Neuroprotective Effect Of Astaxanthin On Traumatic Brain Injury And Study Of Its Mechanism

Traumatic brain injury（TBI）is the main cause of death and disability in the world,and has become one of the serious threats to human health.In addition to mechanical injury caused by primary injury,the secondary injury,such as cerebral edema,intracranial hematoma,cerebral ischemia,increased intracranial pressure,is also the important pathological basis of TBI.It is reported that the extent of brain edema after TBI is directly related to the prognosis of patients.The Na⁺-K⁺-2Cl^-co-transporter-1（NKCC1）,which has an essential role in cerebral edema,is an intrinsic membrane protein mainly expressed in astrocytes and neurons.Edema can be markedly reduced by the cotransporter inhibitor or genetic deficiency.It has been reported that TBI can trigger and amplify inflammatory responses by participating in specific signal transduction pathways（eg NF-кB pathway）,which play an important role in the development and prognosis of injury.Also some studies have reported that astaxanthin is neuroprotective in central nervous system diseases,including ischemic brain injury and subarachnoid hemorrhage.However,it remains unclear that whether axtaxanthin is a potential neuroprotectant in TBI.In this study,we investigated the neuroprotective effect and mechanism of astaxanthin on TBI in vitro and in vivo using a controlled cortical impact（CCI）model and a model 94A Cell Injury Controller.To explore the neuroprotective effect of astaxanthin,we constructed the TBI model using a Pin Point^TM（Model PCI3000）controlled cortical impact device and then treated mice with astaxanthin.The Garcia score and the rotarod test were used to assess the neurological impairment of the injured mice.The Morris water maze（MWM）was employed to assess the learning and memory function of mice.To verify whether astaxanthin can reduce brain edema by downregulating the expression of NKCC1,brain water content was estimated using the wet-dry method.Blood–brain barrier（BBB）disruption was evaluated using Evans blue dye extravasation.Real-time PCR,Western blot and immunofluorescence staining were used to evaluate the expression of NKCC1.To prove whether astaxanthin can reduce the expression of NKCC1 by inhibiting inflammation,we injured astrocytes by Cell Injury Controller II（CICⅡ）and then treated astrocytes with astaxanthin or a corresponding inhibitor.Cell Counting Kit 8 assays were performed to evaluate cell viability.TUNEL staining and Western blot were used to evaluate the apoptosis of astrocytes.The expression of NF-кB p65 and pro-inflammatory cytokines,including IL-1β、IL-6、TNF-α,were investigated by real-time PCR and Western blot.Real-time PCR,Western blot and immunofluorescence staining were used to evaluate the expression of NKCC1.Our results showed that astaxanthin administration at 30 minutes after TBI improved neurological function,space learning and memory ability and cognitive ability.Astaxanthin reduced cerebral edema and Evans blue leakage at day 3.Additionally astaxanthin treatment within 1 h following TBI alleviated cerebral edema significantly at day 3.However,axtaxanthin treatment at 3 h after TBI had no effect on cerebral edema.We also found NKCC1 m RNA and protein expressions in the peri-contusional cortex were reduced by astaxanthin in a dose-dependent manner after TBI.Stretch injury in cultured astrocytes decreased cell viability time-dependently,which was significantly attenuated by pretreatment with ATX.Stretch injury increased the expression of apoptotic proteins,resulting in the apoptosis of astrocytes.Furthermore,stretch injury both significantly increased the gene and protein expressions of pro-inflammatory cytokines and induced the expression and nuclear translocation of NF-κB p65.Pretreatment with ATX significantly inhibited the trauma-induced activation of NF-κB p65,expressions of pro-inflammatory cytokines,and cell apoptosis.Moreover,stretch injury markedly elevated the gene and protein expressions of NKCC1,which was partly blocked by co-treatment with ATX or NF-κB inhibitor.Apoptotic protein activity was partially reduced by NF-κB inhibitor or NKCC1 inhibitor.Our study suggests that astaxanthin improves TBI-related neurological impairment in mice by ameliorating NKCC1-mediated vasogenic edema.We also prove in vitro that astaxanthin inhibit NKCC1 expression by reducing the expression of NF-κB-mediated proinflammatory factors.