| Objective: A model of ischemic stroke in the rat was established after transient middlecerebral artery occlusion(MCAO)followed by reperfusion.Here we observe the changes of autophagy after cerebral ischemic injury in rats,the angiogenesis effect of Longzhi Decoction and study the relationship between each other.Method:(1)The model of middle cerebral artery occlusion in SD rats was replicated by suture method.The ultrastructure and autophagy activation of ischemic cortex were observed under transmission electron microscopy at 12 h,24h,3d and 5d after ischemia.(2)The rat model of MCAO was duplicated and administered with low,medium and high doses of Longzhi Decoction.The neurological deficit scores were measured in rats,and the microvessel density was calculated by immunohistochemistry to detect CD31 protein expression in cerebral ischemic area.(3)90 healthy adult male SPF SD rats were randomly divided into sham operation group(saline+sham operation),model group(saline+MCAO),Longzhi Decoction group(Longzhi Decoction+ MCAO),autophagy agonist group(Rapa Group,saline+MCAO+rapamycin),autophagy inhibitor group(3-MA group,saline +MCAO + 3-MA),18 in each group.After ischemiafor 2 hours and reperfusion for 24 hours,the neurological deficit scores(Zea-longa score)were obtained by Zea-longa neurological deficit score.The ratio of cerebral infarction volume was determined by TTC staining.The HE staining method was used to observe each group.Histopathological changes of cortical tissue in cerebral ischemic area of rats;vascular endothelial cell marker CD31 and calculate microvessel density;Western Blot method was used to detect the expression of autophagy-related proteins LC3 and Beclin-1 in each group.Result:(1)Transmission electron microscopy showed that mitochondria and endoplasmic reticulum were swollen in the early stage of ischemia,and autophagosomes were produced.The number of lysosomes increased and peaked at 24 h.In the late ischemic period,autophagosomes were still observed under the microscope.Mainly manifested as apoptosis or necrosis.(2)Intervention of MCAO model rats with low,medium and high doses of Longzhi Decoction can reduce the neurological deficit scores of rats,up-regulate the expression of CD31 protein,and increase the density of microvessels.The dose group was more effective than the model group(P<0.05).There was no significant difference between the middle dose group and the high dose group(P>0.05),but it was significantly higher than the Longzhi Decoction.Low dose group(P<0.05).(3)Compared with the sham operation group,the model group had significant neurological deficit after cerebral ischemia-reperfusion,with a large range of cerebral infarction,swelling of neuronal cells,increased CD31 expression,increased microvascular density,and autophagy-associated marker proteins.LC3 and Beclin-1 were up-regulated,the neurological deficit scores of the Longzhi Decoction group and the rapamycin group were significantly reduced,and the cerebral infarction volume was reduced.The nerve cellsshowed mild ischemic changes,the degree of cell swelling was mild,the expression of CD31 autophagy-associated marker proteins LC3 and Beclin-1was significantly up-regulated.The difference was statistically significant(P<0.05);Compared with the Longzhi Decoction group,the neurological deficit score of the 3-MA group was increased,the volume of cerebral infarction was expanded,and the cortical neurons in the ischemic cortex were moderately ischemic,the microvessel density and the expression of LC3 and Beclin-1 were significantly decreased(P<0.05).Conclusion:(1)Focal ischemic brain damage in rats can enhance the autophagy level in cerebral ischemic area.(2)Longzhi Decoction can improve the cerebral ischemic angiogenesis,improve the symptoms of cerebral ischemia in rats,reduce the volume of cerebral infarction,and play a protective role in the brain;(3)Longzhi Decoction up-regulates the focal brain of rats,the level of autophagy after ischemia may be one of the mechanisms that promote angiogenesis. |
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