The Neuroprotective Effects Of Bexarotene On Mice After Traumatic Brain Injury

Part Ⅰ The Influence of Bexarotene on the Restoration ofNeurological Functions of Mice Following Traumatic Brain InjuryObjectiveThe aim of this study was to determine the role of bexarotene in neurobehavioral recovery,neuro-inflammation and cell apoptosis around the injury site in mice cerebral cortex following traumatic brain injury(TBI).MethodsExperiment one: the effects of bexarotene treatment on the motor function,learning ability and spatial memory of mice following TBI.Male C57BL/6 mice(n= 90)were used to establish the controlled cortical injury(CCI)model to simulate TBI according to following parameters,velocity: 5.0 m/s,depth: 2.0 mm,and dwelling time: 100 ms.Afterward,the mice were respectively dosed with bexarotene(5mg/kg/day)or vehicle.On the 1~st,3~rd,7~th,14~th day following CCI,the motor function was assessed by neurological severity score(NSS),rotarod test and wire-griping test.From 15~th to 20~th day following TBI,the learning ability and spatial memory were evaluated by morris water maze test.Experiment two: the effects of bexarotene treatment on inflammation around the injury site in mice cerebral cortex following TBI.On the 1~st,3~rd,7~th day following CCI,about 10 mg mice cerebral cortex around the injury site was taken out to collect the total protein.Then the levels of interleukin(IL)-1b,IL-6,and tumor necrosis factor(TNF)-α were determined by enzyme-linked immunosorbent assay(ELISA).In addition,the brains were made into frozen sections and processed according to the standard immunofluorescent staining procedure to detect the level of Iba-1 protein which is the specific marker of microglia.Experiment three: the effects of bexarotene treatment on cell apoptosis around the injury site in mice cerebral cortex following TBI.On the 1~st,3~rd,7~th day following CCI,about 50 mg mice cerebral cortex around the injury site was taken out to collect the total protein.Then the levels of cleaved caspase 3 was determined by western blot.In addition,on 7~th day following TBI,the brains were made into frozen sections and paraffin sections.The expression of cleaved caspase 3 also was determined by immunofluorescent staining.The ruptured DNA fragments were detected by terminal deoxynucleotidyl transferase–mediated d UTP nick end labeling assay(TUNEL).Experiment four: the side effects of consecutive bexarotene treatment on mice.On 21~st day following TBI,the venous blood of mice was harvested to determine the concentration of plasma triglyceride.The livers were taken out to be made into paraffin sections.Hematoxylin–eosin staining was carried out to observe the micro structure of liver.ResultsExperiment one:The NSS was higher in vehicle group compared to bexarotene group,and a significant difference was detected on 3~rd,7~th,14~th days following TBI between the two groups(P < 0.05).The duration in rotarod test was significantly longer in bexarotene group compared to vehicle group on 1~st,3~rd,7~th,14~th days following TBI(P < 0.05).A significant difference of the wire-gripping performance was detected between vehicle and bexarotene groups,but this difference was only observed on the 1~st following TBI(P < 0.05),compared to other testing days which showed no difference.Compared with vehicle group,bexarotene administration did not significantly shorten the latency on 15~th,16~th,17~th,18~th,19 th days following TBI(P > 0.05).As compared to vehicle group,bexarotene group had significantly more dwelling time,longer path length in quadrant 4,and more times to pass over the platform(P < 0.05).These results indicated that bexarotene administration significantly improved the motor function and spatial memory of mice following TBI.Experiment two:Compared with the vehicle group,bexarotene administration significantly decreased the concentration of IL-1b and IL-6 only on 7~th day following TBI(P < 0.05),but not on 1~st and 3~rd day following TBI(P > 0.05).Bexarotene administration significantly decreased the TNF-α concentration on 1~st and 7~th day following TBI(P < 0.05),but not on 3~rd day following TBI(P >0.05).The Iba-1 positively staining cells around the injury site were significantly less in bexarotene group compared to vehicle group on 7~th day following TBI(P <0.05).Experiment three:Bexarotene administration significantly decreased the protein level of cleaved caspase3 around the injury site on 1~st,3~rd,7~th day following TBI(P <0.05).The number of positive TUNEL staining cells around the injury site was prominently increased in both vehicle and bexarotene groups compared to sham group(P <0.05).Compared with vehicle group,the number of positive TUNEL staining cells were significantly less in bexarotene group on 7~th day following TBI(P <0.05).Experiment four:The concentration of plasma triglyceride was higher in bexarotene group compared to vehicle group,but the difference was not statistically significant(P >0.05).The(liver/body weight)×100 in bexarotene group was not significantly different from that of both vehicle and sham groups(P >0.05).The microstructure of the liver in both vehicle and bexarotene groups was unchanged compared with that of sham group,the hepatic lobule was in physical arrangement and no fiber accumulation was detected in any liver section.ConclusionThe present study shows that the anti-cancer drug bexarotene can improve the motor function,strengthen the spatial memory,reduce inflammation,and inhibit cell apoptosis around the injury site in cerebral cortex following TBI.The results may present a novel application for bexarotene and may reveal a promising new therapy for TBI patients.Part Ⅱ The role of long non-coding RNA Neat1 in the therapeutic effect of bexarotene on traumatic braininjury in miceObjectiveTo explore the possible roles of long non-coding RNA(lnc RNA)Neat1 in bexarotene’s neuroprotective effects in mice following TBI.MethodsExperiment one: the best dose of bexarotene for cell treatment and its influence on RXR isoformsThe immortalized mouse hippocampal neuron cell line(HT22)was cultured and respectively treated with gradient dose of bexarotene: 0.1μM,0.3μM,0.9μM,2.7μM,8.1μM,24.3 μM.The HT22 cells were subjected to oxygen/glucose-deprivation(OGD)conditions(lasting for 6 h),and the cell viability was measured by MTT assay.The total protein of HT22 cells dealt with respective conditions was collected and used to perform western blot to measure the protein level of RXR isoforms.Experiment two: the binding relationship between RXR-α and the promoter zone of Neat1 geneThe bio-informatic analysis was carried out to predict the relationship between RXR-α and the promoter of Neat1 gene.The chromatin immunoprecipitation(Ch IP)was performed to capture the DNA fragment binding with RXR-α.The polymerase chain reaction(PCR)was carried out to detect the sequence of the Neat1 promoter.Experiment three: the influence of RXR-α on the transcription of Neat1 geneDual-luciferase reporter gene assays was carried out to verify the influence of RXR-α on the transcription of Neat1 gene.Then the HT22 cells were treated with bexarotene or subjected to OGD condition,and its total RNA was extracted.TBI model was established using C57BL/6 mice,and the total RNA around the injury site was extracted.The respective total RNA was used to measure the level of Neat1 lnc RNA.Experiment four: the protein directly captured by Neat1 lnc RNA in HT22 cellsThe lnc RNA Neat1 and its full-length antisense RNA chain were constructed and incubated with the total protein of HT22 cells.The protein pulled down by Neat1 lnc RNA was separated by electrophoresis.The targeted bands were subjected to mass spectrometry to identify the protein category.Experiment five: the in vitro effects of Neat1 lnc RNA on apoptosis and inflammation,and the role of PIDD1 in Neat1 lnc RNA’s functionThe adenoviruses were constructed to silence the transcription of Neat1 and PIDD1 gene,and to over-express Neat1 gene.The HT22 cells were subjected to OGD condition to induce cell apoptosis.The cell line of microglia(BV2 cell)was treated with lipopolysaccharide(LPS)to induce inflammation response.The HT22 cells and BV2 cells were infected with respective adenovirus to measure the inflammation and cell apoptosis under various conditions.Experiment six: the in vivo effects of Neat1 lnc RNA on apoptosis and inflammationThe adenovirus solution was stereotactically injected into the right lateral ventricle of C57BL/6 mice.Afterward,TBI model was established,and the production of inflammatory cytokines,microglia accumulation,in situ cell death and cleaved caspase 3 were measured around the injury site in cerebral cortex.ResultsExperiment one:The cells’ viability was not significantly influenced by bexarotene at a low drug concentration(from 0.1 to 8.1 μM)(P>0.05).Instead,the cells’ viability was significantly decreased when the concentration of bexarotene was ≥24.3 μM(P<0.05).When HT22 cells were subjected to OGD condition,the dose of 8.1 μM bexarotene could bring the most significant improvement of cell viability.Only RXR-α was significantly increased responding to bexarotene treatment(P<0.01),neither RXR-β nor RXR-γ(P>0.05)in both HT22 cells and mice cerebral cortex.Experiment two:RXR-α could bind to the promoter zone of Neat1 gene,and the binding site was "TGACGTCA," located on chr19:5,845,979-5,846,077.Experiment three:RXR-α prominently enhanced the activity of the Neat1 gene promoter.Under normal conditions,the level of Neat1 lnc RNA was significantly up-regulated by bexarotene treatment in HT22 cells(P<0.05)and in cerebral cortex of C57BL/6 mice(P<0.05)partially through an RXR-α-dependent mechanism.Both OGD condition and TBI insult significantly up-regulated the level of Neat1 lnc RNA(P<0.05),and bexarotene could further prominently enhance this up-regulation(P<0.05).Experiment four:The protein pulled down by Neat1 lnc RNA was separated by electrophoresis and four targeted bands were collected.The mass spectrometry determined 20 kinds of non-paraspeckle proteins which were directly captured by Neat1 lnc RNA,including PIDD1.Experiment five:When PIDD1 level was significantly decreased,the production of inflammatory cytokines and cell apoptosis were significantly inhibited(P<0.05),indicating its pro-inflammation and pro-apoptosis roles.Neat1 over-expression significantly inhibited the production of inflammatory cytokines and cell apoptosis(P<0.05),but Neat1 knockdown significantly promoted it(P<0.05).Interestingly,the increase of inflammatory cytokines and cell apoptosis induced by Neat1 knockdown was significantly inhibited by PIDD1 knockdown(P<0.05),indicating the close relationship between Neat1 and PIDD1 in regulating the production of inflammatory cytokines and cell apoptosis.Experiment six:TBI insult induced significant cell apoptosis surrounding the injury site(P<0.01).Compared to the control group,Neat1 knockdown further increased cell apoptosis on 7~th day following TBI(P<0.01),but Neat1 over-expression significantly decreased it(P<0.05).Inflammatory cytokines were significantly increased by TBI insult(P<0.01).On the 1~st,3~rd and 7~th days following TBI,Neat1 knockdown significantly further increased the production of inflammatory cytokines around the injury site in cerebral cortex compared to the control group(P<0.05),but Neat1 over-expression significantly inhibited it(P<0.05).Moreover,Neat1 over-expression significantly promoted the recovery of motor function,learning ability and spatial memory of mice following TBI,but Neat1 knockdown blocked it.ConclusionBexarotene up-regulates the level of Neat1 lnc RNA through a RXR-α-dependent mechanism,and this up-regulated Neat1 lnc RNA inhibits cell apoptosis and suppresses inflammation by restricting PIDD1 protein,resulting in better functional recovery following TBI.