The Role Of NOX2/NOX4 On Early Brain Injury After Experimental Subarachnoid Hemorrhage And Its Mechanism Research

Part Ⅰ: The expression of NOX2/NOX4 in the brain of rats after subarachnoid hemorrhage Objective To investigate the expression of NOX2 / NOX4 which belong to the nicotinamide adenine dinucleotide phosphate oxidase(NADPH oxidase)family after the subarachnoid hemorrhage(SAH)and the relationships with EBI.1.Experimental animals were divided into healthy adult male Sprague-Dawley(SD)rats,were randomly divided into sham operation group,SAH 3h group,SAH 12 h group,SAH 2h group,SAH 48 h group a total of 5 groups,each group 12 Rat only.2.SAH group model was established: the extraction of autologous arterial blood from rats,into the visual crossover.Rats were sacrificed at 3 hours,12 hours,24 hours,48 hours after surgery and the brain was perfused.3.The changes of NOX2 / NOX4 protein in brain tissue of rats were detected by Western-blot.The levels of NOX2 / NOX4 in neurons and astrocytes and the relationship between the expression of NOX2 / NOX4 and the apoptosis of brain cells were observed by immunofluorescence staining.4.In vitro experiment: Prepare the dish 1 day in advance with sterile 12-well plates,take the brain of 16th–18th days fetal rats,digest and wash,treated with 200 mesh filter.Changing the neurol basil medium every 2 days,each time for half the liquid.Treat with si RNA and over-expression m RNA(Ribobio,Guangzhou,China)and hemoglobin after 14 days.Extract the extracellular medium,proteins and coverslips at the certain time after treatment according to the experimental design.Cells cultured for 14 days-18 days,the cells were divided into 2 groups: normal group,Oxy Hb group.Western blot results shows the outcomes of the regulation of NOX2/4 expression,TUNEL staining show the apoptosis rate of neurons,LDH assay shows the degeneration rate of neurons.Results1.Compared with sham group,the expression of NOX2/NOX4 in brain tissue began to increase at 3 hours after SAH,and reached the peak at 12 hours after SAH(P <0.01),then decreased after 48 hours after SAH The expression level of NOX2/NOX4 protein was still significantly higher than that of sham operation group(P <0.01).2.The expression of NOX2/NOX4 in the neurons and astrocytes of the temporal basal ganglia was significantly higher than that in the sham operation group at 12 hours after SAH.In the NOX2/NOX4 high expression of brain cells showed TUNEL positive,NOX2 / NOX4 low expression of brain cells showed TUNEL negative.3.In the in vitro experiment,the western blot analysis showed that levels of NOX2 and NOX4 expression in Oxy Hb group were higher than in the sham group(p<0.01);Oxy Hb group shows higher apoptosis rate(p<0.01)and degeneration rate(p<0.01).ConclusionsNOX2 / NOX4 reached a peak at 12 to 24 hours after SAH and was within EBI(within 72 hours).After SAH,NOX2/NOX4 protein levels in brain tissue,especially neurons and astrocytes,The expression of NOX2/NOX4 was associated with the apoptosis of brain cells,suggesting that NOX2/NOX4 may be involved in the development of EBI after SAH.Part Ⅱ: Effects of NOX2/NOX4 on EBI after SAH in rats.ObjectiveTo investigate the effect of NOX2/NOX4 on EBI after SAH in rats and whether it can reduce the level of EBI after SAH by regulating NOX2/NOX4.Methods1.Animals were divided into two groups: healthy adult male SD rats,were randomly divided into sham operation group,SAH group,SAH + NOX2 inhibitor gp91 ds-tat Peptide2 group,SAH + NOX4 inhibitor GKT137831 group,SAH + NOX2 & 4 inhibition Ingredients apocynin group,12 per group.2.Administration: In the SAH + gp91 ds-tat Peptide2 group,the NOX2 inhibitor gp91 ds-tat Peptide2 was dissolved in physiological saline and intraventricular injection was performed at a dose of 100 ng / kg per rat;in the SAH + GKT137831 group,NOX4 inhibitor GKT137831 was dissolved in normal saline and subcutaneously at a dose of 60 mg / kg body weight.In the SAH + apocynin group,the NOX2 & 4 inhibitor apocynin was dissolved in normal saline and injected intraperitoneally at a dose of 50 mg / kg body weight.All drugs were performed 30 minutes before SAH modeling.The vehicle group was injected with the same amount of saline in the lateral ventricle,subcutaneously and intraperitoneally 30 minutes before the SAH model.3.SAH group model was established: the rat autologous femoral artery blood,skull bone through the stereotactic instrument after the non-heparinized arterial blood into the visual crossover front pool.In the sham operation group,the same skull drilling and femoral artery were punctured,but the arterial blood was not injected into the optic chiasm.4.The rats in each group were sacrificed 24 hours after the model,and then the rats were sacrificed and the brain tissue of the temporal cortex was taken.The degeneration of neurons in SAH cells was analyzed by FLUORO-JADE B fluorescence staining.The content of albumin in brain tissue was detected by Western-blot.The integrity of blood-brain barrier was analyzed.TUNEL staining was used to analyze the cerebral cortex And the cerebral edema after SAH was measured by dry and wet method.5.In vitro experiment: Prepare the dish 1 day in advance with sterile 12-well plates,take the brain of 16th–18th days fetal rats,digest and wash,treated with 200 mesh filter.Changing the neurol basil medium every 2 days,each time for half the liquid.Treat with si RNA and hemoglobin after 14 days.Extract the extracellular medium,proteins and coverslips at the certain time after treatment according to the experimental design.Cells cultured for 14 days-18 days,the cells were divided into six groups: normal group,Oxy Hb group,Oxy Hb+NC group Oxy Hb +si NOX2 group,Oxy Hb +si NOX4 group,Oxy Hb +si NOX2&4 group.Western blot results showed the outcomes of the regulation of NOX2/NOX4 expression and the UNEL staining show the apoptosis rate of neurons,LDH assay shows the degeneration rate of neurons,Results1.The FLUORO-JADE B fluorescence staining showed that the three NOX2 / 4 inhibitors inhibited neuronal degeneration in different degree compared with the sham-operated group,and the apocynin effect was the most significant in 24 hours after SAH2.In vivo.Significantly.TUNEL test results showed that TUNEL positive cells were absent in the cerebral cortex of sham operation group,and SAH group was scattered in more TUNEL positive cells.The number of TUNEL positive cells decreased after three kinds of NOX2 / 4 inhibitors pretreated,and the apocynin effect was the most significant.(P <0.01).3.After pretreatment with three kinds of NOX2 / 4 inhibitors,the contents of albumin in brain edema and brain tissue were decreased,and the effect of apocynin was significantly increased(P <0.01),and the content of albumin in brain tissue was significantly increased after SAH Most notable.4.Compared with SAH group,the neurotoxicity of three kinds of NOX2 / 4 inhibitors pretreated in vivo was significantly improved,and apocynin was the most significant.5.The results of Western Blot showed that the expression of NOX2 and NOX4 protein was significantly up-regulated(P <0.01)after 24 hours of Oxy Hb treatment in vitro,and the results were consistent with the first part.Can significantly down-regulate the protein levels of NOX2 and NOX4(P <0.01).6.The survival rate of neuronal cells was significantly higher than that of Oxy Hb + NC group(P <0.05)after silencing NOX2 / 4 by small interfering RNA in vitro.7.The number of TUNEL positive cells in neuronal cells was significantly lower than that in Oxy Hb + NC group(P <0.01)after silencing NOX2 / 4 by small interfering RNA in vitro.8.The content of lactate dehydrogenase(LDH)in the supernatant of neuronal cells was significantly lower than that of Oxy Hb + NC group(P <0.01)by small interfering RNA silencing NOX2 / 4 in vitro.ConclusionsIn vivo and in vitro SAH model,neuronal apoptosis and degradation can be alleviated by using NOX2 / NOX4 inhibitors and small interfering RNA,as well as brain edema and blood-brain barrier damage;NOX2 / NOX4 is involved in the apoptosis of neurons after SAH Degeneration,and brain edema and blood-brain barrier damage process.Part Ⅲ: The mechanism of NOX2 / NOX4 involved in action of EBI study after SAH ObjectiveTo investigate its possible mechanism of NOX2 / NOX4 which participated in the EBI after SAH Methods1.Experimental animal groups: 60 healthy adult male SD rats were randomly divided into 5 groups respectively as control group,SAH group,SAH + gp91 ds-tat Peptide2(NOX2 inhibitor)group,SAH + GKT137831(NOX4 inhibitor)group,SAH + apocynin(NOX2/4 inhibitor)group,12 rats in each group.2.SAH model: the optic chiasma pool before blood law into SAH animal model.All SAH rats by autogenous femoral artery of extraction of heparinization pool before injection of optic chiasma,SAH model,24 hours after SAH kill rats.Control rat femoral artery blood and skull drilling,but without blood injection,within 24 hours after the treatment to be put to death in the rat.3.The dosing method: gp91 ds-tat Peptide2 was dissolved in normal saline,with 100 ng/kg dose of each rat(lateral ventricle injection);GKT137831 was dissolved in normal saline,with 60 mg/kg body weight(subcutaneous injection);apocynin was dissolved in normal saline,with doses of 50 mg/kg body weight(abdominal cavity injection).All drugs are made 30 mins before SAH models.SAH group injectedsame amount of saline solution at the same time.4.The detection index: SAH animal model rats were executed after 24 hours.The bottom of the temporal cortex of rats’ brain tissue were removed as specimens.The ROS kits measured ROS content of brain tissue around hematoma.Western blot was used to analyze tissue marker protein of endoplasmic reticulum stress(CHOP)and endoplasmic reticulum stress mediated apoptosis protein(active-caspase-12).5.SAH model in vitro: using oxyhemoglobin(Oxy Hb)to deal with the generation of rat cerebral cortex neurons.Divided into normal control group,Oxy Hb group,Oxy Hb + NC group,Oxy Hb + si NOX2,Oxy Hb + si NOX4 group and Oxy Hb + si NOX2 & four groups,each experiment are independent repeated three times.Oxy Hb groups: Oxy Hb processing was added in the neurons in vitro cell culture medium for 24 hours.Oxy Hb + NC groups: the Control Si RNA treatment.si NOX2 group,si NOX4 group and si NOX2 & 4 groups: the three groups of transient transfection in vitro cultured neurons respectively si NOX2,si NOX4 and si NOX2/4,and then accept Oxy Hb processing testing for 24 hours.Results1.In the body: three NOX2/4 inhibitors could reduce levels of ROS after SAH and the effect of apocynin was the most significant(P < 0.05).2.Inhibition of NOX2 / NOX4 could decrease protein levels of CHOP and active-caspase-12 around hematoma after SAH in rats(P < 0.05).3.In vitro: NOX2 / NOX4 si RNA interference could reduce and active-caspase-12 protein levels(P < 0.05).ConclusionROS produced in the brain tissue around hematoma after SAH increased.Endoplasmic reticulum stress after SAH was activated and its mediated apoptosis occurred.NOX2 and NOX4 could induce endoplasmic reticulum stress via activating ROS generation,promoting apoptosis and exacerbating the EBI.