The Protective Effects Of Annaopingchong Decoction On Neurons After Cerebral Hemorrhage Was Studied On The BDNF-Nrf2 Signaling Pathway

Objective:Intracerebral hemorrhage is a common critical disease in neurology.The death of nerve cells after intracerebral hemorrhage is an important cause of secondary brain injury.In order to clarify the effect of Annaopingchong Decoction on injured neurons after cerebral hemorrhage,this study established the hemoglobin-inducded neuronal injury model and observed the regulation effects of Annaopingchong Decoction on ferrous ion overload and excessive oxidative stressand alteration on BDNF-Nrf2 signaling pathway in hemoglobin-damaged neurons.Methods:The primary cultured cortical neurons were intervented by hemoglobin of 10μM、15μM、20μM、25μM、30μM for 24h.To confirm the optimal concentration of hemoglobin to neuronsthe morphology of neurons were observed by microscope and cell viability were detected by CCK-8.Further more,hemoglobin induced of injured neurons were intervented correspondingly by 5%,10%,20%drug-containing serum of Annaopingchong Decoction and by 10μM,20μM,30μM methylthio deferoxamine.After 24h,observed the morphology,and detecting cell viability to confirm the optimal concentration of Annaopingchong Decoction containing serum and methylthio deferoxamine,The neurons were laid on a 96-well plate,and divided into 5 groups（the normal group,the model group,the blank serum group,the Annaopingchong-Decoction-containing serum group,the Methylthio Deferoxamine group.）the 5 groups were intervened by group for 24h.Separately detecting the content change of Neuroferrous ion and the level of reactive oxygen in each group by Specific Fluorescent probe for Fe²⁺and Reactive Oxygen Species;Extract the total protein in each group,through western blot to detecting the expression level of BDNF and Nrf2.Results:（1）Compared with the normal group,the results of morphological observation and CCK-8 kit showed that,after 24h intervention of 25μM hemoglobin,the cell survival rate was 48%（P<0.01）,and the number of living cells decreased,the number of synaptic network decreased.When Compared with the model group,the result of each group’s intervene function shows that,10μM Methylthio Deferoxamine increased the cell survival rate of hemoglobin injured neurons to 85%（P<0.01）,while 10%of Annaopingchong Decoction could promote the cell survival rate of hemoglobin injured neurons to 95%（P<0.01）.（2）The data detected from Specific Fluorescent probe for Fe²⁺showed that,compared with the normal group,the fluorescence value of ferrous ion in each drug group and model group was significantly higher（P<0.01）,and compared with the model group,the fluorescence value of ferrous ion in Annaopingchong Decoction decreased（P<0.01）.ROS fluorescence staining data showed that ROS fluorescence value of model group was higher than that of normal group（P<0.01）,and ROS fluorescence value of each drug group was lower than that of model group（P<0.01）.（3）WB detection results of BDNF and Nrf2 in hemoglobin injured neurons showed that,the expression of BDNF and Nrf2 in model group was higher than that in normal group（P<0.05）.Compared with the model group,the expression of BDNF and Nrf2 in Annaopingchong Decoction formula and Methylthio Deferoxamine group was significantly higher（P<0.05）,and the expression of BDNF and Nrf2 in Annaopingchong Decoction formula group was better than that in Methylthio Deferoxamine group.Conclusion:Annaopingchong Decoction can protect neurons by up regulating the expression of bdnf-nrf2 protein,regulating the overload of ferrous ion and excessive oxidative stress in neurons damaged by hemoglobin.