| [Background]Stroke is the second leading cause of death after ischemic heart disease worldwide.As the Chinese economy develops and the way of life changes,it has become the first fatal disease in our country,among which ischemic stroke accounts for 69.6%to 77.8%,causing serious loss and heavy economic burden to both society and family.The main treatment for acute stroke is early rt-PA thrombolysis or mechanical thrombolysis to reopen the blood vessel.However,the treatment efficiency needs to be further improved due to 3-4.5-hour time window and the complications of cerebral edema,intracranial hemorrhage and hemorrhagic transformation caused by ischemia-reperfusion(I/R)injury after vascular recanalization.Cerebral I/R injury is involved in complex pathogenesis including energy metabolism deficiency,inflammation,oxygen free radical injury,calcium overload,nitric oxide and nitric oxide synthase,excitatory amino acid toxicity,blood-brain barrier(BBB)damage,cell necrosis and apoptosis.Among them,BBB damage and inflammatory response play a key role in the pathological process of ischemia-reperfusion injury.Microglia,as a component of neurovascular units,contributes to maintaining the structural integrity and function of the BBB as well as the initiation and regulation of neuroinflammation.As the major active ingredient of traditional Chinese herb Gastrodia elata Blume,phenolic glucoside gastrodin(GAS)has been widely used in clinical practice.It has neuroprotective effects against various neurological diseases through multiple ways such as anti-oxidative stress,anti-neuroinflammatory response,regulation of neurotransmitters and neural remodeling,anti-apoptosis and anti-autophagy.Currently,little is known to the protective mechanisms of gastrodin on the BBB during I/R injury as well as neuroinflammation caused by I/R injury of microglia cells.Therefore,this study aimed to investigate the protective effect of gastrodin on neurobehavioral and volume of cerebral infarction in rats with cerebral I/R injury and to evaluate the effect of gastrodin on BBB and related proteins.Besides,it explored the regulation and mechanism of gastrodin on neuroinflammation induced by I/R injury of microglia cells,providing a new potential therapeutic target and idea for the treatment of ischemic stroke.Part Ⅰ Study on the neuroprotective effect of gastrodin pretreatment on cerebral ischemia-reperfusion injury in ratsObjective:To establish a Sprague-Dawley(SD)rat model of ischemia-reperfusion in vivo.Additionally,an oxygen-glucose deprivation/reperfusion(OGD/R)model using BV-2 microglia was established in vitro to simulate I/R injury.This study aimed to access the effects of gastrodin on neurobehavioral,the volume of cerebral infarction and brain water content in rats with I/R injury by gastrodin pretreatment and to evaluate its effect on BBB.This research was also designed for studying regulation of gastrodin on BBB-associated proteins in microglia in vivo and in vitro.Methods:Middle cerebral artery occlusion(MCAO)model of SD rats were established using suture method for the experiment of I/R in vivo.I/R injury was simulated in vitro by OGD/R of BV-2 microglia cells.The model groups were pretreated with the following doses of gastrodin(in vivo:50 mg/kg,100 mg/kg,200 mg/kg;in vitro:20μmol/L,40 μmol/L,80 μmol/L)to be compared with the positive control group nimodipine(NIM)as well as negative control group.Garcia JH score was used to evaluate the neurobehavioral changes in each group;TTC staining was adopted for measuring the volume of cerebral infarction;The change of the BBB permeability was evaluated by determining Evans blue(EB)as well as cerebral water content in brain tissues and HE staining test;Western blot and immunofluorescence staining were applied for detecting the expression levels of matrix metalloproteinases(MMP2 and MMP9),water channel protein aquaporin-4(AQP4)and zonula occludens protein 1(ZO-1)in rats and microglia in each group.Results:We showed that the neurological scores of rats in MCAO group pretreated with different concentrations of GAS and NIM were significantly improved compared with the MCAO group(P<0.001),and the group with GAS at a dose of 100 mg/kg had the highest score among all treatment groups.Moreover,the volume of cerebral infarction in the GAS pretreatment groups at doses of 100 mg/kg and 200 mg/kg was significantly reduced(P<0.001)in comparison with MCAO group.Besides,Evans blue leakage volume was significantly increased in MCAO group,whereas it was significantly reduced in GAS pretreatment group and NIM group compared with that in MCAO group(P<0.01,P<0.001).The GAS pretreatment group at a dose of 100 mg/kg showed the most obvious decrease trend.Furthermore,the water content of brain tissue was significantly increased in MCAO group,but significantly reduced in GAS pretreatment group(100mg/kg)(P<0.01).Additionally,Western blot results showed that the expression of MMP2,MMP9 and AQP4 in the MCAO group were significantly increased after 72 hours of I/R injury(P<0.01),while the expression of ZO-1 was decreased obviously(P<0.01).Meanwhile,the expression of MMP2,MMP9 and AQP4 in GAS pretreatment group(100 mg/kg)and NIM pretreatment group(4 mg/kg)were significantly reduced compared with those in MCAO group(P<0.01),while the expression of ZO-1 was increased.Immunofluorescence showed a similar trend in microglia cells in brain tissue.Furthermore,Western blot results showed that the protein changes of OGD/R model in BV-2 cells were most obvious at 3h and 6h time points.The increasing trend of MMP2,MMP 9 and AQP4 after OGD/R were inhibited in GAS pretreatment group(40 μmol/L,80μmol/L)and the expression of ZO-1 was increased at 3h.Conclusion:The present results have shown that SD rats still had neurologic impairment,the increased BBB permeability and water content in brain after 72 hours of cerebral I/R.Nevertheless,gastrodin pretreatment significantly compensated for the neurological behavior defects in rats of MCAO group,reduced the infract size and reversed BBB impairment.Gastrodin inhibited the expression of MMP2,MMP9 and AQP4 in vivo and in vitro of microglia cells at doses of 100 mg/kg and 40 μmol/L,respectively and increased the expression of ZO-1 to exert its protective effect on MCAO and OGD/R models with I/R injury.Part Ⅱ Regulation of gastrodin on SOX4 in microglia with ischemia-reperfusion injuryObjective:An animal model and a cell model of ischemia-reperfusion injury were established in the experiment.Western blot and immunofluorescence assay were applied for examining the changes of SOX4 expression in microglia after I/R injury in vivo and in vitro.Meanwhile,the study aimed to investigate the effect of gastrodin on SOX4 expression in microglia by interfering with the animal model and cell model of ischemia-reperfusion injury.Gastrodin was used to intervene SOX4-overexpressed BV-2 cells and the changes of MMP2 and MMP9,AQP4 and ZO-1 expression in BV-2 cells were detected to study the protective mechanism of gastrodin on ischemia-reperfusion injury.Methods:A Sprague-Dawley(SD)rat model of ischemia-reperfusion was established using suture method.Primary microglia in rats were extracted,purified and then cultured for passage.Following this,OGD/R models using B V-2 microglia and primary microglia were established in vitro to simulate I/R injury.SD rats and microglia were randomly divided into control group,model group and GAS(in vitro:50 mg/kg,100 mg/kg,200 mg/kg;in vivo:20 μmol/L,40 μmol/L,80 μmol/L)pretreatment groups.SOX4 basal expression in rat brain tissue and two microglial cell lines and its change after MCAO,OGD/R stimulation and GAS intervention was detected by Western blot and immunofluorescence assay.SOX4 overexpression and control plasmids were constructed;Lentivirus vector system was used to package SOX4 lentivirus with GFP fluorescent and control no-load virus;BV-2 cell lines stably infected by SOX4 lentivirus and control virus were established.Then the transfected cell line OGD/R model was intervened by gastrodin,and Western blot and immunofluorescence staining were used to detect the changes of SOX4,MMP2 and MMP9,AQP4 and ZO-1 expression.Results:SOX4 protein was basically expressed in BV-2 immortalized microglia cell line,primary microglia cell line of newborn SD rats and little in microglia in the corpus callosum of SD rats.Besides,Western blot results showed that the expression of SOX4 in brain tissue of MCAO group was increased after 72 hours(P<0.01).Meanwhile,the increasing trend of SOX4 expression can be inhibited by GAS treatment at various concentrations.SOX4 expression was reduced notably at dose 100 mg/kg.Immunofluorescence showed a similar trend in microglia cells in brain tissue.Furthermore,Western blot results showed that SOX4 was upregulated most significantly at 3h of OGD/R BV-2 cells and primary microglia cells,yet GAS(40μmol/L)pretreatment could inhibit its upward trend at 3h.Moreover,SOX4,MMP2,MMP9 and AQP4 expression were decreased and ZO-1 expression was increased after GAS(40 μmol/L)pretreatment in OGD/R BV-2 microglia.However,GAS(40 μmol/L)pretreatment could not reduce the expression of MMP2,MMP9 and AQP4 of increase ZO-1 expression in SOX4-overexpressed cell lines.Conclusion:SOX4 protein was basically expressed in BV-2 immortalized microglia cell line,primary microglia cell line of newborn SD rats and little in microglia in the corpus callosum of SD rats.Additionally,SOX4 expression was significantly increased in microglia both in vivo and in vitro with OGD/R and MCAO,while its upward trend would be inhibited by GAS pretreatment.Finally,SOX4 overexpression could reverse the effect of GAS on the MMP2,MMP9,AQP4 and ZO-1 expression in OGD/R microglia,indicating that GAS regulated MMP2,MMP9,AQP4 and ZO-1 expression via SOX4 to exert neuroprotective effects. |
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