| In our country,stroke is one of the leading causes of death and severe long-term disability in adults.The occurrence rate of stroke tends to be increasing year by year and the age of onset to incline to the young.And ischemic stroke is the most common type of stroke,accounting for about 75%of all cerebrovascular disease.The pathogenesis of cerebral ischemia is complicated with limited therapeutic options.The incidence,mortality,isability rate and recurrence rate of cerebral infarction are increasing year by year.Ischemic stroke brings a heavy economic burden to the social and family because of its long recovery time and unsatisfactory prognosis.Intravenous thrombolysis is considered to the most effective treatment for acute cerebral infarction,but due to the strict treatment time window and bleeding complications,intravenous thrombolysis rate is not high.Among a variety of treatment,nerve protective treatment has been studied extensively.However,the clinical effect is not so satisfied as that achieved in animal experiments.Therefore,it is particularly important to find new therapeutic targets and provide a reliable basis for the treatment of cerebral infarction.In recent years,the concept of the‘neurovascular unit’has emerged as a new paradigm for understanding the pathology of CNS disease,including stroke.This modular concept is defined at an intercellular level that comprises dynamic interactions between cerebral endothelial cells,glia,neurons and the extracellular matrix.This concept offers new research and therapeutic targets for treating neurological disorders.Perhaps,preventing neuronal death per se may not be enough.In order to truly rescue brain tissue and function,one may have to rescue all the complex signals and interactions between a network of multiple cell types,including neurons,astrocytes and microvascular endothelial cells.Angiogenesis refers to the generation of new blood vessels from existing vascular endothelial cells（ECs）in order to deliver nutrients and oxygen to various organs and tissue.Thus,angiogenic vessels in the ischemic boundary zone may contribute to recovery of tissue-at-risk by restoring cellular metabolism in surviving neurons as well as provide the essential neurotrophic support to newly generated neurons.The analysis of post mortem brain tissues obtained from patients with varying survival times following stroke revealed increased cerebral microvessel density in the penumbral areas in comparison with the contralateral normal hemisphere.Of note,stroke patients with greater cerebral blood vessel density appear to make better progress and survive longer than patients with lower vascular density,suggesting that active post-ischemic angiogenesis may be beneficial for neurological functional recovery.Therefore,after stroke,it is necessary timely and effective to promote angiogenesis.It can restore the blood supply of ischemic penumbra,provide good nutrition support for the newly born neurons,effectively promote nerve functional recovery,reduce infarct volume and improve the prognosis of patients with cerebral ischemia,prolonging the survival period.Moi et al.（1994）discovered a new transcription factor denominated Nuclear factor erythroid 2-related（Nrf2）which is a protein with a molecular weight of 95-110 kilodalton belonging to the basic leucine zipper transcription factor.Nrf2 is a member of the cap’n’collar（CNC）family of transcription factors,which plays an extremely important role in protecting the body and defensing against oxidative stress.Acute responses of brain tissue to cerebral ischemia and its chronic pathogenic progression involve many pathways,with accumulating evidence implicating reactive oxygen species（ROS）and inflammation as pivotal mediators.Then ROS triggered a series of cellular and molecular events,including nitroso protein oxidation/nitrification,lipid peroxidation and DNA damage,which increased cell damage after cerebral infarction.At the same time,ROS reactive activation of Nrf2,which transfer into the nucleus.Nrf2 mediates the induction of a battery of antioxidant defense enzymes,including heme oxygenase 1（HO-1）,NAD（P）H:quinone oxidoreductase-1（NQO1）,glutathione S-transferase（GST）,which improve the oxidation resistance and detoxification ability in the body.Our previous results suggested that activation of Nrf2 pathway promoted neurological recovery in acute ischemic stroke.However,the role of Nrf2 activation in the long-term recovery of cerebral ischemia is still unclear.Recently,Florczyk et al.discovered that Nrf2 played an important role in physiological,non-inflammatory neovascularization in vitro,which suggested that Nrf2,in addition to its anti-oxidant function,might also promote angiogenesis in vivo.Tert-butylhydroquinone（tBHQ）is widely used as food additives,with high efficiency,low toxicity and so on.Tert-butylhydroquinone（tBHQ）is a known inducer of Nrf2,which can delay the degradation rate of Nrf2 and promote Nrf2 nuclear transfer and the expression of antioxidant enzymes.It has powerful antioxidant capacity,so as to play the role of cell protection.Studies have confirmed that tert-butylhydroquinone played a protective role in acute phase of cerebral ischemia,but it didn’t know whether tert-butylhydroquinone had effect on angiogenesis after cerebral ischemia.Unilateral neocortical ischemia was induced in adult male CD-1 mice and Nrf2 gene mice by permanent occlusion of the distal middle cerebral artery（dMCAO）and ipsilateral common carotid artery.Tert-butylhydroquinone was administered intraperitoneally once a day for 14 days initiated 1 day after stroke.After administered with tert-butylhydroquinone,infarct volume and neurological deficit scores were measured to observe whether tBHQ has protective effect on cerebral ischemia.Microvessel density in the ischemic penumbra of each group though immunofluorescence were measured to observe whether tBHQ promote angiogenesis on cerebral ischemia.After treatment of tBHQ,expression of Nrf2/HO-1 pathway and angiogenic growth factors were measured to explore the possible mechanism of promoting angiogenesis,and to provide theoretical support and experimental basis for the further study and clinical application of tBHQ.This study is divided into three parts,each part of the contents are summarized as follows.Part one The neuroprotective effect of tert-butylhydroquinone in themice of focal cerebral ischemiaObjective:To observe the effect of tert-butylhydroquinone in the mouse model of focal cerebral ischemia through evaluating neurological deficit scores and infarct volumes.Methods:1 Male,healthy adult CD-1 mice were used as the objects,permanent occlusion of the distal middle cerebral artery（dMCAO）and ipsilateral common carotid artery in mice were established.CD-1 mice were randomly divided into five groups:Sham group,Vehicle group,L-tBHQ group,M-tBHQ group,H-tBHQ group.Mice in Sham group received the sham operation and intraperitoneal injection of 3%ethanol/isotonic saline solution once a day for14 days initiated 1 day after sham operation.An equal volume of 3%ethanol/isotonic saline solution was administered by intraperitoneal injection to Vehicle group for 14 consecutive days initiated 1 day after stroke.Mice in L-tBHQ group received the operation of dMCAO and intraperitoneal injection of tBHQ at 20mg/kg for 14 consecutive days initiated 1 day after stroke.Mice in M-tBHQ group received the operation of dMCAO and intraperitoneal injection of tBHQ at 30mg/kg for 14 consecutive days initiated 1 day after stroke.Mice in H-tBHQ group received the operation of dMCAO and intraperitoneal injection of tBHQ at 40mg/kg for 14 consecutive days initiated1 day after stroke.Neurological functional evaluation was performed using rotarod test.In all animals,the rotarod test was performed at d1,d7,d14,d28（10 mice per group）after cerebral ischemia.2 According to the results of the behavioral evaluation,the appropriate concentration of tert-butylhydroquinone was selected.Then we explored whether tert-butylhydroquinone played a role through the Nrf2 signaling pathway.Nrf2 gene mice were used as the objects,permanent occlusion of the distal middle cerebral artery（dMCAO）and ipsilateral common carotid artery in mice were established.All Nrf2+/+and Nrf2-/-mice were randomly divided into six groups:（1）Sham Nrf2+/+group(sham operation with 3%alcohol treated Nrf2+/+mice);（2）Vehicle Nrf2+/+group(equal volume of 3%alcohol treated Nrf2+/+mice after stroke);（3）tBHQ Nrf2+/+group(tBHQ at 40 mg/kg treated Nrf2+/+mice after stroke);（4）Sham Nrf2-/-group(sham operation with3%alcohol treated Nrf2-/-mice);（5）Vehicle Nrf2-/-group(equal volume of3%alcohol treated Nrf2-/-mice after stroke);（6）tBHQ Nrf2-/-group(tBHQ at40 mg/kg treated Nrf2-/-mice after stroke).And tBHQ（Sigma,St Louis,MO,USA）was dissolved in 3%alcohol.The tBHQ was first dissolved in pure alcohol and saline was added to final volume.The mixture was administered to corresponding group at 40 mg/kg for 14 consecutive days through intraperitoneal injection as early as 1 day postoperatively.An equal volume of vehicle solution was also administered by intraperitoneal injection to the corresponding group for 14 consecutive days.In all animals,the rotarod test was performed at d1,d7,d14,d28（10 mice per group）after cerebral ischemia.3 Nrf2 gene mice were used as the objects,permanent occlusion of the distal middle cerebral artery（dMCAO）and ipsilateral common carotid artery in mice were established.All Nrf2+/+and Nrf2-/-mice were randomly divided into six groups:Sham Nrf2+/+group,Vehicle Nrf2+/+group,tBHQ Nrf2+/+group,Sham Nrf2-/-group,Vehicle Nrf2-/-group,tBHQ Nrf2-/-group.At d7after dMCAO,the brains of each group（6 mice per group）were removed quickly under deep anesthesia to measure the infarct volume using TTC staining.Results:1.The effect of tert-butylhydroquinone on the neurological deficit scores of CD-1 miceThe rotarod test was performed on 1,7,14,and 28 days after the operation in Sham group,Vehicle group,L-tBHQ group,M-tBHQ group and H-tBHQ group.There was no significant neurological deficit in Sham group.One day after cerebral ischemia,the dMCAO model is successfully established,and each group of mice have different degree of neurological deficit,the difference was not statistically significant（P>0.05）.Compared with Vehicle group,the neurological deficit scores of H-tBHQ group in d7（rotarod test）improved significantly,and continue to 28 days after cerebral ischemia,and the difference of each time was statistically significant（P<0.05）.2.The effect of tert-butylhydroquinone on the neurological deficit scores of Nrf2 gene miceThe rotarod test were performed on d1,d7,d14 and d28 after the operation in Sham Nrf2+/+group,Vehicle Nrf2+/+group,tBHQ Nrf2+/+group,Sham Nrf2-/-group,Vehicle Nrf2-/-group,tBHQ Nrf2-/-group.There was no significant neurological deficit in Sham Nrf2+/+group and Sham Nrf2-/-group.One day after cerebral ischemia,the dMCAO model is successfully established,Vehicle Nrf2+/+group,tBHQ Nrf2+/+group,Vehicle Nrf2-/-group,and tBHQ Nrf2-/-group have different degree of neurological deficit,and the difference was not statistically significant（P>0.05）.Compared with Vehicle Nrf2+/+group,the neurological deficit scores（rotarod test）of tBHQ Nrf2+/+group improved significantly from d7 to d28after cerebral ischemia,and the difference of each time was statistically significant（P<0.05）.Compared with Vehicle Nrf2+/+group,the neurological deficit scores（rotarod test）of Vehicle Nrf2-/-group decreased significantly from d7 to d28after cerebral ischemia,and the difference of each time was statistically significant（P<0.05）.There was no significant improvement after treatment of tBHQ.3.The effect of tert-butylhydroquinone on infarct volume（d7）No infarction was observed in Sham Nrf2+/+group and Sham Nrf2-/-group.On d7 after stroke,there were different size of infarction in the Vehicle Nrf2+/+group,tBHQ Nrf2+/+group,Vehicle Nrf2-/-group and tBHQ Nrf2-/-group.On d7 after stroke,in Vehicle Nrf2+/+group,the infarction of cerebral cortex was observed,and the infarct volume was 12.04%±0.78%.Compared with the Vehicle Nrf2+/+group,the infarct volume of tBHQ Nrf2+/+group decrease significantly(7.90%±0.79%;Vehicle Nrf2+/+vs.tBHQ Nrf2+/+,P<0.05).The infarct volume of Vehicle Nrf2-/-group was 15.24%±1.37%.After treatment of tBHQ,the infarct volume of tBHQ Nrf2-/-group were unchanged.The infarct volume of tBHQ Nrf2-/-group was 14.61%±1.75%.Conclusion:Tert-butylhydroquinone improved the neurological function,reduced infarct volume through activation of Nrf2 signaling pathway,which played a pivotal role in neuroprotective process.Part two The angiogenesis effect of tert-butylhydroquinone in the miceof focal cerebral ischemiaObjective:Nrf2 gene mice were used as the objects,permanent occlusion of the distal middle cerebral artery（dMCAO）and ipsilateral common carotid artery in Nrf2+/+mice and Nrf2-/-mice were established.We observe the angiogenesis effect of tBHQ in the mouse model of focal cerebral ischemia through evaluating microvessel density and endothelial cell proliferation.We explore whether the treatment of tBHQ promote angiogenesis and neurological functional recovery though Nrf2 signaling pathway.We explore the related mechanism of tBHQ promoting angiogenesis in the mice of focal cerebral ischemia though observing the effect of tBHQ in Nrf2/HO-1 pathway and angiogenic growth factors.It provides theoretical support and experimental basis for the further study and clinical application of tBHQ.Methods:Nrf2 gene mice were used as the objects,permanent occlusion of the distal middle cerebral artery（dMCAO）and ipsilateral common carotid artery in mice were established.All Nrf2+/+and Nrf2-/-mice were randomly divided into six groups:（1）Sham Nrf2+/+group(sham operation with 3%alcohol treated Nrf2+/+mice);（2）Vehicle Nrf2+/+group(equal volume of 3%alcohol treated Nrf2+/+mice after stroke);（3）tBHQ Nrf2+/+group(tBHQ at 40mg/kg treated Nrf2+/+mice after stroke);（4）Sham Nrf2-/-group(sham operation with 3%alcohol treated Nrf2-/-mice);（5）Vehicle Nrf2-/-group(equal volume of 3%alcohol treated Nrf2-/-mice after stroke);（6）tBHQ Nrf2-/-group(tBHQ at 40 mg/kg treated Nrf2-/-mice after stroke).And tBHQ was dissolved in 3%alcohol.TBHQ was first dissolved in pure alcohol and saline was added to final volume.The mixture was administered to corresponding group at 40 mg/kg for 14 consecutive days through intraperitoneal injection as early as 1 day postoperatively.An equal volume of vehicle solution was also administered by intraperitoneal injection to the corresponding group for 14 consecutive days.We calculated the microvessel density and endothelial cell proliferation of each group on d7,d14 and d28 to explore whether the treatment of tBHQ promote angiogenesis and neurological functional recovery though Nrf2 signaling pathway.Western blot were employed to detect the expression of Nrf2,HO-1 and VEGF in each group on d7 and d14 after cerebral ischemia.We explore the related mechanism of tBHQ promoting angiogenesis.Results:1.The effect of tBHQ on angiogenesis in the mice of focal cerebral ischemiaThe microvessel density（MVD）of each group was detected by immunofluorescence stain on d7,d14 and d28 after cerebral ischemia.On d7after cerebral ischemia,in Nrf2+/+mice,compared with Vehicle Nrf2+/+group,after the treatment of tBHQ,the microvessel density of tBHQ Nrf2+/+group increased significantly（P<0.05）.Whereas in Nrf2-/-mice,compared with Vehicle Nrf2-/-group,after the treatment of tBHQ,there was no increase in microvessel density（P>0.05）.The change of microvessel density in d14 and d28 after cerebral ischemia were consistent with d7.To determine endothelial cell proliferation,the number of endothelial cells positive for both BrdU and CD31 was also quantified on day 14.And the tBHQ Nrf2+/+group was significantly increased compared to the vehicle Nrf2+/+group on d14.2.The effect of tBHQ on Nrf2 nuclear translocationIn Western blot analysis,compared with Vehicle Nrf2+/+group,the nuclear protein Nrf2 of t BHQ Nrf2+/+group in the right cortex were significantly increased at d7 and d14 after cerebral ischemia（P<0.05）.Compared with Vehicle Nrf2+/+group,there was a statistically significant decreased in Vehicle Nrf2-/-group.After treatment of tBHQ,the nuclear protein Nrf2 of tBHQ Nrf2-/-group were unchanged,and the difference was not statistically significant（P>0.05）.3.The effect of tBHQ on the expression of HO-1 and VEGF in the mice of focal cerebral ischemiaIn Western blot analysis,compared with Vehicle Nrf2+/+group,the expression level of HO-1 and VEGF in tBHQ Nrf2+/+group significantly increased on d7,d14 after cerebral ischemia（P<0.05）.However,the similar role of tBHQ on promoting angiogenesis in Nrf2-/-mice was blocked.Compared with Vehicle Nrf2-/-group,the protein expression of HO-1 and VEGF in tBHQ Nrf2-/-group did not increase in d7 and d14,and the difference was not statistically significant（P>0.05）.Conclusions:1.Tert-butylhydroquinone increased microvessel density and endothelial cell proliferation,promoted angiogenesis and neurological functional recovery though Nrf2 signaling pathway.2.After cerebral ischemia,treatment of tBHQ increased the Nrf2 nuclear protein and its downstream factors,simultaneously increased angiogenic growth factors VEGF.Whereas in Nrf2-/-mice,after the treatment of tBHQ,there was no increase in the expression of HO-1 and VEGF.This suggested that tBHQ increased the expression of VEGF through activating of Nrf2/HO-1signaling pathway,which promoted angiogenesis and neurological functional recovery after cerebral ischemia.Part three Delayed administration of the GLP-1 analogue liraglutidepromoting angiogenesis after focal cerebral ischemia in miceObjective:To observe the effect of liraglutide in the mouse model of focal cerebral ischemia through evaluating neurological deficit scores and infarct volumes.To observe the angiogenesis effect of liraglutide and the related mechanism in the mouse model of focal cerebral ischemia through evaluating microvessel density and endothelial cell proliferation,simultaneously assessing the angiogenic growth factors.Methods:CD-1 mice were used as the objects,permanent occlusion of the distal middle cerebral artery（dMCAO）and ipsilateral common carotid artery in mice were established.In the first set of studies,mice were randomly assigned to four groups:sham group,normal saline group（NS group）,L-Lirglutide group（100μg/kg）,H-Liraglutide group（200μg/kg）.The dosage regimen of liraglutide was determined based on previous study.The day of surgery was defined as day 0.And liraglutide was dissolved in NS and administered to the liraglutide group at 100μg/kg or 200μg/kg for 14consecutive days through intraperitoneal injection as early as 1 day postoperatively.The sham group and NS group received an equal volume of NS in the same way.These mice were used for the rotarod test and finding the optimal dose of the liraglutide in the following experiments.In the second set of studies,mice were randomly assigned to three groups:sham group,NS group,liraglutide group（200μg/kg）.These mice were administrated by intraperitoneal injection with 5-bromo-20-deoxyuridine（BrdU）once daily for 14 consecutive days as early as 1 day postoperatively.At 7,14 and 28 days after stroke as the experimental design,the mice were killed and the brain samples were collected for further study.We calculated the microvessel density and endothelial cell proliferation of each group to explore whether the treatment of liraglutide promote angiogenesis and neurological functional recovery.Western blot were employed to detect the expression of VEGF in each group on d7 and d14 after cerebral ischemia.We explore the related mechanism of liraglutide promoting angiogenesis.Results:1.The effect of liraglutide on the neurological deficit scoresThe rotarod test was performed on 1,7,14,and 28 days after the operation in Sham group,NS group,L-Liraglutide group and H-Liraglutide group.There was no significant neurological deficit in Sham group.One day after cerebral ischemia,the dMCAO model is successfully established,and each group of mice have different degree of neurological deficit,the difference was not statistically significant（P>0.05）.Compared with NS group,the neurological deficit scores of L-Liraglutide group improved significantly from d7 to d28 after cerebral ischemia,but the difference was not statistically significant（P>0.05）.The neurological deficit scores of H-Liraglutide group in d7（rotarod test）improved significantly,and continue to 28 days after cerebral ischemia,and the difference of each time was statistically significant（P<0.05）.2.The effect of liraglutide on infarct volume（d7）No infarction was observed in the sham group.The infarct volume of the liraglutide group was obviously smaller than that of the NS group on day 7.3.The effect of liraglutide on the angiogenesis process in the mice of focal cerebral ischemiaThe microvessel density（MVD）of each group was detected by immunofluorescence stain on d7,d14 and d28 after cerebral ischemia.On d7after cerebral ischemia,compared with NS group,after the treatment of liraglutide,the microvessel density of liraglutide group increased significantly（P<0.05）.To determine endothelial cell proliferation,the number of endothelial cells positive for both BrdU and CD31 was also quantified on day 14.And the liraglutide group was significantly increased compared to NS group.4.The effect of liraglutide on the expression of VEGFIn Western blot analysis,compared with NS group,the expression level of VEGF in liraglutide group significantly increased on d7,d14 after cerebral ischemia（P<0.05）.Conclusions:1.Liraglutide improved the neurological function and reduced infarct volume,which played a pivotal role in neuroprotective process.2.Liraglutide increased microvessel density and endothelial cell proliferation.Liraglutide also promoted angiogenesis though increasing VEGF,which promoted neurological functional recovery after cerebral ischemia.Conclusions:1.Tert-butylhydroquinone improved the neurological function,reduced infarct volume through activation of Nrf2 signaling pathway,which played a pivotal role in neuroprotective process.2.This suggested that tBHQ increased the expression of VEGF through activating of Nrf2/HO-1 signaling pathway,which promoted angiogenesis and neurological functional recovery after cerebral ischemia.3.Liraglutide improved the neurological function and reduced infarct volume,also promoted angiogenesis though increasing VEGF. |
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