Resolvin D1 Ameliorates Cognitive Impairment Following Traumatic Brain Injury Via Protecting Astrocytic Mitochondria

Cognitive impairment is one of the most common and devastating neuropsychiatric sequelae after traumatic brain injury(TBI).Secondary injuries include neuroinflammation,blood–brain barrier(BBB)dysfunction,mitochondrial dysfunction and glutamate excitotoxicity.Together,these events lead to synaptic and neuronal loss and dysfunction,which are recognized as the main causes of cognitive.Neuroinflammation has been regarded as an important therapeutic target for cognitive impairment caused by TBI in animal and human studies.And the ideal outcome of neuroinflammation is resolving.Astrocytes,the largest population of glial cells,are responsible for metabolic support of neurons.Astrocyte dysfunction amplifies both neuroinflammation and glutamate excitotoxicity,leading to a vicious cycle that contributes to neuronal loss.Alterations in mitochondrial function are critical components of the secondary injury cascade initiated by TBI.After TBI,an increased generation of mitochondria-derived reactive oxygen species(mito-ROS)leads to redox imbalance and bioenergetic failure,culminating in cell death.Injury to astrocytic mitochondria and an increase in the generation of mito ROS lead to gliosis and release of proinflammation cytokins,which causes extensive neuroinflammation.Considering that neuroinflammation and mitochondrial dysfunction are crucial secondary injuries following TBI that can also impair cognition directly,we propose that a treatment that can both control neuroinflammation and improve the supportive function of astrocytes via protecting astrocytic mitochondria may be a promising therapeutic option for TBI,especially for TBI-associated cognitive impairment.Resolvin D1(RvD1),an important endogenous specialized pro-resolving mediator,can effectively inhibit neuroinflammation and play a protective role in various diseases of central nervous system.RvD1 has recently been reported to exert a potent protective effect on mitochondria.This suggests that RvD1 may suppress neuroinflammation and protect astrocytic mitochondria at the same time to play further neuroprotective roles.Based on the above study,we carried out two experiments.In the first part of this study,C57BL/6 mice subjected to TBI using a controlled cortical impact(CCI)device were used for in vivo experiments.And observe the effect of RvD1 on cognitive impairment after TBI.Our results revealed that RvD1 significantly ameliorated cognitive impairment,suppressed gliosis and alleviated neuronal loss in the hippocampus.To explore the mechanism underlying this activity,we verified that RvD1 can induce a higher level of mitophagy to remove damaged mitochondria and eliminate extra mitochondria-derived reactive oxygen species(mito ROS)by activating ALX4/FPR2 receptors in astrocytes.To further verify the above results,in the second part of this work,cultured primary mouse astrocytes and an N2 a mouse neuroblastoma cell line were used for in vitro experiments.we further confirmed that RvD1 can protect mitochondrial morphology and membrane potential in astrocytes and thereby enhance the survival of neurons.Meanwhile,RvD1 was also shown to increase the expression of brain-derived neurotrophic factor(BDNF),Trk B(receptor of BDNF),glutamate aspartate transporter(GLAST)and GLT-1(Glutamate transporter-1)in the hippocampus following TBI,which indicates a possible way by which RvD1 increases the supportive function of astrocytes.Part ? Effects of RvD1 on cognitive impairment and neuroinflammation after TBIAIM: The moderate TBI model was performed on C57BL/6 mice using CCI device to investigate the role of RvD1 in cognitive impairment and neuroinflammation after TBI.METHODS: 1.TBI model was performed on 8 weeks old female C57BL/6 mice using CCI device.2.FCT(Fear conditioning test)was used to evaluate the effect of RvD1 on cognitive impairment in mice after TBI.3.The beam walking test was used to evaluate the motor coordination ability of mice after TBI.4.immunofluorescence staining was taken for Neu N to evaluate the effect of RvD1 on neuron loss in the hippocampus of mice after TBI.5.RT-q PCR(Real-time q PCR)was used to evaluate the effect of RvD1 on synaptic loss in the hippocampus of mice after TBI.6.Immunofluorescence staining was taken for ALX4/FPR2,Neu N,Iba1 and GFAP,and the expression of ALX4/FPR2 on neurons,microglia and astrocytes was investigated by confocal laser microscopy.7.Immunofluorescence staining was taken for Iba1 and GFAP to evaluate the effect of RvD1 on activation of glial cells in the hippocampus after TBI.8.EB(Evans blue)extravasation was used to detect the effect of RvD1 on BBB damage after TBI.9.WB(Western blot)and RT-q PCR were used to detect the effect of RvD1 on neuroinflammatory cytokines(NLRP3/ASC/IL-1?,TNF?,IL-6,and MCP1)in the hippocampus after TBI.RESLUTS:1.RvD1 treatment significantly improves hippocampal-dependent learning and memory impairment after TBI.2.Motor coordination did not recover during Day 1 to Day 7 after TBI.3.RvD1 receptors are mainly expressed on astrocytes in the hippocampus after TBI.4.RvD1 alleviates the loss of neurons and synapses in the hippocampus after TBI.5.The activation of glial cells in the hippocampus significantly increased after TBI,while gliosis was significantly inhibited after RvD1.6.RvD1 alleviates exudation of EB dye after TBI and down-regulates the expression of neuroinflammatory cytokines in the hippocampus.7.The expression of p-GSK3? was reversed after RvD1 treatment,but the expression levels of p-AKT and p-CREB in the upstream and downstream signalling of p-GSK3? were not significantly changed compared to TBI+vehicle group.CONCLUTIONS:1.RvD1 significantly improves cognitive impairment and loss of neurons and synapses in the hippocampus after TBI.2.RvD1 receptors are mainly expressed on astrocytes in the hippocampus after TBI.3.RvD1 alleviates cerebral oedema after TBI and inhibited neuroinflammation in the hippocampus after TBI.4.RvD1 has an indirect effect on p-GSK3? and AKT/GSK3? /CREB signaling pathways.Part ? Effects of RvD1 on mitochondrial function of astrocytes and survival of neuronsAIM: TBI model was performed on C57BL/6 mice using CCI device to evaluate the effect of RvD1 on mitochondrial function in the hippocampus after TBI.Primary astrocytes and neurons were cultured in vitro to investigate the protective effect of RvD1 on mitochondrial function of astrocytes and survival of neurons.METHODS:1.The effect of RvD1 on mitochondrial energy state in the hippocampus after TBI was assessed by ATP content assay kit.2.Primary astrocytes were extracted for in vitro experiments,and were treated in groups with LPS(1 g/ml),RvD1(250 n M)with or without RvD1 receptor inhibitor Boc2(10 M)of RvD1,for subsequent experiments.3.WB was used to detect the expression levels of PINK,Parkin and LC3II/I,proteins related to mitophagy in the hippocampus after TBI.4.The effect of RvD1 on mitophagy damage induced by LPS was observed by transmission electron microscopy.5.WB was used to detect the expression levels of TRX2.6.Mito ROSspecific fluorescent probe mito SOX and laser confocal microscopy were used to observe the effect of RvD1 on the accumulation of mito ROS in LPS-induced astrocytes.7.N2 a cell line were cultured in vitro and WB was used to detect the expression levels of PINK,Parkin in LPS-induced N2 a cell line.8.The effects of RvD1 on LPS-induced mito ROS accumulation were observed by mito SOX staining and laser confocal microscope imaging.9.The mitochondrial fluorescent dye JC-1 detected by flow cytometry was used to evaluate the effect of RvD1 on mitochondria membrane potential in astrocytes.10.Mitochondrial fluorescent probe,Mito Tracker Red was used to investigate the effect of RvD1 on mitochondrial morphology in astrocytes.11.Transwell system was used to co-culture primary astrocytes and N2 a cell lines,and PI staining detected by flow cytometry was performed on the neurons after treatments to evaluate the effect of RvD1 on neuronal survival and whether it works through its receptor.12.WB was used to detect the expression levels of BDNF and its receptor Trk B.13.WB was used to detect the expression levels of neuroglial metabolic coupling associated proteins MCT1,MCT4,GLUT1 and GLUT3,as well as the effect of RvD1 on the expression of the above proteins.14.WB and q PCR analysis were used to dectect the expression of GLT-1,GLAST,GS,GLS1,Glu R1 and Glu R2 in the hippocampus after TBI.15.WB and immunofluorescence analysis were used to detect the expression and location of PKM2 in the peri-contusional cortex after TBI.16.WB analysis was used to evaluate the effect of shikonin on NLRP3/ASC/Caspase1/IL-1? signaling in the peri-contusional cortex after TBI.17.WB analysis was used to evaluate the effect of shikonin on the expression of PGC1?and DRP1 in the peri-contusional cortex after TBI.18.FCT was used to evaluate the effect of shikonin on cognitive impairment after TBI.RESLUTS:1.The ATP level was decreased after injury,which was reversed by RvD1 treatment.2.The expression level of PINK was significantly increased after TBI,while it was inhibited after RvD1 treatment.Compared with TBI group,Parkin was further significantly increased but LC3II/I showed no signicificant difference among groups after RvD1 treatment.3.The number of mitophagosomes increased in astrocytes induced by LPS,and further increased after RvD1 treatment,but the effect of RvD1 was significantly blunted by Boc2.4.The expression level of anti-mitochondrial oxidative stress protein,TRX2 increased after TBI and reversed by RvD1 treatment.5.LPS induced mito ROS accumulation in astrocytes,RvD1 significantly inhibited the increase of mito ROS,but Boc2 significantly inhibited the effect of RvD1.6.The expression of PINK and Parkin were increased in LPS-induced neurons,but no significant effect was observed after RvD1 treatment.7.LPS induced mito ROS accumulation in LPS-induced neurons,but no significant effect was observed after RvD1 treatment.8.Mitochondrial membrane potential decreased in LPS-induced astrocytes and RvD1 reversed the level of mitochondrial membrane potential,while the effect of RvD1 was significantly blunted by Boc2.9.The number of damaged mitochondria increased in LPS-induced astrocytes,and the number of damaged mitochondria decreased after RvD1 treatment,while the effect of RvD1 was significantly blunted by Boc2.10.LPS can induce neuronal death,while RvD1 plus primary astrocyte treatment can significantly reduce neuronal death.However,RvD1 alone did not significantly reduce neuronal death.11.RvD1 can further improve the expression level of BDNF and its receptor Trk B in the hippocampus after TBI.12.For the astrocyte-neuron lactate shuttle transporters the expression level of MCT2 in the hippocampus was significantly decreased after TBI,and was significantly recovered after treatment with RvD1;For astrocyte-neuron glucose metabolism transporters,RvD1 can further improve the expression of GLUT1 and restore the expression of GLUT3.13.RvD1 can restore the expression level of GLAST and GLT-1 which related to glutamate toxicity after TBI.14.The expression of PKM2 was significantly increased and PKM2,colocalizing with Iba1 were mainly expressed in the pericontusional cortex after TBI.15.SK signicantly inhibited the activation of NLRP3/ASC/Caspase1/IL-1? in the peri-contusional cortex after TBI.16.The expression of PGC1? and DRP1 were significantly increased but reversed by SK treatment.17.SK treatment significantly improves hippocampal-dependent learning and memory impairment after TBI.CONCLUTIONS: 1.RvD1 can protect mitochondria in astrocytes via improving the level of mitophagy in astrocytes and inhibiting the accumulation of mito ROS by activating the ALX4/FPR2 receptor.2.RvD1 protects mitochondria in astrocytes and improves neuronal survival.3.The possible underlying mechanism of RvD1 improves neuron survival: RvD1 improve the support function of astrocytes by increasing the expression of BDNF and restoring the expression of GLT-1 and GLAST.4.Drugs like RvD1 such as SK that have the dual effects of controlling neuroinflammation and improving mitochondria founction may be potential therapies for ameliorating cognitive impairment after TBI.