The Role And Mechanisms Of Acetyl-CoA Carboxylase 1 And Tetramethylpyrazine In Regulating Ischemic Brain Injury

Part Ⅰ: The role and mechanism of acetyl-CoA carboxylase 1(ACC1)in regulating neuronal survival in ischemia-reperfusion(I/R)brain injury.Objective: Ischemic stroke is an acute cerebrovascular disease with high morbidity and disability,and the mechanism of I/R brain injury has been the focus of research.ACC1 is a key enzyme in fatty acid metabolism that catalyzes the production of Malonyl-CoA.In addition,ACC1 is involved in inflammatory response.Previous studies have shown that ACC1 can aggravate I/R brain injury by promoting peripheral inflammatory response.However,it has not been reported whether ACC1 directly affects neuronal survival after ischemia by regulating metabolism.The purpose of this part study is to investigate the effect of ACC1 on the survival of neurons treated with oxygen glucose deprivation reperfusion(OGD/R)and its mechanism.Methods: OGD/R treatment was performed on mouse cortical neurons,human neuroblastoma SH-SY5Y cell line and mouse neuroblastoma Neuro-2a(N2a)cell line in vitro to mimic neurons in I/R brain injury.The activity of ACC1 in neurons was inhibited using TOFA,and the expression of ACC1 in SH-SY5Y cell line and N2 a cell line was knocked down using siRNA interference.Expression of ACC1,p-CREB,and CREB was analyzed by western blot,cell viability was measured by CCK8 to assess cell survival,and the levels of Malonyl-CoA in cells were measured by enzyme-linked immunosorbent assay(ELISA).Results: During OGD/R,the expression of ACC1 in neurons decreased first and then recovered.Inhibition of ACC1 activity by TOFA improved the cell viability of OGD/R-treated-neurons.Inhibition of ACC1 activity and knockdown of ACC1 expression both reduced the level of Malonyl-CoA,and exogenous supplementation of Malonyl-CoA reversed the neuroprotective effect by ACC1 inhibition.Furthermore,inhibition of ACC1 activity upregulated the expression of p-CREB in OGD/Rtreated-neurons,while exogenous supplementation of Malonyl-CoA further downregulated the expression of p-CREB in OGD/R-treated-neurons.Conclusion: After OGD/R treatment,ACC1 expression was decreased in neurons,leading to a decrease in Malonyl-CoA level and an increase in CREB phosphorylation level,which ultimately promoted neuronal survival.This study for the first time elucidated the effect and mechanism of ACC1,a key enzyme of lipid metabolism,on the survival of oxygen and glucose deprivation neurons,and revealed the role of malonyl-COA,a lipid metabolism intermediate,in the survival of oxygen and glucose deprivation neurons.These results provide potential therapeutic targets for reducing I/R brain injury.Part Ⅱ: Application of tetramethylpyrazine(TMP)in ischemic brain injury.Objective:Ischemic stroke caused by middle cerebral artery occlusion can lead to a massive cerebral infarction requiring decompressive craniectomy to reduce intracranial pressure.After decompression,the damaged dura need to be repaired with a dura graft.However,common dural grafts may result in epidural adhesions and scar tissue with no further neuroprotective effect.This part study aims to prepare a nanofiber dura mater(NDM)with anti-adhesion and neuroprotective effects using TMP,PLGA and chitosan(CS)as raw materials based on electrospinning technology,as a potential choice for dura mater graft.Methods: PLGA NDM,PLGA/CS NDM and PLGA/TMP/CS NDM were prepared by electrospinning,EDC/NHS crosslinking and coaxial electrospinning.The morphology of nanofibers was characterized by scanning electron microscopy(SEM),and the chemical structure of nanofibers was characterized by Fourier transform infrared spectroscopy(FTIR).The absorbance of NDM solution and immersion solution at wavelength 280 nm was detected by a full-wavelength microplate analyzer to calculate the encapsulation efficiency(EE)and release profiles of TMP.Cell viability of fibroblasts and SH-SY5Y cell line was detected by CCK8,and LDH release level of SH-SY5Y cells was detected by lactate dehydrogenase(LDH)detection kit.Cell morphology of fibroblasts and SH-SY5Y cell line were observed by Phalloidin-i Fluor 488 and DAPI staining.The proliferation of fibroblasts was analyzed by Ki-67 and DAPI staining.GAP43 and DAPI staining were performed on SH-SY5Y cell lines to calculate the expression of GAP43 protein.Results: SEM and FTIR results showed that PLGA NDM,PLGA/TMP NDM and PLGA/CS NDM were successfully prepared.The EE(%)of TMP is(57.95±2.46)%.In Vitro TMP release profiles showed that TMP underwent burst release in the first 8 h,and then released steadily until 14 d,with a cumulative release ratio of about 80%.The results of CCK8,Phalloidin-i Fluor 488 and Ki-67 staining showed that PLGA NDM and PLGA/CS NDM inhibited the proliferation of fibroblasts.In vitro experiments of SH-SY5Y cells showed that both PLGA/CS NDM and PLGA/TMP/CS NDM at 1,4,7 and 14 days promoted the survival of SH-SY5Y cells damaged by OGD/R.PLGA/TMP/CS NDM immersion solution for 7 and 14 days reduced LDH release level of OGD/R-treated SH-SY5Y cells.The immersion solution of PLGA/TMP/CS NDM at 1,4,7 and 14 days promoted the expression of GAP43 protein in OGD/R-treated SH-SY5Y cells,and promoted the repair of damaged neurite.Conclusion: In this study,PLGA/TMP/CS NDM prepared based on electrospinning technology can inhibit the proliferation of fibroblasts,promote the survival,the expression of GAP43 protein,and the repair of damaged neurite of OGD/R-treated SH-SY5Y cells.PLGA/TMP/CS NDM solves the limitations of TMP such as short half-life and poor water solubility to some extent,and has the potential of preventing epidural adhesion and long-term neuroprotection,which is expected to become a potential choice for dura graft.