The Research Of Mild Hypothermia Reduce Hippocampus Neurons Injury By Nrf2 / Keap1 / ARE Pathways

When cardiac arrest attack head, heart, lungs and other organs suffer from ischemia hypoxia. The damage is irreversible. Cerebral injury is the main cause of death of the patients who have already resuscitated from cardiac arrest. Therefore, cerebral resuscitation has become one of the primary task of Cardiopulmonary Resuscitation(CPR). Clinical studies have shown in recent years that mild hypothermia(MHT) can improve the patient’s neurological function,reduce mortality. However the mechanism remains unclear. Study the mechanism of mild hypothermia reduce brain damage has important scientific value and clinical practical significance.The objectives of the present paper are to study the effect of mild hypothermia therapy for treatment in reduce brain damage by establishing newborn rat hippocampal neurons OGD(oxygen and glucose deprivation/ reperfusion, OGD/R) model. To prove that the protection would occur through activation of the Nrf2 / Keap1 / ARE pathways and alleviate the hippocampal neurons injury.Methods1 To explore the effect of Nrf2 in primary rat hippocampal neurons with OGD/R injury1) To extract hippocampal neurons from newborn rat(<1d) and culture in vitro7 days after inoculation. Then to establish respectively OGD 3h R0 h, OGD 3h R6 h, OGD3hR12h model after random allocation;2) To select the optimum medical concentration through MTT assay and activity detection of LDH release method. To detect the Nrf2 activator sulforaphane(SFN)and the inhibitors brusatol(BST) with different rug concentrations cell survival rate, degree of damage;3) To detect the cell oxidative stress related enzymes and metabolites through using related detection kits. To detect each group of cell culture supernatant of SOD activity, GSH and MDA content;4) To observe neuronal apoptosis, DNA fragmentation in each group of cell through the in situ cell death detection kit, TMR red. And the apoptosis index was calculated for each group of cells.2 To investigate MHT in the role of primary rat hippocampal neurons with OGD/R injury1) To extract hippocampal neurons from newborn rat(<1d) and culture in vitro7 days after inoculation. Then to establish respectively OGD 3h R0 h, OGD 3h R6 h, OGD 3h R12 h model after random allocation;2) To detect the cell oxidative stress related enzymes and metabolites through using related detection kits. To detect cell culture supernatant of control group, MHT group and MHT+BST group with SOD activity, GSH and MDA content;3) To observe neuronal apoptosis, DNA fragmentation in each group of cell through the in situ cell death detection kit, TMR red. And the apoptosis index was calculated for each group of cells.3 To explore the mechanism of the MHT protection through activation of the Nrf2 / Keap1 / ARE pathways1) To extract hippocampal neurons from newborn rat(<1d) and culture in vitro7 days after inoculation. Then to establish respectively OGD 3h R0 h, OGD 3h R6 h, OGD 3h R12 h model after random allocation;2) To detect the HO-1, Nrf2 protein expression changes in OGD3 h R0h, OGD3 h R6h,OGD3h R12 h models respectively with control group, SFN group, BST group, MHT group and MHT+BST group by Western blot;3) To detect the m RNA of HO-1, Nrf2 expression changes in OGD3 h R0h, OGD3 h R6h, OGD3 h R12h models respectively with control group, SFN group, BST group, MHT group and MHT+BST group by RT-q PCR.Result1 To explore the effect of Nrf2 in primary rat hippocampal neurons with OGD/R injury1) The optimal drug concentration was screened SFN20μmol /L, BST40nmol/L by comprehensive MTT, LDH release test results;2) Cellular oxidative stress related enzymes and metabolites of the test results showed: Compared with group C, SFN group significantly reduce the oxidative damage of cells, however, BST group increased cellular oxidative damage;3) TUNEL staining showed: SFN group was significantly better than group C in cells state, the apoptosis index is decline,but the BST group was significantly worse than that of group C cell state, its apoptosis index is arise.2 To investigate MHT in the role of primary rat hippocampal neurons with OGD/R injury1) Cellular oxidative stress-related enzymes and metabolites of the test results showed: Compared with group C, the MHT group significantly reduce oxidative damage cells, however, the MHT + BST group increased cellular oxidative damage;2) TUNEL staining showed: the MHT group was significantly better than the group C in cells state, the apoptosis index is decline, but the MHT+BST group was significantly worse than that of group C cell state, its apoptosis index is arise.3 To explore the mechanism of the MHT protection through activation of the Nrf2 /Keap1 / ARE pathways1) Western blot results showed that: the protein expression of HO-1 and Nrf2 in C Group and SFN Group was increased with time. However, it was reduced with time in BST Group. In the experiment of MHT, HO-1 and Nrf2 protein in MHT Group increased compared with the respective periods Group C and Hypo + BST group;2) RT-q PCR results showed that: the m RNA expression of Nrf2 and HO-1 was increased in SFN group and MHT group respectively. However it express reduce and even no expression in BST group and MHT+BST group.ConclusionMild hypothermia could reduce the hippocampal neurons apoptosis induced OGD / R. The protection may be associated with the activation of Nrf2 / Keap1 /ARE pathway. Through exploring the molecular mechanism of mild hypothermia reduce hippocampus neurons injury, to provide new therapeutic targets for CPR, help to improve the survival rate of ROSC patients and improve clinical outcomes with important scientific value and clinical practical significance.