Application of shionone in preparation of medicine for treating cerebral arterial thrombosis
A technology of ischemic stroke and shionone, applied in the field of pharmacotherapeutics, to improve inflammatory response and improve brain function
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Embodiment 1
[0052] In vitro studies demonstrated that asterone attenuates LPS-induced inflammatory responses in microglia:
[0053] 1) Materials and methods
[0054] Primary microglia were obtained from C57BL / 6 mice and cultured in a humidified incubator at 37°C for 10 days, then primary microglia were isolated and re-cultured in 6-well or 12-well plates with cell culture medium DMEM medium (Thermo Fisher, USA) containing 10% FBS (fetal bovineserum Biological Industries, Israel) and 100 μ / ml penicillin and streptomycin was used.
[0055] The primary microglia were grouped according to the same density: control group, LPS-stimulated group, drug and LPS group. The supernatant of the treated primary microglia was discarded, followed by washing with PBS, and the total RNA of primary microglia and tissues was extracted with Trizol reagent (Invitrogen). Then, cDNA was transcribed using PrimeScript RT kit (Vazyme Biotech). qRT-PCR was performed on an ABI 7500 PCR machine (Applied Biosystems) ...
Embodiment 2
[0060] In vitro studies demonstrate that asterone reduces the production of pro-inflammatory factors at the protein level:
[0061] 1) Materials and methods
[0062] The expression levels of inflammatory factors were determined by Western blot. The supernatant was discarded from the primary microglia treated in different ways in Example 1, and then the cells were washed with PBS buffer, and then RIPA was added for protein lysis. RIPA Lysis Buffer, scraped the protein into a centrifuge tube, centrifuged at 1300rpm for 30min, aspirated the supernatant, used the BCA method to quantify the protein, then dissolved it in 5x Loading buffer and boiled for 5min. After electrophoresis on a 4-20% polyacrylamide gel, the protein was transferred to a PVDF membrane, blocked with 5% nonfat milk for 1 hour at room temperature, and the primary antibody (IL1β, RampD Systems, TNF-α, ab215188, Abcam, USA) was added separately. iNOS, 610328, BD Biosciences, USA), overnight at 4°C, horseradish per...
Embodiment 3
[0066] In vivo studies demonstrate the therapeutic effect of asterone on MCAO model mice:
[0067] The MCAO model was prepared by suture method and administered orally or intraperitoneally at a dose of 20 mg / kg / day. It was proved by behavioral studies that asterone has a protective effect on ischemic brain injury.
[0068] 1) Materials and methods
[0069] Neurological deficit score (NSS): The Modified Neurological Severity Score (mNSS) was used to score the degree of neurological deficit in mice, with a total score of 18 points and 1-6 points as mild injury , 7-12 is divided into moderate injury, 13-18 is severe injury;
[0070] Calculation of cerebral infarction volume: After perfusion, the brain tissue was quickly taken out, placed in a -80 ℃ refrigerator for 5-10 minutes, cut into 2 mm thick slices from the frontal pole to the posterior coronal section, and placed in 2% TTC (2, 3, 5- Triphenyltetrazolium chloride) solution, incubate at 37 °C for 30 min in the dark, the w...
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