CN103784974B - Application of interferon regulatory factor 8 (IRF8) in cerebral apoplexy disease - Google Patents

Abstract

The invention discloses the function and application of an IRF8 gene in cerebral apoplexy, and belongs to the field of gene function and application. The present invention takes IRF8 gene knockout mice and nerve-specific IRF8 transgenic mice as experimental objects, and through the middle cerebral artery ischemia-reperfusion model, the results show that compared with wild-type C57 mice, the brain infarct volume of IRF8 gene knockout mice The infarct volume of neurospecific IRF8 transgenic mice was significantly reduced, and the neurological function was significantly improved. This suggests that a function of the IRF8 gene in stroke is mainly reflected in the function of the IRF8 gene in protecting the nervous system, especially the protection of the IRF8 gene in the stroke disease. Aiming at the above-mentioned functions of IRF8, the application of IRF 8 in the preparation of drugs for the treatment of stroke is provided.

Description

interferon regulatory factor 8 ( IRF8 ) Application in stroke disease

technical field

The invention belongs to the field of gene function and application, and particularly relates to the application of an interferon regulatory factor 8 (interferon regulatory factor 8, IRF8) in stroke diseases.

Background technique

Ischemic stroke is currently the leading cause of death and disability in the world. At present, tissue-type plasminogen activator (tPA) fibrinolysis is still the main treatment for ischemic cerebrovascular disease. Blood reperfusion will further aggravate ischemic nerve cell injury. Studies have shown that neuronal protection strategies can improve brain function and reduce neuronal cell loss long after cerebral ischemic injury. Apoptosis is one of the basic mechanisms of cell death during cerebral ischemia/reperfusion, but its regulatory mechanism is still not fully elucidated. Therefore, studying the molecular mechanism of neuronal apoptosis and survival during cerebral ischemia/reperfusion will help provide new therapeutic strategies and methods for neuron protection.

Cerebral ischemia-reperfusion injury can cause a variety of different but overlapping signaling pathways to regulate cell survival or death. In neuronal focal cerebral ischemia, most of the cells in the core area of the infarction undergo necrosis, which is characterized by sharply reduced energy supply leading to cell edema, organelle rupture, and irreversible cell death. The hypoperfused brain tissue around the infarct nucleus is called the "ischemic penumbra". Because it still has metabolic activity, if the cerebral blood perfusion is improved, the cell activity in this area can still be restored. Therefore, saving the ischemic penumbra is of great value for post-stroke treatment. Although tissue plasminogen activator (tPA) has been approved for the treatment of ischemic stroke since 1996, it is still the only thrombolytic drug approved by the US Food and Drug Administration (FDA). Because the risk of bleeding increases over time, the treatment time window of tPA is only 4.5 hours; considering that ischemic and hemorrhagic stroke are not easy to distinguish in early imaging, it further delays the opportunity for patients to receive tPA treatment. Currently, less than 5% of patients with ischemic stroke are treated with tPA thrombolysis. In addition, studies have found that if the brain tissue is severely hypoxic for a long time, even if the cerebral blood flow is restored later, it will still cause irreversible damage to the brain tissue. Treatment strategies for the resulting pathophysiological events. Since the 1990s, the study of therapeutic strategies to protect nerves and brain tissue has been a hot spot in stroke treatment. These strategies can not only prolong the treatment time window of tPA, but also reduce the brain tissue damage induced by ischemia-reperfusion. A variety of neuroprotective drugs have achieved exciting results in animal experiments, but after entering the third phase of clinical trials for stroke, most of them failed to achieve the expected effect. One of the primary reasons for their failure is that most of the known The neuroprotective mechanism works within 4-6 hours after stroke, and it is difficult to implement treatment in such a short time window in clinical practice, so further elucidate the molecular mechanism that promotes or protects brain tissue damage for a long period of time after stroke occurs It is of great significance for the study of effective stroke treatment targets or strategies.

The interferon regulatory factor (interferon regulatory factor, IRF) family has been found to have 10 members, which are composed of IRF1-IRF10. Existing studies suggest that members of the IRF family are involved in a wide range of biological processes, mainly involving innate and acquired immune responses, regulating cell growth and survival, apoptosis and proliferation, participating in hematopoiesis, and anti-tumor formation. IRF8 is an interferon consensus sequence-binding protein (ICSBP) and a member of the IRF family. As a transcription factor, it can regulate DNA transcription and play a role. IRF8 was first discovered in myeloid cells and lymphocytes, and studies have shown that IRF8 plays a central role in immune regulation and myeloid cell differentiation. Many studies have shown that IRF8 may be involved in multiple sclerosis, central nervous system inflammatory demyelinating diseases, peripheral nerve injury and other diseases.

Contents of the invention

In order to solve the defects and deficiencies of the above-mentioned prior art, the primary purpose of the present invention is to provide an application of IRF8 in the preparation of drugs for treating nervous system diseases.

Another object of the present invention is to provide an application of IRF8 in the preparation of medicaments for the treatment of cerebral apoplexy.

The object of the present invention is achieved through the following technical solutions:

The present invention takes IRF8 gene knockout mice and neuron-specific IRF8 transgenic mice as experimental objects, and through the middle cerebral artery ischemia-reperfusion model, the results show that compared with wild-type C57 mice, IRF8 knockout mice have cerebral infarction The neurological function was also significantly worsened, while the infarct volume of the neuron-specific IRF8 transgenic mice was suppressed, and the neurological function also improved. This suggests that the IRF8 gene has the function of protecting the function of the nervous system and can protect the nerve damage caused by cerebral ischemia, which provides a theoretical and clinical basis for the study of new drugs and new strategies for the prevention and treatment of cerebral ischemia.

A function of the IRF8 gene in cerebral apoplexy is mainly reflected in the function of the IRF8 gene in protecting the function of the nervous system, especially the function of the IRF8 gene in protecting the cerebral ischemic disease.

Aiming at the above-mentioned functions of the IRF8 gene, the application of the IRF8 in the preparation of medicines for treating nervous system diseases is provided.

A drug for the treatment of neurological diseases, comprising IRF8.

Aiming at the above-mentioned functions of the IRF8 gene, the application of the IRF8 in the preparation of medicines for the treatment of cerebral apoplexy is provided.

A drug for treating cerebral apoplexy, comprising IRF8.

The research results of the present invention show that in IRF8-KO mice, in the injury caused by middle cerebral artery ischemia-reperfusion, the infarction volume of the mice is obviously increased, the neurological function is obviously deteriorated, and the apoptosis of nerve cells is also obviously increased. It is proved that IRF8 gene plays an important protective role in stroke disease model.

Compared with the prior art, the present invention has the following advantages and effects:

1. The present invention discovers a new function of the IRF8 gene, that is, the IRF8 gene can protect stroke diseases.

2. The role of IRF8 in the protection of stroke diseases, for the preparation of drugs for the treatment of stroke diseases.

Description of drawings

Figure 1 is the construction and identification results of nerve-specific IRF8 transgenic mice

A is the construction diagram of nerve-specific IRF8 transgenic mice;

B is the identification result of nerve-specific IRF8 transgenic mice;

Figure 2 is a diagram of TTC staining results of WT and IRF8-KO mice.

A is the result of TTC staining;

B is a statistical histogram of cerebral infarction volume;

C is the statistical histogram of neurological function score;

Figure 3 is a diagram of the TTC staining results of IRF8-TG and NTG mice.

A is the result of TTC staining;

B is a statistical histogram of cerebral infarction volume;

C is the statistical histogram of neurological function score;

Fig. 4 is a graph showing the results of measuring the apoptosis of neurons in the peri-infarction area of the brain tissue of WT and IRF8-KO mice.

Figure A is Fluoro Jade B detection display graph and statistical result graph;

Figure B is TUNEL Apoptosis diagram and statistical result diagram;

Fig. 5 is a graph showing the results of measuring the apoptosis of neurons in the peri-infarction area of the brain tissue of IRF8-TG and NTG mice.

Figure A is the Fluoro Jade B detection display and statistical results;

Figure B is the TUNEL cell apoptosis diagram and statistical results diagram;

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Experimental Animals and Breeding

Experimental animals: Wild-type mice (WT, purchased from Beijing Huafukang Biotechnology Co., Ltd.), IRF8 knockout mice ( IRF8-KO, C57BL/6J background, purchased from EMMA, catalog number EM: 02414), non-transgenic mice (NTG, Neuron-specific Cre transgenic mice (CaMKIIα-Cre; purchased from Jackson Laboratory, Stock No. 005359)) and nerve-specific IRF8 transgenic mice (IRF8-TG, obtained by crossing IRF8-flox transgenic mice and CaMKIIα-Cre mice, IRF8-flox transgenic mice were constructed by our laboratory, IRF8-flox transgenic mice The build process is described below).

Breeding environment: All experimental mice were raised in the SPF-level experimental animal center of the Institute of Cardiovascular Diseases, Wuhan University. The special feed for mice was provided by the Animal Center of Chinese Academy of Military Medical Sciences. Breeding conditions: room temperature between 22-24°C, humidity between 40-70%, alternating light and dark lighting time for 12 hours, free access to water and food.

[Example 1] Construction of nerve-specific IRF8 transgenic mice

IRF8-flox transgenic mouse construction information:

Transgenic vector construction information: Use the upstream primer, namely 5'-CCAGATTACGCTGATTGTGACCGGAACGGCGGGCG-3' (SEQ ID NO. 1); the downstream primer, 5'-AGGGAAGATCTTGATTTAGACGGTGATCTGTTGAT-3' (SEQ ID NO. 2), to amplify the full length of mouse IRF8 Gene ( NCBI, Gene ID: 15900, NM_008320.3), insert the cDNA into the pCAG-CAT-LacZ vector, which contains a CMV enhancer and a chicken β-actin gene (CAG, chicken β-actin gene) promoter, and is connected to chloramphenicol acetyl transferase gene (CAT, chloramphenicol acetyltransferase), loxP sites are located on both sides of CAT. Expression of IRF8 in neuronal cells is driven by the CAG promoter (Fig. 1A). IRF8-floxed mice: The constructed pCAG-IRF8-CAT-LacZ vector was constructed into fertilized embryos (C57BL/6J background) by microinjection to obtain IRF8-floxed transgenic mice. Neuron-specific IRF8 transgenic mice were obtained by crossbreeding IRF8-flox mice and CaMKIIα-Cre mice.

Genomic DNA was obtained from transgenic mice by cutting their tails and identified by PCR. The primer information for PCR identification was: detection of forward primer 5'- CCAGATTACGCTGATTGTGACCGGAACGGCGGGCG-3' (SEQ ID NO. 3), detection reverse primer 5'- AGGGAAGATCTTGATTTAGACGGTGATCTGTTGAT-3' (SEQ ID NO. 4). Identify the expression level of IRF8 protein in the brains of different transgenic mice by Western Blot: extract the brain tissue proteins of different transgenic mice, and verify the overexpression of IRF8 by polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 1B)

We constructed several strains of neural-specific IRF8 transgenic mice (IRF8-TG). In order to reflect the changes of IRF8 in pathophysiological conditions, we selected IRF8-TG5 mice. Western Blot and quantitative analysis showed that the expression of IRF8 in the brain tissue was about 10.5 times that of normal tissue.

[Example 2] Acquisition of mouse cerebral infarction model (I/R)

1. Grouping of experimental animals: male C57BL/6 strain wild-type mice, IRF8 knockout mice, brain-specific IRF8 transgenic mice and non-transgenic mice, cerebral infarction model was established by middle cerebral artery ischemia-reperfusion (I /R). Randomly divided into 8 groups, 10 mice in each group: C57BL/6 strain wild type mouse sham operation group (WT SHAM) and I/R operation group (WT I/R), IRF8 gene knockout mouse sham operation group (KO SHAM) and I/R operation group (KO I/R), non-transgenic mouse sham operation group (NTG SHAM) and I/R operation group (NTG I/R), neuron-specific IRF8 transgenic mouse sham Surgery group (TG SHAM) and I/R surgery group (TG I/R).

2. I/R operation of cerebral infarction by suture method adopts mouse middle cerebral artery ischemia-reperfusion (middle Cerebral artery Ischemia Reperfusion) model operation process:

(1) Grab the mouse, use 3% isoflurane to anesthetize the mouse, remove the mouse hair on the neck with 8% sodium sulfide, and quickly cut off the mouse hair on the top of the skull with surgical scissors, and disinfect the neck and skull with 3% iodine. Top skin twice, 75% alcohol deiodination once;

(2) Make a transverse incision on the skull top of the mouse to expose the skull, and gently peel off the connective tissue on the surface of the skull with forceps. Fix the fiber optic probe of the laser Doppler blood flowmeter to the position 2mm behind the anterior bregma and 5mm to the left with biological glue;

(3) Fix the mouse supine, make a midline incision in the neck, separate the muscle and fascia along the inner edge of the sternocleidomastoid muscle, and separate the left common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) ). The distal end of the ECA was ligated with 8-0 suture, and the proximal end of the ECA was hung for backup. Temporarily clamp the ICA and CCA with an arteriole clip; then cut a small opening between the ligation at the distal end of the ECA and the hanging thread at the proximal end, send the thread plug into the CCA through the cut, and place the hanging thread at the proximal end of the ECA in the cut. Tie a slipknot at the mouth, the tightness should be that the thread bolt can enter and exit freely but with a slight friction feeling, then loosen the ICA arterial clamp, and send the thread bolt into the ICA, and calculate the distance from the bifurcation of the blood vessel, when the insertion depth is 9- About 11mm until the blood flow drops and stops when it encounters resistance. At this time, lightly fasten the tether with a slipknot around the proximal end of the ECA. During the whole process, the anal temperature of the mouse must be maintained at 37±0.5°C;

(4) Start timing from the time when the thread plug enters the cerebral blood vessel until the blood flow drops and meets resistance. After 45 minutes, loosen the slipknot at the proximal end of the ECA, pull out the thread plug, and fasten the slipknot at the proximal end of the ECA. Loosen the arterial clip at the CCA, and ligate the proximal end of the ECA (in the Sham group, the thread plug was pulled out from when the thread plug entered the cerebral vessel until the blood flow declined and met resistance). Pay attention to the recovery of blood flow, and select mice whose blood flow has decreased by more than 75% and whose blood flow has recovered by more than 70% to be included in the experiment;

(5) Suture the neck and head skin of the mouse, and disinfect the wound with active iodine. After the operation, the mice were placed in an incubator, the temperature of which was maintained at 28°C, and water and feed were given until the materials were collected.

[Example 3] Determination of Cerebral Infarction Volume in Cerebral Infarction Model (I/R) Mice

The evaluation indicators of the severity of cerebral ischemia/reperfusion injury mainly include cerebral infarct volume and neurological function score, and these indicators are positively correlated with the severity of ischemia/reperfusion injury.

(1) Neurological function and morphology scores were performed 24 hours after operation and 72 hours before sampling;

Improved method based on Berderson neurological function score (9-point scale):

0 points: no symptoms of nerve damage;

1 point: The contralateral forelimb is curled up when the tail is raised, or the forelimb on the affected side cannot be fully reached;

2 points: The opposite shoulder is adducted when the tail is raised;

3 points: flat push: the resistance decreases when pushing to the opposite side;

4 points: can move spontaneously in all directions, but only turn to the opposite side when the tail is off;

5 points: turning in circles or only in opposite directions during spontaneous movement;

6 points: No voluntary movement, only movement when stimulated;

7 points: no voluntary movement, no movement when stimulated;

8 points: death related to cerebral ischemia.

(2) Grab the mouse, anesthetize the mouse with 3% pentobarbital sodium intraperitoneally, cut open the mouse chest cavity, and cut the heart to let blood;

(3) Disinfect the skin of the back of the neck with 75% alcohol by volume, cut the skin of the back of the neck, expose the head and neck, cut off the cervical cord from the cervical spine, separate and remove the muscles of the back of the neck, and cut the brain stem and cerebellum longitudinally with ophthalmic scissors For the skull, use the toothed forceps to peel off the skull, separate the dura mater on the surface of the brain, and avoid scratching the brain tissue by the dura mater. When taking the brain, start from the medulla oblongata, and carefully separate the skull base tissue to avoid damage to the brain;

(4) Rinse the removed brain tissue in a Petri dish containing PBS, blot the PBS dry with gauze, put the brain tissue into a 1mm mouse brain model, and store it in a -20°C refrigerator (no more than 4h );

(5) Brain tissue 2,3,5-Triphenyltetrazolium chloride (2,3,5-Triphenyltetrazolium Chloricej, TTC) staining: the brain tissue was taken out from the -20°C refrigerator, and immediately cut into 1mm thick slices, including 4 slices in front of bregma and 3 slices in back, totaling 7 slices. Immediately place the slices in a serum bottle of 10mL 2% TTC solution, and incubate at 37°C for 10min. Flip the sections from time to time to allow even staining of the tissue. The normal brain tissue was stained bright red, while the infarct area was pale;

(6) Brain tissue fixation: transfer the brain tissue and the solution in the beaker to a marked cup, discard the TTC solution, fix the brain tissue slices with 10% neutral formalin solution, take pictures after 24 hours and use IPP software analysis;

(7) Cerebral infarction volume calculation: infarct volume% = (contralateral cerebral hemisphere volume - infarcted side non-infarcted volume) / (contralateral cerebral hemisphere volume × 2) × 100%;

The total infarct volume was the sum of the results of 7 brain slices.

The results of TTC staining are shown in Figure 2. After 45 minutes of I/R ischemia and 24 hours of reperfusion, the infarct volume of IRF8-KO mice increased compared with that of wild-type mice; and this worsening effect continued 72 hours after I/R , while the neurological function score increased at 24 hours and 72 hours after I/R.

As shown in Figure 3, after I/R ischemia for 45 min and reperfusion for 24 hours, the infarct volume of IRF8-TG mice was significantly reduced compared with that of wild-type mice; and this protective effect still persisted 72 hours after I/R, while Neurological scores were reduced at 24 and 72 hours after I/R.

Example 4. Determination of apoptosis of neurons in the peripheral area of brain tissue infarction

1. Brain Tissue Cryosection Preparation

1) Experimental mice were anesthetized by intraperitoneal injection of pentobarbital sodium at a dose of 50 mg/kg;

2) Open the chest to expose the heart, puncture the left ventricle with an injection needle, and cut the right atrium at the same time;

3) Perfuse with 0.1mol/l PBS (pH7.4) at 100mmHg pressure until the liver becomes pale, then perfuse with 4% paraformaldehyde for 15 minutes;

4) Remove the mouse brain quickly after craniotomy, and fix it with 4% paraformaldehyde at room temperature for 6-8 hours;

5) Resect the olfactory bulb and cerebellum of the brain tissue, then divide the brain into two parts successively along the midline, and then fix with the previous fixative for 15 minutes;

6) Then immerse in phosphate buffer solution containing 30% sucrose, and sink to the bottom overnight in a refrigerator at 4°C;

7) After mixing 30% sucrose and OCT at a ratio of 1:1, pour an appropriate amount into the embedding frame, take out the tissue from the previous step, absorb the liquid on the gauze, soak it in the embedding frame for a while, and then transfer it into the embedding frame. Go to another embedding frame that has been added with 2 drops of OCT, and adjust the position of the tissue so that it is just in the middle of the embedding frame;

8) Move the embedding frame containing the tissue into the dry ice, and make it as horizontal as possible. After a while, continue to add OCT to immerse the tissue to a certain height. After the OCT solidifies, store it in a refrigerator at -80°C middle;

9) Use the standard program of the cryostat to cut 5 μm frozen sections for later use.

2. Apoptosis was detected by TUNEL kit staining.

Apoptosis was detected by TUNEL kit staining. (TUNEL Kit: ApopTag® Plus In Situ Apoptosis Fluorescein Detection Kit (S7111, Chemicon)):

1) Place ice-cut tissue sections in (pH 7.4) 1% paraformaldehyde, fix and hydrolyze at room temperature for 10 minutes;

2) Wash twice with PBS, 5 min each time;

3) Place in pre-cooled ethanol: acetic acid (2:1) solution, soak at -20°C for 5 minutes, remove excess liquid, but be careful not to dry;

4) Wash twice with PBS, 5 min each time;

5) Carefully absorb the excess liquid on the filter paper, immediately add equilibration buffer directly to the slice at 75 μL/5 cm ² , and incubate at room temperature for 1-5 min;

6) Carefully absorb the excess liquid on the filter paper, immediately add TdT enzyme reaction solution directly on the slice at a rate of 55 μl/5 cm ² , and place it in a dark humid box for 1 hour (add the reaction solution without TdT enzyme to the negative control);

7) Place the slices in the stop/wash buffer, shake gently for 15 sec, and incubate at room temperature for 10 min; at this time, prepare an appropriate amount of anti-digoxigenin antibody, preheat to room temperature, and avoid light;

8) Wash with PBS three times, each time for 1 min;

9) The excess liquid was carefully absorbed by the filter paper, and the anti-digoxigenin antibody was directly added to the slice at a rate of 65 μL/5 cm ² , and reacted for 1 hour at room temperature in a light-proof and insulated humid box;

10) Wash four times with PBS, 2 min each time;

11) SlowFade Gold antifade reagent with DAPI (Invitrogen, S36939) for mounting;

12) Observe under a fluorescent microscope and take pictures. If preservation is required, store in a dark and humid box at 4°C. Observed under a fluorescent microscope, took pictures, and counted apoptotic neuron cells. (If preservation is required, store in a dark and humid box at 4°C)

3. FJB (Fluoro Jade B) Dyeing

1) Dry ice-cut tissue slices in an oven for 1 hour;

2) 1% NaOH+80% absolute ethanol for 5min;

3) 70% absolute ethanol for 2 minutes;

4)dd H ₂ O 2min;

5) Flouro jade B diluent (AG310, Millipore, Billerica, MA), at room temperature in the dark for 20 minutes;

6) Wash 3 times with dd H ₂ O for 1min;

7) Dry the slices in an oven for 5-10 minutes;

8) Xylene treatment for 2-3 minutes;

9) Seal the film and take pictures.

See Figure 4 and Figure 5 for the measurement results of neuronal cell apoptosis in the peripheral area of brain tissue infarction. Figure 4 shows the apoptosis of neurons in the peri-infarction area of the brain tissue in IRF8-KO mice and wild-type mice 24 hours after I/R. Fluoro Jade B staining (A) and TUNEL staining (B) were used to detect cell apoptosis, and the results showed that the apoptosis rate of neurons in IRF8-KO mice was higher than that in wild-type mice, further suggesting that IRF8 has a relationship with neuronal cell ischemia/regeneration Death was associated with perfusion. The same figure 5 shows the apoptosis of neurons in the peri-infarction area of the brain tissue of IRF8-TG mice and NTG mice 24 hours after I/R. The results of Fluoro Jade B staining (A) and TUNEL staining (B) show that IRF8-TG The apoptosis rate of neurons in mice was lower than that in NTG mice. These results indicate that promoting the expression of IRF8 can improve brain tissue ischemia/reperfusion injury, and may be closely related to neuronal apoptosis.

Our research results show that in the injury caused by middle cerebral artery ischemia-reperfusion in IRF8 KO mice, we found that after IRF8 knockout, the infarct volume of the mice was significantly increased, the neurological function was significantly deteriorated, and the apoptosis of nerve cells was also significantly increased. It is proved that IRF8 gene plays an important protective role in stroke disease model.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Wuhan University

<120> Application of Interferon Regulatory Factor 8 (IRF8) in Stroke

<130> 1

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 35

<212>DNA

<213> Artificial

<223> IRF8 Upstream Primer

<400> 1

ccagattacg ctgattgtga ccggaacggc gggcg 35

<210> 2

<211> 35

<212>DNA

<213> Artificial

<223> IRF8 downstream primer

<400> 2

agggaagatc ttgattaga cggtgatctg ttgat 35

<210> 3

<211> 35

<212>DNA

<213> Artificial

<223> Detect Forward Primer

<400> 3

ccagattacg ctgattgtga ccggaacggc gggcg 35

<210> 4

<211> 35

<212>DNA

<213> Artificial

<223> Detect reverse primer

<400> 4

agggaagatc ttgattaga cggtgatctg ttgat 35

Claims (1)

1. interferon regulatory factor 8 or the application of its gene in preparation treatment apoplexy disease medicament.