| Ischemic stroke is a brain disease that causes brain tissue damage due to interruption or poor blood circulation in the brain.The main physiological manifestations are stenosis or complete blockage of the blood vessels in the brain,and ischemia,hypoxia,injury and even death in the brain tissue.Ischemic stroke can seriously affect the patient’ s own brain function,and severe hemiplegia,clinical symptoms of delirium and strabismus can lead to death.The treatment of this disease imposes a heavy burden on society and families.Currently,in addition to the application of reperfusion therapy,other treatments are less common or even fail.With the discovery of the study,brain ischemia stimulates brain endogenous neural stem cells to migrate to the ischemic site,proliferate and mature,and this series of reactions can improve the defects and cognitive functions of damaged brain tissue.However,the number of ischemic-induced neonatal nerve cells is very limited,so the mechanism of nerve regeneration after ischemic injury is deeply understood.The clinical intervention to promote ischemia-induced neural regeneration is crucial for the recovery of neurological function after ischemic stroke.significance.Studies have shown that autophagy plays an important role in maintaining the resting state of stem cells and maintaining pluripotent dryness.The processes of self-renewal,differentiation and proliferation of neural stem cells during nerve regeneration are related to autophagy.Therefore,the study of autophagy state of ischemic neural stem cells to promote nerve regeneration provides an effective experimental basis.Yang Huan Wu Tang is a classic prescription for the treatment of stroke,including ischemic stroke.Through the Qing Dynasty Wang Qingren passed the "modern pharmacology research."The medicine of Buyang Huanwu Decoction consists of Astragalus,Chuanxiong,Angelica,Dilong,Peach Kernel and Safflower.Among them,Astragalus and Chuanxiong are high-frequency drugs for treating stroke.The use of Buyang Huanwu Tang is as follows.It shows that you can.Treatment of ischemic stroke drugs Chinese medicine believes that the pathogenesis and cause of ischemic stroke is "lack and stop bleeding." Traditional Chinese medicine pharmacology in vitro research uses direct administration method,but due to the complex composition of traditional Chinese medicine and traditional Chinese medicine,and the direct administration method is difficult to simulate the real environment in the body,so the serum pharmacology method is proposed,that is,the traditional Chinese medicine or Chinese medicine compound is given to the animal.Animal serum was taken as a drug source for in vitro experiments.Serum pharmacology method makes up for the shortcomings of direct drug administration,and provides a new idea for the in vitro research and development of traditional Chinese medicine pharmacology.Based on the above research background,it is now discussed whether the serum of Buyang Huanwu Decoction has protective effect on neural stem cell injury caused by hypoxia/reoxygenation,and its protective mechanism is preliminarily discussed,that is,whether it plays a role in regulating autophagy.ObjectiveIn this study,the neural stem cells of rat young rats with hypoxia/hypoxia/reperfusion injury were used as research models.The effects of Buyang Huanwu Decoction on the proliferation and differentiation of damaged neural stem cells were investigated by serum pharmacological methods,and the possible mechanisms were discussed.methodRats were randomly divided into two groups.They were continuously administered with Buyang Huanwu Decoction and the same amount of distilled water for 7 consecutive days.The dosage was 2 ml/mouse,2 times a day.Blood was taken from the abdominal aorta 1 h after the administration on the 8th day,and the cells were centrifuged to prepare serum,which was inactivated.The experiment was carried out with the serum containing Buyang Huanwu Decoction as the drug source.The rat neural stem cells were cultured for more than three generations after extraction,isolation and identification,and prepared for the next experimental procedure.The model of hypoxia/deficient/reperfusion injury(OGD/R)was used to simulate brain ischemia-reperfusion injury.The neural stem cells after three generations of isolation and culture were divided into three groups.Normal group:Normal culture medium was used to culture neural stem cells and cultured in a normal CO2 incubator;model group:neural stem cells were cultured in a sugar-free medium and placed in a three-gas incubator with hypoxia(1%02,5%CO 2,94%N2)cultured for 2 hours,placed in a normal CO 2 incubator;Bu Yang Huan Wu Tang drug-containing serum group:using sugar-free medium and adding Buyang Huanwu Decoction-containing serum The neural stem cells were cultured and placed in a three-gas incubator for hypoxia(1%O 2,5%C02,94%N2)for 2 h.After cultured for 2 h according to the above conditions,the sugar-free medium was replaced with normal neural stem cell culture medium,and the cells were cultured in a normal incubator.The drug-administered group was still supplemented with Buyang Huanwu Decoction serum,and reoxygenated for 24 h.Carry out follow-up experimental studies.To investigate the effect of Buyang Huanwu Decoction on the proliferation and differentiation of hypoxia-deficient/reperfusion neural stem cells and its possible mechanism,cell viability was detected by CCk-8 method,and dehydrogenation of lactate was detected by enzyme-linked immunosorbent assay(ELISA).Enzyme(LDH)leakage,fluorescence inversion microscope to detect cell proliferation and differentiation;Western Blot to detect Beclinl,Lc3B,p62 levels.RESULTS Immunofluorescence was used to identify the cells isolated from the rats.The cells were full-bodied and spherical.The light-shielding was good under the microscope.It was self-proliferating,the marker staining was positive,and the late differentiation was identified as nerve cells,indicating that the cells were extracted.Neural stem cells.Through the screening of experimental conditions,it was found that Buyang Huanwu Decoction-containing serum inhibited the leakage of lactate dehydrogenase in hypoxic/nose/reperfusion neural stem cells,alleviated the hypoxic injury of cells,and was screened by experimental conditions,hypoxia and hypoglycemia for 2 h reperfusion.24h will significantly reduce cell viability and establish a model,and blank serum and drug-containing serum have no damage to cells.The effect of Buyang Huanwu Decoction serum on the activity of neural stem cells induced by hypoxia/nose hypoxia/reperfusion was determined by cck-8 assay.Immunofluorescence assay showed that the medium and high doses of Buyang Huanwu Decoction significantly increased cell proliferatio Differentiation ability(P<0.05,p<0.01).Compared with the normal group,the expression of Beclinl protein in the model group was significantly increased,and the expression of p62 protein was significantly decreased-Compared with the model group,the expression of LC3B and Beclinl in the Buyang Huanwu Decoction group was significantly decreased,and the expression of p62 protein was significantly decreased.Significantly increased,the difference was significant(P<0.05).ConclusionBuyang Huanwu Decoction can improve OGD/R neural stem cell injury and autophagy,reduce the leakage rate of LDH and regulate autophagy activity,which may be related to down-regulation of LC3-Ⅱ,Beclinl expression and up-regulation of p62 expression. |
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