The Dynamic Expression,Neuroprotective Roles And Mechanism Of Nrf2 After Traumatic Brain Injury In Mice

Objective:Traumatic brain injury(TBI)is defined as a brain damage resulting from an external mechanical force,and it causes structural damage and functional deficits,which contributes to a substantial number of deaths and disability.TBI is one of the most common causes of death and disability worldwide among young adults.An estimated 50 million individuals worldwide are diagnosed with TBI yearly.Both primary and secondary brain injuries compose the complex disorder caused by TBI.The primary injury includes contusion,damage to blood vessels and axonal shearing.Secondary injury is the result of cascades of metabolic,cellular and molecular events that ultimately lead to brain cell death,tissue damage and functional deficits.Numerous studies have provided evidence that several mechanisms are associated with secondary brain injury,including oxidative stress,glutamate excitotoxicity,neuroinflammation and apoptosis.Oxidative stress and inflammatory reaction are recognized as the important mechanisms of TBI-caused secondary injury.Theoretically,improving antioxidant response and reducing inflammation would alleviate TBI-induced secondary brain damage.Nuclear factor erythroid 2-related factor 2(Nrf2),a basic region-leucine zipper(bZIP)transcription factor,has been recognized as a master regulator of endogenous defense against oxidative stress.Under physiological conditions,Nrf2 is generally distributed in the cytoplasm by binding to the homodimeric protein Kelch-like ECH-associated protein 1(Keap1),which presents Nrf2 to ubiquitination and subsequent degradation by the proteasome.In response to oxidative stress,Nrf2 liberates from Keap1 and translocates into the nucleus,forming heterodimers with small Maf or Jun proteins followed by binding to regulatory DNA regions termed antioxidant response elements(AREs)or electrophile response elements(EpREs)to initiate transcription of a network of cooperating genes.Nrf2 was activated post TBI.Emerging evidence indicates that Nrf2 plays an important protective role in brain injury and neurodegenerative diseases,as deletion of Nrf2 exacerbates TBI-induced acute oxidative damage and subsequent neurological deficits in mice.In addition,Nrf2 activation ameliorates TBI induced damage,similar to sulforaphane,melatonin,carnosic acid,and tert-butyl hydroquinone,suggesting that modulating the transcription factor Nrf2 is a potentialstrategy to treat TBI-induced oxidative stress and inflammatory response.Curcumin,a phytochemical compound extracted from Curcuma longa rhizomes,has been extensively studied for its multiple biological activities,including anti-inflammatory and antioxidant properties.Curcumin has been proven to cross the blood-brain barrier and has the ability to attenuate cerebral edema,decrease the inflammatory response,promote energy homeostasis,and influence synaptic plasticity following TBI and cerebral ischemia/reperfusion injury.To our knowledge,the molecular mechanism underlying curcumin effects remains unclear.In vivo and in vitro evidences demonstrate curcumin is an effective activator of Nrf2;however,it is unknown whether the neuroprotective role of curcumin in TBI is through the Nrf2 pathway.Endogenous Nrf2 was activated post TBI.In the experiment,we first investigated the dynamic changes in expression and cell type-specific characteristics,which provides a biological marker for determination of injury time in forensic practice.Then,we examined whether curcumin enhances the expression of Nrf2 and ARE-regulated antioxidant response in mice subjected to TBI.Further,we explored TBI-induced neuronal apoptosis and inflammatory response in Nrf2-KO mice treated with curcumin or not to determine whether the neuroprotective role of curcumin in mouse TBI is dependent on the Nrf2 pathway,to provide evidence for clinical treatment of Nrf2 signaling on TBI.Methods : Male C57BL/6(wild-type,WT)and Nrf2 gene knockout(Nrf2-KO)mice(8–12 weeks;20–26 g)were used in this study.Moderate TBI models were induced according to the Feeney weight-drop contusion model,with slight modifications.Part one: WT mice were assigned to the sham group and TBI groups.The mice were sacrificed at 1,3,7 and 14 days post TBI(dpi),and brains were collected.Nrf2 protein levels were detected by Western blot.Cell type-specific localization of Nrf2 after TBI was detected at different time intervals by double immunofluorescence staining.NeuN,GFAP,IBA1 and NG2 were used as cell type-specific markers to neurons,astrocytes,microglia and NG2 glia,respectively.Part two: WT and Nrf2-KO mice were assigned to three groups,including the sham group with no curcumin treatment(Sham),TBI group with Vehicle treatment(TBI+Veh)and TBI group with curcumin treatment(TBI+Cur).In each group,the mice weresacrificed at 24 hours following surgery and brains were collected.The levels of Nrf2 and its downstream genes(Hmox-1,Nqo1,Gclm,and Gclc)were detected by Western blot and qRT-PCR.In addition,oxidative damage,edema,cell degeneration,cell apoptosis and inflammatory reactions were evaluated in wild type(WT)and Nrf2-knockout(Nrf2-KO)mice to explore the role of Nrf2 signaling after curcumin treatment.Results:Part one: After TBI,Nrf2 protein levels peaked at 1 dpi.Robust transient Nrf2 accumulation was co-localized with neurons,which was predominant at 1 dpi.Continuous weak Nrf2 expression was detected in activated astrocytes,and the number of double positive cells peaked at 7 dpi.Inducible widespread immunostaining of Nrf2 was observed in the nucleus of the microglia,and the number of Nrf2+ microglia peaked at 7 dpi.In addition,we also explored colocalization of Nrf2 in NG2 glia,in which the percentage of Nrf2+ in NG2 glia reached a climax at 3 dpi.Part two: In wild type mice,curcumin treatment resulted in reduced ipsilateral cortex injury,neutrophil infiltration,and microglia activation,improving neuron survival against TBI-induced apoptosis and degeneration.These effects were accompanied by increased expression and nuclear translocation of Nrf2,and enhanced expression of antioxidant enzymes.However,Nrf2 deletion attenuated the neuroprotective effects of curcumin in Nrf2-KO mice after TBI.Conclusion:1.Nrf2 expression in neurons and glias was upregulated post TBI.2.Nrf2 expression in neurons and glias was cell-type specific and time-dependent.3.TBI increases endogenous Nrf2 activity and curcumin potentiates it.4.Curcumin improves the outcome following TBI partly through Nrf2 activation.5.Curcumin exhibits anti-inflammatory,anti-apoptotic and antioxidant effects after TBI partly via Nrf2 signaling.