BML-111 Reduces Cognitive Impairment In Mice With Sepsis Via The SIRT1/NF-κB Signaling Pathway

Part Ⅰ BML-111 reduces cognitive impairment in mice with sepsisObjective: To explore the effect of BML-111 on cognitive impairment in mice with sepsis.Methods: Cecal ligation and puncture(CLP)was used to introduce sepsis.Part A,a total of 120 male C57 mice were subjected to CLP,then the survived mice were divided into 4 groups(n=10):(1)Sham group: Mice received the same operations with CLP group except for cecal ligation and puncture.2 μl normal saline(NS)was intracerebroventricular injected after CLP.(2)CLP + Vehicle group: Mice were subjected to CLP.2 μl NS was intracerebroventricular injected after CLP.(3)CLP + 0.01mg/kg BML-111 group: Mice were subjected to CLP.2 μl BML-111(0.01mg/kg)was intracerebroventricular injected after CLP.(4)CLP + 0.1mg/kg BML-111group: Mice were subjected to CLP.2 μl BML-111(0.1mg/kg)was intracerebroventricular injected after CLP.The survivors underwent three behavioral tasks at 7 days after the surgery: open-field test,novel-object-recognition test,and fear conditioning test.Part B,a cohort of C57 mice were randomly divided into 4 groups:(1)Sham group: Mice received the same operations with CLP group except for cecal ligation and puncture.2 μl NS was intracerebroventricular injected 30 min before and after immediately CLP.(2)CLP + Vehicle group: Mice were subjected to CLP.2 μl NS was intracerebroventricular injected 30 min before and after immediately CLP.(3)CLP + BML-111 group: Mice were subjected to CLP.2 μl NS was intracerebroventricular injected 30 min before CLP,2 μl BML-111 was intracerebroventricular injected after CLP.(4)CLP + BML-111+ Boc-2 group: Mice were subjected to CLP.2 μl Boc-2(50 μg/kg)was intracerebroventricular injected 30 min before CLP,2 μl BML-111 was intracerebroventricular injected after CLP.After 24 h,48h the tissues of cortex and hiippocampus were tested by western blotting,enzyme-linked immunosorbent assay,tunel assay,immunofluorescence assay.After 7d,the survivors underwent behavioral tasks : Open-field test,novel-object-recognition test,and fear-conditioning test.Results: BML-111 at a dose of 0.1 mg/kg was effective at increasing the recognition index and freezing behaviors when mice were performing the novel-objectrecognition and fear-conditioning tests.Therefore,a BML-111 dose of 0.1 mg/kg was used in the subsequent experiments.CLP did not influence the motor or exploratory activity.Mice in the CLP + vehicle group spent less time investigating the new object,resulting in the recognition index not differing between the new object and the familiar one,which suggests that sepsis resulted in cognitive impairments.Administering BML-111 reduced the cognitive impairment,which presented as an improvement in recognition index compared with that for the familiar object.In addition,pretreatment with Boc-2(which is an antagonist of ALX)before CLP and BML-111 treatment can abolish the effects of BML-111.The freezing times in the contextual and tone conditional tasks were significantly lower in the CLP + vehicle group than in the sham group.BML-111 significantly increased the freezing behaviors when compared with CLP + vehicle group.Administering Boc-2 before CLP and BML-111 treatment significantly decreased the freezing time compared to that in the BML-111-treated group.CLP significantly reduced the expression levels of PSD95 and Synapsin1 compared to the sham group.When BML-111 was administered to the CLP mice,the levels of PSD95 and Synapsin1 were increased sharply in the cortex and hippocampus.After administering Boc-2(an antagonist of ALX),the protein levels of PSD95 and Synapsin1 were significantly decreased in the cortex and hippocampus.Compared with the sham group,large numbers of TUNEL-positive cells were observed in the cortex and hippocampus of mice subjected to CLP.BML-111 decreased the number of TUNEL-positive cells,while pretreatment with Boc-2 increased the number of TUNEL-positive cells in the cortex and hippocampus compared with BML-111 treatment alone.Large numbers of Iba1-and GFAP-positive cells were observed in the cortex and hippocampus of CLP mice,whereas very few such cells were visible in the sham-operated group.It should be emphasized that BML-111 decreased the numbers of Iba1-and GFAP-positive cells in the cortex and hippocampus relative to the CLP + vehicle group.Administering Boc-2 before CLP and BML-111 treatment increased the numbers of Iba1-and GFAP-positive cells in the cortex and hippocampus relative to the BML-111-treatment group.The presence of sepsis up-regulated the proinflammatory mediators,TNF-αand IL-1β,in the cortex and hippocampus relative to the sham group.Administering BML-111 after CLP significantly reduced the increases in these mediators relative to mice subjected to CLP.Administering Boc-2 before CLP and BML-111 treatment was effective at increasing the expression levels of TNF-α and IL-1β.Graph Pad Prism software(version 5 for Windows,San Diego,CA,United States)was used for all statistical analyses.All values were expressed as mean ± SEM(?x±SEM).The move time and distance,fear-conditioning data,Western-blot results,TUNEL positive cells,microglia cells,astrocytes cells and Elisa data were analyzed using one-way ANOVA with the Student–Newman–Keuls tests.The novel-objectrecognition data were analyzed using two-way ANOVA with the Bonferroni tests.Statistical significance was accepted when P < 0.05.Conclusion: BML-111 reduces neuroinflammation and cognitive impairment in mice with sepsis.Part Ⅱ BML-111 reduces cognitive impairment in mice with sepsis via the SIRT1/NF-κB signaling pathwayObjective: To investigate the possible mechanisms of BML-111 on cognitive impairment in mice with sepsis.Methods: CLP was used to introduce sepsis.All C57 mice were randomly divided into 4 groups:(1)Sham group: Mice received the same operations with CLP group except for cecal ligation and puncture.2 μl NS was intracerebroventricular injected every 2 days for a total of three times prior to performing the CLP operation.2 μl NS was intracerebroventricular injected after immediately CLP.(2)CLP + Vehicle group: Mice were subjected to cecal ligation and puncture(CLP).2 μl NS was intracerebroventricular injected every 2 days for a total of three times prior to performing the CLP operation.2 μl NS was intracerebroventricular injected after immediately CLP.(3)CLP + EX527 group: Mice were subjected to CLP.2 μl EX527 was intracerebroventricular injected every 2 days for a total of three times prior to performing the CLP operation.2 μl NS was intracerebroventricular injected after immediately CLP.(4)CLP + BML-111 group: Mice were subjected to CLP.2 μl NS was intracerebroventricular injected every 2 days for a total of three times prior to performing the CLP operation.2 μl BML-111 was intracerebroventricular injected after CLP.(5)CLP + BML-111 + EX527 group: Mice were subjected to CLP.2 μl EX527 was intracerebroventricular injected every 2 days for a total of three times prior to performing the CLP operation.2 μl BML-111 was intracerebroventricular injected after CLP.After 24 h,48h the tissues of cortex and hippocampus were tested by western blotting,enzyme-linked immunosorbent assay,tunel assay,immunofluorescence assay.After 7d,the survivors underwent behavioral tasks:Open-field test,novel-object-recognition test,and fearconditioning test.Graph Pad Prism software(version 5 for Windows,San Diego,CA,United States)was used for all statistical analyses.All values were expressed as mean ± SEM(?x±SEM).The move time and distance,fear-conditioning data,Western-blot results,TUNEL positive cells,microglia cells,astrocytes cells and Elisa data were analyzed using one-way ANOVA with the Student–Newman–Keuls tests.The novel-objectrecognition data were analyzed using two-way ANOVA with the Bonferroni tests.Statistical significance was accepted at P < 0.05.Results: sepsis led to a significantly decrease in cytoplasmic p65 relative to the sham group.Meanwhile,neuroinflammation caused by sepsis significantly up-regulated the protein levels of nuclear p65 relative to the control group.A particularly notable observation was that BML-111 treatment significantly increased the expression of cytoplasmic p65 relative to the CLP + vehicle group and decreased the expression of nuclear p65 relative to the CLP + vehicle group.When Boc-2 was administered before CLP and BML-111 treatment,the protein levels of cytoplasmic p65 was significantly reduced relative to the CLP + BML-111 group and the protein level of nuclear p65 was significantly increased relative to the BML-111-treatment group.Additional,neuroinflammation also increased the protein levels of Ac-NF-k B compared with sham group.BML-111 treatment significantly decreased the expression of Ac-NF-k B relative to the CLP + vehicle group.Administrating Boc-2 before CLP and BML-111 treatment was effective at increasing the protein levels of Ac-NF-k B relative to the BML-111-treatment group.CLP decreased the SIRT1 level in the cortex and hippocampus relative to the sham group.As expected,post-treatment with BML-111 up-regulated the expression of SIRT1 relative to the CLP + vehicle group.Administering Boc-2 before CLP and BML-111 treatment down-regulated the expression of SIRT1 relative to the BML-111-treatment group.EX527,which is an antagonist of SIRT1,was utilized in the experiments.The protein levels of SIRT1 decreased in CLP + Vehicle group and CLP + EX527 group relative to the sham group.Administering BML-111 increased the protein levels of SIRT1 relative to the CLP + Vehicle group.EX527 significantly inhibited the expression level of SIRT1 relative to the CLP + BML-111 group.EX527 down-regulated cytoplasmic p65(P < 0.001;P < 0.001),up-regulated nuclear p65,increased Ac-NF-κB,increased the levels of TNF-α and IL-1β in the cortex and hippocampus,increased the numbers of activated microglia and astrocyte cells,increased the number of TUNEL positive cells relative to CLP + BML-111 group.EX527 made the recognition index for the new object not differing from that for the familiar one and decreased the freezing time compared with the CLP + BML-111 group.Conclusion: BML-111 reduces the neuroinflammation and cognitive impairment induced by sepsis via SIRT/NF-k B signaling pathway.