The Neuroprotection And Mechanism Of TSA Combined With BMSCs On Spinal Cord Injury Mice

BackgroundSpinal cord injury?SCI?is a serious injury of the central nervous system.The main clinical manifestations are the loss of sensation,complete loss of motor function and incontinence below the plane of injury.The incidence and the rate of disability are increasing year by year,causing a heavy burden to the family and the society.At present,there are no effective treatment methods.Stem cells have the characteristics of self-renewal,high proliferation and multiple differentiation potential.Therefore,stem cell replacement therapy has become a new starting point for the treatment of central nervous system diseases.Bone marrow mesenchymal stem cells?BMSCs?are widely used in stem cell replacement therapy because of their convenience,no immunogenicity and no ethical problems.However,transplantation of stem cells for the treatment of central nervous system diseases also has the disadvantages of low survival rate,low differentiation efficiency and high incidence of neuralgia after transplantation.Epigenetic regulation is a means of regulation of gene expression based on chromatin.One of the more extensive types of research is histone acetylation.The study found that the level of histone acetylation was associated with a variety of neurological diseases.It has been reported that histone deacetylase inhibitors?HDACi?can promote neural differentiation of stem cells and have extensive neuroprotective effects.Studies have found that HDACi can activate SDF1/CXCR4 pathway,promote the migration of stem cells to the lesion area,and HDACi can make Nrf2acetylation,activate Nrf2/ARE pathway,and effectively alleviate the inflammatory reaction after injury.Trichoderin A?TSA?is a stable and efficient HDAC inhibitor.It has not been reported whether TSA has neuroprotective function after central nervous system injury.In this study,the effect of TSA combined with BMSCs on neuroprotection in mice after SCI was explored and discuss the action mechanism.ObjectiveIn vitro,the prevention and treatment of oxidative damage of Hippocampal neuronal cell line?HT22?by TSA was detected.In vivo,the neuroprotective effect of TSA combined with BMSCs on SCI mice and its mechanism were studied.MethodsPart one:The Protective Effect of TSA on Oxidative Damage of HT22 CellsThe experiment was divided into NC control group,H₂O₂ injury group and TSA+H₂O₂ treatment group:1.the level of cell proliferation was detected by CCK-8;2.cell apoptosis was measured by flow cytometry,Transwell was used to detected cell migration;3.the expression of SOD and MDA were detected by ELISA kit,the ROS level were detected by ROS kit.Part two:The Neuroprotection and mechanism of TSA Combined with BMSCs on spinal cord injury miceThe mice model of SCI was established by modified Allen's method.Experimental mice were randomly divided into Veh group,TSA group,BMSCs transplantation group and TSA+BMSCs combined treatment group:1.BMS?Basso Mouse Scale?and inclined plate were performed to observe the ability of hind limb motor function in mice,tail suspension test and sucrose preference test were used to evaluate depression in mice;2.detection of damage volume by CV staining and the determination of spinal water content in the injured part of mice by dry wet weighing method to evaluate the spinal edema;3.qRT-PCR was used to detect the expression of neurotrophic factors BDNF,NT3 and NGF,as well as neuronal differentiation related factors:DCX,MAP2 and NSE.Immunofluorescence staining was used to detect the expression of GFAP,MBP and NeuN in the injured site of the spinal cord;4.the expression of TNF-?and IL-1?of inflammation related factors was detected by ELISA kit.Het staining was used to detect the expression of reactive oxygen species at the injured site.PI staining was used to detect the number of necrotic cells in the injured site of the mice.The number of apoptosis cells at the injured site was detected by TUNEL kit;5.Western blot was used to detect the expression of Nrf2/ARE pathway related proteins.ResultsPart one:The Protective Effect of TSA on Oxidative Damage of HT22 Cells1.Compared with the H₂O₂ group,TSA could increased the cell proliferation and migration,reduced cell apoptosis caused by H₂O₂ damage?P<0.05?;2.Compared with the H₂O₂ group,the SOD activity in the TSA treatment group increased significantly,the MDA level and the accumulation of ROS decreased.TSA treatment can significantly reduced the oxidative damage caused by H₂O₂?P<0.05?.Part two:The Neuroprotection and mechanism of TSA Combined with BMSCs on spinal cord injury mice1.TSA+BMSCs treatment significantly improved the motor function and emotional state of the hind limbs of mice,reduced the damage volume,increased the weight and reduced the water content of the spinal cord?P<0.05?;2.TSA+BMSCs treatment increased the expression of neurotrophic and neural differentiation factors,alleviated the inflammatory response,reduced the expression of GFAP,the activation of astrocytes and the expression of reactive oxygen species?P<0.05?;3.TSA+BMSCs treatment increased the expression of neuron cells and reduced the damage of white matter,decreased the number of necrotic cells and apoptotic cells?P<0.05?;4.Western blot results showed that TSA increased Nrf2 expression,decreased Keap1 expression,and increased the expression of SOD1 and NQO1 in downstream protein,indicated TSA could activate Nrf2/ARE pathway?P<0.05?.Conclusion1.Treatment with TSA could reduced H₂O₂-induced oxidative damage to HT22cells;2.TSA combined with BMSCs can reduced the inflammatory response and oxidative damage in the lesion site of SCI mice,reduce cell necrosis and apoptosis,and play a role in neuroprotection by activating the Nrf2/ARE pathway and reducing the accumulation of ROS.