The Effects Of Rhynchophylline On HMGB1 And CDK5 MRNA Expression Of Oxygen Glucose Deprivation Astrocytes In Rats

ObjectiveTo investigate the mRNA expression of astrocytes High Mobility Group Box 1?HMGB1?and Cyclin-dependent Kinase 5?CDK5?under the condition of ischemia hypoxia,and observe the effects of Rhynchophylline?Rhy?on HMGB1 and CDK5mRNA expression of ischemia-hypoxia astrocytes.MethodsThe hippocampus of newborn SD rats?within 1-3 day?was taken for primary As culture,remove other cells by cell exchange and cell subculture,then taken the 3rd generation cells for cell crawling,and performded As glial fibrillary acidic protein?GFAP?immunofluorescence identification,the cells can be used for subsequent experiments when the positive rate is obove 90%-95%.The experiment was divided into four groups:blank control group,ischemia-hypoxia group,Rhy low-dose group(0.02mg.ml^-1),and Rhy high-dose group(0.2mg.ml^-1).The cell culture flask was placed in an anaerobic tank to establish an in vitro model of ischemia and hypoxia in cells.The cells in each group were collected to extract total RNA from each group,and real-time quantitative PCR was used to detect As HMGB1 and CDK5 mRNA expression in each group.Results1.In the primary culture of astrocytes for 6-7 days,the cells have basically covered the bottom of the cell culture bottle,which is mainly astrocytes which irregular morphology.The subcultured As can be seen to be irregular in shape,mostly polygonal,and the cell volume increase.The cytoplasm is rich,the nucleus is skewed to the sides of the cell body,and is round or oval,with low refractive index,and the protrusions of some cells become slender.After 6-7 days,the cells basically covere the bottom of the bottle;after 6 hours of oxygen-glucose deprivation,the cells lose their original shape and become long spindle-shaped,with edema in the cell body,fine particulate matter can be seen in the nucleus,the protrusions became more elongated,and the cells are intertwined into a network.The morphology of GFAP-positive cells is irregular under a fluorescent microscope,it is flat,the edges of the cells are clear,and there are some large coarse protrusions.Most of the cells showed green fluorescence,and the positive rate of As is about 90%-95%.2.The expression of HMGB1 and CDK5 mRNA in the ischemia and hypoxic cells was significantly higher than that in the blank control group?p<0.05?;3.The expression of HMGB1 and CDK5 mRNA of astrocytes in the low-dose and high-dose groups was significantly lower than that in the ischemia and hypoxia group?p<0.05?.It was not obvious that the down regulation of rhynchophylline in high dose group than that in low dose group?p>0.05?.Conclusions1.The hippocampus can be purified by trypsiniation to obtain higher purity of As after removing other cells through primary culture,liquid exchange and subculture.2.The expression of As HMGB1 and CDK5 mRNA is up-regulated after oxygen-glucose deprivation;the intervention with Rhy can down-regulate the expression of HMGB1 and CDK5 mRNA in astrocytes after oxygen-glucose deprivation,suggesting that Rhy maybe has an inhibitory effect on HMGB1 and CDK5.