Application of small molecule compound in preparation of medicine for treating cerebral arterial thrombosis
A small molecule compound, a technology for ischemic stroke, applied in the application field of small molecule compounds in the preparation of drugs for the treatment of ischemic stroke, can solve problems such as poor blood-brain barrier permeability, and achieve strong blood-brain barrier permeability. Transient, inhibiting pro-inflammatory phenotype transition, inhibiting the effect of downstream cytokine release
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Embodiment 1
[0028] In vitro studies demonstrate that CP-07 inhibits microglial cytokine production in vitro:
[0029] 1) Materials and methods
[0030] Primary microglia culture: Primary microglia were cultured in the cerebral cortex of newborn 1-2 day old C57BL / 6J mice. Mice were euthanized by carbon dioxide asphyxiation, followed by rapid dissection of mouse brain tissue with ice-cold saline. The meninges were removed, and the cerebral cortex was digested with 0.25% trypsin-EDTA for 10 min. Digestion was stopped with an equal amount of 10% fetal bovine serum (FBS, Gibco) containing Dulbecco's modified Eagle's medium (DMEM, Gibco) and centrifuged at 800 rpm for 10 min. Primary microglia were resuspended in DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin and stored in T75 flasks (Corning) for 10-12 days. Reseeding floating primary microglia at appropriate density into 6-well plates at 37 °C, 5% CO 2 incubator.
[0031] BV2 microglia culture: BV2 microglia were c...
Embodiment 2
[0058] In vivo studies demonstrate the therapeutic effect of CP-07 on post-MCAO mice
[0059] The C57BL / 6J mouse side middle cerebral artery infarction model, namely the MCAO model, was created by the suture method, and CP-07 was injected intraperitoneally 30 minutes before the operation. It has been proved by behavioral studies that CP-07 has a protective effect on ischemic brain injury.
[0060] 1) Materials and methods
[0061] The MCAO mouse model was established by suture method. The experiment was divided into 2 groups: MCAO+Vehicle group and MCAO+CP-07 group (CP-07 injection volume was 1 mg / kg / time). 10 mice were randomly selected from each group. By intraperitoneal injection.
[0062] Detection of cerebral blood flow in mice after MCAO: Laser Doppler flow imaging was used to measure cerebral blood flow in mice after MCAO in resting state and during ischemia-reperfusion period.
[0063] Volume of cerebral infarction: mice after MCAO were killed by inhalation of isofl...
Embodiment 3
[0072] In vivo studies demonstrate that CP-07 attenuates post-ischemic stroke inflammatory response and inhibits the differentiation of microglia to pro-inflammatory type
[0073]1) Materials and methods
[0074] The MCAO mouse model was established by suture method, and the experiment was divided into 3 groups: SHAM group, MCAO+Vehicle group, MCAO+CP-07 group (CP-07 injection volume was 1 mg / kg / time), 10 mice were randomly selected from each group mice, by intraperitoneal injection.
[0075] Detection of cytokine mRNA expression levels: RT-PCR analysis was performed on the brain tissue of the ischemic penumbra of mice after MCAO to detect the transcription levels of cytokines such as IL-1β, IL-6, iNOS and TNF-α.
[0076] Immunofluorescence staining: After 24 hours of reperfusion, mice were perfused transcardially with frozen 1×PBS and 4% PFA (pH7.4). Brain tissue was quickly removed, fixed with 4% PFA overnight at 4°C, and dehydrated with 30% sucrose solution for at least 4...
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