CN113813260A - Use of roxasistat for relieving ischemic stroke-mediated synaptic injury - Google Patents

Abstract

The invention provides application of roxasistat in medicines for treating cerebral arterial thrombosis reperfusion-mediated synaptic injury. The roxasistat has a neuroprotective effect on cerebral ischemia-reperfusion therapy, and can effectively relieve synaptic plasticity injury mediated by ischemic stroke. Is expected to become a medicament for clinically treating cerebral arterial thrombosis reperfusion-mediated synaptic injury.

Description

Use of roxasistat for relieving ischemic stroke-mediated synaptic injury

Technical Field

The invention belongs to the field of new application of medicines, particularly relates to application of roxasistat in relieving cerebral ischemic stroke-mediated synaptic injury, and particularly relates to a nerve protection effect of roxasistat after cerebral ischemia reperfusion to relieve synaptic injury.

Background

Ischemic stroke is a disease that seriously endangers human health. The treatment means is mainly to implement thrombolytic therapy within an effective time window and to recover blood as soon as possible to prevent the ischemic region from spreading. Once the effective time window is exceeded, irreversible damage and even death of brain cells can result, which is life threatening and results in ischemic stroke reperfusion injury. At present, calcium channel antagonists, EAA antagonists and the like are mainly used clinically for resisting cerebral ischemic injury, but the treatment effect is poor.

After cerebral ischemia occurs, the damaged area of the brain expands in a gradient manner from the center of the damage to the periphery. As cerebral blood flow continues to decline, nervous system activity is inhibited and subsequent metabolic activity is affected. At the same time, the relevant signal transduction pathways that are activated trigger a series of events, such as: oxidative inflammatory reaction, apoptosis, etc. Under certain external stimuli or pathological conditions, the structure and density of dendritic spines are significantly altered, and remodeling of synaptic signaling may be activated thereby, controlling the brain's functions of learning, memory, cognition and motor coordination. After cerebral ischemia for 20min, the dendritic spine is changed in shape and reduced in number, resulting in failure of nerve conduction function, which indicates that neuron death and dendritic spine deletion are major nerve injuries caused by cerebral ischemia.

Rosemastat is an oral hypoxia inducible factor prolyl hydroxylase inhibitor (HIF-PHI) developed by the cooperation of Fabo and Asricon in China, which is approved for the treatment of anemia in CKD dialysis patients, including hemodialysis and peritoneal dialysis patients. The roxasistat can activate the transcription of related genes by stabilizing HIF, inhibiting the degradation of the HIF, generating corresponding physiological response, moderately increasing the concentration of erythropoietin, improving the sensitivity of Erythropoietin (EPO) receptors, coordinating the generation of red blood cells, reducing the level of hepcidin, increasing the content and activity of transferrin receptors and promoting the absorption and utilization of iron.

The structural formula of the roxasistat is shown as follows:

disclosure of Invention

The invention provides application of roxasistat in drugs for treating cerebral arterial thrombosis reperfusion-mediated synaptic injury. Solves the problem of synapse damage in the reperfusion therapy of iron-deficiency cerebral apoplexy in the prior art.

The technical scheme of the invention is realized as follows:

application of roxasistat in medicines for treating cerebral arterial thrombosis reperfusion-mediated synaptic injury.

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

the roxasistat can improve the perfusion-mediated synaptic injury of ischemic stroke. Is expected to become clinically used for treating and improving the synapse damage caused by the ischemic stroke perfusion treatment.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1-A is a graph of the body weight change before and after treatment of Rosemastat with ischemic stroke reperfusion injury;

FIG. 1-B is a graph of TTC staining of cerebral infarct volume after treatment of reperfusion injury in ischemic stroke by Rosxastat;

FIG. 1-C comparative graph of analysis of cerebral infarction volume after treatment of reperfusion injury by Rosxastat in ischemic stroke;

FIG. 1-graph of rat mortality following treatment of ischemic stroke reperfusion injury with Rosesarta;

FIG. 2-A is a graph of HE staining of brain histopathology following treatment of ischemic stroke reperfusion injury with Rosxastat;

FIG. 2-B is a graph of the rate of necrotic cells in the cortical region following treatment of reperfusion injury from stroke with ischemia;

FIG. 2-C is a graph of the rate of necrotic cells in the hippocampal region following treatment of reperfusion injury by Rossastat;

FIG. 2-graph of hippocampal CA1 zone pyramidal cell layer thickness following treatment of ischemic stroke reperfusion injury with Rosesartal;

FIG. 2-E graph of hippocampal CA2 zone pyramidal cell layer thickness after treatment of ischemic stroke reperfusion injury by Rosesarta;

FIG. 2-graph of Hippocampus CA3 zone pyramidal cell layer thickness after treatment of ischemic stroke reperfusion injury with Rosesartal;

FIG. 3-A shows an electrophoretogram of HIF-1. alpha. protein after treatment of reperfusion with Rosxastat in ischemic stroke;

FIG. 3-B is a graph comparing HIF-1 α expression levels after treatment of stroke with rospastat;

FIG. 3-C shows an electrophoretogram of VEGF protein after treatment of reperfusion with Rosxastat in ischemic stroke;

FIG. 3-graph comparing VEGF expression levels after treatment of ischemic stroke reperfusion with Rosxastat;

FIG. 4-A shows the protein electrophoresis patterns of CaMK II, PSD95 and CRBE after treatment of reperfusion of stroke with rospashead;

FIG. 4-B is a graph comparing the expression levels of CaMK II after reperfusion therapy of stroke with rosloxacin;

FIG. 4-C is a graph comparing the expression of PSD95 after treatment of Rosxastat with reperfusion of ischemic stroke;

FIG. 4-graph comparing CRBE expression levels after treatment of reperfusion with Rosesarta;

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

This example demonstrates the use of rosxastat in the treatment of cerebral arterial ischemic reperfusion-mediated synaptic injury.

1. Laboratory animal handling and grouping

Uniformly dividing 75 SD rats into 5 groups by using a random digital table method, wherein each group comprises 15 rats, constructing an ischemic stroke reperfusion model by using a middle cerebral artery embolization method, treating the rats with different doses of Rosemastat, and then taking brain tissues for detection: (1)2, 3, 5-triphenyltetrazolium chloride (TTC) staining to evaluate the cerebral infarction volume; (2) daily body weight and mortality of rats were monitored; (3) western blot is used for detecting the expression conditions of HIF-1 alpha and VEGF related proteins of an HIF-1 alpha signal pathway; expression levels of synaptic related proteins CaMK ii, PSD95, and CRBE. Thereby determining the effect of the roxasistat on synaptic plasticity damage mediated by cerebral arterial ischemic reperfusion. The specific grouping is as follows:

2. experimental methods

2.1 construction of Central cerebral artery embolism (MCAO) reperfusion injury model

One end of a nylon wire with the diameter of 0.23mm is repeatedly immersed into the molten paraffin for about 5 times, and after the paraffin on the surface of the nylon wire is solidified, the suppository wire is immersed into the heparin for 30min and then dried for later use. After anesthetizing (0.35mL/100g) rats by intraperitoneal injection of 10% chloral hydrate, they were fixed supine on the operating table with the rubber band, and the median incision of the neck was taken, the fascia was cut, the right sternocleidomastoid muscle and sternohyoid muscle were blunt-isolated, and the right Common Carotid Artery (CCA) and vagus nerve were exposed and isolated. The ECA, ICA were isolated and the ECA was ligated along the bifurcation of the right External Carotid Artery (ECA) and Internal Carotid Artery (ICA). Clamping the CCA and the ICA by using an artery clamp, cutting a small opening at a position 5mm away from the artery bifurcation of the ECA by using an ophthalmic scissors, slowly inserting the thread plug into the ICA, removing the ICA artery clamp when the ICA and the ECA are intersected, adjusting the thread plugging angle, continuously and slowly pushing the thread plug towards the ICA cranium direction until the insertion depth is 18 +/-0.5 mm. Ligating and fixing the thread with ECA, loosening the CCA artery clamp, cutting off the redundant tail end of the thread bolt, and finally suturing fascia and skin. After 2h of ischemia, the suppository was withdrawn, the ECA proximal bifurcation was ligated, and the Sham group (Sham group) was operated in the same manner as the model group except that no insertion was performed.

2.2 Experimental treatment

Performing different treatments according to the groups, namely, constructing a model with right cerebral ischemia for 2h and reperfusion for 7 days by using a sham operation group with only the cervical artery exposed and no inserting wire for molding, constructing a model group with right cerebral ischemia for 2h, constructing a positive control group, performing edaravone treatment on a model rat according to 5mg/kg after molding, preparing a low-dose group of rosmarinstat according to 10mg/kg after molding, performing rosmarinstat treatment on a model rat according to 10mg/kg after molding, and preparing a high-dose group of rosmarinstat according to 20mg/kg after molding.

2.3 rat brain tissue fixation

The rats are anesthetized by intraperitoneal injection of 10% chloral hydrate, the thoracic cavity is opened, the rats are fixed by cardiac perfusion of 4% paraformaldehyde for 15min, then the rats are perfused by normal saline for 10min, brain tissues are taken out, and the rats are continuously soaked in the 4% paraformaldehyde for 24h and can be used for paraffin embedding sections.

2.4 TTC staining

After the model is made for 24 hours, anesthetizing a rat, taking the brain, freezing for 30min at the temperature of-20 ℃, continuously cutting the rat brain into coronal sections from the frontal pole to the back, cutting a layer by about 2mm, placing the brain slice into a 1% TTC solution, incubating for 20min at 37 ℃ in a dark place, then placing the brain slice into a 4% paraformaldehyde solution for fixing for 24 hours, and scanning by a scanner to obtain an image. Normal brain tissue appears bright red, and infarcted brain tissue appears white. The infarct area and total area of each tablet were measured by Image J software, and the infarct volume in each layer was the infarct area x the layer thickness, and the total infarct volume was the sum of the infarct volumes in each layer. Infarct volume percentage infarct area volume/total brain volume x 100%.

As shown in fig. 1, rosxastat can significantly reduce the volume of cerebral infarction mediated by ischemic stroke reperfusion and control mortality.

2.5 HE staining

The paraffin sections were baked in a thermostat at 60 ℃ for 1 h. Dewaxing and hydrating: xylene 2 × 15min → absolute ethanol 2 × 5min → 90% ethanol 5min → 70% ethanol 5min → 50% ethanol 5min → distilled water 5 min; and (3) carrying out hematoxylin nuclear staining: hematoxylin staining for 5-10min (the specific time can be adjusted according to different tissue staining results and requirements) → recovering the staining solution, washing with tap water until the section turns blue → separating with 1% hydrochloric acid and alcohol solution for color separation, removing the excess staining solution → washing with tap water; eosin staining: placing the slices into 1% eosin solution for infection for 5-10 min; and (3) dehydrating and transparency: ddH2O washed off eosin dye solution → 70%, 80%, 90% ethanol for color separation and dehydration, 5min → absolute ethanol 2X 3min → xylene 2X 3min each time; sealing with neutral gum, and storing at room temperature for a long time.

The results are shown in fig. 2, and the results show that the roxasistat can reduce the apoptosis number of nerve cells and remarkably relieve the damage caused by cerebral arterial thrombosis reperfusion.

2.6 Western blot detection

(1) Extraction of total tissue protein

1) Taking about 50-100mg of ischemic lateral cortex brain tissue, putting the ischemic lateral cortex brain tissue into a homogenizer, adding 200-400uLRIPA lysate (containing a phosphatase inhibitor), and homogenizing the slurry in ice bath until no large-particle precipitate exists. The homogenate was transferred to a 1.5mLEP tube, incubated in an ice bath for about 15min for complete lysis, transferred to a cryo-centrifuge and centrifuged at 12000rpm for 5 min.

2) And (3) subpackaging the protein supernatant into 0.5mLEP tubes, and storing in a refrigerator at-20 ℃.

(2) Measurement of tissue protein content (BCA method)

1) Diluting a protein standard (5mg/mL) by 10 times by using RIPA lysate, namely adding 10uL of the protein standard into 90uL of RIPA lysate, adding the diluted protein standard into a 96-well plate according to the proportion of 0uL, 1uL, 2uL, 4uL, 8uL, 12uL, 16uL and 20uL, and then supplementing the solution to 20uL per well by using the RIPA lysate.

2) 19uLRIPA lysate was added to 96-well plates, and 1uL of the protein of interest was added, and triplicate wells were assayed for each sample.

3) Preparing a BCA working solution: according to the formula of the solution A: and preparing solution B at a ratio of 50:1, adding 200uL of BCA working solution into each hole, and then placing in an incubator at 60 ℃ for 30 min.

4) OD572 is measured by a microplate reader, a standard curve is drawn, and the concentration of the target protein is calculated.

(3) Western Blot analysis

1) Preparing loading protein: taking 6 mu L of protein Sample to be detected (50 mu g) and SDS Reducing Sample Buffer (5 x) to an EP tube, supplementing RIPA lysate to a 30 mu L system, vortex, shaking, mixing uniformly, carrying out water bath at 95 ℃ for 5min to denature the protein, cooling to room temperature, and storing at-20 ℃ for later use.

2) Preparing glue: preparing 12% separation gel: 30% Acrylamide mix 8mL, 1.5M Tri HCl (pH 8.8): 5mL, ddH 2O: 6.6mL, 10% SDS: 0.2mL, 0.2mL of 10% APS, TEMED; 0.008 mL. Mixing, pouring into a glass plate for gel making, flattening the liquid surface with 1ml ddH2O, and gelling at room temperature for 60 min.

3) Preparing 5% concentrated glue: 30% Acrylamide mix 1mL, 1M Tri HCl (pH 6.8): 0.75mL, ddH 2O: 4.1mL, 10% SDS: 0.06mL, 0.06mL of 10% APS, and 0.01mL of TEMED. Pouring off water on the separation gel, sucking dry with filter paper, adding concentrated gel, inserting into comb with 5mm aperture, gelling at room temperature for 40min, and taking out the comb after the concentrated gel is formed.

4) Electrophoresis: the gel plate was placed in a vertical electrophoresis tank, electrophoresis buffer was poured in, and the prepared loading protein and MAKER were sampled by a microsyringe into loading wells at 25. mu.l per well. And (5) carrying out constant-voltage 100V electrophoresis until bromophenol blue is specially moved to the bottom of the separation gel, and stopping electrophoresis.

5) Electrotransfer: and (3) cutting the PVDF membrane and the filter paper with the same size as the separation gel, firstly soaking the PVDF membrane in methanol for several seconds, then soaking the PVDF membrane and the filter paper in the electric transfer liquid, clamping the PVDF membrane and the filter paper by an electric transfer clamp (the PVDF membrane is close to a white plate) in the sequence of the filter paper-the PVDF membrane-the separation gel-the filter paper, putting the PVDF membrane and the filter paper into an electric transfer groove, pouring the electric transfer liquid, and carrying out 350mA constant current electric transfer in an ice bath for about 75 minutes.

6) Blocking and incubating the antibody: immersing the PVDF membrane in 5% skimmed milk powder, and sealing for 2h in a shaking table at room temperature; pouring off the blocking solution, adding primary antibody diluted with the blocking solution, incubating at 4 deg.C overnight, incubating at room temperature for 30min the next day, recovering the primary antibody, and storing at-20 deg.C. Washing the membrane with TBST for 3 × 10min, adding HRP secondary antibody diluted by blocking solution, incubating for 2h at room temperature in a shaking table, and washing with TBST for 3 × 10 min.

7) Developing and exposing: uniformly mixing a stable peroxidase solution and an enhancement solution in an ECL luminescent reagent according to the volume ratio of 1:1, dripping the mixture onto a PVDF membrane, removing redundant luminescent substrates after reaction for a plurality of minutes to obviously emit light, wrapping the luminescent substrates with a preservative film, tabletting and exposing an X-ray film, washing the film with a developing solution and a fixing solution, drying the film, and scanning by a scanner.

8) And analyzing the gray value of the film by using Image J software, and calculating the relative content of the target protein by using the ratio of the target protein to the internal reference beta-actin.

The results are shown in fig. 3 and fig. 4, and it can be seen that the rosxastat can significantly increase the expression of HIF-1 α, VEGF, CaMK ii, PSD95 and CRBE, which indicates that the rosxastat can alleviate ischemic stroke reperfusion-mediated neurosynaptic injury by regulating the expression of synapse-related protein on the basis of regulating the activity of HIF-1 α pathway.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (2)

1. Application of roxasistat in medicines for treating cerebral arterial thrombosis reperfusion-mediated synaptic injury.

2. Use according to claim 1, characterized in that:

the dosage of the roxasistat is 10 mg/kg-20 mg/kg.

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