| Objective: Intracerebral hemorrhage is an important clinical type of cerebrovascular accident,which is simply less than the ischemic stroke,accounting for 20%?30% of all stroke cases.The occurrence and development of intracerebral hemorrhage include occupations caused by enlarged hematoma,brain edema,local hemodynamic changes and toxic effects of products after the hematoma separation.Due to its characteristics of rapid onset and high fatality rate,although it has received much attention in public,therapy methods are still extremely limited.At present,the treatment of intracerebral hemorrhage mostly adopts impatient surgery and maintenance therapy,and there are few interventions in the clinical treatment of brain protection and neurological function recovery.With the progress of medical crafts,a variety of treatment strategies and drugs with neuroprotection have been gradually put into clinical use.Anesthetic drugs are widely used in patients with cerebral hemorrhage due to their involvement in clinical operations and sedation of critically cared patients.As a commonly used anesthetic and sedative drug,propofol has been clinically proved to own the advantages of reducing local cerebral blood flow,slowing down the progress of hemorrhage,reducing inflammatory response,faster postoperative recovery of the patients,better recovery of nerve function,etc.But its mode of action and mechanism of action are still unknown.This study intends to conduct a neuronal injury model on HT22 mice hippocampal neurons with hemin,which is the main components of breakdown products of hemoglobin,simulating the the toxic effects of hemoglobin secret by hematoma,and at the same time,the application of different concentrations of propofol were trained,aiming to explore the neuroprotective function of propofol and its possible mechanism.Methods: Using CCK8 method,hemin application scheme with 50% mortality of HT22 cells was screened out in order to construct a neuronal injury model.Morphological changes of cells in each group were observed under inverted microscope.According to the selected time and concentration,the experimental groups were set as blank control group(NC),solvent control group(Vehicle),model group(Hemin),low dose propofol treatment group(P25)and high dose propofol treatment group(P50).Cell slides were stained by Hoechst staining and apoptosis was observed under fluorescence microscope.Cell suspension was stained with PI staining,and cell necrosis was observed and evaluated under fluorescence microscope.DCFH-DA probe was used to conduct active staining of ROS in cell slides to evaluate the level changes of reactive oxygen species in each group.Western blot was used to detect the expression changes of apoptotic proteins Caspase3,cleaved-Caspase 3,Bcl2,Bax,oxidative stress-related proteins Nox4.The expression changes of ROS and MDA related to oxidative stress were detected by kit quantitative method.Results: With the increase of hemin application concentration and the extension of application time,the damage degree of HT22 cells gradually worsened,and the mortality rate reached about 50% at the place where hemin at 50 uM acted for 6h.Therefore,this scheme was selected for subsequent injury model construction.Compared with the blank control group,there was no significant injury in the solvent control group,and the cell damage in the model group was significant,which was positively correlated with the dose distribution.Hoechst results showed that compared with the control group,the number of Hoechst positive cells in the model group increased,that is,the proportion of apoptotic cells increased.Compared with the model group,the proportion of apoptotic cells decreased in the propofol treatment group,and the proportion of apoptotic cells decreased significantly in the high-dose treatment group.PI staining showed that compared with the control group,the number of necrotic cells in the model group increased;compared with the model group,the number of necrotic cells in the treatment group decreased,but it was still higher than that in the blank control group;and the number of necrotic cells in the high-dose treatment group was lower than that in the low-dose treatment group.Western blot showed that compared with the control group,the expression levels of apoptosisrelated proteins Caspase3,cleaved-Caspase3 and Bcl2 were increased in the model group,the expression levels of oxidative stress-related proteins Nox4 and Nrf2 were increased,and the expression levels of Bax were decreased.Compared with the model group,the levels of Caspase3,cleaved-Caspase 3,Nox4,Nrf2 in the treatment group were decreased,and the expression levels of Bax were increased.Compared with the control group,ROS expression increased in the model group,decreased in the treatment group,and decreased more in the high-dose group.Compared with the control group,the expression level of MDA,the product of lipid peroxidation,increased in the model group;there was no statistical difference in the expression level of MDA in the low-dose treatment group compared with the model group.The expression level of MDA decreased significantly in the high-dose treatment group compared with the model group.Conclusion: Propofol has protective effect on hemin injured HT22 hippocampal neurons,slowing down its oxidative stress response and reducing the occurrence of necrotic and apoptotic injury.NADPH oxidase pathway and Nrf2 signaling pathway can upregulating or downregulating the expression levels of ROS and MDA and other effecting components by regulating the oxidative stress response of cells,and also regulate the inflammatory response,or regulate Caspase3,Bcl2,Bax and other classic response pathways which are related to cell apoptosis and necrosis.Propofol may show protective effect on neurons in hemorrhagic injury through NADPH oxidase pathway and Nrf2 signaling pathway. |
PDF Full Text Request