| BackgroundsIn recent years,with the continuous development of the construction and transportation,the incidence of traumatic brain injury(TBI)increases year by year.TBI is caused by the direct or indirect effects of external forces on the head,which is a pathological event characterized by axonal destruction,neuronal loss and demyelination,is mainly manifested as headache,nausea,disturbance of consciousness,paralysis,etc.In severe cases,cerebral hernia may occur and threaten patients’ life.TBI,characterized by high medical costs,high morbidity and mortality,is the leading cause of death and disability among young people around the world and is considered a major public health problem worldwide.According to 2005 statistics,the annual incidence rate is 2 to 2.5 million in the United States alone.TBI-induced damage can be divided into primary injury and secondary injury according to the pathophysiology process.Primary injury is a transient injury caused by the direct effect of mechanical external impact.Secondary brain injury is a complex pathological process that occurs within hours or days after the primary brain injury,including oxidative stress,generation of superoxide radicals and neuroinflammatory reaction,etc,resulting in neuronal injury and apoptosis.Therefore,neuronal apoptosis is an important part of secondary injury.TBI survivors not only have physical disabilities,but also have neurobehavioral dysfunction,which increases their susceptibility to neurodegenerative diseases such as Alzheimer disease and Parkinson’s disease.According to the World Health Organization,TBI is now recognized as a global public health epidemic and is expected to become the leading cause of neurological disability in all age groups by 2020.In the United States,an estimated 1.1 million TBI patients receive treatment each year,124,000 of whom have long-term disability and 50,000 deaths,making TBI a leading cause of premature death.In addition,long-term health care and loss of impose considerable social and economic burdens,which remains an unresolved public health issue.The current TBI clinical treatment strategy focuses on the management of intracranial pressure,cerebral perfusion,cerebral blood flow,brain metabolism and cerebral vascular autoregulation,and there is no specific neuroprotective drug,therefore,to explore drugs with neuroprotective effects on secondary TBI injury is a very important research direction for TBI treatment.Dexmedetomidine(DEX)is a highly selective a-2 adrenergic receptor agonist with analgesic,sedative and inhibitory sympathetic activity,and is currently widely used in the treatment of sedation in critically ill patients.DEX was developed and studied in the 1980s and 1990s.In recent years,a large number of studies have shown that DEX has anti-inflammatory,anti-oxidative stress and anti-apoptotic effects,and exerts neuroprotective effects in nervous system diseases.The previous experimental results of our research group also showed that dexmedetomidine pretreatment,with an optimal dose of 100ug/kg,can inhibit TBI-induced neuronal apoptosis,but the mechanism remains unclear.Silent information regulator family protein 1,SIRT1,is a conserved nicotinamide adenine dinucleotide(NAD+)-dependent histone deacetylase that is involved in a variety of intracellular signals such as senescence,inflammation,apoptosis and autophagy,showed neuroprotective effects in various animal models,including ischemic brain injury,traumatic brain injury,subarachnoid hemorrhage and neurodegenerative diseases.Studies have shown that inhibition of SIRT1 expression can aggravate TBI-induced mitochondrial damage and promote neuronal apoptosis.Therefore,DEX pretreatment and inhibition of SIRT1 protein expression in vivo were used in this study,to determine whether dexmedetomidine inhibits neuronal apoptosis after TBI through SIRT1 signaling pathway,providing pharmacological mechanism for dexmedetomidine treatment of TBI.Objective:The TBI model of SD rats was constructed by Feeney free fall method to investigate whether DEX inhibits neuronal apoptosis in TBI rats by regulating SIRT1 signaling pathway,providing a theoretical basis for the clinical application of DEX in the treatment of TBI.Methods:(1)The TBI model of SD rats was constructed by Feeney free fall method.The experimental animals was divided into 4 groups:Sham group(sham operation group,no drug treatment),TBI+NS group(peripheral injection of normal saline 10 min before model establishment),TBI+DEX group(DEX 100 ug/kg was intraperitoneally injected 10min before molding),and TBI+DEX+EX527 group(EX527,a SIRT1-specific inhibitor,was intraperitoneally injected at a dose of 5mg/kg every 2 days before TBI modeling,for a total of 4 injections.DEX 100 ug/kg was intraperitoneally injected at 10min before TBI modeling).(2)The mNSS scoring were used to evaluate the neurological function of rats in each group 24 hours after modeling.(3)After the mNSS scoring,the following experiments were performed:1.The morphological changes of brain tissue in the cortical injury area of each group were observed by HE staining.2.Immunofluorescence assay was used to detect the expression of SIRT1 and Cleaved-caspase3 in the cortical injury area of each group.3.Western blotting was used to detect the expression of SIRTI,Bax,Cleaved-caspase3 and Bcl-2 protein in cortex injury area of rats.Results:(1)Modified neurological severity score(mNSS)showed that the neurological deficits in each group after TBI were significantly higher than those in Sham group(P<0.05).Compared with the TBI+NS group,the mNSS of the TBI+DEX group was significantly improved(P<0.05).And the mNSS of the TBI+DEX+EX527 was significantly higher than the TBI+DEX group(P<0.05).(2)HE staining:①Compared with Sham group,shrinkage necrosis,cell body shrinks and deep stained,degeneration and edema of some neurons,cytoplasmic vacuole formation and karyopyknosis were observed in the injured cerebral cortex of the TBI+NS group.②Compared with TBI+NS group,TBI+DEX group showed reduced cell degeneration,edema,cytoplasmic vacuoles and neuron damage.?Compared with the TBI+DEX group,the neurons impairment in the TBI+DEX+EX527 group were much more serious.(3)Immunofluorescence detection:1)expression of the apoptotic protein Cleaved caspase-3:①Compared with the sham group,the fluorescence expression of Cleaved caspase-3 protein was significantly increased in the other three groups(P<0.05).②Compared with TBI+NS group,the fluorescence expression of Cleaved caspase3 protein in TBI+DEX group was significantly reduced(P<0.05),and the fluorescence expression of Cleaved caspase3 protein in TBI+DEX+EX527 group was slightly decreased,but there was not statistically significant(P>0.05).③Compared with TBI+DEX group,the fluorescence expression of Cleaved caspase-3 protein in TBI+DEX+EX527 group was significantly increased(P<0.05).2)SIRT1 protein expression:①Compared with sham group,the fluorescence expression of SIRT1 protein in TBI+NS group and TBI+DEX group was significantly increased(P<0.05).②Compared with TBI+NS group,the fluorescence expression of SIRT1 protein in TBI+DEX group was significantly increased(P<0.05).③Compared with TBI+DEX group,the fluorescence expression of SIRT1 protein in TBI+DEX+EX527 group was significantly reduced(P<0.05).(4)Western blot assay:1)Expressions of pro-apoptotic factors Cleaved caspase-3,Bax and anti-apoptotic factor Bcl-2 in injured cortical area of each group after TBI:①Expression levels of Cleaved caspase-3 and Bax protein in TBI+NS group were significantly increased compared with Sham group(p<0.05).②Compared with TBI+NS group,the expression levels of Cleaved caspase-3 and Bax in TBI+DEX group were significantly reduced(P<0.05).③Compared with TBI+DEX group,the expression of Bax protein in TBI+DEX+EX527 group was significantly higher(P<0.05),and the expression of Cleaved caspase-3 protein was slightly higher than that in TBI+DEX group,but there was no statistical difference(P>0.05).The expression trend of Bcl-2 protein was similar to Bax.2)SIRT1 expression in injured cortical areas of each group after TBI:①Compared with Sham group,SIRT1 protein expression level in TBI+NS group was increased(p<0.05).②Compared with TBI+NS group,SIRT1 protein expression in TBI+DEX group was significantly increased(P<0.05).③Compared with the TBI+DEX group,the expression of SIRT1 protein in the TBI+DEX+EX527 group was significantly decreased(P<0.05),Conclusions:(1)Dexmedetomidine can alleviate the secondary damage of TBI in rats model,thus playing a neuroprotective role.(2)Dexmedetomidine can reduce neuronal apoptosis after TBI by regulating the SIRT1 signaling pathway. |
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