Protective Effect And Mechanism Of Butylphthalide Against Oxidative Stress And Excitatory Intoxication Of Nerve Cells And Hemorrhagic Brain Injury

BackgroundCerebral hemorrhage is a subtype of stroke,accounting for 10%-15%of all strokes.It is characterized by acute onset,high mortality and morbidity.The mortality rate of cerebral hemorrhage is about 40%.At the same time,some patients with cerebral hemorrhage leave serious neurological disorders.At present,cerebral hemorrhage is mainly treated by symptomatic support and surgical treatment,such as antihypertension,dehydration,nerve nutrition and so on,but the therapeutic strategies are not sufficiently effective.Therefore,the problem that finding effective drugs for the treatment of cerebral hemorrhage is urgently need to be solved in stroke.Local edema and inflammation of intracerebral hemorrhage causes intracellular environment metabolism disorders,such as oxidative stress,endoplasmic reticulum stress,mitochondrial damage,excitatory poisoning,calcium influx and so on,which ultimately determine the degree of neurological damage.There are mainly three types of neuronal death,autophagy,apoptosis and necrosis,which of them can change from one type of death to the other with no clear boundary.At present,studies have shown that cerebral hemorrhage induces autophagy to cause neuronal damage,while autophagy inhibitor（3-MA）can inhibit autophagy and protect neurons.As a neurotrophic drug,butylphthalide can reduce neuronal damage by improving circulation and reducing brain edema.It has been widely used in treating ischemic stroke in clinical practice.According to the research that NBP can inhibit autophagy and apoptosis by activating Akt/m TOR signal pathway and improving perfusion injury after cerebral ischemia,but its role in autophagy in cerebral hemorrhage has not been reported.Therefore,we selected H₂O₂oxidative stress model,glutamate excitotoxicity model and intracerebral hemorrhage model of mice to study whether butylphthalide can protect neurons by regulating autophagy and explored its application value in cerebral hemorrhage.ObjectiveTo investigate the protective effect and mechanism of butylphthalide on oxidative stress and excitatory intoxication of nerve cells and hemorrhagic brain injury.Method1.SH-SY5Y were selected for the experiment.（1）Treated with 50μM,100μM,200μM,300μM and 400μM of H₂O₂,SH-SY5Y cells were dynamically observed the morphological changes under microscope,and the effect of H₂O₂ on the survival of SH-SY5Y cells was detected by MTT method,（2）SH-SY5Y cells were directly treated with different concentrations of 10μM,20μM,40μM,80μM and 100μM butylphthalide,then we observed the changes of SH-SY5Y cells under microscope.MTT was used to detect the effect of NBP on the survival of SH-SY5Y and to detect the cytotoxicity of NBP,（3）SH-SY5Y were preincubated with NBP for 3h,and then the oxidative stress model of SH-SY5Y was stablished with 200μM H₂O₂ for 2h.We observed the changes of cells under microscope and detected the changes of cell vitality by MTT,（4）Divided into 8 groups:DMSO,NBP 40μM,NBP 80μM,NBP 100μM,H₂O₂+DMSO,H₂O₂+NBP 40μM,H₂O₂+NBP 80μM,H₂O₂+NBP 100μM,SH-SY5Y cells were directly treated with DMSO/NBP in the first four groups for 5h,and DMSO/NBP in the latter four groups were preincubated for 3h,while H₂O₂ model was established for 2h.Then we collected the Protein samples and detected the expression of Beclin-1,p62,LC3B,Caspase3 and Caspase6 by western blot.2.Cerebellar granule neurons of SD mice（CGNs）were selected for theexperiment.（1）CGNs was treated with different concentrations of glutamate（50μM,100μM,200μM）in 25K medium for 12h.We observed the change under microscope and counted the nuclear pyknosis Hochest33258 staining,（2）CGNs were treated with different concentrations of NBP（50μM,100μM,200μM）in 25K medium for 12h.We observed the change of CGNs under microscope and counted nuclear pyknosis by Hochest33258 staining,（3）Glutamate excitotoxicity model was established by using 100μM glutamate.CGNs was treated with NBP and glutamate of 50μM,100μM and 200μM for 12h.We observed the change of cells under microscope and counted the rate of apoptosis after Hochest22358 staining,（4）After CGNs were treated with 25μM,50μM,100μM,200μM NBP and glutamate12h,we detected the protein expression of autophagy and apoptosis-related proteins by western blot.3.The cerebral hemorrhage model of C57 mice was established.（1）C57 mice were divided into three groups:Sham group,ICH group and ICH+NBP group.After injecting 0.5μl collagenase,mice were injected intraperitoneally with vegetable oil or NBP for three days.After 3 days,the mice were evaluated by Garcia neurobehavioral score and the area of intracerebral hematoma,（2）The homogenate of brain tissue around blood was taken out,and the expression of autophagy and apoptosis-related proteins around intracerebral hemorrhage was detected by Western Blot,（3）The brain tissue was fixed after perfusion,and the expression of LC3BⅡwas detected by immunofluorescence.Result1.In the oxidative stress injury model of H₂O₂,NBP inhibits autophagy andprotects SH-SY5Y.（1）The death rate of SH-SY5Y cells increased with the increase of H₂O₂concentration.MTT results showed that the survival rate of SH-SY5Y cells decreased with the increase of concentration,and the difference was statistically significant（P<0.05）;（2）NBP directly acted on SH-SY5Y cells,but there was no significant change in cell morphology and survival rate（P>0.05）,suggesting that NBP at this concentration had no drug toxicity and did not affect proliferation;（3）In H₂O₂ oxidative stress injury model of SH-SY5Y,NBP could reduce the death of SH-SY5Y cells in a concentration-dependent manner,and the difference was statistically significant;（4）Comparing with the control group,H₂O₂ increased the protein expression of Beclin-1 and LC3B,but there was no significant change in p62,but Caspase-related splices were not detected,while compared with H₂O₂model group,NBP treatment group could reduce the expression of Beclin-1 and LC3BⅡ.2.In glutamate excitotoxicity model,NBP inhibits autophagy and apoptosis to protect cerebellar granule neurons.（1）With the increase of glutamate concentration,the cell body atrophy of CGNs increased in a concentration gradient.The nuclear pyknosis rate was calculated by Hochest33258,and the apoptosis rate in glutamate treated group increased in a concentration-dependent manner.The difference was statistically significant（P<0.05）;（2）The cell morphology and nuclear pyknosis rate of NBP groups changed no significantly（P>0.05）;（3）In the CGNs glutamate excitotoxicity model,NBP reduced the cell body atrophy of CGNs,and the apoptosis rate was significantly lower than that of the glutamate model group（P<0.05）;（4）Compared with the control group,Cleaved-Caspase3/Caspase3,LC3BⅡ/LC3BⅠ,Beclin-1 increased and p62 decreased in the glutamate model group,while Cleaved-Caspase3/Caspase3,LC3BⅡ/LC3BⅠ,Beclin-1 and p62 increased at first and then decrease in the NBP treatment group compared with the glutamate model group.3.In the mouse model of intracerebral hemorrhage,NBP can inhibit autophagy and apoptosis and improve cerebral hemorrhage.（1）The model of cerebral hemorrhage was successfully constructed.;（2）The Garcia score in ICH group was lower than that in Sham group,and the sensory and motor nervous system score was impaired in NBP+ICH group,but the Garcia score in NBP+ICH group was higher than that in ICH group,but the difference was statistically significant;（3）Compared with Sham group,the protein expression of Beclin-1,LC3BⅡ/LC3BⅠand Cleaved-Caspase3 increased in ICH group,but Beclin-1,p62,LC3BⅡ/LC3BⅠand Cleaved-Caspase3 decreased in NBP+ICH group while comparing with ICH group,suggesting NBP regulate autophagy and apoptosis;（4）Immunofluorescence results showed that LC3B increased significantly and LC3B puncta that gathered around the nucleus per cell in ICH group increased while LC3B puncta aggregation expression decreased in ICH+NBP group.ConclusionButylphthalide can improve nerve cell injury and hemorrhagic brain injury by inhibiting autophagy.