Based On PI3K/AKT/ENOS Pathway To Explore The Mechanism Of Electroacupuncture Combined With Rich Rehabilitation Training In The Regulation Of Focal Cerebral Ischemic Angiogenesis

Objective:Taking the PI3K/AKT signal pathway as the entry point,observe the effect of electroacupuncture combined with extensive rehabilitation training on the expression of PI3K/AKT signal transduction pathway-related factors in rats with acute middle cerebral artery ischemia（MCAO）,and explore its effect Mechanism of angiogenesis in the brain to promote neural function recovery.Methods:SPF-grade healthy male SD rats were randomly divided into a sham operation group（n=27）and a model group（n=81）.Four hours after model establishment,model rats with a neural function score of 1-3 were randomly selected They were divided into model group,acupuncture group,and inhibitor group（inhibitor combined with acupuncture method）,27 in each group,and each group was divided into three subgroups according to cerebral ischemia 3d,7d,and 14d.An animal model of focal middle cerebral artery ischemia was established with reference to Zea Longa suture method.The acupuncture group was treated with electroacupuncture and rehabilitation at 4h after operation.1 time daily.In the inhibitor group,AKT blocker was injected into the lateral ventricle,and electroacupuncture combined with rehabilitation training intervention was performed.1 time daily.There was no intervention in the sham operation group and the model group.After intervention of rats in each group for 1d,3d,7d,and 14d,the neurological impairment of each group was observed with neurological deficit scores;HE staining was used to observe the histopathological changes in the hippocampal CA1 region of rats;TTC staining was used to measure the area of cerebral infarction;Immunohistochemical method was used to detect the expression of PI3K,Akt,VEGF,VEGFR₂positive cells in ischemic lateral brain tissue of rats;Western blot was used to detect the expression of PI3K,Akt,VEGF,VEGFR₂in ischemic lateral brain tissue of rats;RT-q PCR method The relative expressions of PI3Km RNA,Akt m RNA,VEGFm RNA,VEGFR₂m RNA,and hippocampus in the ischemic area of rats were detected.The microvascular density of cerebral infarction area was measured by Weidner method.Results:1.Neurological deficit scoreThe scores of neurological deficits in the sham operation group were all 0 points.Compared with the sham operation group,the scores of neurological deficits at 4h,3d,7d,and 14d after surgery were significantly higher than those of the sham operation group（P<0.01）;compared with the model group After two hours of intervention,4h and 3d,there was no statistically significant degree of inhibition of neurological deficits in the dose group and the acupuncture group;after two time points of intervention,7d and 14d,the neurological deficit scores in the inhibitor group and the acupuncture group were significantly lower.In the model group,the difference was statistically significant（P<0.01 or P<0.05）.Compared with the inhibitor group,the difference between the acupuncture group and the inhibitor group was statistically significant after intervention at 7 and 14 days（P<0.01 or P<0.05）.2.Cerebral infarction volume detectionNo infarct area was found in the sham operation group,and the cerebral infarction volume was 0 on 3d,7d,and 14d after operation.Compared with the sham operation group,the intervention group had statistically significant differences in cerebral infarction volume after 3d,7d,and 14d time points（P<0.01）;Compared with the model group,the volume of cerebral infarction in the acupuncture group was significantly smaller than that in the model group after three time points:3d,7d,and14d.The volume of cerebral infarction in the inhibitor group was smaller than that in the model group（P<0.01）.Compared with the inhibitor group,the volume of cerebral infarction after the intervention of 3,7 and 14 days was statistically significant（P<0.01）.3.Histopathological observationThe results of HE staining showed that the brain tissue of the hippocampal CA1area in the sham operation group was edema-free,the layers were clear,the neurons were not degenerated and necrotic,the cell structure was normal,the cells were arranged neatly,and the cells were basically free of lesions or necrosis.Significant edema was found in the brain tissue of hippocampal CA1 region,a large number of neurons degenerated and necrotic,atrophied,the boundaries between cell membranes and nuclear membranes were unclear,the gaps between cells were widened and arranged irregularly,and the nucleus was broken or disappeared.Compared with the model group,the neuroedema in the hippocampal CA1 area of rats in each intervention group was alleviated to varying degrees,the number of neurons and the number of new blood vessels increased,and the structure and shape of nerve cells were significantly different after 7 days and 14 days after operation in each intervention group.Large improvement,gradually becoming intact,suggesting that acupuncture is beneficial to promote angiogenesis;compared with the inhibitor group,the number of neuron degeneration and necrosis in the hippocampal CA1 region of rats in the acupuncture group is significantly reduced,and the structure and shape of nerve cells are regular.And the number of neurons improved significantly in the inhibitor group.4.Immunohistochemical detection of PI3K、Akt、VEGF、VEGFR₂positive expressionCompared with the sham operation group,the positive expressions of PI3K,Akt,VEGF,and VEGFR₂in the ischemic penumbra of the model group were significantly increased after intervention at three time points:3d,7d,and 14d（P<0.01）;compared with the model group,the acupuncture The positive expressions of PI3K,Akt,VEGF,and VEGFR₂were significantly higher in the Kang group after 3d,7d,and 14d intervention than in the model group（P<0.01 or P<0.05）.The inhibitor group was positive for PI3K,Akt,VEGF,and VEGFR₂after 7d,14d The expression difference was statistically significant（P<0.01）;compared with the inhibitor group,the PI3K,Akt,VEGF,and VEGFR₂positive expression differences were statistically significant at three time points:3d,7d,and 14d after intervention in the Acupuncture Group（P<0.01）.5.Western Blot detection of PI3K、Akt、VEGF、VEGFR₂proteinCompared with the sham operation group,the relative expression of PI3K/Akt/VEGF/VEGFR₂at three time points（3d,7d,14d）on the ischemic side of the model group significantly increased（P<0.01）;compared with the model group,The relative expression levels of PI3K/Akt/VEGF/VEGFR₂in the Kang group rats were significantly higher than those in the model group after 3d,7d,and 14d intervention（P<0.01 or P<0.05）,and the inhibitor group after 7d and 14d intervention.The relative expression of PI3K/Akt/VEGF/VEGFR₂was statistically significant（P<0.01）.Compared with the inhibitor group,the relative expression of PI3K/Akt/VEGF/VEGFR₂was significantly different after 3 days,7 days,and 14 days of intervention.（P<0.01）.6.RT-q PCR detection of PI3K/Akt/VEGF/VEGFR₂m RNACompared with the sham operation group,the relative expression of PI3K/Akt/VEGF/VEGFR₂m RNA at the three time points（3d,7d,14d）on the ischemic side of the model group significantly increased（P<0.01）;compared with the model group,The relative expression levels of PI3K/Akt/VEGF/VEGFR₂m RNA in the Kang group rats were significantly higher than those in the model group（P<0.01 or P<0.05）after 3d,7d,and 14d intervention.The relative expression levels of PI3K/Akt/VEGF/VEGFR₂m RNA were statistically significant（P<0.01）;compared with the Acupuncture group and the inhibitor group,the relative expression levels of PI3K/Akt/VEGF/VEGFR₂m RNA were significantly different after 3d,7d,and 14d intervention（P<0.01）.7.Measurement of microvessel density in ischemic areaCompared with the sham operation group,the microvascular density of the model group rats at three time points（3d,7d,14d）was significantly increased（P<0.01）;compared with the model group,the rats in the acupuncture group were intervened for 3d,7d After 14 days,the microvessel density was significantly higher than in the model group（P<0.01 or P<0.05）.After intervention at 7 and 14 days,the MVD in the inhibitor group was significantly different（P<0.01）;compared with the inhibitor group,In comparison,the microvessel density of the Acupuncture group was significantly different after 3d,7d,and 14d intervention（P<0.01）.Conclusions:1.Electroacupuncture combined with extensive rehabilitation training can effectively promote the recovery of nerve function in MCAO rats and reduce the volume of cerebral infarction.2.Electroacupuncture combined with extensive rehabilitation training can improve the pathological changes of MCAO rat brain tissue,increase the expression of PI3K/Akt/VEGF/VEGFR₂in ischemic lateral brain tissue of rats,thereby increasing MVD.This may be the combination of electroacupuncture and enriched rehabilitation training to activate PI3K/AKT Signal pathways are one of the mechanisms that induce angiogenesis,thereby restoring neural function after cerebral ischemia and exerting brain protective effects.