Regulation Of HIF-1α And Effect Of Quercetin In Early Brain Injury Of An Experimental SAH Model

Mortality and morbidity following subarachnoid hemorrhage (SAH) is high, but themechanism of SAH is complicated. In addition to cerebral vasospasm (CVS) well-known72hours after SAH, which causes severe brain injury (delayed brain injury, DBI), recentstudies have found that early brain injury (early brain, EBI) within72hours, includingdecrease of cerebral perfusion pressure (CPP), ion imbalance, glutamate toxicity, oxidativestress, inflammation, endothelial damage, platelet activation and aggregation, permeabilitychanges, activation of cell death pathways, and other series of pathophysiologicalresponses lead to cognitive disorders, neurological defects. They are critical to the overallprognosis of SAH, and the molecular mechanism study in EBI is hotspot. Hypoxiainducible factor-1(HIF-1) is an oxygen sensitive transcription factor, which regulatesmore than70downstream target genes such as vascular endothelial growth factor (VEGF),erythropoietin (EPO), glucose transporter (GLUT), etc. HIF-1includes two subunits ofHIF-1and HIF-1β. The expression HIF-1has been shown upregulated in an animalmodel of focal cerebral ischemic injury, which can play a neuroprotective role. But howabout the regulation of HIF-1expression and the role it plays in SAH is not clear. Theinhibitor of HIF-1is a compound called YC-1. Except that low oxygen environment,several experiments proved the quercetin can upregulate HIF-1expression. Quercetin,which have a clear formula, is one of the components of flavonoids. Different chemicalbonds play different roles. It has the abilities of scavenging oxygen free radicals (ROS),inhibiting lipid peroxidation (LPO), anti-oxidative stress, chelating calcium, iron and otherions, protecting DNA from damage. In Europe and other countries, it has been served as a drug in the market. In domestic application, the most important ingredient of ginkgobiloba extract and gingko injection drugs is quercetin. But the role and mechanism ofquercetin in SAH have not been studied well. Therefore, in order to strengthen the brainprotection, deep study of the mechanisms of HIF-1, quercetin and their interactions inEBI after SAH is needed considerably.The study is divided into five experiments. The first experiment is about theprotective effect of HIF-1against hippocampal apoptosis and cognitive dysfunction in anexperimental rat model of SAH. A mature, stable, reproducible rat model of SAH (cisternamagna double injection model) was established. Rats were divided into a total of threegroups including the sham group, the SAH model dimethyl sulfoxide (DMSO) controlgroup (SAH+vehicle) and the YC-1inhibitor group (SAH+YC-1). YC-1is a specificinhibitor of HIF-1.30min before24h after SAH, the rats in the YC-1group were givena2mg/kg of YC-1(dissolved in DMSO) by intraperitoneal injection. The same dose ofDMSO in the vehicle group, and no drugs in the sham group. At48h after SAH, theexpression of HIF-1and Caspase-3in the hippocampal CA1region of rats in each groupwere observed by immunohistochemistry, and then the protein of Caspase-3, HIF-1andits downstream target molecules VEGF, EPO and GLUT1in the hippocampus by westernblot. Further, hippocampal cell death DNA fragments detected by enzyme-linkedimmunosorbent assay (ELISA). Finally, time and distance to escape, time and distance inthe target quadrant was measured through the Morris water maze (MWM) to show thecognitive changes of rats in each group. The results showed that at48h after the SAHmodel established, the expression of HIF-1and its downstream molecules VEGF, EPOand GLUT1increased in the hippocampus (P <0.01). Compared to the vehicle group, theydecresead in the YC-1group (P <0.01). At48h after SAH, Caspase-3positive cells in therat hippocampal CA1region increased (P <0.01), Caspase-3protein and cell death DNAfragments increased (P <0.01). Compared to the vehicle group, Caspase-3positive cells inthe rat hippocampal CA1region increased further (P <0.01), Caspase-3protein and celldeath DNA fragments increased further (P <0.01). At48h after SAH, the time to escapeand distance traveled increased and time spent and distance traveled in the target quadrant decreased (P <0.01). Compared to the vehicle group, time to escape and distance in theYC-1group increased and time spent and distance traveled in the target quadrantdecreased further (P <0.01). The experiment results showed that at48h after the SAHmodel, the expression of HIF-1and its effector molecules VEGF, EPO and GLUT1inthe hippocampus increased, and the inhibition of their expression deterioratedhippocampal cells apoptosis and reduce the cognitive ability, suggesting that in the earlyphase of SAH, HIF-1may be involved in the protection of hippocampal apoptosis andcognitive function impairment.The second experiment is about the effect of HIF-1on BBB in an experimental ratmodel of SAH. Rats were divided into a total of three groups including the sham group,the SAH+vehicle group and the YC-1group, the administration programs were same asthe first experiment. At48h after SAH, the protein expression of occludin and claudin-5in the brain were detected by western blot. Further, Evans blue in the brain was assessedby spectrophotometer. The results showed that at48h after the SAH model established,the protein expression of occludin and claudin-5increased in the hippocampus (P <0.01),Compared to the vehicle group, they decresead in the YC-1group (P <0.01). At48h afterSAH, Evans blue staining in the brain was obvious, and quantitative analysis showed thatthe content of Evans blue increased in the brain (P <0.01). But it was dodge in the YC-1group, and quantitative analysis showed that the content of Evans blue decreased in thebrain compared to the vehicle group (P <0.01). The experiment results showed that at48h after the SAH model, the reduction of HIF-1expression reduced the permeability ofBBB, and reduced BBB damage, which suggested that HIF-1may be involved in thedeterioration of BBB on the early phase after SAH. The first two experiments suggestedthat HIF-1played a dual regulatory role on the early phase after SAH.The third experiment is about the protective effect of quercetin against oxidativestress and brain edema in an experimental rat model of SAH. Rats were divided into atotal of four groups including the sham group, the SAH+vehicle group, the SAH+quercetin (10mg/kg) group and the SAH+quercetin (50mg/kg) group.30min,12h,24hafter SAH, the rats in the quercetin (10mg/kg) and quercetin (50mg/kg) group were given a dose of10mg/kg and50mg/kg of quecetin (dissolved in0.9%sodium chloride solution)by intraperitoneal injection respectively. The same dose of0.9%sodium chloride solutionin the vehicle group, and no drugs in the sham group. At48h after SAH, the neurologicalfunction score table was used for observing neurological deficit (the higher score, themore severe neurological dysfunction). The activities of CuZn-SOD and GSH-Px, also thelevel of MDA in the cerebral cortex of rats in each group were observed by ELISA. Then,the expression of Caspase-3in the hippocampal CA1region of rats in each group wereobserved by immunohistochemistry, and the protein of Caspase-3in the hippocampus bywestern blot. Finally, the brain water content was determined using the wet/dry method.The results showed that at48h after the SAH model established, neurobehavioral deficitswere observed in the vehicle group (P <0.01). No signifcant difference was observedbetween the quercetin10group and the vehicle group (P>0.05). Neurobehavioral scoreswere significantly decreased in the quercetin50group compared to the vehicle group (P <0.01). At48h after SAH, CuZn-SOD and GSH-Px activities in the rat cerebral corticeswere markedly decreased, and MDA level increased in the SAH＋vehicle group (P <0.01).No signifcant difference was observed between the quercetin10group and the vehiclegroup (P>0.05). CuZn-SOD and GSH-Px activities increased, and MDA level decreasedin the quercetin50group compared to the vehicle group (P <0.01). At48h after SAH, theexpression of Caspase-3in the hippocampal CA1region increased (P <0.01). Nosignifcant difference was observed between the quercetin10group and the vehicle group(P>0.05). The expression of Caspase-3were significantly decreased in the quercetin50group compared to the vehicle group (P <0.01). At48h after SAH, the brain watercontent in the vehicle group was signifcantly increased (P <0.01). No signifcantdifference was observed between the quercetin10group and the vehicle group (P>0.05).The brain water content were significantly decreased in the quercetin50group comparedto the vehicle group (P <0.01). The experiment results showed that at48h after the SAHmodel, treatment with50mg/kg dose of quercetin could enhance the antioxidant activityand reduce the degree of lipid peroxidation in the rat brain, and it could reducehippocampal apoptosis, brain edema and neurological dysfunction. But10mg/kg dose of quercetin did not play these effects. It suggested that the appropriate dose of quercetin onthe EBI after SAH may play a neuroprotective effect through inhibiting oxidative stress,neuronal apoptosis and brain edema, etc.The fourth experiment is about the effect of of quercetin against basilar artery (BA)vasospasm in an experimental rat model of SAH. Rats were divided into a total of fourgroups including the sham group, the SAH+vehicle group, the SAH+quercetin(10mg/kg) group and the SAH+quercetin (50mg/kg) group.15d before (once a day) and30min,12h,24h after SAH, the rats in the quercetin (10mg/kg) and quercetin (50mg/kg)group were given a dose of10mg/kg and50mg/kg of quecetin by intraperitoneal injectionrespectively. The same dose of0.9%sodium chloride solution in the vehicle group, and nodrugs in the sham group. At72h after SAH, the lumen diameter and wall thickness of BAwas observed by HE staining, and the area of BA was statistically calculated. Then, theprotein of eNOS of the BA in each group were observed by western blot. Finally, theconcentration of eNOS in the rat serum in each group was observed by ELISA. The resultsshowed that at72h after the SAH model established, BA area decreased in the vehiclegroup (P <0.01). No signifcant difference was observed between the quercetin10groupand the vehicle group (P>0.05). BA area increased significantly in the quercetin50groupcompared to the vehicle group (P <0.01). At72h after SAH, the protein expression ofeNOS in the BA decreased (P<0.01). No signifcant difference was observed between thequercetin10group and the vehicle group (P>0.05). The protein expression of eNOS in theBA increased in the quercetin50group compared to the vehicle group (P <0.01). At72hafter SAH, the concentrationof eNOS in the rat serum decreased (P<0.01). No signifcantdifference was observed between the quercetin10group and the vehicle group (P>0.05).The concentration of eNOS in the rat serum increased in the quercetin50group comparedto the vehicle group (P <0.01). The experiment results showed that at72h after the SAHmodel, treatment with50mg/kg dose of quercetin could increase the protein expression ofeNOS in rat BA and the concentration in serum, and it could reduce CVS. But10mg/kgdose of quercetin did not play these effects. It suggested that the appropriate dose ofquercetin treatment on the EBI after SAH may play a anti-CVS effect through enhancing the protein expression of eNOS in rat BA and improving the concentration in serum, etc.The fifth experiment is about the effect of quercetin on HIF-1expression in anexperimental rat model of SAH. Rats were divided into a total of three groups includingthe sham group, the SAH+vehicle group and the quercetin group.15d before (once a day)and30min,12h,24h after SAH, the rats in the quercetin group were given a dose of50mg/kg of quecetin by intraperitoneal injection respectively. The same dose of0.9%sodium chloride solution in the vehicle group, and no drugs in the sham group. At48hafter SAH, the expression of HIF-1in the hippocampal CA1region of rats in each groupwere observed by immunohistochemistry, and then the protein of HIF-1in thehippocampus by western blot. The results showed that at48h after the SAH modelestablished, the morphological expression of HIF-1increased in the hippocampal CA1region (P <0.01). Compared to the vehicle group, they incresead further in the quercetingroup (P <0.01). At48h after SAH, the protein expression of HIF-1increased in thehippocampus (P <0.01). Compared to the vehicle group, they incresead further in thequercetin group (P <0.01). The experiment results showed that at48h after the SAHmodel, quercetin can upregulate the expression of HIF-1in the hippocampus, suggestingthat quercetin may play a neuroprotective effect by HIF-1pathway on the early phaseafter SAH.In this paper, we firstly observd the effect of HIF-1on the hippocampal apoptosis,cognitive and BBB function, also firstly detected the effect of quercetin on the cerebraloxidative stress, brain edema and CVS. It was also the first time to study the regulation ofquercetin on HIF-1in the early phase in an experimental rat model of SAH. The resultsshowed that on the early phase after SAH, HIF-1played a dual regulatory role, and theappropriate dose of quercetin treatment may play a neuroprotective effect possibly throughHIF-1pathway. Great efforts are still needed to study in vitro and the interactionmechanisms between HIF-1and quercetin for providing possible targets in order toprotecting EBI after SAH.