Research On The Expression And Function Of High Mobility Group Box1 After Experimental Subarachnoid Hemorrhage In Rats

Part 1:Time course analysis of HMGB1 and Rage expression after SAHObjective: To investigate the dynamic changes of HMGB1 and its receptor Rage in the temporal cortex after SAH.Methods: In vivo,48 SD rats were randomly divided into 8 groups of 6 rats each: sham group,SAH-2h group,SAH-12 h group,SAH-1d group,SAH-3d group,SAH-5d group,SAH-7d group,SAH-2w group.Rat SAH model was induced by injection of 0.3ml fresh arterial,nonheparinized blood into the prechiasmatic cistern.All the rats were killed at different time points after SAH.The brain cortex near the blood clots in each group was extracted and used for double immunofluorescence analysis and western blot analysis.Results: According the results of Western blot,SAH induction increased the protein levels of HMGB1 and Rage in the cortex.HMGB1 expression reached the peak at 12 h after SAH,Rage expression reached the peak at 1d after SAH.2.Compared with sham group,both HMGB1 and Rage expression was significantly higher on day 14 after SAH.3.HMGB1 was observed to be widely present in the neuronal nuclei in the sham group,however,on day 14 after SAH,HMGB1 moved from the nucleus into the cytoplasm in neurons.Conclusion: 1.The protein levels of HMGB1 and Rage were up-regulated as early as 2h after SAH induction and reached peak at early stage of SAH;2.The expression of HMGB1 and Rage was not significantly decreased and reached another peak at late phase of SAH;3.Extracellular release of HMGB1 in neurons was detected at 14 d after SAH.Part 2: Effects of HMGB1 on neurovascular remodeling in the late phase of SAHObjective: To explore the significance of high expression of HMGB1 and its function in the late phase of SAH.Methods: 90 SD rats were randomly divided into 5 groups of 18 rats each: sham group,SAH-14 d group,SAH+ Vehicle group,SAH+ ethyl pyruvate(EP)group,SAH+ glycyrrhizin acid(GA)group.Rat SAH model was induced by injection of 0.3ml fresh arterial,nonheparinized blood into the prechiasmatic cistern.Ethyl pyruvate and glycyrrhizin acid are inhibitors of HMGB1,they were administered via intraperitoneal injection once daily on days 7–14 after SAH induction.At 14 d after SAH,all the rats were exsanguinated and brain samples were taken for analysis.We used western blot to analyse the protein levels of HMGB1、 Rage、 NGF、 BDNF and VEGF.Immunofluorescence analysis evaluate the expression and distribution of HMGB1 in neurons in the cortex.Brd U labels cells in S-phase of cell cycle,and DCX is a marker of new neurons.Nissl’s staining show the morphology and number of neurons in the hippocampus and cortex.Enzyme-linked immunosorbent assay(Elisa)measure the levels of TNF-α in serum and cerebrospinal fluid.The dry-wet weight method was used to measure brain water content.Results: 1.On day 14 after SAH,levels of NGF,BDNF and VEGF and numbers of DCX-and Brd U-positive cells were significantly higher in the cortex near the blood clots than those in the SAH+ Vehicle group.Both EP and GA were found to decrease the levels of HMGB1 along with the down-regulated expression of Rage、NGF、BDNF and VEGF and decreased numbers of DCX-and Brd U-positive cells;2.Nissl staining suggested that there were fewer surviving neurons after EP and GA treatment than in the SAH-Vehicle group.3.Inhibition of HMGB1 by EP and GA increased the levels of TNF-α in the serum and Cerebro-Spinal Fluid(CSF)of rats relative to the vehicle group;4.Brain water content in EP and GA groups were also higher than in the vehicle group.Conclusion: In the late phase of SAH,HMGB1 is able to promote the expression of neurotrophic factors,contribute to neural regeneration,alleviate inflammatory response and brain edema.Part 3: Effects of different redox status of HMGB1 and its interaction with Rage in the late phase of SAHObjective: Explore the redox status of HMGB1 and its interaction with Rage in the rat cortex in the late phase of SAH and study its impact on inflammation,neurotrophin expression and cell proliferation activity.Methods: 126 SD rats were randomly divided into 7 groups of 18 rats each: sham group,SAH-14 d group,SAH+ rHMGB group,SAH+ oxy-HMGB group,SAH+ FPS-ZM1 group,SAH+ oxy-HMGB + FPS-ZM1 group.Rat SAH model was induced by injection of 0.3ml fresh arterial,nonheparinized blood into the prechiasmatic cistern.FPS-ZM1 is an antagonist of Rage,oxidized HMGB1 was made from the oxidation of rHMGB1.Saline、rHMGB、oxy-HMGB were administered via prechiasmatic injection at day 7,day 10,day 13.FPS-ZM1 was administered via intraperitoneal injection once daily on days 7–14 after SAH induction.At 14 d after SAH,all the rats were exsanguinated and brain samples were taken for analysis.western blot analyse the protein levels of HMGB1、 Rage、 NGF、 BDNF and VEGF.Immunofluorescence analysis evaluate the expression and distribution of HMGB1 in neurons in the cortex.Brd U labels cells in S-phase of cell cycle,and DCX is a marker of new neurons.Nissl’s staining show the morphology and number of neurons in the hippocampus and cortex.Enzyme-linked immunosorbent assay(Elisa)measure the levels of TNF-α in serum and cerebrospinal fluid.The dry-wet weight method was used to measure brain water content.Results: Both protein levels of HMGB1 and Rage in the r HMG group and oxy-HMG weresignificantly higher than in the vehicle group,oxidized HMGB1 increased NGF,BDNF,and VEGF expression and numbers of DCX-and Brd U-positive cells in the cortex relative to the vehicle group.The expression of NGF,BDNF,and VEGF and numbers of DCX-and Brd U-positive cells were lower in the r HMG group than those in the vehicle group;2.Administration of Rage antagonist FPS-ZM1 suppressed the expression of Rage on day 14 post-SAH along with the decreased expression of NGF,BDNF,and VEGF and numbers of DCX-and Brd U-positive cells;3.Nissl staining indicated that there were significantly fewer surviving neurons in the r HMG group than in the vehicle group,but the numbers remained relatively higher in the oxy-HMG group;4.Serum and CSF levels of TNF-α were markedly higher in the r HMG group than in the vehicle group,but addition of oxidized HMGB1 in the subarachnoid space caused no significant increase in the late phase of SAH.Inhibition of Rage by FPS-ZM1 increased the serum and CSF levels of TNF-α in comparison with the vehicle group;5.In the r HMG group and FPS-ZM1 group,brain water content was higher than in the vehicle group.Conversely,no remarkable difference was found between the oxy-HMG group and the vehicle group.Conclusion: HMGB1 act via Rage to promote brain recovery 14 days after SAH,oxidized HMGB1 might be the prominent form in the late phase of SAH.