The Neuroprotective Effects Of Luteolin Against Secondary Brain Insults In Models Of Traumatic Brain Injury

Traumatic brain injury(TBI)is a common disease in modern society.It is mostly happened in traffic accident,construction injury,sport injury and battal field.In US,?823 people per 100,000 suffer TBI annually,and?91 of which are admitted to the hospital.The mortality of hospitalized TBI patients is up to 18%,and?30%of them receive lifelong disability.Besides the primary insult happened at the moment of TBI,the secondary insult include excitoxity,oxidative stress,neuroinflammation,cerebral vasospasm and blood-brain barrier disruption.These pathologic changes fanally lead to subacute or chronic neuron death,exacerbating the primary insult.They are not independent processes,but have much interaction with each other,which makes TBI a very complex disease.Nuclear factor erythroid-2 related factor 2(Nrf2)is an important transcript factor to keep the balance of oxidoreduction,it works by the downstream antioxidant response elements(ARE).Under the normal condition,Nrf2 is located in the cytoplasm with its inhibitor Keap-1,while translocates to the nuclei when been activated.Neclear factor-kappa B(NF-?B)plays key roles on regulation of inflammatory response in animals.It combines with inhibitor of NF-?B(I?B)in the cytoplasm.Upon inflammatory stimulation,NF-?B enters to the nuclei,upregulating downstream genes,such as cellular factors,cell adhesion molecule,matrix metalloproteinase and proinflammatory protease.Now it is clear that NF-?B is activation after TBI,and evolves in the neuroinflammation process.Autophagy is an evolutionarily conserved process in which intracellular dysfunctional protiens,organelles and bacteria are enclosed by autophagosome,a double membrane structure appeared when autophagy initiates.The autophagosome is then fuse with lysosome and the cargos are degraded by the lysosomal protease.Stresses such as oxidative stress,infection,and hypoxia are the conditions which can induce autophagy.Recent years,people found that elevated autophagy is found in tumor,aging and neurodegeneration diseases.Besides,regulation of this process could influent different pathological changes,such as oxidative stress and inflammation.Luteolin is proven to confer neuroprotective effects in various neurological diseases,namely Alzheimer,Parkinson disease and ischemic stroke,but its role in TBI still not clear.This study is aimed to clarify the role and the mechanisms of luteolin in TBI,using cell culture and animal models.Based on previous studies,we will focus on Nrf2-ARE antioxidative stress pathway,NF-?B antiinflammation pathway and autophagy.Part ?:Firstly we elucidate the role of luteolin in TBI,then we clarify the relationship between the neuroprotection effect of luteolin and its antioxidative property.Especially where Nrf2-ARE pathway is involved.Male CD1 mice were divided into six groups:sham,TBI,TBI+vehicle,TBI+luteolin(10mg/kg),TBI+luteolin(30mg/kg)and TBI+luteolin(50mg/kg).We use weight-drop model to induce TBI model.Measure the brain water content at 1 day after TBI,and the movement ability at Day 1,Day 3,Day 7 using grip test.The results showed luteolin can ameliorate brain edema and improve movement performance.Furthermore,our observations showed that luteloin can promote Nrf2 transfering to nuclei,upregulate downstream genes and reduce oxidative stress level after TBI using biochemical studies,western blot,immunohistochemistry and real time PCR.All the results were confirmed by the in vitro studies.In addition,we found that the neuroprotection of luteolin was attenuated in Nrf2-/-mice.From this part,we conclude that luteolin provides neuroprotection effects against TBI,which is attribute to its antioxidative property,and Nrf2-ARE pathway plays a key role in this process.Part ?:Based on the first part,we want to continue to explore the mechanisms of luteolin's neuroprotection in TBI.We focus on neuroinflammation and NF-?B pathway after TBI.Male CD1 mice were divided into four groups:sham,TBI,TBI+vehicle,TBI+luteolin(30mg/kg).We detect the permeability of the blood brain barrier and the neuronal degeneration at Day 1 after TBI.We found that luteolin can protect them from these secondary brain insults.Further,by using western blot,immunostaining,ELISA and RT-PCR,we proved that luteolin can inhibit NF-?B pathway and reduce the production of proinflamatory factors.This effect was also proved in primary microglia culture studies.All these results indicate that luteolin can protect the BBB and reduce the neuronal degeneration after TBI,and the potential mechanism is related to NF-?B pathway.Part ?:We speculate the effect of luteolin on Nrf2-ARE pathway and NF-?B are all related with autophagy.Here we examine the autophagy changes after TBI and after luteolin administration.To elucidate the changes of autophagy after TBI,male CD1 mice were divided into sham group and groups of 3h,6h,12,24h and 48h after TBI.Western blot was used to detect autophagy markers Beclinl and LC3II.We found both Beclin1 and LC3? were upregulated after TBI and peaked at 24h.To measure the effect of luteolin on autophagy after TBI,mice were divided into sham,TBI,TBI+vehicle and TBI+luteolin(30mg/kg).We found luteolin can further upregulate autophagy markers Beclin1 and LC3? after TBI.Here we conclude that luteolin upregulates autophagy after TBI,and may have a deep relationship with its antioxidative and antiinflammation properties.Taken together,luteolin provides neuroprotection against TBI,which is due to its antioxidation property via Nrf2-ARE pathway and modulation of NF-?B.In addition,luteolin can also regualate autophagy after TBI,the relationships between them need further study.