton X100, blocked with 5% BSA, and incubated with primary antibodies APR3, S100β, Iba1, and Olig2 diluted in 5% BSA at 4°C overnight. After washing with 1xPBS, the mice were incubated with fluorescent secondary antibodies, and the nuclei were stained with DAPI. The images were taken with a confocal microscope, and the ratio of positive signal cells to nuclear positive cells was counted. The experiment was repeated three times, and the co-labeling of cells in the control group and CFA experimental group was statistically analyzed. Example 2: Construction of Aldh1l1-cre;APR3f / f conditional knockout mice and related behavioral tests In order to detect the effect of knocking out APR3 in astrocytes on the pain threshold of mice, the specific operation is as follows: 1) The G0 generation was mated with wild mice to obtain the G1 generation heterozygotes, the G1 generation heterozygotes (flox / +) were mated with each other to obtain the G2 generation homozygotes (flox / flox), the G2 generation homozygotes (flox / flox) were mated with astrocyte-specific tool mice to obtain the G3 generation (flox / +, Cre), and the G3 generation (flox / +, Cre) was mated with each other to obtain the G4 generation (flox / flox, Cre), i.e., the cKO homozygotes. The mated mice were killed and the samples were collected. The sections were immunofluorescently stained and the protein gel was verified by western blot.

[0023] Pain behavior test: The mechanical withdrawal threshold of mice was tested using the classic up-down test method. Before the first formal test of the animals, the animals were allowed to adapt to the test environment 2-3 times, each time for 30 minutes, in order to prevent the animals from affecting the test results due to behaviors such as exploring the new environment. Before each formal test in the future, the animals also need to have an adaptation time of about 30 minutes. The formal test can only be carried out after the animals are quiet. After ensuring that the experimental environment is quiet and the lighting is appropriate, the mice to be tested are placed on a metal grid with a pore size of 2 mm×2 mm. Each mouse is separated by a plexiglass box of a fixed size (6 mice can be placed at a time). After the mice have adapted for 0.5 hours, the mechanical withdrawal reflex threshold (PWT) of the mice is measured. Von Frey Filaments (vFFs) of different stimulation intensities are used to stimulate the interdigital skin of the third and fourth toes of the mice to observe the possible withdrawal reaction of the mice. The stimulation intensity generally starts from 0.4 g. If the vFFs fiber is bent more than 90°, if the mouse still does not lift its foot, it is considered to be unresponsive. At this time, the adjacent fiber with a higher stimulus intensity should be given; if there is a reaction, the adjacent fiber with a lower stimulus intensity should be replaced. The time interval between each stimulation must be greater than 10 s until the fiber that can cause a 50% paw lift reaction is found. The maximum stimulus intensity in this experiment is 2 g. The fiber stimulus intensity that can cause a 50% paw lift reaction of the tested mouse is recorded, which is the mechanical paw withdrawal reflex threshold (g). The results are calculated according to the formula to obtain the data of the mechanical paw withdrawal threshold (PawWithdraw Threshhold). Each mouse was measured 3 times, and the experiment was conducted on three days, starting at 9:00 am every day. The infrared thermal conductivity meter was used for heat pain test. The mouse was adapted to the room for 30 min; the heat pain meter was adjusted to 30%, and a transparent perforated glass was placed around the heat source; the mouse was placed on the hot plate and the timing was started at the same time; the mouse licked its hind foot, jumped on the spot, or obviously retracted its legs as a positive reaction, and the timing was stopped immediately, and the reaction latency (latency) was recorded. Each mouse repeated the test 3 times, and the experiment was conducted on three days, starting at 9:00 am every day.

[0024] The elevated plus maze test (EPM) is an internationally recognized classic method for measuring anxiety reactions. Before the experiment, ensure that the entire elevated plus maze device is clean, remove urine, feces, etc. left in the body by the previous animal, and create a clean, odorless ideal experimental environment as much as possible. Adapt to the environment for at least 3 hours to reduce animal tension. Take the experimental animal (rat or mouse) out of the cage, with the experimental animal facing away from the experimenter as much as possible, and gently place the animal in the central area of ​​the instrument body, with the animal facing the open arms, and then the experimenter quickly and quietly leaves; Open the SuperMaze animal behavior analysis software, track the animal's trajectory in the elevated plus maze instrument, and automatically calculate the indicators. The experiment lasts for 5 minutes. After the experiment, take out the experimental animal, put it in the breeding cage, and mark the animal after the experiment. At the same time, clean the maze with alcohol and paper towels. The software is used to analyze the time when the mouse enters the open arm and the distance it moves in the open arm.

[0025] The open field test detects the spontaneous activity and exploratory behavior of rats or mice, and evaluates the autonomous behavior, exploratory behavior and tension of experimental animals in a new environment. Before starting, make sure that the experimental box is clean and odorless, and pay special attention to cleaning the feces and urine left by the animals in the last experiment at the bottom of the experimental box. Set the corresponding parameters in the software and record the animal number, date, and status light information; gently take the experimental animal out of the cage and turn its back to the experimenter; quickly place the experimental animal in the central area of ​​the experimental box and leave immediately; open the animal behavior analysis software to automatically record the animal's activities in the box. The experimental time is usually 15 minutes. After the experiment, put the experimental animal in other cages prepared in advance. Spray the instrument with alcohol to remove odors and wipe it dry with a paper towel. Software analysis, central zone retention time - reflects the anxiety of the rat; total horizontal movement distance - reflects the movement of the rat.

[0026] Example 3: Electrophysiological status of knockout mice In order to detect whether the APR3 gene knockout mice in astrocytes have an effect on the excitatory and inhibitory neuronal current signals, electrophysiological techniques were used for detection. The specific operations are as follows: Techniques that use electrophysiological instruments, electrodes, voltage clamps, and patch clamp techniques to record or measure changes in electrical potential, conduction velocity, and ion channel activity in whole animals or isolated organ tissues, nerves, and cell ion channels. For brain slice preparation, cool 1 L aCSF on dry ice until the temperature is less than 4°C, and add 250 ml aCSF to the brain slice storage room.

[0027] Set up the vibratome by filling the buffer chamber with chilled aCSF solution, setting the desired thickness (500 µm) and adjusting the cutting speed to the desired setting. Place the brain in the pre-chilled aCSF solution and mount the brain on the vibratome specimen tray using super glue, adjusting the specimen angle so that the cortex faces the cutting blade. Use a transfer pipette to transfer individual brain slices containing the region of interest from the buffer tray to a clean Petri dish pre-filled with chilled aCSF. Cut the brain in half along the midline and transfer the individual brain slices to the holding chamber pre-filled with aCSF. Keep the brain slices at room temperature for 1 hour to allow the brain tissue to recover from the mechanical shock of slicing. Fill the bottles with aCSF solution and the test compound solution. Fill both bottles with mixed oxygen and adjust the flow rate of the aCSF solution to approximately 5 ml / min using a metal spiral clamp. Place the brain slice into the recording chamber using a small brush and secure the slice with a reference electrode.

[0028] Fill the glass recording microelectrode with intracellular solution using the microelectrode filler, making sure the solution is all the way down at the end of the microelectrode. Connect the microelectrode to the electrode clamp on the top stage of the patch clamp amplifier and adjust the position. Using the fine-tuned micromanipulator, lower the recording microelectrode to the ACC region of the brain slice. Once the microelectrode contacts the neurons in the brain slice, apply negative pressure to the microelectrode using a 1 ml syringe. Monitor the resistance of the seal on an oscilloscope or computer.

[0029] After the seal resistance exceeds 1 GΩ, an amplifier and computer software are used to compensate for transients and apply further negative pressure to rupture the cell membrane and obtain whole-cell neurons. Computer-controlled software performs current-voltage relationship tests to obtain neuronal health and assess active membrane conductance. Computer-controlled software is used to monitor the response of test compounds and perform electrophysiological tests such as current-voltage relationship and evoked excitatory or inhibitory postsynaptic potentials. Software analysis obtains corresponding data.

[0030] Example 4: Transcriptome sequencing revealed the specific pathway by which APR3 regulates pain.

[0031] In order to understand the specific pathway of pain regulation by this gene, transcriptome sequencing was performed on the ACC brain region after CFA modeling. Analysis showed that APR3 regulates the progression of pain through the endocannabinoid signaling pathway.

[0032] RT-PCR was used to verify the genes in this pathway. RT-PCR was used to detect the effects of knockout mouse CB1R and GABRA2 transcription levels. The tissue was immersed in Trizol, ultrasonically lysed, placed at room temperature for 5 minutes, centrifuged at 12,000 rpm, 4°C for 10 minutes, the supernatant was aspirated, and 200 μL of chloroform was added to the supernatant, gently inverted for 2 minutes to mix, placed at room temperature for 5 minutes, centrifuged at 12,000 rpm, 4°C for 15 minutes, 400 - 500 μL of the upper aqueous phase was carefully aspirated, 500 μL of pre-cooled isopropanol was added, placed at -20°C for 20 minutes, and then centrifuged at 12,000 rpm, 4°C for 10 minutes. At this time, white precipitate can be seen at the bottom of the tube, the supernatant is discarded, 1 mL of 75% ethanol is added, centrifuged at 12,000 rpm, 4°C for 5 minutes, the supernatant is discarded, the EP tube mouth is opened and dried for 10 minutes, and after drying, 30 μL of double distilled water / DEPC water is added to the precipitate and dissolved at 55°C for 6 minutes. After removing genomic DNA using a kit, reverse transcription was performed, and RT-PCR analysis was performed using the SYBR two-step method. Three plates of cells were prepared for each control group and experimental group, and one plate of cells corresponded to one sample. Three replicate wells were prepared for each sample corresponding to the target gene primer and the internal reference β-actin primer. RT-PCR was performed on the machine, and the 2- ΔΔCt The values ​​were statistically analyzed to determine the changes in transcription levels of related genes; The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0033] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention. In summary, the present invention provides a target for regulating chronic inflammatory pain and discovers its possible molecular mechanism for treating and relieving chronic inflammatory pain, which can provide new ideas for the treatment of chronic pain.

Claims

1. Application of APR3 gene in the treatment of sexual inflammatory pain.

2. The use according to claim 1, Features APR3 is involved in suppressing pain through the endocannabinoid signaling pathway.

3. Application of APR3 gene in the preparation of drugs or health products for inhibiting chronic inflammatory pain.

4. A medicine or health product for treating or preventing chronic inflammatory pain, Features Including inhibiting the expression of APR3 gene in the brain.

5. A medicine or health product for treating or preventing chronic inflammatory pain according to claim 1, Features The gene is a clear target.

6. A medicine or health product for treating or preventing chronic inflammatory pain according to claim 1, Features It also includes pharmaceutically acceptable carriers that directly reach the target site.

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