Neuroprotective Effects Of HUC-MSCs With HDAC1 Gene Silencing In A Mouse Model Of TBI

Traumatic brain injury(TBI)can lead to neurological disorders such as cognition,exercise and sensations,especially to death in severe cases.Due to the complex regulatory mechanisms,the traditional surgery,drug and hyperbaric oxygenation are less effective,so it is seriously important to look for new drugs or methods to intervene.Stem cells are a kind of cell population with self-renewal,highly variable proliferation and multidirectional differentiation potential.Stem cell replacement therapy has become one of the most promising methods for the treatment of nervous system diseases such as brain injury.However,the further researches find that only a small part of MSCs transplanted can differentiate into neuronal cells.Therefore,how to improve the differentiation ability and efficiency of MSCs after transplantation is the key to improve the clinical therapeutic effect of MSCs.In recent years,the role of epigenetics in the pathogenesis of nervous system diseases has gained more and more attention.Histone deacetylases HDACs and its inhibitors play an important role in regulating the proliferation and directional differentiation of embryonic stem cells,neural stem cells,tumor stem cells and mesenchymal stem cells.Histone acetylation modification is involved in the development of many neuro-related diseases,such as Alzheimer's disease,stroke,cerebral ischemia injury.The use of HDAC inhibitors can change the degree of histone acetylation of proteins associated with the site of injury,and play an important role in nerve protection and promote nerve regeneration.However,thesynergistic effect and molecular mechanism of hUC-MSCs with HDAC1 deleted transplantion into TBI mice have not been clearly reported.ObjectiveThis research is first to evaluate the role of MSC-siHDAC1 in improving the cognitive function and regeneration after it was transplanted to mice in a mouse TBI model.Furthermore,it is to analyze the effect of signaling pathway PTEN/PI3K/AKT during the process mentioned above.MethodsIn this experiment,a C57BL/6 moderate TBI model was established by stereotaxic instrument and craniocerebral impact device.Sixty mice were randomly divided into vehicle group,MSC group and MSC-siHDAC1 group(n = 20).On day 0,1,3,7,14,21 and 28 after operation,the weights changes of the three groups of mice were detected,and the neurological deficit symptom is observed by m NSS score.The long-term exercise and depression in mice were detected by tail suspension test,forced swimming test and sugar water preference test.The volume of brain injury and the morphological changes of neurons in the CA3 area of hippocampus were evaluated by CV staining.Luxol evans blue(EB)staining was used to detect the degree of openness of blood brain barrier(BBB).Cell necrosis and generation of ROS in the lesion area were detected via PI staining and HEt staining respectively.Oxidative stress and symptom factors in serum were measured by Elisa.Luxol fast blue staining and Myelin basic protein(MBP)immunofluorescence staining were used to evaluate the changes of myelin demyelination.qRT-PCR and immunofluorescence assay were used to detect the expression of NSE,MAP2 and DCX mRNA and neural trophic factors factors in the brain of each group mice.Western bolt method was used to examine the express of related pathways in PTEN/PI3K/AKT.Finally,LY294002 was used to reversely verify whether MSC-siHDAC1 plays a neuroprotective role in TBI mouse neuron through the PTEN/PI3K/AKT pathway after traumatic brain injury.Results1.After 3 days of TBI,the injured volume of TBI mice(p<0.05)was decreased after MSC transplantation compared with Vehicle group,and EB staining showed that BBB of mice in MSC-siHDAC1 group was lower than in Vehicle group and MSC group.After 3 days,the mNSS of mice in each group was significantly decreased,and the recovery of nerve in MSC-siHDAC1 group was better than that in Vehicle group and MSC group(p<0.05).MSC-siHDAC1 transplantation treatment can increase the degree of glyphosate in mice and reduce the mouse suspension time and immobility time,relieve the symptoms of depression(forced swimming and tail suspension test),the difference was statistically significant(p<0.05).2.After 3 days of TBI,the percentage of Luxol fast blue and MBP-positive area in MSC-siHDAC1 group was significantly higher than other group(p<0.05)compared with Vehicle group.MSC-siHDAC1 transplantation was beneficial in reducing myelin damage(p<0.05).In the Vehicle group,Luxol fast blue staining and MBP immunofluorescence myelin staining showed that the structure of corpus callosum was disordered in the brain,and the positive region was abnormal.The staining of striatum became less.3.After 3 days of TBI,MSC-siHDAC1 transplantation could reduce neuronal loss in hippocampal CA3 area and prevent neuronal degeneration.Compared with the Vehicle group,the expression of Bcl-2 and Caspase3 protein increased and the expression of Cleaved Caspase3 decreased in MSC-siHDAC1 group,in addition to the number of apoptotic cells decreased significantly(p<0.05).4.Compared with Vehicle group,MSC-siHDAC1 could increase the antioxidant capacity and the activity of SOD,GSH and GSH-Px,decrease the contents of MDA and ROS(p<0.05).MSC-siHDAC1 increased the release of IL-4 and IL-10 despite decreased IL-1? and TNF-? release(p<0.05).5.The expression of neuronal markers(Iba1,GFAP,Ki67,DCX and NeuN)in MSC-siHDAC1 group was better than that in vehicle group and MSC group(p<0.05).MSC-siHDAC1 transplantation can regulate hippocampal neurotrophic factor synergistically,promote the survival of the dentate gyrus of the hippocampus,repair and regeneration,accelerate the recovery of nerve function.6.Compared with Vehicle group,the levels of p-PTEN,p-AKT1 and p-GSK3?were increased after MSC-siHDAC1 transplantation(p<0.05),but the total PI3 K,total GSK3? and AKT1 levels did not change(p 0.05).The use of MSC-siHDAC1 transplantation could increase the expression of p-PTEN.The use of AKT pathway inhibitor LY294002 could reverse the neuroprotective effect of MSC-siHDAC1(p<0.05).Conclusion1.MSC-siHDAC1 transplantation can improve the motor function and cognitive function of TBI mice,reduce brain tissue loss,the openness of BBB,degeneration and necrosis of hippocampal neurons and myelin damage,improve the damage site microenvironment,and promote neuroregeneration in the SGZ region of the brain.2.MSC-siHDAC1 plays the role of protecting neuro by activating PTEN/PI3K/AKT.And hence,these results suggest that hUC-MSCs with HDAC1 gene silencing have better neuroprotective effects in TBI mice.