The Study On The Mechanism That HDAC4 Regulate The Expression Of MKK7 With The Neuronal Apoptosis And Early Brain Injury

Subarachnoid Hemorrhage(SAH)is a clinical syndrome resulted from cerebral blood vessel rupture of diverse causes,of which 70% are intracranial aneurysms and cerebral vascular malformations,with large amount of blood directly flowing into the subarachnoid space.Although SAH only accounts for 5% of all stroke cases,there are about 10/106 people suffer from SAH every year according to autopsy reports.About 12-15% of SAH patients died before receiving effective treatments,and another 40% died within one month after admission.Disability rate of survivors is 50%.As the extremely high mortality and disability rate,the treatment of SAH have received great attention.Current pharmacotherapeutic researches are devoted to improving cerebral vasospasm and reducing sequelae.Although surgical techniques,radiology and anesthesiology have made significant progresses,the mortality and morbidity rate of SAH has not been significantly reduced.In recent years,the concept of Early Brain Injury(EBI)has been proposed.EBI refers to the pathophysiological changes that occur within 72 hours after SAH,which is the main cause of death and poor prognosis of SAH patients.Therefore,EBI has become a new focus for the search of SAH drug therapy.In the mechanisms of EBI,on the one hand,SAH can rapidly activate inflammatory reaction,excitotoxicity and other pathways,causing disruption of blood-brain barrier,neurons edema and even cell necrosis.On the other hand,the release of cytokines,stress response induced by intracranial pressure change after hemorrhage,and ischemia caused by vasospasm account for continuous neuronal apoptosis of the cortex or hippocampus in more than 80% SAH patients.Randomized Controlled Trials(RCTs)from different institutions have demonstrated that Fasudil(a Rho-kinase inhibitor)or steroids(such as Hydrocortisone and methylprednisolone)fail to significantly improve the outcomes of inflammation-induced EBI.Neither did the anti-oxidative stress and platelet responses treatments.Studies based on SAH experimental animals demonstrated that inhibition of neuronal apoptosis can significantly increase neurological scores,suggesting that excessive neuronal apoptosis may be an important pathological cause of EBI.Therefore,inhibiting neuronal apoptosis could be a potential therapeutic approach of SAH.The MAPK pathway plays a key role in EBI-induced neuronal apoptosis.Inhibiting the activity of JNKs at both cellular and animal level can effectively suppress neuronal apoptosis and improve neurological function.In the MAPK pathway,JNKs are mainly activated by MKK4 and MKK7.The studies on Gene Knock-out Mice have demonstrated that JNKs is activated by MKK7 but MKK4 when neurons are challenged by apoptotic stimulus.Therefore,we are committed to finding effective drug targets for the purpose of treating EBI and improving prognosis by inhibiting MKK7.Our previous studies have demonstrated that HDACs transcriptionally regulate the expression of MKK7 in glioma cells.And we are wondering whether inhibiting HDACs could affect the activity of MKK7 in neurons? Therefore,in this paper we mainly discuss the following aspects:1.In CGNs,whether inhibition of HDACs could suppress neuronal apoptosis via downregulation of MKK7?2.To suppress the expression of MKK7,which member in the HDACs family is inhibited?3.Whether inhibiting this very HDAC member could exert anti-apoptosis effects in neurons by suppressing MKK7 expression?4.In the rat SAH model,whether inhibiting HDACs could improve early neurological functional impairment by suppressing MKK7?Method and result 1.In CGNs,inhibiting HDACs suppreses the expression of MKK7.1.1.In CGNs,the MKK7-JNK-c-Jun apoptosis pathway is activated on the stress of 5K.Using primary culture cerebellar granule neurons of SD newborn rats as objects of study in vitro experiments.The natural electrolyte environment was simulated by making medium with the concentration of K+ at 25 m M(25K),while the concentration of K+ at 5m M(5K)was used to induce the neuronal apoptosis.The results of western blot show the expression of MKK7,JNK and c-Jun all increased at different time point.The results indicate that the MKK7-JNK-c-Jun apoptosis pathway was activated on the stress of 5K.1.2.In CGNs,inhibiting HDACs suppresses the expression of MKK7.The CGNs were dealt with different HDACI at the condition of 5K.The western blot results show that the expression of MKK7 are declined after using HDACI.It demonstrate that HDACs prevent MKK7 from expressing.2.Inhibiting HDAC4 suppresses the expression of MKK7.2.1.HDAC4 inhibitor suppresses the expression of MKK7 and p-c-Jun.CGNs were treated respectively with different concentrations of the HDAC4 inhibitor(LMK-235)in 25 K and 5K.Western blot showed the expression of MKK7,p-JNK,and p-c-Jun were suppressed depending on the change of LMK-235 under 25 K and 5K conditions.Trizol method was used to extract RNA.PCR and agarose gel electrophoresis showed that the m RNA expression of MKK7 in neurons after LMK-235 treatment was reduced under 5K.It was demonstrated that in CGNs,LMK-235 inhibited the expression of MKK7 at the transcriptional level.2.2.si RNA of HDAC4 reduce the expression of MKK7 and p-c-Jun effectively.we design small interferon RNA to suppress the expression of HDAC4.Because of the low transfection efficiency of CGNs,we choose rat glioma cell C6 to verify the effectiveness of the small interferon RNA.The expression of HDAC4 are declined in cells which had been transfected si HDAC4-1,si HDAC4-2 compared to control group.The results show that small interferon RNA of HDAC4 reduce the expression of HDAC4 effectively.We cotransfect GFP with si HDAC4 to mark the CGNs which had been successfully transfected si HDAC4.And then using fluorescence detection to show the expression of MKK7 and p-c-Jun.The results show that the expression of HDAC4,MKK7 and p-cJun are declined significantly in the group of si HDAC4-1 and si HDAC4-2 when compared with the control group,the differences have statistical significance(p<0.05).It validates the small interferon RNA of HDAC4 reduce the expression of HDAC4 effectively again,and indicates that suppressing the expression of HDAC4 reduce the expression of MKK7,p-c-Jun.3.Suppressing the expression of HDAC4 protects CGNs from apoptosis by suppressing the expression of MKK7.3.1.HDAC4 Inhibitor decreases Neuronal Apoptosis Treatment of CGNs with different concentrations of HDACs inhibitor(LMK-235)at 5K.Apoptosis rate of cells was analyzed by Hoechst staining.It showed that apoptosis rate of LMK-235-treated neurons is significantly reduced.These differences have statistical significance(p<0.05).Apoptosis rate decreased most when LMK-235 concentration was 1?M.The results showed that inhibition of HDACs inhibited neuronal apoptosis by inhibiting MKK7 expression under low potassium conditions. 3.2.si RNA reduce neuronal apoptosis by inhibiting HDAC4.We cotransfect GFP with si HDAC4 to mark the CGNs which had been successfully transfected si HDAC4.Then,CGNs were stimulated with 25 K or 5K for 12 hours.Further,we apply Hoechst method to dyeing nucleus of apoptosis cells.Last,the apoptosis cells were counted and conducted statistical analysis.The results show that the apoptosis rate reduce effectively at the condition of 5K when compared with control group,these differences have statistical significance(p<0.05).It indicates that suppressing the expression of HDAC4 protect CGNs from apoptosis by suppressing the expression of MKK7.4.In the rat model of subarachnoid hemorrhage,inhibiting HDAC4 improves early neurological impairment of brain effectively by suppressing the expression of MKK7.4.1.In the rat model of subarachnoid hemorrhage,inhibiting HDAC4 decreases the expression of MKK7 and P-c-Jun effectively.Choose female adult SD rats at a weight of 300 g as objects.And modelling subarachnoid hemorrhage after injecting LMK-235 or Vechile through ventricle for 24 hours.Then,we choose those rat's brain tissues which had been successfully modeled subarachnoid hemorrhage after another 12 hours.Further,we extract the frontal lobes protein of rat and apply western blot to detect the expression of MKK7,JNK,p-JNK,c-Jun,p-c-Jun.We use gray-scale to analyze the western blot result with the GAPDH as the reference.The analysis shows that the expression of MKK7,p-JNK,p-c-Jun are decreased and these differences have statistical significance(P<0.05).Last,we make frozen sections from above rats brain tissues and use immunofluorescence to detect the p-c-Jun expression of frontal lobes.The results of fluorescence show that the expression of p-c-Jun of LMK-235 treatment group decreased significantly(P<0.05)compared to control group.Above results indicate that injecting LMK-235 through ventricle decreases the expression of MKK7,JNK,c-Jun effectively in the rat model of subarachnoid hemorrhage.4.2.In the rat model of subarachnoid hemorrhage,inhibiting HDAC4 improves early neurological impairment of brain effectively.Choose female adult SD rats at a weight of 300 g as objects.And modelling subarachnoid hemorrhage after injecting LMK-235 or Vechile through ventricle for 24 hours.Further,we record and conduct statistical analysis the neurobehavioral scores of rats which had been successfully modeled subarachnoid hemorrhage after another 12 hours.The results show that compared with vechile treatment group,neurobehavioral scores were decreased significantly in LMK-235 treatment group(P<0.05).The results indicate that injecting LMK-235 through ventricle improves early neurological impairment of brain effectively by suppressing the expression of MKK7.Conclution 1.Inhibiting HDACs suppresses the expression of MKK7.2.Inhibiting HDAC4 suppresses the expression of MKK7.3.Suppressing the expression of HDAC4 protects CGNs from apoptosis by suppressing the expression of MKK7.4.In the rat model of subarachnoid hemorrhage,inhibiting HDACs improves early neurological impairment of brain effectively by suppressing the expression of MKK7.