WO2012108595A1 - Proteinase-3 as a target for stroke treatment - Google Patents

Abstract

The present invention relates to proteinase-3 (PR3) as a target for stroke treatment. In detail, proteinase-3, which is a proteinase existing in great quantities in neutrophil, plays a significant role in damage to nerve cells of the brain and in inflammatory response to a stroke, and therefore may be used as a target for stroke treatment.

Description

Pronerase-3 as a Stroke Treatment Target

The present invention relates to a proteinase-3 as a therapeutic target for stroke. More specifically, proteinase-3 (PR3), a protease enzyme present in large quantities in neutrophils, plays an important role in initiating brain nerve cell damage and inflammatory response in stroke. To be used as a therapeutic target for stroke.

When ischemic injury occurs, the brain responds to acute and chronic inflammatory processes, which are mainly due to the activation of microglia, the production of inflammatory mediators, neutrophils, T cells, and monocyte / macrophage. do. From peripheral blood within several hours, and primarily the leucocytes from entering the brain substantial portion to be penetrated into the case primarily the neutrophils (neutrophil) (Jin et al J Leukoc Biol 87 (5):.. 779-789, 2010). It is reported that these peripheral neutrophils are induced into brain tissue within a few hours and play an important role in the destruction of the blood brain barrier (BBB) due to the induction of vascular damage, secondary invasion of other inflammatory cells and initiation of inflammatory responses in brain tissue (Yilmaz and Granger, 2008; Kriz, 2006).

Until now, vascular damage response by neutrophil matrix metalloproteinase, ROS generation and cytokine chemokine production by activating NADPH oxidase and myeloperoxidase have been suggested to be important for control of inflammatory response and cell damage (Gidday et al. Am J Physiol Heart Circ Physiol. 289 (2): H558-68, 2005; Rosell et al. Stroke. 39 (4): 1121-1126, 2008), and there is no clear evidence of the possibility of stroke control by their control.

There is also much debate about the specificity of these substances as drug development targets. Recently, a decrease in cerebral infarction, a decrease in brain edema, and a decrease in BBB damage have been observed due to the inhibition of neutrophil infiltration using CD47 knockout mice, and attention has been focused on the importance of neutrophil in stroke (Jin et al. Exp Neurol 217 (1): 165-170, 2009). Despite these experimental evidence, studies on proteolytic enzymes, which are thought to play an important role in the regulation of neutrophil inflammatory responses, are relatively weak (Matayoshi et al. Brain Res. 1259: 98-106, 2009; Shimakura et. al. Brain Res . 858 (1): 55-60, 2000).

Cell damage mechanisms due to ischemia and reperfusion of ischemic vascular diseases include excitatory cell damage by blocking oxygen supply, increased free radical species, and apoptosis signaling pathways. Different strategies for doing so should be reviewed in various ways. For example, ion channel blockade, mitochondrial function control, free radical species blockage, and apoptosis blockage may be useful for blocking acute phase damage. On the other hand, in order to block the damage in the chronic phase, other mechanisms such as suppressing the influx of inflammatory cells, inhibiting late cell death, inhibiting cytokines, glial activation, and controlling central inflammatory responses should be considered for the development of therapeutic agents.

Indeed, studies conducted by many researchers have shown that the prevention of secondary (chronic) brain damage is more evident than the prevention of primary (acute) brain injury, and has a long-term inhibitory effect on brain damage. Therefore, it is expected that the suppression of chronic brain damage will be effective in minimizing the damage after the onset and in the whole rehabilitation process, whereas the inhibitors of the acute phase have only one-time effects.

Therefore, understanding the mechanisms of initiation, maintenance, expansion, and extinction of reactions that cause chronic inflammatory neurological damage is considered to be a key research area for stroke medicine development. Recently reported research papers suggest that inflammatory cell invasion and activation from blood vessels play a very important role in the initiation and maintenance of inflammatory injury response, which will ultimately lead to the activation of inflammatory response inside the central nervous system. Is considered.

Proteinase-3 (hereinafter referred to as 'PR3') is a neutrophil-derived serine protease such as neutrophil elastase, cathepsin G, etc. The primary function is to break down extracellular proteins at the site of inflammation, but they are excessively long or persist Inadequate proteolytic ability, such as, has a harmful effect on the human body. Although they are structurally similar, PR3 is the target antigen of an autoantibody called anti-neutrophil cytoplasmic antibldies (ANCA), and the cell cycle (Csernok et al.Clin Exp Rheumatol26 (3 Suppl 49): S112-117, 2008), differentiation (Dublet et al.J Biol Chem 280 (34): 30242-30253, 2005), apoptosis (Yang et al.Am J Pathol 149 (5): 1617-1626, 1996) and is a multifunctional serine protease that has recently been known to modulate immune function (Sugawara S.Crit                                  Rev                                  Immunol. 2005; 25 (5): 343-360, 2005). PR3 has both a soluble form and a membrane-associated form and also acts as an autoantigen in systemic vascular diseases such as Wegener's granulomatosis and is a danger / alarm against external stimulation in dendritic cells by protease-activated receptor-2 (PAR-2). It acts as a signal. Activated in acidic environment and inactive under normal conditions. PR3 has recently been shown to be able to bind and activate IL-32, which is known to play an important role in innate and adaptive immune responses.

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems and the need for the above, and an object of the present invention is to provide a new stroke treatment target.

In order to achieve the above object, the present invention comprises the steps of culturing the proteinase-3 in microglia;

Before, during or after this treatment step, contacting the cell with the compound to be screened;

Treating the culture of the cells with nerve cells to measure free oxygen or cell death; And

Among the compounds to be screened, a compound which inhibits or prevents the increase in free radicals or apoptosis of nerve cells by contact with the compound is adopted as a leading compound and the compound which does not inhibit or prevent the increase in free radicals or cell death is rejected. Provided are methods for screening compounds to identify compounds useful for treating or preventing stroke, including.

In one embodiment of the invention, the proteinase-3 (proteinase) -3 preferably has an amino acid sequence as shown in SEQ ID NO: 1 or more substitutions, deletions, inversions or translocations, etc. All mutants exhibiting the desired effect of the present invention by inducing mutations of are also included in the scope of the protein of the present invention.

In another aspect, the present invention provides a composition for identifying a compound useful for the treatment or prevention of stroke comprising proteinase-3 as an active ingredient.

The present invention also provides a method of measuring proteinase-3 levels from a sample isolated from a subject;

Measuring proteinase-3 levels in normal control samples; And a subject's stroke comprising comparing the proteinase-3 level of the subject and the proteinase-3 level of the normal control group to indicate the presence of stroke when the proteinase-3 level is increased compared to the normal sample. An in vitro method of measuring stroke markers is provided to provide the information necessary for diagnosis.

The present invention also provides a method of inducing stroke by injecting proteinase-3 into the brain.

Hereinafter, the present invention will be described.

In the present invention, proteinase-3 (PR3), a protease enzyme present in large quantities in neutrophils, will play an important role in initiating inflammatory reactions and neuronal cell damage in stroke. It is prepared.

The inventors have found that proteinase-3 (PR3) is increased several-fold with the invasion of vascular-derived cells in animal models of inflammatory and inflammatory models, and this increase is due to invasion and activation of inflammatory cells, initiation of inflammatory responses, and damage to neuronal cells. The present invention has been completed by confirming that it brings.

The present invention provides an increase in proteinase-3 in the brain by neutrophil infiltration and induction of inflammatory cell activation and neuronal cell death in animal models of stroke, a brain neurological disease.

Hereinafter, the present invention will be described in detail.

We performed middle cerebral artery occlusion (hereinafter referred to as 'MCAo') with 300 g of SD male rats to create a stroke model and section the brain to neutrophil infiltration and the resulting proteinase-3 (PR3). The increase was confirmed using immunohistostaining. As a result, it was confirmed that PR3 was increased in MCAo animal model by confirming that elastase, which is a neutrophil marker, was increased in the striatum portion, and PR3 was increased in the portion (see FIGS. 1 and 2).

Thereafter, it was confirmed by Western blotting whether PR3 protein increased from brain tissues of animals subjected to MCAo and brain tissues injected with inflammatory substances such as LPS (lipopolysachharide). As a result, it was confirmed that PR3 expression was significantly increased in 5 days and 7 days in the cortex by LPS injection. In the brain of MCAo-treated rats, PR3 expression was significantly increased from 15 hours to 24 hours after striatum. (See FIGS. 3 and 4), it can be seen that PR3 mediates neuronal cell death and inflammatory responses due to stroke.

The present inventors performed Western blotting with brain tissues obtained 6 hours and 24 hours after MCAo to confirm the increase of neutrophil infiltraion and PR3 with time after MCAo. As a result, the expression of neutrophil markers such as elastase and MPO (myelinperoxidase) increased from 6 hours after MCAo and PR3 also increased from 6 hours to the highest increase at 24 hours (see Fig. 5). This was done first, and thus it was confirmed that PR3 expression was increased more.

Subsequently, microglial cells and astrocytes were first cultured from the cerebral cortex of rats to investigate the activation of inflammatory cells by PR3. As a result, it was confirmed that reactive oxygen species (ROS, active oxygen) in the cells were increased in a concentration-dependent manner by the concentration-specific treatment of PR3 in cultured microglia and astrocytes. PR3 also increased protein expression of inflammatory mediators such as inducible nitric oxide synthase (iNOS) and matrix metalloprotease-9 (MMP9) in glial cells and increased inflammatory cytokines such as IL-1β and IL-6. 6 and 7 it was found that PR3 can activate the inflammatory cells in the brain and increase the inflammatory mediators.

The present inventors processed DCFH2-DA by increasing the intracellular ROS increase by treating conditioned media obtained by treating glial cells with primary cultured cells to determine whether neural cell death is induced by activation of microglia by PR3. Was confirmed by treatment, and MTT analysis was performed to confirm neuronal cell death. In addition, we confirmed the cleavage of caspase-3 as a marker of epoptosis to confirm whether the death of neurons is a form of apoptotic cell death. As a result, conditioned media obtained from microglia increased concentration-dependent ROS in neurons, induced neuronal cell death, and increased expression of cleaved caspase-3 (see FIGS. 8 and 9). It was found that the microglia, which are cells, were activated, thereby inducing the death of neurons as a form of epoptosis.

Subsequently, direct microinjection of PR3 into the brain was used to stain neuronal cell death and microglial activity in striatum and substantia nigra using TH (Tyrosine hydroxylase), OX-6 and CD11b antibodies. As a result, the number of activated microglial cells stained with CD11b and OX6 was increased in striatum and substantia nigra of brain from PR3 injected animals, and neuronal cell death increased with decreasing neurons stained with tyrosine hydroxylase or NeuN. This was observed. In addition, hematoxylin / eosin staining was observed to increase apoptosis by PR3 injection (see FIGS. 10 and 11), it was confirmed that PR3 can activate microglia in the brain and increase neuronal cell death.

Through the above results, the present inventors demonstrated that neutrophil infiltration was induced after MCAo and the expression of proteinase-3 was increased, which induced neuronal cell death by activating microglia and increasing the expression of inflammatory mediators.

Based on the above results, the present invention provides that proteinase-3 can be a target substance for brain diseases such as stroke.

As can be seen from the present invention, the present invention confirmed that proteinase-3 (PR3), a protease enzyme present in large amounts in neutrophils, plays an important role in initiating an inflammatory response in stroke and inducing brain neuron damage. It can be usefully used to establish therapeutic targets for stroke and to develop bionew drugs.

1-2 are immunostained as a result of neutrophil infiltration and increased PR3 expression in the brain by stroke.

Figure 1 shows the expression of neutrophils (ELastase) and PR3 in the striatum of the animal model brain of the stroke, Figure 2 is merged after staining with antibodies to neutrophils (ELastase) and PR3 in the striatum of the animal model brain of the stroke Represent one picture.

3-4 is a diagram showing the results of Western blotting as a result of the increase in the expression of PR3 in the brain in animal models of stroke and inflammation-induced animal models.

Figure 3 shows the results of Western blotting to obtain the protein of each part of the brain by time from the rat injected with inflammatory lipopolysaccharide (LPS), Figure 4 shows the protein of each part of the brain by time from MCAo rats Obtained shows the result of Western blotting.

Figure 5 shows the results of Western blotting as a result of neutrophil infiltration and PR3 expression in the brain from an animal model of stroke.

6-7 is a diagram showing the results of activation of microglia (central inflammatory control cells) by PR3, Figure 6 shows the intracellular ROS after treatment of the concentration of PR3 in cultured microglia and astrocytes Results are shown, Figure 7 shows the results confirmed by RT-PCR changes in inflammatory mediators that change after treatment with each concentration of PR3 in cultured microglia.

Figure 8-9 is a diagram showing the neuronal cell death result (in vitro) by the activation of microglia (microglia, central inflammatory control cells) by PR3, Figure 8 is a microglia (microglia, central inflammatory control cells) by PR3 MTT assay and ROS measurement results of neuronal cell death caused by activation are shown. FIG. 9 is a diagram showing an increase in apoptosis in neurons by activation of microglia (CG) by PR3.

10-11 is a diagram showing microglia activation and neuronal cell death in vivo by PR3. FIG. 10 is a result obtained by immunostaining neuronal cell death and microglia activity by PR3 microinjection. Figure 11 shows the results of immunostaining of neuronal cell death by PR3 microinjection using H & E staining and NeuN (neuron-specific marker) antibodies.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are merely to illustrate the present invention is not to be construed as limited by the following examples.

Example 1: Neutrophil Infiltration and Increased PR3 Expression in Brain by Stroke

Middle cerebral artery occlusion (hereinafter referred to as 'MCAo') in 300 g SD male rats was performed to section the brain to determine whether neutrophil infiltration and the resulting proteinase-3 (PR3) are increased. It investigated by the following method.

<1-1> Surgery of Middle cerebral artery occlusion (MCAo) as an animal model for stroke

Ten week old 300 g SD male rats were prepared and anesthetized with Rompun / ketamine (1: 2, 2 ml / kg, i.p.). External carotid artery (ECA) and internal carotid artery (ICA) were exposed from the right common carotid artery. ECAs were tied and lingual and maxillary branches cut. ICA was exposed along the pterygopalatine branch and 4-0 nylon suture was inserted into the ICA along the ECA stump to block the middle cerebral artery. After 1.5 hours ischemia, nylon suture was removed and used for the experiment after 24 hours reperfusion. Animals were maintained at 37 ° C. ± 1 ° C. during surgery (FIGS. 1 and 2).

<1-2> Neutrophil infiltration and PR3 expression after MCAo: Immunohistochemical screening

After MCAo, neutrophil infiltration and PR3 expression were examined by immunohistochemical staining using antibodies against neutrophil markers elastase and PR3. Anesthetize the rats and perform perfusion before the heartbeat stops. Wash the blood in all tissues, including the brain, with sterile PBS and immediately add 4% Paraformaldehyde (PFA) in PBS to confirm that the body has hardened. Opened the brain out. The extracted brain was soaked in 4% PFA in PBS solution and fixed at 4 ° C. for 2 hours, and then placed in sterile 15% sucrose for 16 hours. The next day, after soaking for 20 seconds in iso-pentane, which had been put at 70 ℃ in advance, it was stored at 70 ℃ until sectioning. Tissue sections were performed using a microtome (microme, Walldorf, Germany) in the coronal direction (40 μm thick). The cut tissue sections were placed on a poly L-lysine coated slide (ESCO, New Hampshire, USA) and placed in 4% PFA in PBS for 15 minutes to fix the brain tissue on the slide, followed by PBS-Triton X-100 ( PBST) was washed three times for 5 minutes. Blocking with 10% normal horse serum (Lfe Tech., Califonia, U.S.A.) in PBST for 2 hours and immediately reacted the primary antibody (elastase, PR3) overnight at 4 ℃. The next day, washed three times with PBS-Triton X-100 (PBST) three times for 10 minutes and then attached a fluorescent secondary antibody for 1 hour. After washing repeatedly with PBS-Triton X-100 (PBST) for about 3 minutes for 10 minutes, 20 ul mounting solution was dropped and the cover-slip was covered and observed under a fluorescence microscope to confirm the expression of protein.

As a result, it was confirmed that elastase, which is a neutrophil marker, was increased in the striatum portion of the MCAo-administered brain and that the PR3 was increased in the portion (FIGS. 1 and 2). At least 5 animals per group were used in these experiments.

<1-3> Comparison of PR3 Expression in Brain Tissues of Rats Treated with LPS and MCAo

Western blotting was performed by MCAo-treated rats and rats injected with liposomal lipopolysaccharide (LPS) by microinjection through stereotaxic surgery.

For stereotaxic surgery, 300 g of SD male rats were prepared and anesthetized with Rompun / ketamine (1: 2, 2 ml / kg, ip), and the anesthetized rat was placed on a stereotaxic frame (Stoelting, Wood Dale, IL). Fixed with. LPS (5 ug / 5ul) was injected into the corpus callosum (anterior (A), +0.7; lateral (L), +1.8; ventral (V), +3.2) and compared with the PBS control group.

Western blotting was performed to confirm the expression changes of PR3 in the proteins obtained from brains obtained by MCAo and LPS injection. Western blotting was performed by the following method.

Proteins were extracted from the tissues of the brain (cortex, striatum, hippocampus) and quantified by BCA protein quantification. 30 ug of protein was obtained, sample buffer was added and electrophoresed using 10% SDS-polyacrylamide gel. Electrophoresed proteins were electrically transferred to nitrocellulose membranes and blots were blocked using 5% skim milk powder. PR3 was added as the primary antibody and reacted with aubonite at 4 ° C. Secondary antibody with horse radish peroxidase (HRP) was added for 1 hour. After washing three times with PBS three times a minute and after the last wash, the expression level of the protein was confirmed using the LAS-3000 instrument using Enhanced chemilumnescence (ECL) method and quantified by Image J program.

As a result, it was confirmed that PR3 expression was significantly increased on the 5th and 7th day in the cortex where the inflammatory substance was mainly induced by LPS injection.In the brain of the MCAo-treated rats, the PR3 expression was found in the striatum part where the main injury occurred. There was a marked increase in time after 24 hours and at least 4 animals were used in the experiment (FIGS. 3 and 4).

<1-4> Neutrophil infiltration and PR3 expression by MCAo

To confirm neutrophil infiltration, brain tissues were obtained at 6 and 24 hours after MCAo. Protein expression levels were confirmed by Western blotting using antibodies to neutrophil markers, elastase and MPO. Proteins were extracted from the striatum of the brain and quantified by BCA protein assay. 30 ug of protein was obtained, sample buffer was added and electrophoresed using 10% SDS-polyacrylamide gel. Electrophoretic proteins were transferred to nitrocellulose membranes electrically and blots were blocked using 5% skim milk powder. Elastase and MPO (myeloperoxidase) were added as primary antibodies and reacted overnight at 4 ° C. Secondary antibody with horse radish peroxidase (HRP) was added for 1 hour. After washing three times with PBS three times, and after the last wash, the expression level of the protein was confirmed by the LAS-3000 instrument using Enhanced chemilumnescence (ECL) method and quantified by Image J program.

As a result, the expression of neutrophil markers such as elastase and MPO was increased from 6 hours after MCAo and PR3 was also increased from 6 hours to the highest increase at 24 hours through Western blot (FIG. 5). Was implemented.

Example 2: activity of microglia and neuronal cell death by PR3 in vitro )

To investigate the effects of PR3 activation and neuronal cell death by PR3, we obtained the cerebral cortex from the rat brain and cultured neurons and microglial cells. Proceeded.

<2-1> Microglial Activation by PR3

The cerebral cortex was isolated from the brains of rats and cultured microglia and astrocytes, and the activation of PR3 was confirmed by ROS measurement and expression changes of inflammatory mediators such as iNOS, mmp9, IL-1b, and IL-6. Specifically, primary astrocytic and microglial cultures of rat brain were performed as follows. Two-day-old rat cerebral cortex was isolated and dissociated with 0.1% trypsin, and then cells were cultured in T-75 flask coated with poly-D-lysine. The cells were cultured in a medium containing 10% FBS, harvested with trypsin and then cultured in appropriate containers and applied to the study after 7 days. To obtain microglia, confluent astrocytes were shaken at 250 rpm for two hours to collect the cells, and then cultured in a poly-D-lysine-coated culture vessel and applied to the next day experiment. The cultured microglia and astrocytes were treated with concentrations of 10, 20, 50, 100, 200, 500 and 1000 ng / ml, and then treated with H ₂ DCF-DA, a reagent for measuring ROS, in the medium of the cells. The activity was measured and the experiment was repeated at least three times.

Intracellular ROS measurement method is as follows. 50 uM of H ₂ DCF-DA was added to measure the amount of total ROS, and the intensity of fluorescence after 20 minutes of incubation was measured at an excitation 485 nm emission of 530 nm using an ELISA reader (Molecular device, Spectramax Gemini EM). . H ₂ DCF-DA is a reagent that does not fluoresce. When it enters a cell and encounters ROS, diacetate is removed and it is a reagent that fluoresces as DCF (dichlorofluorescence).

As a result, it was confirmed that intracellular ROS increased in a concentration-dependent manner in the microglia and astrocytes (FIG. 6).

Confirmation of activation of microglia by PR3 was confirmed by RT-PCR for changes in inflammatory mediators. Reverse transcription-polymerase chain reaction (RT-PCR) method is as follows. After treatment with cultured microglia cells by concentration of PR3 24 hours later, total RNA was isolated using Trizol. CDNA was synthesized using superscript II enzyme from RNA, and primer sets such as MMP-9, iNOS, IL-6, IL-1β, GAPDH (MMP-9 Forward 5 '-TAA GCT ATT CAG TTA CTC CTA CTG GAA-3 '(SEQ ID NO: 2), Reverse 5'-CCT CTC TAG CAC ACA TGC ACT T-3' (SEQ ID NO: 3); iNOS Forward 5'-GTG TTC CAC CAG GAG ATG TTG-3 '(SEQ ID NO: 4), Reverse 5'-CTC CTG CCC ACT GAG TTC GTC-3' (SEQ ID NO: 5); IL-6 Forward: 5'- TTG TGC AAT GGC AAT TCT GA-3 '(SEQ ID NO: 6), Reverse: 5'-TGG AAG TTG GGG TAG GAA GG-3' (SEQ ID NO: 7); IL-1beta Forward: 5'-AAA ATG CCT CGT GCT GTC TG-3 '(SEQ ID NO: 8), Reverse 5'-CTA TGT CCC GAC CAT TGC TG-3' (SEQ ID NO: 9); PCR using polymerase using GAPDH Forward 5'-GTG AAG GTC GGT GTG AAC GGA TTT-3 '(SEQ ID NO: 10), Reverse 5'-CAC AGT CTT CTG AGT GGC AGT GAT-3' (SEQ ID NO: 11) Amplified by machine. The amplified gene was analyzed by electrophoresis on 1% agarose gel and quantified and the experiment was repeated at least three times.

As a result, it was confirmed that inflammatory cytokines such as IL-1β and IL-6 also increased (FIG. 7).

<2-2> Effect of PR3 on ROS Production and Apoptosis in Primary Cultured Neurons ( in vitro)

After treating PR3 to microglia, the conditioned media obtained 24 hours later was treated with primary cultured neurons to investigate the effects on ROS production and apoptosis. Primary neuron culture in rat brain was performed as follows. At 18 days of gestation, fetuses were collected from the abdominal cavity of rats, and the brains of the fetuses were extracted to separate cerebral cortex. The cells were digested with trypsin to obtain single cells, and cells were cultured using Neuro Basal Medium (NBM) containing B27 and used for the study. In order to measure the toxicity of neurons MTT (3- [4,5-dimethylthiazol- 2-yl] -2,5-diphenyl-tetrazolium bromide) was measured and the resulting formazan using the reagent of intracellular ROS is measured H ₂ DCF-DA was used.

As a result, the conditioned media obtained from PR3-treated microglia cells increased concentration-dependent ROS in neurons and induced cell death (FIG. 8).

Then, the present inventors confirmed the activity of caspase-3, a marker of epoptosis, by Western blotting to determine the form of neuronal cell death by PR3. Proteins were extracted from neurons treated with conditioned media obtained from PR3 treated microglia and quantified by BCA protein assay. 30 ug of protein was obtained, sample buffer was added and electrophoresed using 12% SDS-polyacrylamide gel. Electrophoretic proteins were transferred to nitrocellulose membranes electrically and blots were blocked using 5% skim milk powder. Caspase-3 was added as a primary antibody and reacted overnight at 4 ° C. Secondary antibody with horse radish peroxidase (HRP) was added for 1 hour. After washing for 10 minutes with PBS three times, after the last wash, the expression level of the protein was confirmed using the LAS-3000 instrument using Enhanced chemilumnescence (ECL) method and quantified by Image J program, and the experiment was repeated at least three times.

As a result, it was confirmed that PR3 increased the activity of caspase-3, a marker of epoptosis, by increasing the expression of cleaved caspase-3 (FIG. 9). Through this, PR3 activates microglia and activated microglia secrete various inflammatory mediators to induce neuronal death. However, it was confirmed that apoptosis appears as apoptosis.

Example 3: Neuronal cell death by PR3 microinjection ( in vivo )

After microinjection of 0.5ug PR3 with striatum, the brain was fixed on day 5, and neuronal cell death and microglia activity in striatum and substantia nigra were immunostained using TH (Tyrosine hydroxylase), OX-6 and CD11b antibodies.

For stereotaxic surgery, 300 g of SD male rats were prepared and anesthetized with Rompun / ketamine (1: 2, 2 ml / kg, ip), and anesthetized rats were placed on stereotaxic frame (Stoelting, Wood Dale, IL). Fixed to. Proteinase-3 (1.5 ul; 0.5 ug) was injected into striatum (anterior (A), + 0.7; lateral (L), + 2.1; ventral (V),-5.0) at 0.5 ul / minute and PBS injection control. Compared with. In the method of Example 1-2, TH (Tyrosine hydroxylase), OX-6 and CD11b antibodies were immunostained as primary antibodies in striatum and substantia nigra of rat brain tissue. TH is a marker of dopaminergic neurons of striatum and substantia nigra and OX-6 / CD11b is a marker of microglia. At least three animals per group were used in the experiment.

As a result, the number of activated microglial cells stained with CD11b and OX6 in the striatum and substantia nigra of the brain was increased in the animals injected with PR3 compared with the control group, and neuronal cell death increased with decreasing neurons stained with tyrosine hydroxylase. Was observed (FIG. 10).

Later, the inventors immunostained the neuronal cell death using NeuN, a neuronal marker for PR3 injection, and investigated the hematoxylin / eosin (H / E) staining. We observed a decrease in the number of neurons stained with NeuN in striatum and substantia nigra in PR3-injected animals compared to the control group, and the death of neurons by H / E staining was reduced by PR3 injection ( 11).

Therefore, in cerebral neurological diseases such as stroke, PR3 was increased by neutrophil infiltration and activated neuronal cell death by activating inflammatory cells such as microglia.

Claims (8)

1. Culturing the microglia followed by treatment with proteinase-3;

   Before, during or after this treatment step, contacting the cell with the compound to be screened;

   Treating the culture of the cells with nerve cells to measure free oxygen or cell death; And

   Among the compounds to be screened, a compound which inhibits or prevents the increase in free radicals or apoptosis of nerve cells by contact with the compound is adopted as a leading compound and the compound which does not inhibit or prevent the increase in free radicals or cell death is rejected. A method of screening a compound to identify a compound useful for treating or preventing a stroke.
2. The method of claim 1, wherein the proteinase-3 has the amino acid sequence set forth in SEQ ID NO: 1, to screen for a compound to identify a compound useful for the treatment or prevention of stroke.
3. Composition for identifying a compound useful for the treatment or prevention of stroke comprising proteinase-3 as an active ingredient.
4. According to claim 3, wherein the proteinase (proteinase-3) has a amino acid sequence of SEQ ID NO: 1 composition for identifying a compound useful for the treatment or prevention of stroke.
5. Measuring proteinase-3 levels from a sample isolated from the subject;

   Measuring proteinase-3 levels in normal control samples;

   The presence of stroke when the proteinase-3 level of the subject is compared with the proteinase-3 level of the normal control group compared to the normal sample. An in vitro method for measuring stroke markers to provide information necessary for diagnosing stroke in a subject comprising indicating.
6. 6. The in vitro method of claim 5, wherein the proteinase-3 has the amino acid sequence set forth in SEQ ID NO: 1 to measure the stroke marker to provide information necessary for diagnosing stroke in a subject.
7. How to induce stroke by injecting proteinase-3 into the brain.
8. 8. The method of claim 7, wherein the proteinase-3 has the amino acid sequence set forth in SEQ ID NO: 1.

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